Vitamins and Minerals Biofortification of Edible Plants (New York Academy of Sciences) [1 ed.] 1119511119, 9781119511113

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Vitamins and Minerals Biofortification of Edible Plants

Vitamins and Minerals Biofortification of Edible Plants Edited by

Noureddine Benkeblia

Department of Life Science and Laboratory of Tree Fruit and Aromatic Crop, The Biotechnology Centre The University of the West Indies Mona Campus Kingston, Jamaica

This edition first published 2020 © 2020 John Wiley & Sons Ltd All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions. The right of Noureddine Benkeblia to be identified as the author of the editorial material in this work has been asserted in accordance with law. Registered Office(s) John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, USA John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial Office Boschstr. 12, 69469 Weinheim, Germany For details of our global editorial offices, customer services, and more information about Wiley products, visit us at www.wiley.com. Wiley also publishes its books in a variety of electronic formats and by print‐on‐demand. Some content that appears in standard print versions of this book may not be available in other formats. Limit of Liability/Disclaimer of Warranty While the publisher and authors have used their best efforts in preparing this work, they make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives, written sales materials or promotional statements for this work. The fact that an organization, website, or product is referred to in this work as a citation and/or potential source of further information does not mean that the publisher and authors endorse the information or services the organization, website, or product may provide or recommendations it may make. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for your situation. You should consult with a specialist where appropriate. Further, readers should be aware that websites listed in this work may have changed or disappeared between when this work was written and when it is read. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Library of Congress Cataloging‐in‐Publication Data Names: Benkeblia, Noureddine, editor. Title: Vitamins and minerals bio-fortification of edible plants / edited by   Noureddine Benkeblia. Description: First edition. | Hoboken, NJ : Wiley, 2020. | Includes index. Identifiers: LCCN 2019051491 (print) | LCCN 2019051492 (ebook) | ISBN   9781119511113 (hardback) | ISBN 9781119511137 (adobe pdf) | ISBN   9781119511151 (epub) Subjects: LCSH: Crop improvement. | Food crops–Biotechnology. | Plants,   Edible. | Enriched foods. Classification: LCC SB106.I47 V58 2020 (print) | LCC SB106.I47 (ebook) |   DDC 635/.2–dc23 LC record available at https://lccn.loc.gov/2019051491 LC ebook record available at https://lccn.loc.gov/2019051492 Cover Design: Wiley Cover Images: Prevor Drake/Getty Images, © Shinyfamily/Getty Images, © CasarsaGuru/Getty Images, ©Andrii Yalanskyi/Shutterstock Set in 9.5/12.5pt STIXTwoText by SPi Global, Pondicherry, India Printed and bound by CPI Group (UK) Ltd, Croydon, CR0 4YY 10  9  8  7  6  5  4  3  2  1

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Contents List of Contributors  vii Foreword  xi   1 Biofortification of Edible Plants: Set the Stage for Better Nutrition  1 Noureddine Benkeblia   2 Food Fortification: What’s in It for the Malnourished World?  27 Barbara Poniedziałek, Kinga Perkowska, and Piotr Rzymski   3 Modern Biotechnologies and Mineral Biofortification of Edible Crops  45 Noureddine Benkeblia and Kathleen L. Hefferon   4 Biotechnologies and Vitamins Biofortification of Edible Crops  71 Noureddine Benkeblia and Kathleen L. Hefferon   5 Carotenoids Biofortification of Sweet Potatoes  87 Noureddine Benkeblia, Elisabete M. Pinto, and Marta W. Vasconcelos   6 Improving Iron Nutrition in Plant Foods: The Role of Legumes and Soil Microbes  103 Mariana Roriz, Marta Barros, Paula M. L. Castro, Susana M. P. Carvalho, and Marta W. Vasconcelos   7 Biofortification of Carotenoids in Agricultural and Horticultural Crops: A Promising Strategy to Target Vitamin A Malnutrition  123 Hulikere Jagdish Shwetha, Shivaprasad Shilpa, Bangalore Prabhashankar Arathi, Marisiddaiah Raju, and Rangaswamy Lakshminarayana   8 Agronomic Biofortification from a Stakeholder’s Viewpoint: Evidence from Studies on Iodine-Enriched Foods in Uganda  163 Solomon Olum, Joshua Wesana, Walter Odongo, Joseph Mogendi, Collins Okello, Dominic Webale, Anselimo Makokha, Duncan Ongeng, Xavier Gellynck, and Hans De Steur

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Contents

  9 Biofortification of Cereals through Foliar Application of Minerals  191 Shahid Hussain, Ayta Umar, Mamoona Amir, and Muhammad Aon 10 NAS Overexpression and Rice Zinc Biofortification: An Insight, Current Knowledge, and Outlook  223 Yuta Kawakami and Navreet K. Bhullar Index  235

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List of Contributors Mamoona Amir Faculty of Agricultural Sciences and Technology, Institute of Food Science and Nutrition, Bahauddin Zakariya University, Multan, Pakistan Muhammad Aon Department of Soil Science, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Pakistan Bangalore Prabhashankar Arathi Department of Biotechnology, Bangalore University, Bengaluru, Karnataka, India Marta Barros CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal Noureddine Benkeblia Department of Life Science and Laboratory of Tree Fruit and Aromatic Crop, The Biotechnology Centre, The University of the West Indies, Kingston, Jamaica Laboratory of Crop Science, Department of Life Sciences, The University of the West Indies, Kingston, Jamaica

Navreet K. Bhullar Department of Biology, Institute of Molecular Plant Biology, ETH Zurich (Swiss Federal Institute of Technology), Zurich, Switzerland Susana M. P. Carvalho CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal GreenUPorto & DGAOT, Faculty of Sciences, University of Porto, Vairão, Portugal Paula M. L. Castro CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal Xavier Gellynck Department of Agricultural Economics, Ghent University, Ghent, Belgium Kathleen L. Hefferon Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada Shahid Hussain Department of Soil Science, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Pakistan

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List of Contributors

Yuta Kawakami Department of Biology, Institute of Molecular Plant Biology, ETH Zurich (Swiss Federal Institute of Technology), Zurich, Switzerland

Elisabete M. Pinto CBQF ‐ Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal

Rangaswamy Lakshminarayana Department of Biotechnology, Bangalore University, Bengaluru, Karnataka, India

Barbara Poniedziałek Department of Environmental Medicine, Poznan University of Medical Sciences, Poznań, Poland

Anselimo Makokha Department of Food Science and Technology/Nutrition, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya Joseph Mogendi African Population and Health Research Center, Nairobi, Kenya Walter Odongo Department of Rural Development and Agribusiness, Gulu University, Gulu, Uganda Collins Okello Department of Biosystems Engineering, Gulu University, Gulu, Uganda Solomon Olum Department of Agricultural Economics, Ghent University, Ghent, Belgium Department of Food Science and Postharvest Technology, Gulu University, Gulu, Uganda Duncan Ongeng Department of Food Science and Postharvest Technology, Gulu University, Gulu, Uganda Kinga Perkowska Department of Environmental Medicine, Poznan University of Medical Sciences, Poznań, Poland

Marisiddaiah Raju Department of Botany, Molecular Biology Laboratory, Bangalore University, Bengaluru, Karnataka, India Mariana Roriz CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal Piotr Rzymski Department of Environmental Medicine, Poznan University of Medical Sciences, Poznań, Poland Shivaprasad Shilpa Department of Biotechnology, Bangalore University, Bengaluru, Karnataka, India Hulikere Jagdish Shwetha Department of Biotechnology, Bangalore University, Bengaluru, Karnataka, India Hans De Steur Department of Agricultural Economics, Ghent University, Ghent, Belgium Ayta Umar Department of Soil Science, Faculty of Agricultural Sciences and Technology, Bahauddin Zakariya University, Multan, Pakistan

List of Contributors

Marta W. Vasconcelos CBQF ‐ Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal Dominic Webale School of Agricultural and Environmental Sciences, Mountains of the Moon University, Fort Portal, Uganda

Department of Rural Development and Agribusiness, Gulu University, Gulu, Uganda Joshua Wesana Department of Agricultural Economics, Ghent University, Ghent, Belgium School of Agricultural and Environmental Sciences, Mountains of the Moon University, Fort Portal, Uganda

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Foreword Minerals and vitamins deficiencies form one of the most important global health issues, and according to the World Health Organization (WHO), over 2 billion people are suffering from at least one of these different types of minerals and/or vitamin deficiencies. Consequently, different diseases are resulting from these deficiencies particularly physical and mental development in children, causing more vulnerability or exacerbating these diseases. Unfortunately, most of these deficiencies are not visible, and this “hidden hunger” affects mostly the developing countries particularly African ones. Among the numerous minerals and vitamins required daily, the most prevalent deficiencies are vitamin A, vitamin D, folate, iodine, iron, zinc, and calcium. With the growing population, this hidden hunger is expected to worsen, and therefore, it is urgent to plan and develop successful and sustainable oriented strategies to prevent or alleviate these deficiencies and these strategies require fundamental and applied scientific knowledge. One of the most promising, efficient, sustainable, and cost‐effective strategies in combating this hidden hunger is increasing minerals and vitamins content through biofortification targeting widely consumed staple foods. The biofortification of these crops through different strategies such as cultural practices, breeding programs, or modern biotechnologies (molecular engineering) will contribute undoubtedly in the underlying problem of mineral and vitamin deficiencies by increasing the daily‐intake amount of minerals and vitamins through food systems. Although considerable and significant progress has been made in developing high mineral and vitamin contents crops, the consumers’ acceptance still remains a barrier and a real challenge in generalizing biofortified crops, particularly in countries where deficiencies are prevalent. In this book, after a general overview, the chapters describe different approaches and opportunities for the biofortification edible plants to achieve the goal aiming to improve the diet and setting the stage for malnutrition eradication or alleviation through sustainable agriculture. In addition to the description of the biofortification approaches, specific crops biofortification are described with different examples of biofortified crops or specific biofortification minerals and vitamins of edible crops.

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1 Biofortification of Edible Plants Set the Stage for Better Nutrition Noureddine Benkeblia Department of Life Science and Laboratory of Tree Fruit and Aromatic Crop, The Biotechnology Centre, The University of the West Indies, Kingston, Jamaica Laboratory of Crop Science, Department of Life Sciences, The University of the West Indies, Kingston, Jamaica

1.1 ­Introduction The human organism requires a large number of organic and mineral nutrients that are crucial for the growth, development, and the prevention of diseases and disorders. These nutrients, required at relatively high levels – macronutrients – or low to very low levels – micronutrients – are supplied either by our plant or animal food intakes. However, often the daily diet is balanced in calories and quantity, but does not provide the required amount of these nutrients, leading to malnutrition. Consequently, this malnutrition – or nutrients deficiency – can lead to a variety of metabolic and health problems such as digestive problems (Brodeur et  al. 1993), skin disorders (Prendiville and Manfredi 1992), stunted or defective bone growth (Branca and Ferrari 2002) … etc. To compensate these low levels of nutrients in plants, biofortification of edible plants is becoming one the most efficient strategies to overcome malnutrition, and many foods such as cereals, fruits, and vegetables are being fortified with nutrients that are needed to ­prevent minerals and vitamins deficiencies (Nestel et al. 2006). From ancient times, agriculture has been the primary source of food and all nutrients for human, and the production systems have been subject to numerous changes to ensure quantitative and qualitative food supplies. With human development, the Industrial Revolution, and then the Green Revolution, food and nutrition turned to agriculture and agro‐processing as a primary mean to mitigate, if not eradicating, nutrients deficiencies and malnutrition (Welch 2005). On another hand, the development of novel life science technologies and biofortification of edible plants is regarded as a powerful tool to reduce malnutrition and improve dietary intake of essential minerals and vitamins in staple foods (Figure 1.1). New discoveries in biochemistry and molecular biology have led to incredible development of advanced biotechnology and great promises for improving the output of bioavailable micronutrients from agricultural systems (Welch 2005). However, we need to pragmatically assess whether the biofortification is compatible with the diet diversification Vitamins and Minerals Biofortification of Edible Plants, First Edition. Edited by Noureddine Benkeblia. © 2020 John Wiley & Sons Ltd. Published 2020 by John Wiley & Sons Ltd.

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Figure 1.1  Biofortified crops generated by different approaches: transgenic, agronomic, and breeding. Staple cereals, most common vegetables, beans, and fruits have been targeted by all three approaches. Some crops have been targeted by only one or two approaches depending on its significance and prevalence in the daily human diet. (From Garg et al. 2018. Open access publication under Creative Commons Attribution License [CC BY] terms with free permission).

and how it might impact agricultural biodiversity for long‐term sustainability (Bouis and Welch 2010; Johns and Eyzaguirre 2007). This chapter provides a general overview of the different approaches and opportunities for the biofortification of edible plants to achieve the goal of improving human diet and setting the stage for the eradication or alleviation of malnutrition through sustainable agriculture.

1.2 ­Biofortification and Nutrition To alleviate nutritional issues and nutrient deficiencies, biofortification of edible plants is considered the most appropriate approach. Modifying dietary customs of the population runs into likely resistance from communities. By contrast, biofortification focuses on

1.2  ­Biofortification and Nutritio

improving the nutritional content of region’s current agricultural biodiversity, preserving its habits and customs (Johns and Eyzaguirre 2007). Biofortification of food crops thus has the potential of reaching all the population and communities, particularly rural poor and vulnerable ones with no or limited access to industrially biofortified foods, or where conventional biofortification is difficult or cannot be implemented for technical, economic, or social reasons, and where large quantities of staple rather than nutrition foods crops is consumed. In recent decades, action has been taken to address malnutrition and micronutrient intakes issues in developing countries. Biofortification technology has been identified as a priority initiative; however, it became evident that this technology might also benefit developed countries consumers as well. Therefore, biofortification became more common in developing countries and evidence of malnutrition mitigation nutrient, bioavailability enhancement and biofortified crops acceptability, are raising more interests. Recent data are showing that 150 different varieties of biofortified crops belonging to 10 different crop species have been released in 30 different countries, of which 27 are developing countries, while 12 other crop species are under evaluation for release in 21 other different countries (Bouis and Saltzman 2017; Bouis et al. 2006; Global Panel 2015). In this regard, many examples can be cited, and some of the studies have confirmed the nutritional value and cost‐ effectiveness of high‐iron bean, orange‐flesh sweet potato, cassava, maize, rice, and pearl millet (Global Panel 2015). Vitamin A deficiency (VAD) is the most prevalent nutrient deficiency in young children in the developing countries, with more than 200 million children under the age five worldwide (WHO 2009). VAD has been rated as the first public health problem in more than 70 countries, and this deficiency affects about 33% of children aged between six months and five years in 2013, with 48% found in sub‐Saharan Africa and 44% in South Asia (WHO 2009). In this regard, different studies showed the role of biofortified crops in alleviating VAD deficiency and improving the nutritional status of the population. In developing countries, a study carried out in sub‐Saharan Africa showed that the daily vitamin A needs of young children can be covered by the intake of 100 g of orange‐fleshed sweet potato (OFSP), a carotenoid‐biofortified tuber (Low et al. 2017). Similar observation was noted in Zambia, where inadequate vitamin A intake prevalence was reduced by 3% (Lividini and Fiedler 2015), and diarrhea was reduced by biofortified crops as well (Jones and de Brauw 2015). Mineral deficiencies are also a major concern, and studies have shown that biofortification might be one of the most cost‐effective approaches in alleviating this public health issue (Broadley et  al. 2008, 2009). Using Zn‐biofortified wheat, valuable increases in Zn absorption have been achieved (Rosado et  al. 2009). Feeding two‐ year‐olds with zinc and iron biofortified pearl millet more than adequately enhanced the absorption of both minerals to meet the dietary requirement (Kodkany et al. 2013), and the iron status of schoolchildren (12 to 16 years old) and women fed iron‐biofortified beans and pearl millet was significantly improved (Finkelstein et al. 2015; Haas et al. 2016). Nevertheless, assessment of the efficacy of biofortified foods for enhancing human nutritional status and alleviating malnutrition requires further research in the laboratory,

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as well as community‐based trials. Research should consider the impacts of ­biofortified crops for larger groups of different gender and age and for long‐term consumption (Bouis and Saltzman 2017). Furthermore, additional research is required to assess the nutrients bioavailability of various genotypes of biofortified crops, in particular genetic engineered crops, using different in‐vitro and/or in‐vivo tests. Additionally, trials need to be carried out to evaluate the agricultural, environmental, and socioeconomic impacts, and these trials must include the communities, the stakeholders, and the policy makers as well (King 2002). Biofortification of food plants also causes potential alterations of the plants metabolism, and these alterations should be thoroughly assessed beyond the few studies that have already analyzed these alterations. The alteration of these different metabolic pathways might affect growth, development, and productivity of these plants, and it is imperative to determine to what extent these alterations can be minimized or even avoided. However, recent development in omics, particularly metabolomics and related techniques, is significantly contributing to deciphering the potential alterations in plants caused by biofortification (Hall et al. 2008).

1.3 ­Cultural Practices and Plants Biofortification Agricultural practices are more likely the simplest and readily most accessible to farmers to overcome the problem of nutrients deficiency of edible plants, and these agronomic‐based strategies might be interesting alternative solutions. Indeed, agronomic‐based strategies have shown be efficient in improving nutrient contents by many folds. Although the application methods differ, soil fertilization and/or foliar application of fertilizers at different stages of plant growth have shown to increase the nutrients levels of many crops, and fertilization as agronomic strategy for plants biofortification is considered to be a good and effective way (Mao et al. 2014; Zhao and Shewry 2011) (Figure 1.2) (Table 1.1). Another strategy consisting of intercropping between dicots and gramineous species to increase mineral contents of crops (Zuo and Zhang 2009). Worldwide, iron (Fe) is the most prevalent deficient nutrient. Different attempts to increase iron contents of food crops have been conducted, and the results have been encouraging. The application of iron fertilizers, either inorganic or chelated forms, did not show any Fe increase in cereal. However, improved nitrogen nutrition of plants increased Fe content (Aciksoz et al. 2011; Cakmak 2012; Cakmak et al. 2010), and this might be due to the contrasting abilities of cereals to acquire Fe because the phyto‐siderophores chemistry is species‐specific and determines iron absorption by plants (Bashir et  al. 2006). Similarly, foliar application of Fe‐fertilizers had shown no or little positive effect on grain‐ Fe content (Aciksoz et al. 2011); however, when urea (Aciksoz et al. 2011) or boron (Jin et al. 2008) were incorporated to foliar fertilizers, grain Fe concentration was increased by threefold. Hence, nitrogen plant status deserves special attention in Fe‐crops biofortification (for further details, see Chapter 6). Zinc, which is often associated to iron, is the most ubiquitous micronutrient deficiency issue in plants, and this deficiency is to a large extent caused by soils factors

1.3  ­Cultural Practices and Plants Biofortificatio Foliar spray

Fertilization

Figure 1.2  Foliar application of mineral solution for corps biofortification.

including Zn deficiency (Alloway 2009; Cakmak 2008). Many plants, particularly cereals, might be fortified with Zn using agronomic approaches. Trials showed that foliar application of ZnSO4 to wheat crop increased grain Zn concentration by threefold (Cakmak 2008). However, trials on Zn fertilization did not increase the concentration of Zn in rice grain (Wissuwa et al. 2008). Other studies showed that the application of inorganic Zn salts ameliorates crops Zn deficiency (Takkar and Walker 1993), while when high Zn concentrations in grains are desired, studies showed that soil fertilization combined with foliar application is the most effective method of Zn application (Yilmaz et al. 1997). These discrepancies between the different studies on Zn fertilization and Zn biofortification of plants might be explained by the zinc chemistry and its behaviour in soils, its concentration, soil pH, calcite and inorganic matter, and the concentration of other minerals such as Na, Ca, Mg (Alloway 2009) (for details, see Chapter 10). By adding a small amount of selenium (Se) to fertilizers, its concentration was increased in many plants in Finland, which was the first country to adopt this agronomic approach (Hartikainen 2005), and a similar strategy was adopted in the UK, where Se concentration in crops was increased by about tenfold (Broadley et al. 2010). Addition to iodine (I) to fertilizers also showed that plant can be fortified with higher concentration of iodine. Trials iodine fertigation (iodination of irrigation water) increased significantly iodine intake through foods intake (Cao et  al. 1994; DeLong et al. 1997).

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Table 1.1  Studies of crops biofortification using agronomic approaches. Class

Crops

Nutrients

References

Cereals

Rice

Iron

Fang et al. (2008), He et al. (2013), Wei et al. (2012a), Yuan et al. (2013)

Zinc

Boonchuay et al. (2013), Fang et al. (2008), Guo et al. (2016). Jiang et al. (2008), Mabesa et al. (2013), Ram et al. (2016), Shivay et al. (2008, 2015), Wei et al. (2012b)

Selenium

Chen et al. (2002), Fang et al. (2008), Giacosa et al. (2014), Liu and Gu (2009), Premarathna et al. (2012), Ros et al. (2016), Xu and Hu (2004)

Iron

Aciksoz et al. (2011)

Zinc

Cakmak and Kutman (2018), Cakmak et al. (2010), Yang et al. (2011)

Selenium

Aro et al. (1995)

Maize

Zinc

Alvarez and Rico (2003), Fahad et al. (2015), Lopez‐Valdivia et al. (2002), Wang et al. (2012), Zhang et al. (2013)

Soybean

Selenium

Yang et al. (2003)

Wheat

Legumes

Chickpea

Vegetables

Zinc

Shivay et al. (2015)

Selenium

Poblaciones et al. (2014)

Pea

Iron

Poblaciones and Rengel (2016)

Common Bean

Zinc

Ibrahim and Ramadan (2015), Ram et al. (2016)

Cow pea

Iron

Márquez‐Quiroz et al. (2015)

Potato

Zinc

White et al. (2012b)

Selenium

Cuderman et al. (2008), Poggi et al. (2000)

Sweet Potato

β‐carotene

Laurie et al. (2012)

Carrot

Iodine

Smolen et al. (2016)

Selenium

Smolen et al. (2014)

Lettuce Fruits

Tomato

Iodine

Smolen et al. (2014)

Selenium

Carvalho et al. (2003), Smolen et al. (2014)

Iodine

Landini et al. (2011)

Source: From Garg et al. (2018) with some modifications. Open access publication under Creative Commons Attribution License [CC BY] terms with free permission.

1.4 ­Conventional Breeding and Crops Biofortification For a long time, plant breeding was limited in crossing individuals of interesting and targeted traits and thereafter selecting the interesting varieties. This approach changed with the discovery of the genetic and the sexual propagation of plants. The twentieth century has seen the introduction of hybrid technology and boosted breeding of many crops (Poletti and Sautter 2005). Vitamins and micronutrients biofortification of crops through breeding

1.5  ­Molecular Engineering and Crops Biofortificatio

has been considered for decades (Graham et al. 1998, 1999). This strategy is based on two different approaches: 1) Explore the genetic diversity of the existing species by identifying the parental genotypes potentially interesting for crosses. 2) Identify the existing varieties or germplasms (Welch and Graham 2004). However, and before contemplating this strategy, some criteria should be considered. Among these breeding criteria, the first to consider is the productivity or the yield of the bred crop (Bouis 1996; Graham et al. 1998), the biofortification level, the stability of the enriched mineral or vitamin across cropping, the varying cropping and environmental conditions, and the nutritional bioavailability of the nutrient (Ortiz‐Monasterio et  al. 2007; Welch and Graham 2004). Many attempts have been successful in increasing vitamins and minerals by conventional breeding, including increasing the concentration of β‐carotene and carotenoids (Champagne et al. 2013; Pixley et al. 2013; Suwarno et al. 2014), iron and zinc (Pixley et al. 2011; Velu et al. 2007), as well as other minerals such as selenium (Graham et al. 1999, 2005). However, crops biofortification through breeding has shown some limits and constraints. One example is the inverse correlation noted between the increase of iron and zinc content of crops and the yield due to a dilution effect caused by enhanced starch content (Table 1.2) (Bänzinger and Long 2000). Indeed, crop breeding for biofortification has targeted widely used staple food like maize, cassava, sweet potato, banana, and some legumes. Vitamin A–rich orange sweet potato is likely the most successful development in biofortification resulting from crop breeding. Although many food crops bred, especially fruits, are providing sufficient levels of nutrients to targeted populations, a greater emphasis is being laid on transgenic research. However, breeding is much accepted by the greater consumers compared to transgenic crops. Unfortunately, crops biofortification through conventional breeding have known limited success in general, because this approach requires years to achieve significant enhancement in adapted varieties. The absence of key vitamins and minerals in many crops reflects the fact that the corresponding metabolic pathways are absent, truncated, or inhibited in the targeted species. Therefore, it is obvious that in order to enhance the biosynthesis vitamins pathways and minerals accumulation, genes encoding key enzymes of vitamins biosynthesis and minerals accumulation should be introduced using transgenic methods (Christou and Twyman 2004; Khan et al. 2013; Zhu et al. 2007).

1.5 ­Molecular Engineering and Crops Biofortification During the last few decades, molecular engineering has entered an exciting phase of rapid development and discovery. More generally, genomics tools have been and are still being developed and applied to improve plant crops for human benefit. Most of the edible crops are now subject of in‐depth molecular engineering investigations driven by a need for support of biofortification programmes to enhance nutritional quality of food‐ plants. Indeed, biofortification of crops using genetic techniques focuses on genes with

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Table 1.2  Studies of crops biofortification using conventional breeding approaches. Class

Crops

Nutrients

References

Cereals

Rice

Iron

Sperotto et al. (2012)

Zinc

Gregorio et al. (2000)

Iron

Cakmak et al. (1999, 2004), Monasterio and Graham (2000), Welch et al. (2005)

Zinc

Velu et al. (2014)

Vitamin A, Provitamin A and Carotenoids

Maqbool et al. (2018), Palmer et al. (2016), Pixley et al. (2013)

Vitamin E

Goffman and Böhme (2001), Muzhingi et al. (2016)

Iron

Reddy et al. (2005)

Zinc

Reddy et al. (2005)

β‐carotene

Reddy et al. (2005)

Iron

Rai et al. (2012), Velu et al. (2007)

Zinc

Rai et al. (2012), Velu et al. (2007)

Iron

Kumar et al. (2016)

Zinc

Kumar et al. (2016)

Wheat

Orange Maize

Sorghum

Millet

Legumes

Lentils Cow pea Bean

Vegetables

Potato

Iron

Santos and Boiteux (2013)

Zinc

Santos and Boiteux (2013)

Iron

Beebe et al. (2000), Gelin et al. (2007), Hoppler et al. (2014)

Zinc

Beebe et al. (2000), Blair et al. (2009), Gelin et al. (2007)

Iron

Brown et al. (2010), Burgos et al. (2007), Haynes et al. (2012)

Zinc

Brown et al. (2010), Burgos et al. (2007), Haynes et al. (2012)

Copper

Haynes et al. (2012)

Manganese

Haynes et al. (2012)

Sweet Potato

See Chapter 4

Cauliflower

β‐carotene

Muthukumar (2016)

Cassava

β‐carotene Carotene

Chavez et al. (2005), Maziya‐ Dixon et al. (2000), Peninah et al. (2014)

Iron

Chavez et al. (2005), Maziya‐ Dixon et al. (2000)

Source: From Garg et al. (2018) with some modifications. Open access publication under Creative Commons Attribution License [CC BY] terms with free permission.

1.6  ­Conclusions and Future Perspectiv

different expression patterns targeting for development into breeding new varieties of crops with greatest levels of nutrients. Moreover, the efficiency in genetic techniques has been enhanced through the use of diagnostic DNA‐based markers to develop biofortified crops and the approach of using modern technologies might be viewed as one of the key decisions that breeders make concerning the objectives of biofortification programmes to achieve this goal. The genetic transformation of food‐plants is considered a faster method to achieve the nutritional improvement of crops, and the transgenic approaches might be a good and more realistic alternative, particularly where breeding approaches have not been successful or to overcome its limitation (Brinch‐Pedersen et al. 2006; Kumari et al. 2014; Zhu et al. 2007). Consequently, the number and genetically modified and biofortified crops with different vitamins and minerals is much larger compared to the number of biofortified crops using agronomic or conventional breeding. Furthermore, spectacular advances in genes and genotyping had led at dissecting in depth the architecture of important traits providing the most advanced genomic platforms and analysis methods available for food‐ plants. These techniques combined to new advances in genomic research have led to unprecedented access into the structure and function of crop genomes. From a genetic engineering perspective, different transgenic crops have been developed because of the identification of the respective genes encoding the biosynthesis of vitamins and minerals accumulation in plants (Table 1.3) (White et al. 2012a,b; Zhang et al. 2009). For example, many cereals have been successfully biofortified, such as β‐carotene, vitamin B9 (folate), iron and zinc biofortified rice, high provitamin A and iron content wheat, provitamin A, vitamin E, ascorbic acid and iron biofortified maize, high zinc content barley, and β‐carotene biofortified sorghum. Furthermore, many other legumes, fruits, and vegetables have been biofortified with minerals and vitamins using transgenic approaches (Table 1.4). Nevertheless, crops biofortification using transgenic approaches is constrained by two limitations. The first limitation is the knowledge gap in the genes’ functions and their interaction with the environment, and without this knowledge the transgenic transformation of plants remains still limited to some nutrients or vitamins and to some species as well. The second and perhaps more constraining limitation is the regulatory issues restricting the development and commercialization of transgenic biofortified crops (Johnson et al. 2007; Powell 2007; Ramessar et al. 2009). Moreover, in order to select appropriate crops for minerals and vitamins biofortification using transgenic transformation, two criteria are essential. First, the selected crop for transformation should be of large consumption and economically interesting. Second, the accumulation of the targeted nutrient should not limit the accumulation of another nutrient nor the physiology and development of the crop.

1.6 ­Conclusions and Future Perspective Conclusively, biofortification of edible crops for food is considered the most useful and efficient approach to supplement diets and alleviate malnutrition. Agricultural research, both fundamental and applied, is aimed at developing biofortified crops. The relationship between diet and health has long been demonstrated and numerous scientific studies indicate that food components affect our body and health by influencing the physiological

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Table 1.3  Studies in which chromosomal loci (QTL) have been identified in crop plants that affect the concentrations of vitamins and essential mineral elements most commonly lacking in human diets. Crop species

Tissue

Elements

References

Rice (Oryza sativa)

Grain

Fe, Zn

Garcia‐Oliveira et al. (2009), Gregorio et al. (2000), Lu et al. (2008), Norton et al. (2010), Stangoulis et al. (2007), Zhang et al. (2011)

Provitamin A

Paine et al. (2005)

Oilseed rape (Brassica napus)

Seed

Fe, Zn

Ding et al. (2010)

Potato (Solanum tuberosum)

Tuber

Fe, Zn

Subramanian (2012)

Carotenoids

Ducreux et al. (2005), Diretto et al. (2007)

Sweetpotato

Tuber

β‐carotene

Cervantes‐Flores (2006)

Cassava

Root

Carotenoids

Welch et al. (2010)

Cauliflower (Brassica oleracea)

Leaf

Zn

Broadley et al. (2010)

Carotenoids

Crisp et al. (1975), Dickson et al. (1988), Li et al. (2001)

Canola (Brassica napus)

Seed

Carotenoids

Fujisawa et al. (2009), Ravanello et al. (2003)

Brassica rapa

Leaf

Fe, Zn

Wu et al. (2008)

Wheat (Triticum spp.)

Grain

Fe, Zn

Distelfeld et al. (2007), Genc et al. (2009), Peleg et al. (2009), Shi et al. (2008)

Barley (Hordeum vulgare)

Grain

Zn

Lonergan et al. (2009), Sadeghzadeh et al. (2009), Zeng et al. (2016)

Maize (Zea mays)

Grain

Fe, Zn

Jin et al. (2013), Lung’aho et al. (2011), Simić et al. (2012)

Carotenoid Provitamin A

Burt et al. (2011), Chandler et al. (2013), Harjes et al. (2008)

Fe, Zn

Beebe et al. (2000), Blair et al. (2009, 2010), 2011), Cichy et al. (2005, 2009), Gelin et al. (2007), Guzman‐Maldonado et al. (2003)

Bean (Phaseolus vulgaris)

Seed

Source: From White et al. (2012a) with modifications. Open permission, Hindawi Publishing Corporation.

processes. Thus, biofortified crops are of increasing interest in the prevention of malnutrition and its related diseases. For years, it was thought that plant‐based foods simply provide energy and essential nutrients, but medical and nutrition sciences have demonstrated that many other micronutrients are required although some are at low levels. Food crops are known to have different nutritional profiles and the dietary insufficiency of one or more micronutrients, and the deficiency of one or more nutrient remains a major concern in populations that have an unbalanced diet, leading to nutritional deficiencies and metabolic diseases and disorders. Furthermore, most of the plant‐based and staple crops consumed in many regions do not contain many essential nutrients or their levels are not sufficient to meet minimum daily intake.

Table 1.4  Studies of crops biofortification using molecular engineering approaches. Class

Crops

Nutrients

References

Cereals

Rice

β‐carotene

Beyer et al. (2002), Burkhardt et al. (1997), Datta et al. (2003), Paine et al. (2005), Ye et al. (2000)

Vitamin B9

Blancquaert et al. (2015), Storozhenko et al. (2007)

Iron

Goto et al. (1999), Lee and An (2009), Lee et al. (2012), Lucca et al. (2002), Masuda et al. (2012, 2013), Paul et al. (2012), Takahashi et al. (2001, 2016), Vasconcelos et al. (2003), Wirth et al. (2009), Zheng et al. (2010)

Zinc

Lee and An (2009), Masuda et al. (2008)

Iron

Borg et al. (2012), Sui et al. (2012)

Provitamin A Carotenenoids

Cong et al. (2009), Wang et al. (2014)

Barley

Zinc

Ramesh et al. (2004)

Maize

Provitamin A Carotenoids

Aluru et al. (2008), Decourcelle et al. (2015), Zhu et al. (2007)

Wheat

Legumes

Vegetables

Fruits

Oilseeds

Vitamin E

Cahoon et al. (2003)

Vitamin C

Chen et al. (2003), Levine et al. (1995)

Sorghum

Provitamin A

Lipkie et al. (2013)

Soybean

β‐carotene

Kim et al. (2012), Pierce et al. (2015), Schmidt et al. (2015)

Vitamin E

Van Eenennaam et al. (2003)

Lentils

Manganese

Ates et al. (2018)

Potato

β‐carotene Zeaxanthin

Diretto et al. (2006), Ducreux et al. (2005), Lopez et al. (2008), Romer et al. (2002), Song et al. (2016), Van Eck et al. (2007)

Zinc

Burgos et al. (2007), Brown et al. (2010), Haynes et al. (2012)

Copper

Haynes et al. (2012)

Manganese

Haynes et al. (2012)

Sweet Potato

See Chapter 4

Cauliflower

β‐carotene

Lu et al. (2006)

Lettuce

Iron

Goto et al. (2000)

Carrot

Calcium

Morris et al. (2008), Park et al. (2004)

Cassava

β‐carotene Provitamin A

Telengech et al. (2015), Welch et al. (2010)

Banana

β‐carotene

Waltz (2014)

Vitamin A

Davey et al. (2008)

Provitamin A

Paul et al. (2017)

Tomato

Folate, β‐carotene Lycopene Provitamin A Carotenoid

Apel and Bock (2009), Davuluri et al. (2005), Dharmapuri et al. (2002), Enfissi et al. (2005), Fraser et al. (2007), Huang et al. (2013), Rosati et al. (2000), Wurbs et al. (2007)

Linseed

Carotenoids

Fujisawa et al. (2008)

Canola

β‐carotene

Fujisawa et al. (2009), Ravanello et al. (2003), Shewmaker et al. (1999), Wei et al. (2009), Yu et al. (2008)

Source: From Garg et al. (2018) with some modifications. Open access publication under Creative Commons Attribution License [CC BY] terms with free permission.

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Malnutrition and deficiencies or hidden hunger have been significantly alleviated in many regions of the world, particularly in the developing and poor countries as results of programs that aim to fortify food crops. Nevertheless, much remains to be done regarding biofortification of staple food crops in the poor countries because these approaches have not always been successful due of the limited agricultural resources. Demand for biofortified food will likely increase in the future due to numerous issues faced by the growing population, including the possible effects of climate change on food production and food quality. Therefore, it will be even more important to educate consumers on the benefits of biofortified foods. Nowadays, the real challenge is no longer the science of biofortification. We know it works and have showed its efficiency in dealing with nutritional deficiencies; but the real challenge is to make biofortified crops common plant‐foods for populations at risk, and to do this, rather than modifying their diet, it is more efficient to provide biofortified staple crops. Farmers, stakeholders and policy‐makers should thus scale up biofortified crops to reach millions of households through institutional, regulatory, and financial policies.

­Summary Large number of nutrients are not only required and crucial for the growth and development of our organisms, but they also help in preventing many diseases and disorders. However, often the daily diet is not balanced and does not provide the required amount of these nutrients. To supply adequate nutrients, biofortification of edible plants is becoming one the most efficient strategies to improve daily diet and overcome malnutrition. For the last few decades, crops biofortification technology using different approaches has been identified as a priority initiative and has shown great potential for mitigating malnutrition and enhancing nutrient bioavailability. To improve the nutritional quality of food crops, agronomic, conventional breeding, and molecular engineering approaches have been used. Technically, each approach has its benefits, disadvantages, and constraints. However, to be viable, each approach should be economically feasible, less time consuming, and readily apparent to the consumers.

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Welch, R.M. (2005). Biotechnology, biofortification, and global health. Food and Nutrition Bulletin 26: 419–421. Welch, R.M. and Graham, R.D. (2004). Breeding for micronutrients in staple food crops from a human nutrition perspective. Journal of Experimental Botany 55: 353–364. Welch, R.M., House, R.A., Ortiz‐Monasterio, I., and Cheng, Z. (2005). Potential for improving bioavailable zinc in wheat grain (Triticum species) through plant breeding. Journal of Agricultural and Food Chemistry 53: 2176–2180. White, P.J., Broadley, M.R., and Greg, P.J. (2012a). Managing the nutrition of plants and people. Applied and Environmental Soil Science 2012: 104826. White, P.J., Broadley, M.R., Hammond, J.P. et al. (2012b). Bio‐fortification of potato tubers using foliar zinc‐fertiliser. Journal of Horticultural Science and Biotechnology 87: 123–129. WHO (2009). Global prevalence of vitamin A deficiency in populations at risk 1995–2005. WHO Global Database on Vitamin A Deficiency. World Health Organization (22 January 2019). http://apps.who.int/iris/bitstream/handle/10665/44110/9789241598019_eng. pdf?ua=1. Wirth, J., Poletti, S., Aeschlimann, B. et al. (2009). Rice endosperm iron biofortification by targeted and synergistic action of nicotianamine synthase and ferritin. Plant Biotechnology Journal 7: 631–644. Wissuwa, M., Ismail, A.M., and Graham, R.D. (2008). Rice grain zinc concentrations as affected by genotype, native soil‐zinc availability, and zinc fertilization. Plant and Soil 306: 37–48. Wu, J., Yuan, Y., Zhang, X. et al. (2008). Mapping QTLs for mineral accumulation and shoot dry biomass under different Zn nutritional conditions in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Plant and Soil 310: 25–40. Wurbs, D., Rup, S., and Bock, R. (2007). Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome. The Plant Journal 49: 276–288. Xu, J. and Hu, Q. (2004). Effect of foliar application of selenium on the antioxidant activity of aqueous and ethanolic extracts of selenium‐enriched rice. Journal of Agricultural and Food Chemistry 52: 1759–1763. Yang, F., Chen, L., Hu, Q., and Pan, G. (2003). Effect of the application of selenium on selenium content of soybean and its products. Biological Trace Element Research 93: 249–256. Yang, X.W., Tian, X.H., Lu, X.C. et al. (2011). Impacts of phosphorus and zinc levels on phosphorus and zinc nutrition and phytic acid concentration in wheat (Triticum aestivum L.). Journal of the Science of Food and Agriculture 91: 2322–2388. Ye, X., Al‐Babili, S., Kloti, A. et al. (2000). Engineering the provitamin A (β‐carotene) biosynthetic pathway into (carotenoids‐free) rice endosperm. Science 287: 303–305. Yilmaz, A., Ekiz, H., Torun, B. et al. (1997). Effect of different zinc application methods on grain yield and zinc concentration in wheat cultivars grown on zinc‐deficient calcareous soils. Journal of Plant Nutrition 20: 461–471. Yu, B., Lydiate, D.J., Young, L.W. et al. (2008). Enhancing the carotenoid content of Brassica napus seeds by downregulating lycopene epsilon cyclase. Transgenic Research 17: 573–585.

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Yuan, L., Wu, L., Yang, C., and Quin, L.V. (2013). Effects of iron and zinc foliar applications on rice plants and their grain accumulation and grain nutritional quality. Journal of the Science of Food and Agriculture 93: 254–261. Zeng, Y.W., Du, J., Yang, X.M. et al. (2016). Identification of quantitative trait loci for mineral elements in grains and grass powder of barley. Genetics and Molecular Research 15: gmr15049103. Zhang, B., Chen, P., Shi, A. et al. (2009). Putative quantitative trait loci associated with calcium content in soybean seed. Journal of Heredity 100: 263–269. Zhang, X., Zhang, G., Guo, L., and Wang, H. (2011). Identification of quantitative trait loci for Cd and Zn concentrations of brown rice grown in Cd‐polluted soils. Euphytica 180: 173–179. Zhang, Y.Q., Pang, L.L., Yan, P. et al. (2013). Zinc fertilizer placement affects zinc content in maize plant. Plant and Soil 372: 81–92. Zhao, F.‐J. and Shewry, P.R. (2011). Recent developments in modifying crops and agronomic practice to improve human health. Food Policy 36: S94–S101. Zheng, L., Cheng, Z., Ai, C. et al. (2010). Nicotianamine, a novel enhancer of rice iron bioavailability to humans. PLoS One 5: e10190. Zhu, C., Naqvi, S., Gomez‐Galera, S. et al. (2007). Transgenic strategies for the nutritional enhancement of plants. Trends in Plant Science 12: 548–555. Zuo, Y. and Zhang, F. (2009). ‘Iron and zinc biofortification strategies in dicot plants by intercropping with gramineous species’, a review. Agronomy for Sustainable Development 29: 63–71.

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2 Food Fortification What’s in It for the Malnourished World? Barbara Poniedziałek, Kinga Perkowska, and Piotr Rzymski Department of Environmental Medicine, Poznan University of Medical Sciences, Poznań, Poland

2.1 ­Introduction Fortification, or “enhancement,” “enrichment,” in relation to food, refers to strategy aimed at improving the nutritional quality of a food product by increasing the level of particular nutrient(s). The World Health Organization (WHO) defines fortification as the intentional addition of one or more microelements to food products in order to increase their content or prevent particular deficits and to provide health benefits (Allen et al. 2006). In addition, it is possible to enrich the product with active substances, not naturally found in a certain type of food (National Academy of Sciences 2003; WHO 2015). Fortified products can be classified as functional foods only if the content of the added ingredient is high enough to trigger/cause a clinically documented health effect (e.g. reduce the risk of disease). Therefore, not every fortified product fulfills such requirement, e.g. juices enriched with ascorbic acid designed to prevent oxidation reactions (Siró et al. 2008). Nevertheless, the compound with which food is fortified should not exert a negative effect on the absorption and metabolism of other nutrients or be toxic in the dose used (Dwyer et al. 2015). The current interest in the enrichment of food intended for human consumption arose in the twentieth century. However, the first described case of such activities dates back to ancient times; a well‐known example being Doctor Melanpus, who in 400 bce added iron filings to wine to increase the strength of Persian soldiers (Bulusu and Wesley 2011). In the 1820s, French chemist Jean Baptiste Boussingault observed that the population of the Antioquia region of Colombia, consuming salt obtained from an abandoned mine, did not develop goiters. As he later demonstrated, the salt was contaminated with iodine. Therefore, he proposed widespread enrichment of salt with iodine to prevent thyroid diseases (Preedy et al. 2009; Venkatesh Mannar 2011). However, it was not until 1920 that this suggestion was acted upon – the pro‐health effects of enrichment were so significant that the iodination of salt began to be systematically introduced in several countries. A large number of fortified products were introduced during World War II, when the enrichment of margarine with vitamins A and D, milk with vitamin D, and flour and bread with iron, vitamins B1, B2, and B3 began (Bulusu and Wesley 2011; Nathoo et  al. 2005). Vitamins and Minerals Biofortification of Edible Plants, First Edition. Edited by Noureddine Benkeblia. © 2020 John Wiley & Sons Ltd. Published 2020 by John Wiley & Sons Ltd.

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Currently, food fortification is still gaining popularity, and more products of this type are becoming available on the market in developed and developing countries (Black et al. 2012; Das et al. 2013; Martorell et al. 2015; Velu et al. 2014). This chapter reviews food fortification methods encompassing classical, industrial fortification, biofortification, and nutritional enhancement through genetic engineering in the context of nutritional deficiencies, and discusses the benefits as well as limitations of their implementation.

2.2  ­Nutritional Deficiency as a Public Health Problem The main factors influencing the occurrence of nutritional deficiencies include the growing world population, migration of people from rural areas to cities, climate conditions, political and economic conflicts, low level of dietary awareness, and medical conditions affecting absorption and general burden of nutrients (Alkerwi et al. 2015; Awate et al. 1997; Bruins et al. 2018; Müller and Krawinkel 2005; Saunders and Smith 2010). Many of these factors can co‐occur, increasing the difficulty in determining likelihood of food shortages as well as preventing malnutrition. The average global level of energy supplied through diet is estimated at 2903 kcal/day, which is sufficient to cover the average energy demand (FAO 2015). At the same time, one in nine people in the world suffer from malnutrition (UNICEF 2016). The highest degree of deficiency is observed in the developing countries of Africa and Asia. Ethiopia, where hunger affects over 30 million people, and India, with almost 200 million starving people, occupy the highest positions in the ranking of countries with the largest population suffering from food deficits. The problem of undernourishment also affects, to a large extent, Oceania and Latin America (FAO 2015). Almost 50% of deaths among children under the age of five are caused by malnutrition, and one in four children in the world suffers from stunted growth for the same reason (UNICEF 2016). Dietary deficiencies, i.e. decreased intake of calories and/or proteins, as well as other macro‐ and micro‐elements, can eventually lead to the state of malnutrition. Literature distinguishes three extreme types of undernutrition: marasmus, kwashiorkor, and mixed type (Waterlow 1972). Marasmus, or caloric malnutrition, forces the body to utilize structural proteins (in the process of neoglucogenesis) as a source of energy. Occurrence of this type of malnutrition before second year of life may cause irreversible impairment of brain function. The morphological appearance of children affected by this type of malnutrition suggests that they are much older than their real age, and their behaviors are deprived of the energy characteristic for children of this age. Body weight is reduced to less than 60% of the norm for age and height, body wasting, muscle mass loss, and subcutaneous fat tissue are visible (Vanbergen and Appleton 2013). Kwashiorkor is a protein malnutrition along with sufficient supply of calories. The name comes from Ghana, where it means “a disease that affects a child when a new child appears” or “illness of an abandoned child” (Stanton 2001). After the birth of a younger sibling, the older child is weaned, and is thus deprived of the full‐value protein previously provided by the mother’s milk. Protein deficiencies are manifested in edema, growth arrest, and atrophy of hair, eventually leading to loss of capacity and degeneration of the lungs, heart, and brain tissue (Young 2012).

2.2  ­Nutritional Deficiency as a Public Health Proble

Regardless of the type of malnutrition, it causes reduced resistance to infection, reduced thermal insulation, impaired perception, and an increased risk of premature death (Saunders and Smith 2010). Another significant issue is so‐called latent hunger, caused by an insufficient supply of individual macro‐ and micronutrients and vitamins in the diet. Its symptoms are not as strongly manifested as in the case of caloric or protein deficiencies and are specific to a given deficiency. As estimated by the joint statement of the WHO, the World Food Programme (WFP) and the United Nations Children’s Fund (UNICEF) over 2 billion people suffer from micronutrient deficiencies (WHO‐WFP‐UNICEF 2007). The most serious consequences are found in children and pregnant women, because a lack of micronutrients can cause fetal growth disorders, dwarfism, weakened immunity, and mental retardation. Threats of deficiencies encompass the entire world population (Allen 2003; Whatham et al. 2008). Most often, latent hunger is caused by iron deficiency. This problem affects up to a quarter of the world’s population (McLean et al. 2009), mainly women and children, and its frequency is higher in areas with increased incidence of malaria, HIV/AIDS, and other infectious diseases. Iron deficiency causes anemia, miscarriage, and mental retardation (Allali et al. 2017; WHO 2007). Zinc deficiency is also relatively common – it is estimated that up to 17% of people can be affected by it (Wessells and Brown 2012). The cause of nutritional deficiencies, apart from an insufficient quantitative and qualitative supply of food, may also include food allergies and intolerances (Robbins and Uygungil 2017). Therapeutic activity in such cases consists in the elimination of an allergen from the diet, which may lead to an insufficient supply of certain nutrients. In addition, other diseases, both acute and chronic, can lead to deficiencies, which may be due to weakness/weakening of the body, changed nutritional requirements, or appetite and hunger disorders. Considering the global challenges to prevent nutritional deficiencies in different environmental settings, various methods of food fortification may provide, at least partially, the solutions to the issue of latent hunger caused by a deficiency of minerals or vitamins in the diet. This chapter briefly reviews main strategies of fortification (Figure 2.1): industrial fortification, cultivation of plants and mushrooms on enriched substrates to support bioaccumulation of nutrients in edible parts, conventional selective breeding to produce high‐yielding

Fortification

Industrial fortification

Selective breeding

Growth on enriched substrates

Figure 2.1  Types of food fortification introduced to target nutritional deficiencies.

Genetic engineering

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varieties of crops, and genetic engineering to produce crops that contain nutrients not expressed (or expressed to lesser extend) by wild counterparts. The benefits and limitation of each of these approaches are also discussed, along with their potential benefits, limitations, and threats to implementation on various markets.

2.3  ­Types of Fortification Depending on the target group, fortification can take many forms. Mass fortification consists of enriching food that is commonly eaten (such as cereals, condiments, and milk) by the general population and can be considered a public health strategy to increase intake of critical nutrients (WHO 2009a). Targeted fortification is, in turn, used for products intended for a specific group, e.g. complementary foods for infants or vegan foods enriched with vitamin B12 (Kadıoğlu Şimşek et al. 2019; Mäkinen et al. 2016). This type of fortification is obligatory or voluntary, depending on the public health significance of the problem it is seeking to address. Market‐driven fortification (sometimes called open‐market fortification) is aimed at increasing profits and the prestige of producers and is always voluntary, but is governed by regulatory limits (Allen et al. 2006). If one considers process of food fortification, two main approaches can be distinguished: industrial fortification and biofortification. The former is based on addition of particular ingredients during food production process while the latter can be achieved by cultivation of plants and mushrooms on enriched substrates so to support bioaccumulation of nutrients in edible parts, conventional selective breeding to produce high‐yielding varieties of crops as well as genetic engineering to produce crops that contain nutrients not expressed (or expressed to lesser extend) by wild counterparts. All of these approaches are presented in the subsequent subchapters.

2.3.1  Industrial Fortification The classical method of food fortification relies on adding one or several nutrients to the final food product during its production. Added ingredients may include those already occurring in processed food but in smaller amount or when significant loss occurs during food processing, e.g. levels of niacin, riboflavin, and thiamine in refined grains. They may also include compounds not occurring naturally in such product, e.g. adding calcium to orange juice. The added ingredients may have natural as well as artificial origin (Liyanage and Hettiarachchi 2011). Food products are most frequently enriched with iodine, iron, vitamin A and calcium, vitamin D, and B‐group vitamins (particularly B12) because their deficiencies are most frequently observed (Bailey et al. 2015). One should note that industrial fortification is a main alternative to using food supplements. The use of the latter has gained popularity in developed countries, although their efficiency in decreasing nutrient deficiencies is facing a number of issues such as low bioavailability of micronutrients from such source, cross‐ interference between particular ingredients of food supplement, contamination in low‐ quality products, as well as discrepancies between declared and determined content of nutrients (Poniedziałek et al. 2018; Rzymski et al. 2018; Yetley 2007).

2.3  ­Types of Fortificatio

Fortification with iodine of table salt is the most widespread application of industrial fortification in the world. The earliest obligatory fortification with the use of table salt iodine was introduced in the USA (1924), Switzerland (1922), and New Zealand, and presently over 70% of the world population in 130 countries uses iodized salt. However, the level of iodination varies from country to country, from 8 mg per kg of salt to as much as 110 mg per kg. One should bear in mind that implementation of iodine fortification requires regular monitoring to decrease risks of both inadequate and excess iodine intake (Charlton and Skeaff 2011). Industrial iron fortification is mainly used for food products intended for children, women of reproductive age, and pregnant women, as these groups are most affected by this element deficiency. The most serious effects of iron‐deficiency anemia (IDA) are the increased risk of perinatal mortality of mothers and children and mental retardation of children. IDA also causes poor psychomotor development and worse results in cognitive function tests in children of different age groups (Baltussen et al. 2004). Breakfast cereals are an appropriate and often chosen carrier for iron, due to the mass level of consumption, relatively easy production, and high product durability (IFT 2007). In addition, it is possible to use an in‐home iron fortification in which the carer at home adds micronutrient powders (MNPs) containing iron, to the food items consumed by the infants and children. Currently, home food fortification programs are already implemented or planned in nearly 40 countries around the world (Paganini 2016; Paganini and Zimmermann 2017). Fortification of foods with folic acid seems particularly justified in the case of women of reproductive age. Folic acid is important for the development of the fetus. Its deficiency can lead to neural tube defects, the consequence of which is, for example, anencephaly or spina bifida. This vitamin occurs naturally mainly in green vegetables and is sensitive to high temperatures and sunlight, therefore, satisfying daily dietary needs may be a problem. Enrichment of flour into folic acid has beneficial effects in reducing deficiencies of this vitamin among women of childbearing age and the incidence of neural tube defects in their offspring. One should note that folic acid supplementation alone did not appear to succeed in reducing the incidence of neural tube defects in some regions, contrary to introduction of fortified grain products (Abdollahi et al. 2011; De Wals et al. 2007; Hertrampf and Cortés 2004; Persad et al. 2002). The prevention of nutritional deficiencies and associated disorders constitute the main goal of fortification. However industrial fortification can also be targeted to specific occupational groups that, due to difficult living conditions and limited access to food, require special, tailored products that meet their specific needs. Soldiers are one of such groups. During the Second World War, US soldiers were provided with chocolate bars to supplement energy shortages (Henry and Chappell 2000). Currently, soldiers receive food products such as sweets, chocolate bars, and chewing gums enriched with probiotics, vitamins and minerals, which not only prevent nutritional deficiencies but can also be helpful in the prevention of certain diseases, e.g. diarrhea (probiotics), allergies (quercetin), or hypotension (caffeine) (IFT 2008). Iron‐fortified food products were also designed for female soldiers due to diminished status of this element that occurs in this group following high physical activities, e.g. combat trainings (Karl et al. 2010; McClung et al. 2009). Another group of professionals with a specific demand for fortified products are the crews of space stations. The greatest deficiencies are observed in the case of vitamin D as the astronauts

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during expeditions are not exposed to sunlight because space walks are held in overalls and the windows in spacecraft are protected against harmful UV radiation, although they also prevent skin synthesis of vitamin D. Demand for vitamin D is covered by members of the spacecraft crew through the consumption of milk, yogurts, and cereals as well as orange juices enriched with vitamin D. It is also necessary to fortify foods for astronauts with vitamins C and K, iron, and calcium (Smith et al. 2012).

2.3.2 Biofortification Biofortification is the concept of growing plants or mushrooms with an increased nutritional value intended for human consumption. The yields are characterized by higher content of minerals and vitamins in their edible parts. Biofortification can be based on the selection of seed varieties with a high content of microelements or cultivation on enriched substrate. The particular type of biofortification include genetic engineering to increase nutrients levels (Bailey 2007; Garg et al. 2018; Nestel et al. 2006). Growing enriched plants increases access to high‐quality food for individuals who have no access to commercially fortified products (Garg et al. 2018). The increase in the usefulness of the biofortification process is especially noticeable in developing countries, particularly in rural areas, among poor people whose diets are based on rice, maize and wheat, which are naturally deficient in microelements (Bouis and Saltzman 2017; Garg et al. 2018). 2.3.2.1  Biofortification Through Selective Breeding

Plant breeding is based on genetic improvement of the crop to develop new varieties with desirable traits, including higher nutrition content. One of the major programs that implements such strategy is HarvestPlus, initiated by the economist Howarth Bouis. By 2015, as part of this project, 10 food biofortification programs had been implemented in seven countries: rice biofortified with zinc  –  Bangladesh; common bean biofortified with iron – Democratic Republic of the Congo, Rwanda, Uganda; maize biofortified with vitamin A – Zambia, Nigeria; manioc biofortified with vitamin A – Democratic Republic of the Congo, Nigeria; sweet potatoes biofortified with vitamin A – Uganda; pearl millet biofortified with iron – India (Bouis and Saltzman 2003). The prevalence of seeds characterized by a naturally high ability to accumulate higher concentrations of micronutrients contributes to an increased uptake of the deficient element. The potential benefits of biofortified seeds depend on the bioavailability of ingredients contained in the products and the acceptance of the indicated methods of cultivation by farmers and consumers. As estimated over 20 million people in farm households in developing countries are now growing and consuming biofortified crops since HarvestPlus program was implemented (Bouis and Saltzman 2003). 2.3.2.2  Biofortification Through Cultivation on Enriched Substrates

The most common deficient mineral in the world is iron (Horton and Ross 2003). This element has a significant impact on the mental development of children and on the ability to exercise in adults (Allali et al. 2017). Attempts at biofortification of crops are usually made with the use of the common bean (Phaseolus vulgaris) and pearl millet (Pennisetum glaucum)

2.3  ­Types of Fortificatio

(Gregorio 2002). Pearl millet is a basic cultivation plant in India and in the sub‐Saharan region (Parthasarathy Rao et  al. 2006; Vietmeyer and Ruskin 1996). Studies conducted among 22 women in Benin showed that the total amount of iron absorbed from biofortified pearl millet (the soil was enriched with phosphorus, which increased the absorption of iron by the plant) was twofold higher compared to that from locally consumed crops (Cercamondi et al. 2013). Its use in 246 school‐age children in India resulted in changes in the level of hemoglobin, serum ferritin, and total concentration of iron in the body, and confirmed the efficacy of the product (Finkelstein et al. 2015). As shown in a study conducted on a group of women in Rwanda, consumption of iron beans improved their iron status as marked by increased levels of hemoglobin and serum ferritin (Haas et al. 2016). Importantly, the possibility of bean biofortification with iron is limited by naturally occurring substances that act as inhibitors of iron such as phytic acids, calcium, and ascorbic acid, and eventually reduce the bioavailability of the element (Hurrell and Egli 2010). However, bean varieties differ in the content of these inhibitors. One should also take into account that the process of yield enrichment may additionally increase their concentrations. It appears that potential of the common bean as an iron carrier in biofortification process may be somewhat limited although their high consumption at level of 100 g biofortified beans/day may provide approximately 30–50% of daily iron requirement in women (Boy et al. 2015; Petry et al. 2012). Vitamin A deficiency is, epidemiologically, still one of the most common nutritional issue, being a major cause of blindness, growth disorder, diarrhea, and increased risk of measles among children of developing countries (WHO 2009b). A low level of vitamin A in pregnant women increases the risk of death during delivery. In order to decrease vitamin A deficiencies, a number of supplementation programs were introduced (Wirth et al. 2017). This issue was also addressed by the HarvestPlus program using biofortificated maize, manioc (Manihot esculenta) and sweet potatoes (Ipomoea batatas). An orange variety of corn was obtained by crossing five types of seeds (Pixley et al. 2013). Gannon et al. (2014) showed in their study that consumption maize biofortified in provitamin A by Zambian children results in a significant increase in vitamin A. Significant results in this regard were also obtained in a study with the application of roots of selected yellow cassava rich in provitamin A. Vitamin A was improved following its consumption by Kenyan children aged 5–13 (Chávez et al. 2005; Talsma et al. 2016). Orange‐colored potatoes, containing an increased amount of β‐carotene, were also shown to be an effective source of vitamin A, as confirmed by a study of 741 children in Mozambique, whose plasma retinol increased over a two‐year observation period (Low et al. 2007). Zinc deficiency in the human diet causes multiorgan disorders. This element is responsible for regulatory, structural, and catalytic functions, and acts as a co‐factor of over 300 enzymes. It participates in the transformation of carbohydrates, fats, and proteins and affects the production and/or functioning of hormones. The results of zinc deficiency are extensive, clinically not very specific, and include skin problems, loss of hair, growth inhibition and developmental delay, diarrhea, loss of appetite, hypogonadism, and an impaired immune system (Prasad 1985). The zinc deficiencies were also targeted by biofortification approach, namely biofortification of wheat, rice, and maize. For example, the consumption of wheat flour (produced from wheat biofortified with zinc) by a group of 27 women from Mexico revealed an increase in the content of this element in urine output (Rosado

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et al. 2009). An increase in zinc concentration was also observed in the plasma and urine of children in Zambia, consuming corn flour obtained from biofortified maize (Chomba et al. 2015). Importantly, zinc biofortification of crops such as wheat does not lead to an increase in phytic acid nor reduction in bioavailability of other micronutrients such as iron, manganese, or copper (Liu et al. 2017). Other biofortification application includes enrichment of edible part of plants in selenium content. The availability of selenium in foods depends largely on its soil levels, and these are known to be greatly differentiated (Kieliszek and Błażejak 2016). As a result, many human populations are prone to selenium deficiencies (Ge and Yang 1993; Rayman 2009). Given the physiological role of selenium, particularly its organic forms, selenoproteins, it is fully justified to seek efficient methods to increase its intake where necessary. Bañuelos et al. (2016) found that a number of vegetables can be biofortified in selenium content by cultivation of enriched soil. For example, this has been successfully demonstrated for carrots and broccoli. A biofortification program with the use of selenium, through the fertilization of crops, introduced in Finland has contributed to reducing the incidence of deficiencies of this element (Alfthan et al. 2015). Significant results were also obtained during the biofortification of soil with copper in order to increase the content of this element in wheat grains, as well as with silicon in the case of P. vulgaris (Korzeniowska and Kantek 2014; Montesano et al. 2016). Apart from plants, the potential target of biofortification, particularly with minerals, include mushrooms. It is well‐established that mushrooms easily uptake, and translocate various of elements, and accumulate them in fruiting bodies. This process concerns various contaminants such as toxic metals and has received a great deal of attention. However, simultaneously nutritional elements can be uptaken from overgrown soil or substrate, making mushrooms potential candidates for production of mineral‐enriched food (Kalač 2013; Rzymski and Klimaszyk 2018). This is particularly important if one considers that mushrooms are not only considered as delicacy but are increasingly consumed, particularly as cultivated forms, with Agaricus bisporus, Pleurotus ostreatus, and Lentinula edodes being the most often distributed species for human consumption (Mleczek et al. 2017a; Rzymski et al. 2017a; Siwulski et al. 2017). Several studies show that mushrooms such as P. ostreatus, Pleurotus eryngii or A. bisporus can be successfully enriched in selenium, copper, and zinc by cultivating them on specific substrates enriched with these elements (Matute et al. 2011; Niedzielski et al. 2014; Rzymski et al. 2016a, b). Such enrichment can also lead to increase of antioxidant compounds and antioxidant activities revealed in human platelets (Gąsecka et  al. 2016; Poniedziałek et al. 2017). It has also been shown that mushroom can be biofortified in lithium, which is not considered as a nutrient, although some epidemiological studies suggest that its intake may be associated with mood and potentially incidence of suicide. In response, some authors suggest fortifying food with lithium, and methods to cultivate mushrooms for such purpose have been developed (Mleczek et al. 2017b; Rzymski et al. 2017b). One should, however, bear in mind that no studies have been conducted to reveal (i) bioavailability of elements in which content of the fruiting bodies was biofortified, (ii) level of nutrients loss during mushrooms processing, e.g. cooking, and (iii) whether their consumption may actually lead to improvement of mineral status. Further research in this regard is required.

2.3  ­Types of Fortificatio

2.3.2.3  Biofortification Through Genetic Engineering

The development of science and technology opens new possibilities for food production. Numerous studies have investigated the use of genetic engineering in modification of plants and animals intended for consumption, and a number of such applications have been eventually commercialized (Raman 2017). According to the European Parliament and the Council, genetically modified food is defined as “…food containing, consisting of or produced from GMOs” (European Economic and Social Committee 2003). This definition should be supplemented by an explanation of the term GMO, formulated by the European Parliament and the Council on March 12, 2001, which states that “a genetically modified organism (GMO), means any organism, besides a human being, in which the genetic material (DNA) has been altered in a way that does not occur naturally by mating and/or natural recombination” (European Economic and Social Committee 2003). Genetic engineering can be divided into three main approaches: (i) transformation of the activity of naturally occurring genes, (ii) introduction of additional copies of genes present in the particular organism to alter the expression of encoded product, and (iii) complementing the genotype with genes from another species – transgenic organisms. The first transgenic plant, bred in 1983, was tobacco resistant to infections of Agrobacterium tumefaciens (Fraley et al. 1983). China commercialized modified tobacco in 1992 and became the first country to introduce free access to genetically modified vegetation (Clive 1997). In  1994, Calgene received an approval to introduce the first modified food product  – FlavrSavr tomatoes, which were characterized as more resistant to rotting by adding an antisense gene that interferes with the production of the enzyme polygalacturonase (Bruening and Lyons 2000; Raman 2017). The primary goal in using genetic engineering in food production is to introduce herbicide‐resistance and insecticide activity of crops (Brookes and Barfoot 2017). However, these methods can be also be implemented in order to improve resistant to certain environmental conditions such as drought, organoleptic properties or nutritional value (Datta 2013). GMOs are primarily aimed at improving the technological parameters, organoleptic properties, and resistance of food products (Fiedurek 2007). Genetically modified plants for food production may have a significant impact on solving issues related to nutritional deficiencies, mainly by enriching edible parts with essential nutrients (Hefferon 2015). The global market allows products such as GM plants, foods produced with the use of GMO (e.g. baked bread with the use of ­genetically modified yeast), foods containing transgenic plants (e.g. chips obtained from GM potatoes), and products that do not contain GMOs but are derivatives (e.g. soybean oil from transgenic soybean). The most significant example of a use of genetic engineering in order to improve nutritional value of food product is transgenic rice known as Golden Rice. This variety has been developed in order to enrich rice in β‐carotene and to target populations with high incidence of vitamin A deficiencies and related health consequences, including irreversible childhood blindness (Ye et al. 2000). The rice has been chosen because it is a basic food product for more than half of the world’s population (Rejesus et  al. 2012). Therefore, it ensures the regular intake of increased provitamin A via diet. Golden Rice was created by transforming rice with two beta‐carotene biosynthesis genes: psy (phytoene synthase) and

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(crtI (phytoene desaturase). The former originated from initially Narcissus pseudonarcissus, and in the second variety from Zea mays, while the source of the latter gene was a soil ­bacterium Erwinia uredovora. The first field trials of Golden Rice were conducted at Louisiana State University in 2004. In 2005, researchers from Syngenta produced the “Golden Rice 2” variety, which produced 23 times more carotenoids than the original variety (Paine et al. 2005). The effectiveness of golden rice in improving vitamin A status was confirmed in 2009 during clinical trial. After 36 days of the study, the blood level of retinol had increased significantly in studied subjects. It was concluded that a cup of enriched rice consumed every day would meet 50% of the daily requirement for vitamin A in the diet (Tang et al. 2009). To improve the use of golden rice in decreasing the vitamin A deficiencies in poor countries, the cutoff between humanitarian and commercial use was set. No royalties were to be paid as long as a farmer’s profit was less than $10 000 per year – in such case, collection and replanting of seeds was also allowed (Dobson 2000). Despite it all, golden rice was not approved in countries with increased incidence of vitamin A deficiency. In 2018, it was however approved in Australia, New Zealand, Canada, and the United States. Apart from rice, maize has also been genetically modified to successfully obtain a variety with significantly increased beta‐caroten content (Aluru et al. 2008). Moreover, GM canola crops as novel sources of omega‐3 fish oils has been introduced to target low intake of eicosapentaenoic acid (EPA; 20:5n‐3) and docosahexaenoic acid (DHA; 22:6n‐3) in populations and to provide a plant‐based alternative for omega‐3 foods. The cultivation of such canola has been approved so far in Australia and the United States (Napier et al. 2019). The introduction of GM foods, including those containing increased nutritional value, requires mandatory legal approval and is still subject to low public acceptance. A number of reasons have been suggested as being potentially responsible for the reluctant and skeptical attitudes toward GMO‐based technology, including a lack of public understanding of the science, difficulties in defining what GMOs are exactly, ethical or religious beliefs, and little or no perception of the benefits that GMOs can bring (Aerni 2013; Rzymski and Królczyk 2016; Sturgis et al. 2005). Deliberate anti‐GM actions driven by nongovernmental organizations have been widely publicized over the years, adding to a reluctance to accept GMOs from a significant part of the general public (Paarlberg 2014). This, in turn, has influenced political decisions. For example, it is currently reflected in the internal disagreement between member states of the European Union (EU) and problems in reaching a common position toward GMOs, particularly GM crops (Lucht 2015). This also has had an effect on public‐sector research funding (Fedoroff 2015).

2.4  ­Summary of Potential Risks and Benefits of Food Fortification Modern technology, progress, and science drive dynamic changes in the human environment. Innovative methods are also likely to influence the state of nutrition in society. Despite many benefits, both fortification and biofortification, including biofortification through genetic modifications, may be associated with a number of threats or limitations in implementation (Table 2.1).

2.5  ­Conclusion

Table 2.1  Potential benefits and threats associated with implementation of industrial fortification and biofortification. Benefits

Threats

Improved status of micronutrients

Inadequate consumption due to the varied nutrient needs of population groups

Reduced mortality of children and pregnant women

Uneven access to fortified products as a result of underdeveloped industry in poor countries

Reduced incidence of health consequences of micronutrient deficiencies, i.e. anemia, osteoporosis, blindness

Consumption of fortified products by individuals with no such nutritional needs

High availability of the added substance

Incorrect selection of the carrier, resulting in the interaction between added and naturally occurring substances

Low risk of adverse effects due to legal regulations and tests confirming safety due to legal regulations and a series of tests confirming safety

Allergic activity of some ingredients

No need to change eating habits

Organoleptic alterations of fortified products

The opportunity of enriching one food product with many ingredients

Technological limitations of multi‐ component fortifications

Low costs of obtaining through existing methods and tools and organized distribution

Inadequate retention and bioavailability of added nutrients

Supporting the agricultural sector with higher financial outlays and education of farmers

Alterations in the content of the fortified ingredient during technological processing, transport or storage

The possibility of compensating losses in the nutritional value of products

Enrichment of products with unfavorable health properties, e.g. bars, cookies, sweet drinks

High level of acceptance as a result of not using controversial technologies

Lack of health education resulting in no awareness of benefits of fortified food consumption

2.5  ­Conclusions Food fortification enables an increase in nutrient intake in the general population and has a long history of combating vitamin and mineral deficiencies and their health consequences. The majority of applications include foods fortified by classical, industrial fortification, although there is a continuous interest in production of biofortified food. Although genetic engineering may offer an unpreceded possibility to introduce food of enriched nutritional value, the low social acceptance thus far has limited its implementation in various areas. It can be, however, predicted that increased education, public understanding of science, and dietary awareness will eventually lead to global acceptance of GM foods, including those revealing increased nutritional value. In view of significant percentage of human population suffering from nutritional deficiencies and its

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consequences, the implementation of food fortified by methods discussed in this chapter clearly appears to be justified both in developing as well as developed countries.

­Summary Nutritional deficiencies that arise from an insufficient dietary supply of minerals and vitamins are a major public health concern encompassing both poorer and richer world regions, affecting children and adults, and increasing the risk of a number of medical conditions and reproduction alterations. This issue now mostly concerns iron, zinc, calcium, and selenium deficiencies, and decreased intake of vitamin A, vitamin D, thiamine, niacin, and folate. Food fortification is one of many strategies suggested as an aid in reducing malnutrition, designed to increase the intake of a wide range of nutrients (fiber, essential fatty acids, amino acids, trace elements, and vitamins) in the general population. Such enrichment can be achieved by (i) addition of particular ingredients during food production process, (ii) cultivation of plants and mushrooms on enriched substrates so to support bioaccumulation of nutrients in edible parts, (iii) conventional selective breeding to produce high‐yielding varieties of crops, and (iv) genetic engineering to produce crops that contain nutrients not expressed (or expressed to a lesser extent) by wild counterparts. This chapter presented each type of food fortification with its potential risks, benefits, future prospects, and limitations, and discussed whether such strategies could be successful in preventing malnutrition, and as a consequence, lead to a decrease in the frequency of diseases associated with nutritional deficiencies. Considering that incidences of nutritional deficiencies are still a relevant health issue in developing and developed countries, the use of fortification strategies appears to be fully justified.

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3 Modern Biotechnologies and Mineral Biofortification of Edible Crops Noureddine Benkeblia1,2 and Kathleen L. Hefferon3 1 

Department of Life Science and Laboratory of Tree Fruit and Aromatic Crop, The Biotechnology Centre, The University of the West Indies, Kingston, Jamaica 2  Laboratory of Crop Science, Department of Life Sciences, The University of the West Indies, Kingston, Jamaica 3  Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada

3.1 ­Introduction In the human body, as in all living organisms, mineral nutrients have different functionalities and play different but important roles in metabolism and homeostasis. The deficiency of these nutrients and their absence in the diet result in the increased incidence of many diseases and metabolic disorders. In order to improve the nutritional status of the population and enhance mineral intake through a balanced and enriched diet, it is necessary to identify the different sources of these minerals, their quantification, and their bioavailability, as well as biofortification of edible crops to alleviate malnutrition (Stein 2010). Thus, understanding the mineral content in terms of biofortification and bioavailability can significantly enhance the nutritional quality of edible crops. This chapter explores our growing knowledge of the principles regarding the bioavailability and deficiencies of mineral nutrients. Until the past century, interest in mineral nutrients was focused on their levels in edible crops, or their deficiencies and consequences on human health. In nutrition, more than 20 different dietary minerals are likely considered to be essential for the proper functioning of the human body and well‐being (Martínez‐Ballesta et al. 2010). Deficiencies due to poor nutrient levels in the diet or poor malabsorption by the body negatively affect our health. Although animal organisms require numerous mineral nutrients, global‐level deficiencies in iron (Fe), zinc (Zn) and iodine (I) are the most recognized, having a significant negative impact on public health (Ezzati et  al. 2004; Horton et al. 2008). Traditionally and until recently, food supplementation has been the main strategy to fortify the diet with minerals and deficient nutrients. Unfortunately, this strategy was not always successful for numerous reasons, including the bioavailability of the supplemented

Vitamins and Minerals Biofortification of Edible Plants, First Edition. Edited by Noureddine Benkeblia. © 2020 John Wiley & Sons Ltd. Published 2020 by John Wiley & Sons Ltd.

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minerals during processing. To overcome this issue, biofortification has been considered as an alternative solution and can be achieved by three approaches: 1) Mineral fertilization, an agronomic approach 2) Conventional breeding 3) Plant transformation using molecular engineering Crop species possess considerable genetic variation and can be harnessed for sustainable biofortification. Consequently, edible crop species with increased mineral levels have been and/or are being developed, thus far with emphasis on iron, zinc, calcium, and iodine (Amarakoon et al. 2012; Gómez‐Galera et al. 2010; Hirschi 2009; White and Broadley 2005, 2009). In this chapter, mineral nutrients biofortification of edible crops will be discussed, focusing on fruits and vegetables.

3.2 ­Minerals Requirement in Human Nutrition Although more than 20 essential minerals are needed by our body, 4 are considered to be major: calcium (Ca), phosphorus (P), potassium (K), and magnesium (Mg), while others are considered traces or are needed at very low concentrations; these are iron (Fe), copper (Cu), zinc (Zn), iodine (I) and selenium (Se) among others. Although a balanced diet and the intake of different combinations of varieties of foods can provide all the required nutrients, it is difficult to determine the intake range of a particular food because of the variation of the nutrient levels in this food, its bioavailability, its absorption, and also other considerations such as the food availability and socioeconomic factors. Calcium (Ca) is one of the most essential nutrients required for many functions in human body and health, and the normal adult human body contains c. 1.8–2.5% (Forbes et  al. 1953). Ca is the most abundant mineral in the body, and 99% is found in bone and teeth (Arnold and Gaengler 2007). The scientific progress of Ca requirements and nutrition have resulted in considering it to be a key micronutrient. Research focused first on early growth periods of infancy and childhood, then expanded to the entire life cycle from birth through elder years because bone formation and maintenance is a lifelong process (Flynn 2003). Ca metabolism involves other mineral nutrients, particularly phosphorus and vitamin D; research has established that adequate calcium intake considerably reduces the risk of bone fractures and osteoporosis. Other research also reported that Ca is involved in vascular contraction and vasodilation (Kaplan and Meese 1986), muscle functions (Berchtold et al. 2000), nerve transmission causing seizures due to hypocalcemia, intracellular signaling (Gargus 2008), and hormonal secretion (Holl et al. 1988). Phosphorus (P) is the second‐most abundant mineral in human body. It accounts for about 1% of total body weight (Forbes et al. 1953), with 80–85% in bone, 10–15% in soft tissues, and only 1% in extracellular fluid (Lee et al. 1981). Biologically, P is found in all the tissues but most predominantly in the bones and teeth (Wood 2006), where its main function is their formation (Anderson 1996; Heaney 2004). Besides this main function, P plays also important roles in the metabolism of carbohydrates and fats (Obeid 2013), energy production (Kemp et al. 2007), the biosynthesis of proteins, and the mechanisms of bone repair (Adams 1938). On the other hand, other functions have been reported on

3.2  ­Minerals Requirement in Human Nutritio

P  such as kidney function (Canalejo et  al. 2005), and muscle contractions (Davis 1964; Weiner et al. 1990). Potassium (K) is the third‐most abundant mineral in the body (5), and 95% is found intracellularly where it fulfills a role with respect to intracellular water (Patrick 1977), however, K level varies with age, sex, height, and fat (Pierson et al. 1974). In the body, 80% of K is found in muscle cells, and 20% in bones, liver, and red blood cells (Cheng et al. 2013). Biologically, K plays different roles in human metabolism and health. It helps reduce blood pressure (Cappuccio and MacGregor 1991; Whelton et al. 1997) and water retention (Gallen et  al. 1998; Rubini 1961), protects against stroke (D’Elia et  al. 2011; Khaw and Barrett‐Connor 1987), helps prevent osteoporosis (He and MacGregor 2001; Tylavsky et al. 2008) and kidney stones (McNally et al. 2009), protects nerve signals (Pentreat 1982), and regulates arterial smooth muscle (Haddy 1983; Hodgkin and Horowicz 1960). In body tissues, more than 90% of magnesium (Mg) is found primarily in muscles and the skeleton, while less than 2% is found in extracellular fluid and bone ash (Webster 1987). Among its main roles, Mg is a cofactor in activating enzymes that are catalysts for energy production. These enzymes include glucokinase, which plays a role in DNA and RNA and proteins analyses, and also a role in the electrical potential of the nervous system tissues and the cell membranes (Al‐Ghamdi et al. 1994). Mg deficiency causes anorexia, weakness and lethargy, staggering, fatigue, and many other clinical manifestations (Al‐Ghamdi et al. 1994; Nadler and Rude 1995; Rude 1998), while prolonged deficiency may complicate many other diseases (Flink 1981). Iron (Fe) is one of the most important mineral nutrients and has multiple functions in the body. Fe is the part of the structure of hemoglobin, the oxygen carrier of blood, and plays a role in electron chain transport via cytochromes (Fe‐containing enzymes), as well as an integrated part of many enzymatic and catalytic systems of various tissues (Abbaspour et al. 2014; Mascotti et al. 1995). Worldwide, Fe deficiency is considered to be the most common significant dietary shortfall and it is mainly caused by a poor supply of this mineral through the diet (Denic and Agarwal 2007). A negative balance of Fe balance causes an impairment in the production of hemoglobin, iron‐deficient erythropoiesis, and anemia. Fe deficiency has many negative effects, including weakness and reduced working capacity (endurance) (Haas and Brownlie 2001), diminished brain development and function including memory and learning, and compromised immune system (Beard 2003; Sandstead 2000; Zimmermann and Hurrell 2007). Copper (Cu) has raised the interest of scientists after the discovery of its important role in the formation of hemoglobin (Hart et  al. 1930). Although consistent data are readily available on Cu pertaining to its absorption and biochemical cellular role, little is known regarding its physiology and biochemistry, as well as its metabolic fate (Uauy et al. 1998). In the human body, Cu is poorly stored compared with other elements such as zinc and iron. About 25% is stored in bones, 25% in skeletal muscle, 15% in skin, 15% in bone marrow, from 8 to 15% in the liver, and 8% in the brain (Lockitch et al. 1988). The adult body contains less than 100 mg, and the highest concentrations are in the liver, followed by the brain, kidney, and heart (Turnlund 1998). The most important role of copper in human body is its function in metalloproteins, and different investigations have reported that many copper‐containing proteins display oxidative reductase activity in which Cu functions as an electron transfer intermediate in redox

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reactions (Gacheru et  al. 1990; Krebs and Krawetz 1993; Palumbo et  al. 1990; Sánchez‐ Ferrer et al. 1995). Although not thoroughly explained yet, Cu deficiency has been linked to some diseases and disorders. Some studies have reported on Cu deficiency and Menkes syndrome (Kaler 1998), Parkinson and other neurodegenerative diseases (Bandmann et  al. 2015; Montes et al. 2014) and parenteral nutrition (Shike 2009). Zinc (Zn), one of the most important mineral elements in human nutrition, is present in all tissues and fluids (King et al. 2000), and accounts for c. 57% of the skeletal muscle and 29% of the bone (Jackson 1989). From the metabolic point of view, Zn is an essential cofactor of more than 300 allosteric enzymes catalyzing the anabolism and catabolism of the primary metabolites, as well as the metabolism of nucleic acids and other micronutrients. Zn was also found to play a role in the maintenance of cell and integrity by stabilizing the membranes’ molecular structure and in the transcription process and thus in gene expression. Nutritionally, marginally low Zn deficiency is not well established; however, chronic and severe deficiency leads to growth retardation (Nishi 1996), delayed sexuality (Coble et al. 1971), bone maturation (Seo et al. 2010), skin lesions (Kumar et al. 2012), diarrhea (Fischer Walker and Black 2010), alopecia (Betsy et al. 2013), and increased susceptibility to infections mediated via defects in the immune system (Hambidge 2000; Shankar and Prasad 1998). Other effects, such as impaired taste and wound healing, which have been claimed to result from a low Zn intake, are less consistently observed (Lansdown et al. 2007). Iodine (I) is a mineral nutrient having a specific physiological role in the biosynthesis of the thyro‐hormones synthesized by the thyroid glands, and its level is determined by thyroxine hormone production (Utiger 2006). The nutritional status of the population was considerably improved, mainly through the recommendation of the use of iodized salt. Consequently to this program, more than 110 countries have sufficient iodine intake, 30 countries remain iodine‐deficient  –  9 are moderately and 21 are mildly deficient, while none is currently considered severely iodine‐deficient (Pearce et al. 2013). A recent global scale assessment showed that approximately 2 billion people still suffer from I deficiency (ID), of which approximately 50 million present with clinical manifestations (Biban and Lichiardopol 2017). Iodine deficiency results in many disorders and thyroid function abnormalities, including endemic goiter and cretinism, endemic mental retardation, and decreased fertility (Delange 1994; Gunnarsdottir and Dahl 2012; Hetzel 2000; Zimmermann 2011). The significance of selenium (Se) in human nutrition has been revealed during the last few decades (Arthur and Beckett 1994; Foster and Sumar 1997; Reilly 1996). Studies have shown the role of Se in human nutrition resulting from a better understanding of the complex metabolic role of this mineral nutrient. For example, selenium is involved in regulating growth and development, protecting tissues against oxidative stress, and maintaining defenses against some infections (Arthur et al. 2003; Rayman 2000, 2012), while the use of Se with other vitamins and minerals inhibits genetic damage and the development of cancer (El‐Bayoumi 2001). Se deficiency is poorly defined; however, some clinical manifestations have been reported, such as muscular weakness and in several cases myalgia with development of congestive heart failure. In addition, where agricultural soil was low in Se, there was a strong correlation between Se deficiency and Keshan and Keshan‐Beck diseases (Ge and Yang 1993; Levander and Beck 1997).

3.3  ­Bioaccumulation of Mineral Elements by Plant

3.3 ­Bioaccumulation of Mineral Elements by Plants Understanding uptake and bioaccumulation processes by edible plants are important because the accumulated nutrients can directly affect an individual’s health (Streit 1992). However, mineral nutrients are accumulated in different ways and stored in different compartments/organelles by plants species, and are affected by the growing conditions as well as the interactions with other mineral nutrients (Fageria 2006; Rossi et al. 2004). It has been well established that calcium is an ubiquitous messenger in plants and plays a crucial role in signaling. It is recognized that Ca is involved in most biological processes, such as cell division, growth, development, and adaptation to environmental conditions (Mazars et al. 2009). Most of the literature has suggested that Ca is mainly compartmented in the cytosol as a primary sink (Hetherington and Brownlee 2004; Sanders et al. 2002). However, compartments have been also considered as storage organelles, such as chloroplasts (Johnson et  al. 1995), mitochondria (Logan and Knight 2003), and nuclei (Xiong et al. 2006). Phosphorus P is an important plant macronutrient and the second limiting macronutrient for plant growth and development (Schachtman et al. 1998). This macronutrient is a key‐component of numerous vital molecules such as nucleic acids, phospholipids, and ATP, as well as its involvement in the control mechanisms of the regulation of metabolic pathways (Theodorou and Plaxton 1993). From the 1960s, it was recognized that potassium has an enzymatic activator role in plants (Evans and Sorger 1966), and later different studies have widened this role to many other biochemical and physiological ones (Leigh and Wyn Jones 1984). Many plant physiologists have indicated that K accumulates mainly in the vacuole and the cytoplasm (Flowers and Läuchli 1983; Leigh and Wyn Jones 1984). However, the accumulation of the vacuolar and cytosolic K+ pools differ and cytosolic K+ homeostasis differs quantitatively in different cell types as well (Walker et al. 1996). Magnesium under its cationic form Mg2+ plays an essential role in plant physiology such as the function of many catalytic enzymes and ribosomes, aggregation, and protein biosynthesis and bridging phosphorylating and dephosphorylating enzymes to their substrates (Maathuis 2009; Marschner 1995). Magnesium is also the central ion of chlorophyll and its regulation of photosynthetic enzymes in the chloroplasts (Shaul 2002). At the cellular level, studies showed that K accumulates in the vacuoles, which play a key role in its homeostasis (Hermans et al. 2013; Waters 2011). Iron is needed by all green plants and is an essential element for plant metabolism, growth, and development (Briat et al. 1995). When absorbed by the roots under Fe2+, it is oxidized to Fe3+, chelated by citrate, and then transported to the plant top (Brown 1978). Indeed, the mechanisms controlling the compartmentation of Fe are unknown, but it is suggested that Fe is likely bioaccumulated and stored in the vacuole (Lescure et al. 1990). Copper (Cu) one of the micronutrients has been shown to play a role in photosynthesis, respiration, antioxidant activity, cell wall metabolism and hormonal perception (Pilon et al. 2006). In cells, Cu concentrations are under control through an interrelationship of the ATPase and Cu permeases family (Leary and Winge 2007), and most Cu‐ions in cells are either compartmentalized or bound to proteins or metabolites (Lange et al. 2017).

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Zinc is one of the most essential nutrients for plant growth and development, and its essentiality was reported as early as the 1910s (Mazé 1915). Using different localization methods, it was shown that Zn accumulates preferably and mainly in the vacuoles of the epidermal leaf cells as electron‐dense deposits (Vazquez et al. 1992, 1994). Iodine is not a micronutrient. Knowledge about what mechanisms control plant uptake of iodine and where it is stored in plants is still very limited (Weng et al. 2008). Indeed, the metabolic function of iodine in terrestrial plants is not known (Kabata‐Pendias 2011), and little is known on the form in which it is accumulated, its cellular structure, and its metabolism. Nevertheless, increasing studies show that it participates in many physiological and biochemical processes (Gonzali et al. 2017). In higher plants, selenium, which has similar chemical properties to sulfur, is incorporated into proteins in place of cysteine leading to synthesis of selenocysteine and seleno‐ methionine in nonaccumulator plants, while in accumulator ones, Se is sequestered in nonprotein seleno‐amino acids by different mechanisms (Läuchli 1993; Schiavon and Pilon‐Smits 2016).

3.4 ­Biofortification of Edible Plants and Mineral Deficiency Alleviation Mineral deficiencies, considered a hidden malnutrition, affect millions of people, and this malnutrition is worse in the developing countries. It is estimated that over one‐third of the world population is suffering from mineral malnutrition, mainly iron and zinc and/or other (multiple) micronutrient deficiencies (Black 2003), and this issue is more aggravated in low‐ and middle‐income African countries, with 54% of the continental population being deficient in calcium, 40% in zinc, 28% in selenium, 19% in iodine, and 5% in iron (Joy et al. 2014). In Africa, nutrition in Sub‐Saharan regions shows no improvement except for iodine, due to the global use of iodized salt (Sanghvi et al. 2007). This hidden malnutrition is a serious issue and a heavy burden for the governments and the policy makers, due to the health, economic, and social consequences resulting from it (Stein 2010). In the goal of alleviating mineral deficiencies, mitigating its consequences and addressing this hidden malnutrition, one of the most targeted goals of the agronomists is trying to increase the mineral contents of edible plants, especially staple food crops (Khush et  al. 2012; Mayer et  al. 2008). To enhance mineral contents of edible plants through biofortification, various strategies have been and are being used to deal with micronutrient deficiencies, and most of the studies to address this issue have been focusing on five key mineral nutrients – namely, iron, zinc, iodine, calcium, and selenium (Gómez‐Galera et  al. 2010)  –  while emphasis has been placed on the most consumed staple crops namely rice, wheat, maize, beans, cassava, pearl millet, and sweet potato (Khush et al. 2012). In Africa, biofortified crops have been successful in combating and alleviating mineral malnutrition. The release of high‐iron beans in Rwanda and the Democratic Republic of Congo has enhanced the iron status of women and preschool children (Ngozi 2013). In Bangladesh and India, high‐zinc rice provided increased zinc (Ngozi 2013), and it was estimated than zinc biofortified rice save thousands of children life in India annually

3.5  ­Mineral Elements and Agronomic Biofortification Strategies of Edible Plant

(Cakmak 2009), and the iron and zinc status of children was improved when they have been fed with biofortified pear millet as major food staple (Kodkany et al. 2013). In addition to the improved iron status of Indian adolescent (Finkelstein et al. 2015; Scott et al. 2018), iron‐biofortified pearl millet has also improved some of their measures of cognitive performance (Scott et al. 2018). The consumption of high‐iron rice was tested on Filipino women, and surprisingly, the greatest improvement in iron status was seen in nonanemic women. The study concluded that the consumption of iron‐biofortified rice was efficient in improving iron stores of women with iron‐poor diets in the developing world (Haas et al. 2005). Similar food‐based strategies to reduce nutritional iron deficiency in Rwandan women have been used, and the results showed that the consumption of iron‐biofortified beans significantly improved their iron status (Haas et al. 2016). In China in the early 2000s, around 250 million people were affected by iron deficiency, and approximately 100 million people were affected by zinc deficiency. To alleviate these deficiencies, biofortification with improved Fe and Zn varieties was adopted as long‐term strategy (Ma et al. 2008). In Mexico, the quantity of absorbed zinc was increased by biofortifying wheat with Zn (Rosado et al. 2009), and in Sri Lanka, blood selenium concentration was increased after consumption of high‐Se lentils (Thavarajah et al. 2011).

3.5 ­Mineral Elements and Agronomic Biofortification Strategies of Edible Plants Agronomic practices were used earlier to improve the mineral nutrients of edible crops. Practically, the supply of fertilization to the soil or the application of foliar fertilizers during the growth and the development of crops resulted in increasing the levels of many mineral nutrients in many crops. On the other hand, intercropping between dicots and gramineous species has shown interesting results in improving mineral contents of some crop species (Zuo and Zhang 2009). Nevertheless, the efficacy of this approach depends to some extent on the targeted species to be fortified, the form of the fertilizer, and how this fertilizer is applied (Mao et al. 2014). Numerous edible crops have been fortified using fertilizers either by soil or foliar application (Table 3.1). Soil and foliar application of fertilizers have shown to be interesting strategies in increasing mineral nutrients in crops, particularly cereals wheat, maize and rice and other staple crops (de Valença et al. 2017). However, the effectiveness of mineral fertilization for biofortification depends on numerous factors such as the application method, either soil or foliar, the fertilizer formulation, soil properties, the bioavailability and uptake of the nutrient, and the nutrient use efficiency by the targeted species (Lawson et al. 2015; Shuman 1998). On the other hand, the application of fertilizers always leaves concentrations of these minerals in the fruits, seeds, and grains (Dai et  al. 2004; Frossard et  al. 2000). Furthermore, this approach requires repetition and fertilizers should be applied frequently if not annually. Therefore, it is economically not feasible for many farmers, even though biofortified crops reduce the health burden due to human mineral nutrients deficiencies (Wang et al. 2016).

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Table 3.1  Biofortification of edible crops using fertilization strategy. Mineral Nutrient

Biofortified Crop

Form of Fertilizer

Form of Supply

References

Iron

chard shoots

FeSO4•7H2O

Foliar application

Hernández‐Castro et al. (2015)

Rice

Fe amino acid

Foliar application

Yuan (2013)

DTPA‐F Fe‐amino acid

Soil application Kromann et al. (2017)

EDTA‐chelated Fe

Foliar application

FeSO4‐7H2O Fe‐EDTA, FeSO4

Soil application Aciksoz et al. (2011), Ramzani et al. (2016)

Fe‐EDTA, FeSO

Foliar application

ZnSO4•7H2O

Soil application Yilmaz et al. (1997).

Potato

Wheat

Zinc

Wheat

Foliar application ZnSO4, Foliar Zn‐arginine [Zn(Arg)2], application Zn‐glycine [Zn(Gly)2] Zn‐histidine [Zn(His)2]

He et al. (2013) Kromann et al. (2017)

Aciksoz et al. (2011)

Abdoli et al. (2014), Yilmaz et al. (1997), Yuan (2013) Ghasemi et al. (2013).

Rice

ZnSO4•7H2O

Soil application Liu et al. (2016)

Pea

ZnSO4•7H2O

Soil application Poblaciones and Rengel (2016) Foliar application

Selenium

Teff ZnSO4•5H2O (Eragrostis tef)

Soil application Haileselassie et al. (2011)

Common bean ZnSO4•7H2O

Foliar application

Onion

Zn‐complexes

Soil application Almendros et al. (2015)

Potato

Zn‐Amino Acid

Soil application Kromann et al. (2016)

EDTA‐chelated Zn

Foliar application

Kromann et al. (2016)

chard shoots

FeSO4‐7H2O

Foliar application

Hernández‐Castro et al. (2015)

Wheat

Na2SeO4

Soil application Chilimba et al. (2012), Jiang et al. (2018)

Rice

Na2SeO4 and Na2SeO3

Soil application Lidon et al. (2018), Reis et al. (2018)

Na2SeO4 and Na2SeO3

Foliar fertilization

Na2SeO4

Soil application Chilimba et al. (2012)

Na2SeO3

Soil application Wang et al. (2013)

Maize

Foliar application

Ram et al. (2016)

Boldrin et al. (2013).

3.5  ­Mineral Elements and Agronomic Biofortification Strategies of Edible Plant

Table 3.1  (Continued) Mineral Nutrient

Biofortified Crop

Form of Fertilizer

Form of Supply

Carrot

Na2SeO3, Na2SeO4

Soil application Kápolna et al. (2009), Smoleń et al. (2016)

MeSeCys, SeMet,

Iodine

References

Kápolna et al. (2009)

Cucumber

Na2SeO4 and Na2SeO3

Soil application Businelli et al. (2015), Hawrylak‐Nowak et al. (2015)

Tomato

Na2SeO4

Soil application Businelli et al. (2015)

Pak Choi

Na2SeO4, Na2SeO3

Soil application Li et al. (2015)

Lettuce

Na2SeO4, Na2SeO3

Soil application Businelli et al. (2015), Ríos et al. (2008)

Strawberry

Na2SeO4

Soil application Mimmo et al. (2017)

Wild Cabbage KI and KIO3 butterhead lettuce

Foliar application

Lawson et al. (2015)

Radish Spinach

KI

Soil application Dai et al. (2004), Smoleń and Sady (2012)

Potato

KI and KIO3

Soil application Caffagni et al. (2011, 2012) Foliar application

Tomato

KI and KIO3

Caffagni et al. (2012)

Soil application Caffagni et al. (2011, 2012), Kiferle et al. (2013). Foliar application

Caffagni et al. (2012)

Lettuce

KI and KIO3

Soil application Blasco et al. (2008), Kopeć et al. (2015)

Wheat

KI and KIO3

Soil application Caffagni et al. (2012) Foliar application

Barley

Soil application Foliar application

Cabbage

KIO3

Soil application Ren et al. (2008)

Alfalfa

KI

Soil application Altınok et al. (2003) Foliar application

Nectarine

KI

Soil application Caffagni et al. (2012) Foliar application

Plum

KI

Soil application Foliar application (Continued)

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Table 3.1  (Continued) Mineral Nutrient

Biofortified Crop

Magnesium Wheat

Form of Fertilizer

Form of Supply

MgSO4•7H2O

Soil application Ceylan et al. (2016) Foliar application

Potassium

Wild eggplant KCl

References

Ceylan et al. (2016)

Soil application Adu et al. (2018)

African eggplant

3.6 ­Mineral Elements and Breeding Biofortification Strategies of Edible Plants Biofortification through breeding is based on either exploring the genetic diversity of the existing species or identifying the existing varieties or germplasms (Welsch and Graham 2004). Many crops have been fortified by conventional breeding (Graham et al. 1999, 2005; Pixley et al. 2011; Velu et al. 2007). However, this strategy has some constraints, such as low yield of the biofortified crop (Bänzinger and Long 2000) and cost (Bouis 2003). Compared to the other strategies, plant breeding for biofortification did not develop much fortified crops, except for a few cereals and legumes. Crop fortification of fruits and vegetables has garnered little interest; it has only had limited success and is time‐consuming (Table  3.2). However, breeding is much more accepted by consumers than some of the other methods.

3.7 ­Mineral Biofortification Using Transgenic Plants Biofortification of crops can also take place using modern biotechnology techniques, including transgenesis and genome editing. Transgenic crops are under development with increased accumulation of important minerals such as iron, zinc, and calcium within edible tissue. Other transgenic crops have been developed with reduced concentrations of antinutrients, such as phytate, which reduce the bioavailability of minerals by interfering with their absorption in the gut (White and Broadley 2009).

3.7.1  Transgenic Crops Biofortified with Iron and Zinc One of the most well‐studied crop species for mineral biofortification is rice (Oryza sativa), a staple of a large proportion of the world’s poor and lacking in several essential micronutrients. Transgenic rice plants have been used as a model system to enhance the amount of bioavailable iron and zinc that is found in the edible seed (endosperm) of cereals. Plant scientists have discovered that metal transporter proteins found in many crop species can use multiple metal substrates, including iron, zinc, and even cadmium as substrates for

3.7  ­Mineral Biofortification Using Transgenic Plant

Table 3.2  Biofortification of crops using breeding strategy. Mineral Nutrient

Targeted Crops

References

Iron

Pearl millet

Ashok Kumar et al. (2019), Velu et al. (2007)

Zinc

Rice

Stein et al. (2007)

Maize

Bänzinger and Long (2000), Ortiz‐Monasterio et al. (2007)

Cassava

Chavez et al. (2000)

Cowpea

Santos and Boiteux (2013)

Mumgbean

Nair et al. (2013)

Common bean

Beebe et al. (2000), Blair (2013), Hoppler et al. (2014)

Pearl millet

Velu et al. (2007)

Maize

Graham et al. (1999), Ortiz‐Monasterio et al. (2007)

Wheat

Calderini and Ortiz‐Monasterio (2003) Peleg et al. (2008)

Rice

Graham et al. (1999)

uptake from soil into the roots. Researchers found through mutational analysis that loss of function mutants of these transporter proteins creates a loss of uptake of all three of these metals into plant cells (Morrissey and Guerinot 2009). In addition to this, the iron storage protein ferritin can assist in metal accumulation in plant tissue. For example, Masuda et al. (2012) were able to upregulate accumulation of the iron storage protein ferritin and increase iron translocation via the overexpression of the iron (II)‐nicotianamine transporter OsYSL2 within rice endosperm. Transgenic lines were demonstrated to generate higher levels of both iron (6‐fold in the greenhouse and 4.4‐fold in the paddy) and zinc (1.6‐times) using this approach, suggesting that introduction of multiple genes involved in iron and zinc homeostasis would be superior for iron biofortification than merely the introduction of a single gene. Furthermore, Masuda et  al. (2013), were able to significantly increase iron and zinc accumulation all the more through the enhancement of iron uptake and transport using the ferric iron chelator, mugineic acid. For this approach, the authors developed transgenic plants that expressed the ferritin gene from soybean (SoyferH2), and driven by two endosperm‐specific promoters, in addition to the barley nicotianamine synthase gene (HvNAS1), two nicotianamine aminotransferase genes (HvNAAT‐A and ‐B), and a mugineic acid synthase gene (IDS3). The latter was to increase mugineic acid production in rice plants. Transgenic plants that generated using this approach were tolerant to iron‐deficient soil and displayed increased iron accumulation by 2.5‐fold. Under iron‐sufficient conditions, however, transgenic rice lines increased iron accumulation by fourfold as much as lines that had been cultivated in either commercially supplied soil (iron‐sufficient conditions) or calcareous soil (iron‐deficient conditions). Furthermore, transgenic lines expressing both ferritin and mugineic acid biosynthetic genes displayed signs of iron‐deficiency tolerance in calcareous soil, and the iron concentration in polished T3 seeds increased by 4 and 2.5 times, in comparison to nontransgenic lines grown in normal and calcareous soil, respectively.

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Since the same molecular machinery is utilized for transporting iron and zinc into plants, addressing iron deficiency in rice also brings about increased zinc accumulation. As an example, Aung et al. (2013), generated a transgenic line of rice commonly eaten by consumers in Myanmar, where approximately 70% of the populace is iron deficient. This line overexpressed the nicotianamine synthase gene HvNAS1 to enhance iron transport, the Fe(II)‐nicotianamine transporter gene OsYSL2 to transport iron to the endosperm, and the Fe storage protein gene SoyferH2 to increase iron accumulation in the endosperm. The rice plants were shown to accumulate over 3.4‐fold higher iron concentrations, in addition to 1.3‐fold higher zinc concentrations compared to conventional, nontransgenic rice. The results of this study indicate that transgenic rice biofortified for increased iron content could address both iron as well as zinc micronutrient deficiency in the Myanmar population. Milling tends to remove the nutrient‐rich outer layers of the rice embryo, thus reducing iron and zinc concentrations and causing deficiencies in these micronutrients. One solution proposed by Paul et  al. (2014) was to generate transgenic high‐yielding indica rice expressing the ferritin gene derived from soybeans. These plants produced greater than 2.6‐fold higher level of ferritin, even in the fourth generation of rice plants, than their nontransgenic counterparts. Upon milling, transgenic rice grains provided 2.54‐fold and 1.54‐ fold increases in iron and zinc content, respectively. In a similar fashion, the iron transporter gene MxIRT1 taken from apple trees was utilized by Tan et al. (2015) to generate transgenic rice plants that exhibited a threefold increase in iron and zinc accumulation, as well as a decrease in cadmium concentration. Cadmium is thought to compete with iron and zinc for transport and accumulation in the rice endosperm. Lower amounts of cadmium results in a reduced toxicity in rice seed. The authors concluded that the MxIRT1 transporter gene could be used effectively to biofortify cereal crops such as rice. Other approaches have also yielded improvements in iron and zinc biofortification. For example, Trijatmiko et  al. (2016) showed that plants expressing rice nicotianamine synthase (OsNAS2) and soybean ferritin (SferH‐1) genes possessed enriched endosperm Fe and Zn content. A Caco‐2 cellular assay was employed to demonstrate that the increased iron and zinc levels found in these rice plants were also highly bioavailable. Furthermore, Banakar et al. (2017) generated transgenic rice plants that expressed high levels of nicotianamine and 2′‐deoxymugenic acid (DMA). These rice plants were able to accumulate up to fourfold more iron and twofold more zinc in rice endosperm, as well as lower levels of cadmium compared to wild‐type plants. Other crop species have been examined for iron and zinc biofortification using biotechnological approaches. For example, Tan et al. (2018) biofortified the pulse crop chickpea (Cicer arietinum L.) by improving iron transport and storage through a combination of chickpea nicotianamine synthase 2 (CaNAS2) and soybean (Glycine max) ferritin (GmFER) genes. Transgenic chickpea overexpressing these genes were assessed for iron and nicotianamine (NA) levels. A doubling of NA concentration suggested an increase in iron bioavailability. Similarly, Manwaring et al. (2016) examined the potential for the biofortification of pearl millet with iron and zinc by improving the currently available gene pool. The use of transgenesis to biofortify a staple such as pearl millet would be advantageous for poor regions of the world where soil management or supplementation programs are ineffectual. Finally, Narayanan et al. (2015) expressed the iron‐sequestering Arabidopsis AtVIT1 gene in cassava plants, and

3.8  ­Conclusions and Future Direction

were able to demonstrate an increase in iron storage in the crop’s roots. Iron concentration also increased in stem tissues and accumulated in plant cellular vacuoles. These studies demonstrate the ability of biofortification to address micronutrient deficiencies in a variety of crops found in resource‐poor nations (for additional details, see Chapter 8).

3.7.2  Calcium Biofortified Transgenic Plants To date, efforts to improve the calcium content of crops via biotechnology has taken a number of approaches and the outlook is promising. These advances hinge on improved knowledge of how soluble calcium ions found in the soil are transported and accumulate in plant tissue (Dayod et al. 2010). Calcium is used as a nutrient by both plants and animals; it plays a significant role in general cell signaling. How calcium transporters are expressed can thus influence a plant’s ability to withstand stress and ward off pathogens, and can influence the nutritional status of animals and humans. Park et al. have generated transgenic tomato, potato, lettuce, and carrots expressing high levels of calcium transporters have been generated (Park et al. 2004, 2005a, b, 2009). For example, modification of a calcium transporter known as a short cation exchanger (sCAX1) has been demonstrated to increase calcium transport into plant cell vacuoles (Connolly 2008). Consumption of calcium biofortified transgenic carrots in animal models has shown that enhanced absorption of calcium has taken place. Similarly, Sharma et al. (2017) have examined the potential of finger millet, an orphan crop with high calcium content, by studying the mechanisms behind calcium uptake, transport, and accumulation in grain. Information gathered using finger millet as a model system can help to improve other food crop types. It is important to note that climate change may have adverse effects on mineral accumulation in different crop species; this could further limit the nutrients available in food crops for both humans and animals (Martínez‐Ballesta et al. 2010).

3.8 ­Conclusions and Future Directions Despite intensive research activities for crop biofortification, the number of minerals biofortified crops release is limited compared to the number of edible crops. Although mineral biofortification does not change visible edible crop traits, their adoption by farmers and their acceptance by consumers are still not evident. Indeed, biofortified crops should have agronomic properties of interest to farmers, locally adapted, and acceptable to consumers. In the case of genetic engineering biofortification, additional problems such ethical, wholesomeness, and instinctual properties might be constraints to their development. Current GMO regulation have delayed the introduction of the genetically modified crops, adding another financial burden. Mineral biofortification of edible crops might be more successful by redesigning of agricultural technologies and practices. A first approach is the development of new‐generation fertilizers for biofortification, which should be agronomically and economically effective with improved use efficiency by crops, and with less side effects on the yield and the accumulation of other nutrients, particularly other mineral nutrients and proteins, as is the case nowadays.

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Second, fundamental research is important regarding mineral nutrients bioaccumulation by plants to deepen our knowledge of plant physiological processes and mineral nutrient uptake mechanisms, their translocation, compartmentation, and metabolism. These studies will help to decipher the physicochemical packaging of nutrients, optimal amount to supply, and timing of application to meet plant needs for improved uptake and bioaccumulation. Third, even though studies indicate that biofortification strategies show real promise to be a cost‐effective providers of nutrients, consumers and farmers must be educated on the nutritional benefits in order to enhance their acceptance. Fourth, many countries have restricted the use, labeling, and marketing of biofortified crops, segregating enhanced mineral nutrients from that of nonbiofortified foods and requiring different strategies for each biofortified crop to overcome consumers and farmers skepticism and regulatory obstacles.

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4 Biotechnologies and Vitamins Biofortification of Edible Crops Noureddine Benkeblia1,2 and Kathleen L. Hefferon3 1 

Department of Life Science and Laboratory of Tree Fruit and Aromatic Crop, The Biotechnology Centre, The University of the West Indies, Kingston, Jamaica 2  Laboratory of Crop Science, Department of Life Sciences, The University of the West Indies, Kingston, Jamaica 3  Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada

4.1 ­Introduction Crop plants are the primary source of energy and one the main sources of many vitamins. However, the level and composition of edible crops varies significantly, and many are deficient in certain nutrient components, particularly vitamins. Worldwide, cereals, roots, and tubers are major staples, and these crops are rich in carbohydrates, but have very lowquantity or poor-quality proteins and vitamins (Hirschi 2009). In Asia, where people depend to a great extent on rice, they are therefore more prone to vitamin A and vitamin deficiencies due to the lack of these nutrients in rice; thus making them more susceptible to numerous human health problems (Bhullar and Gruissem 2013). Vitamins have a complex biochemistry and play an essential role in human nutrition and health, and their deficiencies result in several disorders such as blindness caused by vitamin A deficiency, beriberi caused by vitamin B1 deficiency, pellagra caused by vitamin B3 deficiency, anemia caused vitamin B6, scurvy caused by vitamin C deficiency, and rickets caused by vitamin D deficiency (Asensi-Fabado and Munné-Bosch 2010). However, vitamin A deficiency is considered especially challenging worldwide, particularly in lowincome countries. About 125–130 million children are at risk of morbidity and infectious diseases mortality (Kramer et al. 2008). For example, some vitamins (A and C) have been shown to possess antioxidant properties in the processes associated with osteoarthritis (OA), and the action of vitamin D, in parallel to the actions of vitamin A and vitamin C, is thought to play an important role in reducing the risk of this disease by inducing bone mineralization and cell differentiation (Sowers and Lachance 1999). In plants, there are different hydro- and lipo-soluble vitamins with a large variation in their content in crops. However, the concentrations of most vitamins in the edible parts of the plants are often low. One of the goals of research during the last two decades were to decipher the physiological, biochemical, and molecular mechanisms of the synthesis, Vitamins and Minerals Biofortification of Edible Plants, First Edition. Edited by Noureddine Benkeblia. © 2020 John Wiley & Sons Ltd. Published 2020 by John Wiley & Sons Ltd.

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translocation, and accumulation in plants (Grusak and DellaPenna 1999). By understanding these mechanisms, different strategies can be developed to manipulate these plants and improve their nutritional qualities and increase their vitamin content. Indeed, vitamins levels in edible crops can be enhanced by different strategies, including agronomic practices breeding, metabolic engineering, and thus enhancing the nutritional quality of crops (Miret and Munné-Bosch 2014). Although these strategies have known some success, still many issues remain to be addressed. First, the biofortified crop should be successful and the increased vitamin level should not affect the yield. Second, the biofortified crop should be at least as nutritious as the conventional crop. Third, the biofortified crop should be accessible and adopted by the farmers and accepted by the consumers (Bouis and Welch 2010).

4.2 ­Vitamins Requirements in Human Nutrition From the discovery in the 1900s of chemical structures, their physiological roles, and their contribution in health and well-being, vitamins have played a major role in human nutrition (Olson 1994). Indeed, there is a great deal of new scientific evidence establishing the role of vitamins in preventing deficiency diseases, diet-related chronic diseases, and major causes of morbidity and mortality. Studies also show the importance of vitamins for immune function, physical work capacity, and cognitive development, including learning capacity in children (FAO-WHO 1998). Despite our current knowledge about vitamins, inadequate vitamin status still abounds in the less industrialized world. With some vitamins, toxicity also poses a problem. The daily dietary intake required for physiological functions is not well defined. Different scientific definitions exist for the daily dietary requirements of vitamins for humans, but the experimental data readily available provides a rough estimate of the minimum daily intake, and discrepancies between national and international bodies have led to a variety of recommendations (Brubacher 1989; Olson 1994; Ross et al. 2011) (Table 4.1). Nevertheless, the average dietary intake of vitamins to meet all known physiological needs varies with gender, age, and the physiological state. Therefore, recommended dietary intakes (RDI) for infants, children, elderly, men, women, and pregnant and lactating women are different, as shown in Table 4.1.

4.3 ­Bioaccumulation of Vitamins in Plants Many vitamins are found in plants, including β-carotene (pro-vitamin A), thiamin (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5), pyrodoxal (vitamin B6), biotin (vitamin B7), folic acid (vitamin B9), ascorbic acid (vitamin C), α-tocopherol (vitamin E), and phylloquinone (vitamin K). From the biochemical point of view, the development of enzymology and microbial biochemistry helped enormously in understanding the biosynthetic pathways of primary and secondary metabolites; many pathways of vitamins biosynthesis in plants have been deciphered and well understood, and many other features are emerging. All the vitamins are

Table 4.1  Recommended vitamins intakes. Vit. A Vit. C (μg RE/d) (mg/d)

Vit. D (μg/d)

Vit. E (mg Vit. K Vit. B1 Vit. B2 Vit. B3 Vit. B6 Vit. B9 Vit. B12 Vit. B5 Vit. B7 α-TE/d)d (μg/d) (mg/d) (mg/d) (mg NE/d)e (mg/d) (μg DFE/d) (μg/d) (mg/d) (μg/d)

0–6 months

375

25

5

2.7

5

0.2

0.3

2

0.1

80

0.4

1.7

5

6–12 months

400

30

5

2.7

10

0.3

0.4

4

0.3

80

0.7

1.8

6

1–3 y

450

30

5

5

15

0.5

0.5

6

0.5

150

0.9

2.0

8

4–6 y

500

35

5

5

20

0.6

0.6

8

0.6

200

1.2

3.0

12

7–9 y

600

35

5

7

25

0.9

0.9

12

1.0

300

1.8

4.0

20

ADOLESCENTS Males 9–18 y

600

40

5

7.5

35–55 1.0

1.0

16

1.2

400

2.4

5.0

25

Females 9–18 y

500

40

5

10

35–55 1.3

1.3

16

1.3

400

2.4

5.0

25

Females 19–50 600 y

45

5

7.5

55

1.1

1.1

14

1.3

400

2.4

5.0

30

Females 51–65 500 y

45

20

7.5

55

1.1

1.1

14

1.5

400

2.4

5.0

30

Males 19–65 y

Life Stage Group

INFANTS CHILDREN

ADULTS

450

5–10

10

65

1.3

1.3

16

1.7

400

2.4

5.0

30

Females >65 y 600

600

45

15

7.5

55

1.1

1.1

14

1.5

400

2.4

5.0

–

Males >65 y

600

45

15

10

65

1.3

1.3

16

1.7

400

2.4

5.0

–

Pregnant Women

800

55

5

–

55

1.4

1.4

18

1.9

600

2.6

6.0

30

Lactating Women

850

70

5

–

55

1.5

1.6

17

2.0

500

2.8

7.0

30

Source: from FAO-WHO (1998).

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synthesized by plants except ascorbic acid (vitamin C), which is specifically synthesized by eukaryotic cells (Ishikawa et al. 2006; Miret and Munné-Bosch 2014; Smith et al. 2007). However, the metabolites flux and their cellular compartmentations are complex because of plastid organelles, which are found exclusively in eukaryotic cells (Miret and MunnéBosch 2014). In plants, several studies have established that many vitamins are biosynthetically mono-compartmentalized principally in the plastids such as B2 (Fischer and Bacher 2006), carotenoids (pro-vitamin A) (Rodriguez-Villalon et  al. 2009), and vitamin E (Hunter and Cahoon 2007). The plastid compartment has been subject to extensive literature and the accumulation of data from decades, which established its numerous essential functions, such as carbon and nitrogen assimilation and biosynthesis of amino acids, fatty acids, and other secondary metabolites. Additionally, plastid organelles communicate and coordinate their various functions with other cellular compartments (Rolland et al. 2012). Vitamins might also be synthesized in other organelles, for example in the mitochondria such as ascorbic acid (vit C) (Bartoli et al. 2000) and pantothenate (vitamin B5) (Chakauya et al. 2006). However, the biosynthesis of other vitamins may be compartmentalized in two organelles such as vitamin K1, which occurs in the peroxisomes (Reumann 2013), and plastids (Sandoval et  al. (2008), while for other vitamins such as folic acid the biosynthesis pathways present a complex spatial organization with three subcellular compartments, namely the cytosol, chloroplasts, and the mitochondria (Figure 4.1) (Rebeille et al. 2006). Extensive literature established that the biosynthesis and accumulation of vitamins is principally chemistry and physiologically based, respectively, and progress in omics technologies has shown that it is possible to consider biotechnological alternatives, including biotransformation using molecular engineering techniques (Vandamme 1992). However, the development of vitamin hyperproducing plants is not as easy as it looks because it

Figure 4.1  Cross-points on the biosynthetic pathways of vitamins in plants. The subcellular location of the reactions in the white boxes is unknown. Shared precursors of several vitamin biosynthetic pathways are depicted in colored boxes. Enzyme activities that divert the shared precursors toward a specific vitamin biosynthetic route (competitive pathways) are numbered: (1) 1-deoxy-D-xylulose-5phosphate synthase; (2) 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase; (3) 5-phosphoribosyl-1- pyrophosphate synthase; (4) 3,4-dihydroxy-2-butanone 4-phosphate synthase; (5) quinolinate synthase; (6) pyridoxal 50-phosphate synthase; (7) phosphomannose isomerase; (8) chorismate mutase; (9) isochorismate synthase; (10) 4-amino-4-deoxychorismate synthase; (11) phytoene synthase; (12) homogentisate geranylgeranyl transferase; (13) geranylgeranyl reductase; (14) homogentisate phytyl transferase; (15) 1,4-dihydroxy-2-naphtoate phytyl transferase; (16) lycopene e-cyclase; (17) lycopene b-cyclase; (18) b-ring hydroxylase; (19) 5-aminoimidazole ribonucleotide carboxylase; (20) THIC protein; (21) GTP cyclohydrolase II; (22) GTP cyclohydrolase I; (23) thiazole synthase (THI1); (24) glycine amide ribonucleotide synthase; (25) hydroxyphenylpyruvate (HPP) dioxygenase. For ease of visualization, the names of the vitamins are displayed next to the corresponding metabolic compounds. Lipid-soluble vitamins are depicted in red, water-soluble vitamins are depicted in blue. The activity of vitamins as cofactors in some enzymatic reactions is indicated in blue. Abbreviations: AIR, 5-aminoimidazole ribonucleotide; HET-P, 4-methyl5-b-hydroxyethyl thiazole phosphate; HMP-PP, 2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate; HPP, hydroxyphenylpyruvate; IMP, inosine monophosphate; PPRP, 5-phosphoribosyl-1pyrophosphate; S-AdoMet, S-adenosylmethionine (Source: from Asensi-Fabado and Munné-Bosch [2010], with permission of Elsevier).

Pyridoxine 5’-P

Pyridoxine

Pyridoxine

Pyridoxamine 5’-P

Pyridoxamine

Hexose-P pool

Pyridoxal 5’-P Gln 6 Glu

Vit B6

Pentose-P/ Triose-P pool Plastid Ribose 5-P

3

Gln Glu PRPP

Glycine Pyridoxal 5’-P

Glyceraldehyde 3-P

1

Pyruvate

Pentose-P/ triose-P pool PEP

B1

Geranylgeranyl-DP 11

HPP 25 C Homogentisate 13 12

α-Carotene Pro-vit A

β-Carotene 18 β-Cryptoxanthin

Thiamin

21

Ribulose 5-P 4

Cell wall polysaccharides GTP 22

Riboflavin

Pimeloyl-CoA D-Galacturonate B6 L-Alanine

Fructose 6-P 7

L-Galactonate Vit B5

Gln Glu H2Pterin

Phytyl-DP

p-Aminobenzoate

15

Pantothenate

H2Pterin-PP Glu

Phylloquinone Vit K1 Dihydroxyacetone phosphate 5 Quinolinic L-Aspartic acid acid

H4 Folate Vit B9

7,8-Diaminopelargonic L-Galactono- GDP-L- GDP-Dacid 1,4-lactone Galactose Mannose cy cOX

Pantoate β-alanine

cyt cred

GDP-Lgulose

Propionate

Uracil Spermidine

H2 Folate B9 (S-AdoMet)

B9 (S-AdoMet)

Vit E

Guanosine-P

Vit B1

1,4-Dihydroxy-2Naphthoate

Tocopherols

Gln Glu

B9 IMP 20

Thiamin-PP

Vit B2

14

19

Thiamin-P

Chorismate 10 8 9 Tyrosine B1

Tocotrienols All-trans-lycopene 16, 17 17

Erythrose 4-P

AIR

HET-P HMP-PP 23 L-cysteine

Shikimate

Isopentenyl DP

Phytoene (C40)

2

24

B9

L-Ascorbate

Ketopantoate B9 α-Ketoisovalerate

Biotin

Vit B7

L-Valine Gln Glu Nicotinic acid mononucleotide

L-Gulono1,4-lactone

Vit C

Vit B3 Niacin Mitochondrion

Myoinositol

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requires the elucidation of their biosynthesis pathways and their metabolic (de) regulation; therefore, further research is required. Tremendous progress of genomics and other related technologies are contributing significantly in elucidating these pathways, though, and are therefore gaining importance in biofortification strategies.

4.4 ­Biofortified Edible Plants and Vitamin Deficiency Alleviation Indeed, the discovery of the effects of vitamin deficiencies were described thousands of years ago. Beri-Beri, caused by B1 deficiency, was reported by the Chinese more than 2500 years ago, and scurvy caused by vitamin C deficiency was reported more than 1100 years ago (McCormick 2006). Early in the twentieth century, major discoveries on the roles of vitamins in nutrition and  the effects of their deficiencies on human health established that a diversified and ­balanced diet with adequate concentrations and combination of vitamins is required. Therefore, plants can be of great help to alleviate these deficiencies and prevent many diseases and disorders resulting from vitamin deficiencies (Fitzpatrick et  al. 2012; Murgia et al. 2013; Strobbe and Van Der Straeten 2018). This approach has the advantage of not requiring drastic changes in dietary habits. One of the most alleviated vitamin malnutrition causes is vitamin A deficiency. Tremendous efforts have been made to combat this deficiency, particularly in developing countries and regions with a high prevalence of poverty. Vitamin A biofortified crops such as golden rice (Beyer 2010) and sweet potatoes (Mitra 2012) have been proposed as a more effective intervention. Similar studies have also shown that biofortified crops have alleviated vitamin A deficiency in many other developing countries, such as Zambia through orange maize consumption (Gannon et  al. 2014), Kenya through cassava consumption (Talsma et  al. 2016), and in Mozambique and Uganda through consumption of orange sweet potato (Hotz et al. 2012a, b). An implemented project in Kenya further demonstrated that an increased consumption of orange-fleshed varieties of sweet potatoes rich in β-carotene significantly alleviated vitamin A deficiency (Hagenimana and Low 2000).

4.5 ­Agronomic Practices for Vitamin Biofortification of Edible Plants Contrary to the mineral biofortification of edible crops using agronomic strategies, no literature has been reported on using agronomic practices and vitamin biofortification of edible crops.

4.6 ­Edible Crop Breeding for Vitamin Biofortification Breeding of edible crops for vitamin biofortification particularly targets the edible parts of staple crops at concentrations high enough to impact human health; however, the existing genetic variation for the given trait is sufficient. In this context, vast and numerous breeding programs are being developed to biofortify major food staples, particularly

4.6  ­Edible Crop Breeding for Vitamin Biofortificatio

in developing countries, with the most important vitamins β-carotene (provitamin A) and folate (B9) (Mayer et al. 2008; Welch 2002). Although crop science has progressed tremendously these last few decades, breeding strategies for biofortifying edible crops still remain questionable due the limited results obtained, although some success has been achieved with β-carotene and folate (Bouis 2002). Indeed, the real challenge in breeding biofortified varieties is to develop new varieties with high vitamin content using on one hand the excellent traits of the existing varieties rich in the targeted vitamin, and on the other hand high vitamin concentration lines coming from distant and different environments but often having low yield and not always suitable for local use (Pixley et al. 2013). However, this strategy is quite frustrating, especially with the vegetatively propagated crop plants, because their lines and varieties are highly heterozygous. Therefore, the varieties are lost during cross-breeding and the crossbred products revert to a wild type, as is the case for many crops such as potato and cassava (Sautter et al. 2006), although later studies showed that a gain on total carotenoids in cassava might be feasible (Ceballos et al. 2013; Njoku et al. 2011). The most outstanding success of vitamins biofortification is the enhancement of β-carotene (provitamin A) of edible crops. Maize (Ortiz-Monasterio et al. 2007; Pixley et al. 2013), wheat (Ortiz-Monasterio et al. 2007), rice (Datta et al. 2006), and sweet potato (Ma et al. 2009) have been the most successful crops improved with high carotenoid content. As just noted, two combined approaches have been used to enhance the β-carotene levels in sweet potato: the evaluation and screening of the parents and the use of molecular markers. This combination is quite complex due to the genetic complexity of sweet potato (Ma et al. 2009). Recently, banana genotypes with high provitamin A carotenoid (pVAC) content have been identified for possible biofortification, and this fruit might be an efficient approach to combat vitamin A deficiency in developing countries (Amah et al. 2019). Folate is the second vitamin that has raised the interest of scientists, and numerous attempts have been conducted to enrich some mass crops such as rice, corn, wheat, potato, and cassava, which contain inadequate folate levels. Contrary to provitamin A, most of these staple foods have low intrinsic concentrations of folate, and studies show that their folate biofortification through breeding strategies has not been successful and only limited enhancement has been achieved (Rebeille et al. 2006). Nevertheless and due to accessibility of well-characterized interspecific introgression lines (ILs) and novel populations, the goal of folate crop biofortification through conventional plant breeding is becoming a more realistic possibility (Strobbe and Van Der Straeten 2017). Indeed, the screening of the large collections of germplasm has revealed greater diversity in different lines, thereby favoring this strategy (Jha et al. 2015) for rice (Dong et al. 2011), potato (Robinson et al. 2015), spinach (Shohag et al. 2011), and dry beans (Khanal et al. 2011). Indeed, biofortification of edible crops through plant breeding has the potential to be more accessible and at lower cost; however, three issues must be considered: 1) Farmers and consumers must be encouraged to adopt these new varieties because often these varieties have differences that are not attractive nor appreciated by the farmers and/or the consumers (Birol et al. 2015; Wolson 2007). 2) Bioavailability of the nutrient can be questionable as reported by literature (Keats et al. 2019; Palmer et al. 2016).

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3) Often, the new variety has a lower yield (Tester and Langridge 2010) or the biofortification negatively affects the bioaccumulation of other nutrients (Welch and Graham 2004).

4.7 ­Vitamins and Transgenic Biofortification Strategies of Edible Crops GM technology also has the potential to reduce the global burden of malnutrition and hidden hunger. Vitamin or mineral enriched GM foods (GM biofortified foods), are considered to be the next generation of genetically modified organisms, GMOs. Non-GM biofortified crops have been widely developed and commercialized, but the applied conventional breeding techniques may be inadequate for crops with a low or absent level of a certain micronutrient (Beyer 2010). A recent review has summarized successful R&D efforts in the field of GMOs with increased micronutrient content in staple crops (De Steur et al. 2015).

4.7.1  Vitamin Biofortified Rice Two of the most well-known, examples of GM vitamin biofortification are golden rice, enriched with pro-vitamin A (β-carotene) (Paine et al. 2005; Ye et al. 2000), and vitamin B9 (folate) enhanced rice (Blancquaert et al. 2014; Storozhenko et al. 2007). In these cases, conventional breeding techniques were not applicable given the absence and low content of these micronutrients in rice. For golden rice, a combination of transgenes from daffodil and Pantoea were used to increase pro-vitamin A levels within rice endosperm (Ye et al. 2000). Golden rice has been improved further to contain up to 23-fold increase in carotenoid content (Paine et al. 2005). Folate biofortified rice was generated through the overexpression of transgenes from Arabidopsis in rice endosperm. Through transgenic breeding, a fourfold increase in folate concentrations in rice were achieved (Storozhenko et al. 2007), and folate stability for long-term storage was improved (Blancquaert et al. 2010). Researchers have examined the nutrition/health effects of GM biofortification, but currently only a single clinical trial on golden rice is available. A randomized trial in the United States resulted in a high bio-conversion factor of β-carotene in golden rice (3.8 : 1), by which 100 g of uncooked golden rice would provide about 80–100% of the estimated average requirement and 55–70% of the recommended dietary allowance (RDA) for adult men and women (Tang et al. 2009). Findings from 15 simulation analyses confirmed the promising effects of GM biofortified crop consumption on dietary intake and nutritional outcomes in humans (De Steur et al. 2017a). In nearly all of these studies, a regular portion of the targeted biofortified crop would provide the daily micronutrient requirements. An example would be the recent simulation analysis of golden rice in Asia (De Moura et al. 2016). Its implementation could reduce the prevalence of dietary vitamin A inadequacy by up to 30% (children) and 55–60% (women) in Indonesia and the Philippines, and up to 71% (children) and 78% (women) in Bangladesh. GM biofortified crops like golden rice (De Moura et al. 2016; Stein et al. 2006) would be highly cost-effective investments to reduce target micronutrient deficiencies such as vitamin A (De Steur et al. 2017c). Unlike GM crops with agronomic traits, GM foods with quality traits (such as health traits) are only starting to be introduced in the global pipeline of GMOs (Parisi et al. 2016; Potrykus 2010).

4.7  ­Vitamins and Transgenic Biofortification Strategies of Edible Crop

Within the context of SDG 2, however, GMOs that address highly prevalent vitamin and mineral deficiencies are not (yet) approved. The main reason is the current regulatory climate and anti-GMO lobbying efforts (Moghissi et al. 2016; Potrykus 2017). Nevertheless, the proof of concept has been realized for various nutritionally enhanced GMOs (De Steur et al. 2015; Van Der Straeten et al. 2017), which triggered an increase in the number of nutritional traits in the global GM crops pipeline over the last two decades and is expected to be further reinforced in the near future (Parisi et al. 2016). Another reason behind the growing interest in output traits relates to the consumer. A meta-analysis of studies on consumers’ willingnessto-pay revealed general positive reactions toward GM biofortified crops. This is in line with conventional biofortified crops (Birol et  al. 2015), indicating that consumers’ opinion on nutritious crops are hardly affected by the applied technology (De Steur et  al. 2017b). Worldwide, they are willing to pay on average 24% more for GM biofortified food as compared to conventional food (De Steur et al. 2017c). Consumers’ optimism, however, can be significantly influenced by information provision, in both ways. While positive information on the nutritional content/benefits or technology increased consumers’ intention to purchase, the opposite was true for negative information on GM technology. This lends support for recognizing how significantly lobbying can polarize public opinion, regardless of the scientific basis of given arguments (Wesseler and Zilberman 2014).

4.7.2  “Golden” Bananas to Combat Vitamin A Deficiency Bananas, a major staple in many African countries, are the world’s most important fruit crop. Banana grows in tropical climates, where vitamin A deficiency tends to be greatest (Amah et al. 2018). The wide number of banana varieties and broad distribution of vitamin A levels make them amenable for biofortification using biotechnology. Unfortunately, the cooking banana East African highland banana (EAHB) consumed in Uganda as a staple has low vitamin A levels. Bananas are difficult to breed conventionally, and genetic engineering of bananas with increased vitamin A content have been developed in this fashion (Paul et al. 2017). Since most of the research to date has been performed on the Cavendish banana, popular in the West, this work has been used as a model system for the EAHB. High levels of vitamin A (20 lg/g dry weight) were found in transgenic banana lines expressing phytoene synthase (derived from the fruit of the Fe’I banana found in Papua New Guinea, which only grows in small bunches) under the control of the banana ubiquitin promoter (Ubi). These transgenic lines are yellow-orange in appearance and will offer improved nutrition to some of the poorest subsistence farmers in Africa. Consumption of merely 300 g of transgenic banana could provide as much as 50% of vitamin A required per person per day (Paul et al. 2017; Waltz 2014). Unfortunately, Uganda has no current regulatory framework for biotechnology (Paul et al. 2018). The bananas could be ready for release as early as 2021.

4.7.3  Biofortified Maize and Cassava Maize also produces β-carotene, but this can vary greatly in amount between different varieties. β-carotene content can be increased using either conventional breeding or genetic engineering strategies. Consumption of transgenic maize biofortified with β-carotene has been demonstrated to improve individuals’ health in clinical trials held in Africa and North

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America (Li et al. 2010, Mugode et al. 2014). Moreover, chickens fed transgenic biofortified maize produced eggs that exhibited increased carotenoid content (Moreno et  al. 2016). However, the fact that the biofortified maize is more orange in color creates a public perception challenge for some African populaces, as orange maize is often associated with animal feed, whereas white maize is traditionally considered to be for human consumption. The BioCassava Plus project specifically targets cassava, a staple that is nutritionally deficient and consumed by a quarter of a million sub-Saharan Africans (Sayre et  al. 2011). Conventional cassava is high in cyanide and low in protein, iron, and β-carotene. Transgenic cassava expressing high levels of β-carotene has been demonstrated to increase vitamin A levels and improve nutritional status in feeding studies (Talsma et al. 2016). Programs such as the BioCassava Plus project could therefore generate cassava crops with lasting nutritional benefits.

4.7.4  β-Carotene Biofortified Sweet Potato β-carotene biofortified sweet potato is one of the most interesting research areas, since the development of high β-carotene sweet potato cultivars were among the priorities for sub-Saharan Africa (Fuglie 2007). To enhance β-carotene content of sweet potatoes, researchers used two combined approaches: the evaluation and screening of the parents and the use of molecular markers. This combination is thought to provide a good theoretical basis in selecting appropriate parents for breeding new sweet potato varieties of high β-carotene levels (Ma et  al. 2009). The identification of QTL (Quantitative Trait Loci) for β-carotene content in a cross of sweet potato was of great help for understanding how these traits are inherited (Cervantes-Flores et al. 2011). This progress was the first step in developing marker-assisted techniques for breeding high β-carotene sweet potato cultivars. After isolating the orange (Or) gene responsible for carotenoids accumulation in plants, white-fleshed sweet potatoes were transformed. β-carotene and total carotenoids levels in the IbOr-Ins transgenic sweet potatoes were tenfold higher compared to that of white-fleshed sweet potatoes (Kim et  al. 2013). Similar results were obtained by Park et  al. (2015), who noted that the overexpression of IbOr-Ins gene increases the carotenoid contents of purple-flesh sweet potatoes.

4.8 ­Conclusions and Future Directions Vitamin biofortification of edible crops is undoubtedly the most efficient and cost-effective strategy to overcome vitamin deficiencies, particularly in the developing countries and poor communities. This strategy should be considered more than required because of the growing population and food security concerns over the horizon of 2050, and these concerns might be exacerbated by climate change and food scarcity in many regions of the world. However, farmers will not use biofortified plants unless they are high-yielding, of good productivity, and profitable. Consumers must also be educated on the safety and benefits of biofortified crops, as they often have different organoleptic traits (color, texture, structure) and are thus not attractive. Therefore, the real challenge is to persuade consumers that these traits are not important

  ­Reference

compared to the benefits of consuming these biofortified crops. Another concern is the bioavailability of nutrients of biofortified crops. Further research is needed to understand the interplay between biofortification and nutrient uptake to ensure maximum bioavailability of the targeted nutrient. Last but not least, the introduction of biofortification programs, particularly through breeding and molecular engineering, for targeted crops should be locally adapted to the agroecosystem. This introduction requires a certain level of research and development capacities at both local and national levels. Although the dissemination of biofortified crops has been put in place in Asia and the Americas for a long time, too much work still remains to be done in Africa, where hunger remains predominant compared to these other regions.

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5 Carotenoids Biofortification of Sweet Potatoes Noureddine Benkeblia1,2, Elisabete M. Pinto3, and Marta W. Vasconcelos3 1 

Laboratory of Crop Science, Department of Life Sciences, The University of the West Indies, Kingston, Jamaica Department of Life Science and Laboratory of Tree Fruit and Aromatic Crop, The Biotechnology Centre, The University of the West Indies, Kingston, Jamaica 3  Universidade Católica Portuguesa, CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Rua Diogo Botelho 1327, 4169–005 Porto, Portugal 2 

5.1 ­Introduction The sweet potato plant, Ipomoea batatas, the most consumed tuber crop in the world, originated from Latin America and is thought to be one of the oldest crops known to the region. This crop has been consumed since prehistoric times, as evidenced by sweet potato relics dating back 10 000 years, discovered in the Peruvian caves. In 1753, Linneaus was the first botanist who described it as Convolvulus batatas. In 1791, Lammarck reclassified it as I.  batatas, based on the detailed description of the flower of the plant (Huaman 1992). Indeed, the term Batatas, an Arawak name, was used long ago in Central and South America as well as in the West Indies, and thus, used to describe the scientific name of sweet potato. There are two main types: (i) the dry-fleshed type with a yellow, ivory, or white flesh and white or purple skin, which is very popular in the Caribbean, and (ii) the moist-fleshed type with a red-skinned and dark-orange flesh (Figure 5.1). Among these types, there are more than 400 varieties known worldwide, differing in skin color and flesh. Sweet potato varieties have individual nutritional profiles, being excellent sources of different phytonutrients, including vitamins. From a nutritional and medical point of view, the role of phytonutrients in protection against vitamin deficiency and disease prevention have been well recognized, and tremendous efforts have been achieved to increase the phytonutrients content in crops, leading to a better food supplementation through crop biofortification. In the last two decades, crops biofortification can be considered as the most powerful biotechnological approach to enhance the phytonutrient content of crops, and thus increasing dietary intake of vitamins, and other nutrients as well, alleviating nutritional deficiencies (Johns and Eyzaguirre 2007; Mackey 2002; Mayer et al. 2008; Nestel et al. 2006). Indeed, many crops have been bred for higher vitamins and other phytonutrient levels using either conventional or molecular engineering approaches, or many Vitamins and Minerals Biofortification of Edible Plants, First Edition. Edited by Noureddine Benkeblia. © 2020 John Wiley & Sons Ltd. Published 2020 by John Wiley & Sons Ltd.

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5  Carotenoids Biofortification of Sweet Potatoes

(a)

(b)

(c)

Figure 5.1  Cultivated sweet potato. (a) growing sweet potato plant, (b) mechanical harvesting of sweet potato, (c) harvested sweet potato ready for curing and storage.

5.2 ­Carotenoids Content of Sweet Potato Cultivar

biofortified crops have been released, while others are still under trial cropping or are being tested (Saltzman et al. 2013). Extensive literature has been published in the topic in the twenty-first century, and the enzymatic engineered pathways clearly described (Figure 5.2). In order to achieve these goals, different molecular engineering strategies have been used (Giuliano 2014; Giuliano et al. 2008): ●●

●●

●●

●●

Strategies consisting of enhancing the metabolic flux upstream of the target compound through the overexpression of specific catalyzing enzymes, and these strategies have been used to enhance β-carotene in roots and tubers such as potatoes (Diretto et al. 2007) and cassava (Welsch et al. 2010). Strategies consist of slowing down the metabolic flux downstream targeting a specific compound or a competing sub-pathway through gene silencing. These strategies have been successful on potatoes by increasing total carotenoids by about tenfold (Diretto et al. 2006; Giuliano 2017; Romer et al. 2002). Strategies based on source-sink relation consist of enhancing the compartmented accumulation of carotenoids through gene overexpression (Li and Van Eck 2007). Strategies consisting of reducing postharvest losses of post-harvest carotenoids focus on handling and storage. For example, in cold-stored engineered potatoes, carotenoid content increased significantly by regulating the capacity of carotenoid sequestration (Li et al. 2012).

A large number of published works have reported on carotenoids biofortification of many crops, but few targeted sweet potatoes compared to other staple crops such as maize, cassava, and other cereals. This chapter describes approaches to enhancing carotenoids of the sweet potato.

5.2 ­Carotenoids Content of Sweet Potato Cultivars Sweet potato, as a starchy tuber crop, possesses the highest energy value after cassava compared to other root and tuber staples. One of the most interesting nutritional qualities of sweet potato is its content in β-carotene (pro-vitamin A). The carotenoid content of sweet potato is very variable, and ranges from low, medium, and high carotenoids cultivars. This content varies from about 5 to 26 mg per 100 g fresh weight, with an average of about 13 mg per 100 g fresh weight. Most of these high carotenoid values have been reported in the orange-fleshed sweet potato (OFSP) varieties (Table  5.1). Indeed, the OFSP is considered the single most successful example of biofortification of a staple crop, and presents a feasible option to address vitamin A deficiency (VAD) (Laurie et  al. 2018). In fact, a food-based approach introducing OFSP increased vitamin A intake and serum retinol concentrations in young children in rural Mozambique (Low et al. 2007). Still, there is large diversity on provitamin A content between varieties. By analyzing the β-carotene content of 25 sweet potato cultivars from seven countries, Takahata et al. (1993) found β-carotene value ranging from 1.1 to 26.5 mg per 100 g fresh weight. The analysis of the β-carotene of 18 cultivars of sweet potato grown in Hawaii showed levels ranging from

89

90

5  Carotenoids Biofortification of Sweet Potatoes Quinones Gibberellins Chlorophylls Tocopherols GGPP PSY

Cytokinins CrtE GGPS

OPP CrtB

OPP DMAPP IDI OPP MEP IPP pathway

15-cis -phytoene PDS ZISO ZDS CrtISO

LCY-b

lutein

HO

OH CCD2

HO

VDE

HO crocetin

O

HO

NCED 9-cis -violaxanthin ABA HO 9-cis -neoxanthin

O

NXD

OH

cantaxanthin

O BKT

CrtZ

CrtZ

CBFD OH HBFD

CBFD

OH

O

O

β-carotene CHY

CYP97

OH DXP

Pyr + DXS G3P

CrtW

LCY-b CrtY

α-carotene

OP

Strigolactones β-Cyclocitral

Crtl

all-trans -lycopene LCY-e

OH O

zeaxanthin CrtO ZEP OH O violaxantin CCS OH O

O

HO

O

OH

astaxanthin OH

O

O capsorubin

HO

neoxanthin

Plant enzyme cassava maize wheat canola banana

rice

carrot orange

silencing

Bacterial enzyme Microalgal enzyme

tobacco

potato soybean pepper lettuce tomato Arabidopsis

plastid transformation

Cyanobacterial enzyme

Figure 5.2  Carotenoid pathway engineering in crop plants. All depicted steps occur in the plastid. Plant enzyme names are in black letters, bacterial in red, cyanobacterial in blue, microalgal in green. Next to each enzymatic step are shown the species in which it has been overexpressed (plain image) or silenced (crossed image). Boxed images represent species in which plastid transformation was performed. DXS: 1-deoxy-D-xylulose 5-phosphate synthase; IDI: isopentenyl diphosphate isomerase; GGPS: geranylgeranyl pyrophosphate synthase; PSY: phytoene synthase; PDS: phytoene desaturase; ZISO: 15-cis-zeta-carotene isomerase; ZDS: zeta-carotene desaturase; CrtISO: prolycopene isomerase; LCY-b: lycopene beta-cyclase; LCY-e: lycopene epsilon-cyclase; CYP97: heme hydroxylase; CHY: non-heme hydroxylase; CBFD: carotenoid b-ring 4-dehydrogenase; HBFD: 4-hydroxy-b-ring 4-dehydrogenase; ZEP: zeaxanthin epoxidase; VDE: violaxanthin de-epoxidase; NXD: neoxanthin deficient; CCD: carotenoid cleavage dioxygenase; CrtE: bacterial geranylgeranyl pyrophosphate synthase; CrtB: bacterial phytoene synthase; CrtI: bacterial phytoene desaturase/isomerase; CrtO: cyanobacterial ketolase; CrtZ: bacterial hydroxylase; CrtW: bacterial ketolase; BKT: ketolase from green algae; Pyr: pyruvate; G3P: glyceraldehyde 3-phosphate; DXP: 1-deoxy-D-xylulose 5-phosphate; IPP: isopentenyl; pyrophosphate; DMAPP: dimethylallyl pyrophosphate; GGPP: geranylgeranyl pyrophosphate; ABA: abscisic acid. For further details, see supplemental material (Giuliano 2017, with permission).

5.2 ­Carotenoids Content of Sweet Potato Cultivar

Table 5.1  β-carotene concentration of different high carotenoids sweet potato cultivars. Cultivar

β-carotene concentration

References

Kyushu 122

14.7 mg/100 g

Okuno et al. (1998)

BARI SP2

6.38 mg/100 g

Alam et al. (2016)

BARI SP8

7.24 mg/100 g

BARI SP9

4.61 mg/100 g

Covin

12.0 mg/100 g

13–17 Clone

12.7 mg/100 g

13–20 Clone

22.6 mg/100 g

TIB4

13 μg/g bun

LO-323

12 μg/g bun

SPV-61

26.5 mg/100 g

Resisto

20.3 mg/100 g

UC700

18.9 mg/100 g

Benihayato

18.7 mg/100 g

L-2-116

18.5 mg/100 g

L-4-89

18.5 mg/100 g

Santo Amaro

15.5 mg/100 g

Caromex

14.9 mg/100 g

Re Jewel

13.8 mg/100 g

Benihayato (mutant)

11.7 mg/100 g

Ejumula

Teow et al. (2007)

Low and van Jaarsveld (2008) Takahata et al. (1993)

Hotz et al. (2012a, 2012b) Br J. Nutr

Kakamega (SPK004), Vita (SPK004/6) Kabode (SPK004/6/6), Resisto Cordner

11.27 6.79

Gabagaba

10.16

MGCL 01

9.32

LO 323

5.02

Kakamega 4 (SPK 004),

Hagenimana and Low (2000)

Zapallo,

4.3 mg/100 g

Japon Tresmesino Selecto

5.5 mg/100 g

W-220

8.4 mg/100 g

TIB 11

8.8 mg/100 g (Continued)

91

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5  Carotenoids Biofortification of Sweet Potatoes

Table 5.1  (Continued) Cultivar

β-carotene concentration

Kamalasundari Bophelo Blesbok excel

Bhuiyan et al. (2008) 6.71 14.0

Implio

5.09

Khano

14.03

Purple Sunset (or 2001_5_2)

11.8

Resisto

15.45

Serolane W-119

References

Laurie et al. (2015) Food Research Int 76: 962–070

5.16 10.46

Source: Benkeblia (2017). © 2017 John Wiley & Sons Ltd., adapted from Table 1, Chapter 11, with permission.

0.1 to 0.6 mg per 100 g fresh weight (Huang et al. 1999). Other studies also reported different levels of β-carotene in sweet potatoes ranging from 0 to 9 mg per 100 g fresh weight (Hagenimana et al. 1998). Because of this nutritional trait, the sweet potato might play an important role in human diets as a value-added product in food and nutrition systems particularly in countries affected by VAD and needing its alleviation (Bovell-Benjamin 2007; Diretto et al. 2007).

5.3 ­Carotenoid Improvement Strategies for Sweet Potato Besides many interesting traits such as yield and resistance to diseases and drought, the improvement of nutritional traits of the sweet potato is aiming to improving human health, particularly in marginal and high poverty areas across developing countries affected by VAD. In this context, the genetic diversity of sweet potatoes is a critical trait for its improvement because this diversity constitutes the primary source of the specific genes for the desired genetic gains (Yada et al. 2015). Therefore, using this genetic diversity is helping to breed enhanced sweet potatoes with targeted nutritional quality attributes as a functional food crop (Toenniessen 2002). However, sweet potato potentials are yet to be untapped, and the crop wild relatives (WRS) of sweet potato need further attention due to their many potentials and their possible contribution in the breeding objectives, and their promising contribution to crop improvement (Khoury et al. 2015).

5.3.1  Agronomic Strategies Macro- and micro-nutrients are critical factors for plant growth and yield, and their impacts depend on the soil nutrient level and bioavailability at different stages of growth

5.3 ­Carotenoid Improvement Strategies for Sweet Potat

and development of the plant, its absorption and transport within the plant, translocation during maturation of the crop, and lastly its content in the harvested crop (de Valença et al. 2017). Incredible accomplishments have been achieved in crop production by developing different agronomic strategies such as fertilization regimes and external application of specific phytohormones. One of the most important fertilizing soil nutrients is nitrogen, which is known to play an important role in diverse biochemical and physiological processes of plants. Beside its role as a constituent of cell walls, cytoplasmic proteins, nucleic acids, chlorophyll, and other parts of the cell (Hay and Walker 1989), nitrogen plays a role in crop improvement (Lea and Miflin 2011). As indicated in the previous section, sweet potatoes might contain high levels of carotenoids, and the level depends on the cultivar, with particularly high levels being found in the orange-fleshed varieties (Islam et al. 2016); and similarly to many other targeted enhancements, fertilization has been used to improve their carotenoids. The first study carried out in the 1980s showed that the application of nitrogen fertilizer significantly increased the carotenoid content of sweet potatoes (Constantin et al. 1984). However, the response to nitrogen application depends greatly on the genotypic and environmental variations (Villagarcia 1996). In other crops, such as spinach, carotenoids were little or not affected by nitrogen addition. Conversely, sulfur fertilization of spinach modestly increased its carotenoid content (Reif et al. 2012). Combined with fertilization, intermediate irrigation (60% of optimal) positively affected the carotenoid content of sweet potatoes when compared to optimal or high irrigation regimes (Laurie et al. 2017). Surprisingly, another factor has been reported to enhance the carotenoid content. When grown at rural village areas, sweet potatoes contained higher carotenoid compared to those grown under optimal condition, and this content increased with harvesting time, indicating that management of cropping system and the harvesting time might be considered as one agronomic approach for crops biofortification (Faber et al. 2012).

5.3.2  Breeding Strategies Plant breeding is the genetic improvement of a given crop using conventional crossing approaches to develop new varieties with desirable traits such as resistance to diseases, higher yield, and better nutrition (including provitamin A). Biofortification of sweet potatoes through conventional breeding, which includes the selection of orange-fleshed varieties, is being done with the intent of controlling VAD in developing countries (Institute of Food Technologists 2008). The objectives of the provitamin A carotenoid breeding projects for sweet potato have been to develop varieties that demonstrate efficacy and effectiveness to improve vitamin A status and were also profitable to farmers and acceptable to consumers (Bouis and Welch 2010). Enhancing sweet potatoes with provitamin A carotenoids has been part of HarvestPlus’s research continuum since the formation of the biofortification project (Tanumihardjo et al. 2017). Breeding for provitamin A has been successful for several crops including maize (Maqbool et  al. 2018) and sweet potato (Tanumihardjo et  al. 2017). From an agricultural perspective, the currently available information reveals a rapid growth in the development and introduction of provitamin A enhanced sweet potato in

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developing countries around the world, such as Mozambique (Mejia et al. 2017) and South Africa (Laurencia et al. 2019). In particular, OFSP production has evolved from home- to small-scale commercial production. In South Africa, for example, 1,300 cuttings of OFSP (22.800 kg) were disseminated in 2016–2017 with public sector and nongovernmental organization (NGO) programs being the primary avenue for scale-out of OFSP (Laurie et al. 2018). Although improvements by conventional breeding have not been subject to regulations, when biofortification becomes expanded by including other techniques, an appropriate regulatory framework will be necessary (Mejia et al. 2017).

5.3.3  Transgenic Strategies Genetic modification using transgenic approaches for nutritional purposes can be very effective, and several reports have been published on genetic modification of sweet potato for multiple purposes (Liu 2017). Prior to any transgenic approach, a necessary condition is a good understanding of the metabolic pathways involved carotenoid ­synthesis and accumulation. Fortunately, in higher plants, the carotenoid metabolic pathway and the function of the biosynthetic enzymes involved have been well studied (Kang et al. 2017). One the first studies that contributed to the genetic improvement of sweet potato toward enhancing provitamin A content is of Kim et al. (2013a). Kim et al. (2013a) isolated the orange (Or) gene, responsible for carotenoids accumulation in plants, from OFSP and analyzed its function in transgenic sweet potato calli. Similarly, they transformed calli of white-fleshed sweet potato using the same orange (Or) gene, and they noted that β-carotene and total carotenoids levels in the IbOr-Ins transgenic sweet potato was tenfolds high compared to that of white-fleshed sweet potato. More recently, similar results were reported by Park et al. (2015) who noted that the overexpression of IbOr-Ins gene increases the carotenoid contents of purple-flesh sweet potato (Table 5.2). Some mechanisms have been described for the enhancement of β-carotene synthesis in sweet potato: in plants, the biosynthesis of carotenoids from lycopene involves the lycopene ϵ-cyclase (LCY-ϵ); hence, carotenoid synthesis via the β-branch-specific pathway yielding β-carotene was enhanced by downregulating the expression of IbLCY-ϵ using RNAi (RNA interference) technology (Kim et al. 2013b). Recently, the role of an auxin response factor was described. It was found that IbARF5 gene from sweet potato (I. batatas (L.) Lam.) line HVB-3 increased the contents of carotenoids and enhanced the tolerance to salt and drought in transgenic Arabidopsis, suggesting that IbARF5 is involved in carotenoid biosynthesis and salt and drought tolerance in transgenic Arabidopsis, and opening new doors for improving carotenoid contents of sweet potato and other plants (Kang et al. 2018a). Further, the lycopene β-cyclase (LCYB) is an essential enzyme that catalyzes the conversion of lycopene into α-carotene and β-carotene in carotenoid biosynthesis pathway. Recently, a new allele of the LCYB gene (IbLCYB2), was isolated from the storage roots of sweet potato line HVB-3 and its overexpression significantly increased the contents of α-carotene, β-carotene, lutein, β-cryptoxanthin, and zeaxanthin in the transgenic sweet potato (cv. Shangshu 19) plants. These genes have the potential to improve carotenoid contents and abiotic stress tolerance in sweet potatoes and other plants.

Table 5.2  SWOT analysis of the different biofortification strategies used to enhance nutrient-density of plant-based foods. S

W

O

T

Biofortification strategies

Strengths

Weakness

Opportunities

Threats

Agronomic

●● ●●

Relatively simple method Immediate results

●●

●●

Success limited to minerals and dependent on several factors Recurrent costs:

●●

Often used as a complement to other strategies

●●

●●

–– Needs regular application –– Expensive

●● ●●

Conventional plant breeding

●●

●● ●● ●●

Genetic engineering

●●

●● ●● ●● ●●

Successful for minerals and vitamins One-off cost Easier distribution Long-term strategy Successful for minerals and vitamins One-off cost Easier distribution Long-term strategy Speeds up process of conventional breeding

●● ●●

●● ●●

●●

Negative environmental impact Reserves exhaustion (e.g. Se)

Difficult distribution Short-term strategy Long-development time Success limited to minerals available in the soil

Long-development time Success limited to minerals available in the soil Interactions among transgenes (may limit the process)

●●

Wide public acceptance Simple legal framework Fast “omics” developments

●●

Fast “omics” developments

●● ●●

●●

●●

●●

●●

Requires genetic variation

Low public acceptance (especially in Europe) Complex regulatory approval Environmental impact (“gene flow”)

Source: Carvalho and Vasconcelos (2013), with permission of Elsevier.

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5.4 ­Sweet Potato Improvement for β-Carotene Increasing β-carotene in the sweet potato is one of the most interesting research areas. According to a survey launched by the CIP (International Potato Centre), scientists in developing countries considered the development of high β-carotene sweet potato cultivars are among the additional priorities for sub-Saharan Africa (Fuglie 2007). To enhance β-carotene levels in sweet potato, researchers used two combined approaches: the evaluation and screening of the parents and the use of molecular markers. This combination is thought to provide a good theoretical basis in selecting appropriate parents for breeding new sweet potato varieties of high β-carotene levels (Ma et al. 2009). However, addressing this issue is quite complex due to the genetic complexity of sweet potato. To analyze this genetic complexity, Hwang et al. (2013) used SSRs techniques (simple sequence repeats) to analyze the genetic diversity and genetic relationships among many cultivars of sweet potato. Their results showed polycross-derived cultivars have higher genetic diversity levels, suggesting therefore the usefulness of poly-cross breeding strategy, even though crossincompatibility is sometimes observed. Cervantes-Flores et al. (2011) have been the first authors who identified QTL (quantitative trait loci) for β-carotene content in a cross sweet potato, which has enhanced one’s understanding of how these traits are inherited in sweet potato. This development was considered the first step in the development of marker-assisted breeding techniques for breeding high β-carotene sweet potato cultivars. As orange-fleshed sweet potatoes provided a remedy for VAD, another approach was used to enhance β-carotene in the sweet potato.

5.5 ­Carotenoids Biofortification of Sweet Potato and Vitamin A Deficiency Alleviation Vitamin A is an essential micronutrient that has several physiological roles including immunity, vision, cellular differentiation, haemopoiesis, growth, and reproductive health (Sizer and Whitney 2017). Dietary sources of vitamin A includes animal-sourced foods, such as oily fish, liver, cheese, and butter, but also vegetable-sourced foods, like sweet potatoes, winter squash, kale, collard, carrot, among others. Even though, VAD is a major public health problem in more than half of the countries worldwide, mainly in Africa and South-East Asia, especially affecting young children and pregnant women in developing countries (Hombali et al. 2019). About one-third of children aged 6–59 months, in low- and middle income countries suffer from VAD. In 2013, 1.7% of all deaths among children