3.4.3. Plant Breeding

Limitations in the long-term e ffectiveness and sustainability of fertilizer approaches necessitates the development of economical and longstanding strategies for increasing micronutrient density in plants. Genetic engineering technology to produce genetically modified plants with desirable traits has been used in corn, rice, wheat, and soybeans. This can be an e ffective approach for crop improvement; however, political opposition to GMOs in many countries, a complex legal framework for the acceptance and commercialization of transgenic crops, along with expensive and time-consuming regulatory processes are the major limitations of this method [100,160,161]. For example, golden rice has been available since the early 2000s and has the potential to deliver more than 50% of the estimated average requirement for vitamin A, but unfortunately it has not been commercially introduced in any country to date due to risk factors involved in the regulatory approval processes [162,163]. In developed and developing countries, several groups endorse the arguments made by Greenpeace that approval of golden rice will allow multinational corporations to control developing countries' food supplies and pose risks to human health and the environment (https://gmo.geneticliteracyproject.org/).

Restrictions on the use of genetically modified crops in many countries prompted HarvestPlus to take the initiative to address micronutrient deficiencies through conventional plant breeding [20,22]. Biofortification through plant breeding is a cost-e ffective and sustainable approach that can improve the health status of low-income people globally [19,21,147]. This approach has been used to control deficiencies of micronutrients including carotenoids, Fe, and Zn [96,164].

HarvestPlus was started in 2003 by the initiative of the Consultative Group on International Agricultural Research (CGIAR) to target several major food crops, including rice, common beans, cassava, maize, sweet potatoes, pearl millet, and wheat in Asia and Africa, to enrich them with three major nutrients, Fe, Zn, and vitamin A through an interdisciplinary and global alliance of scientific institutions and implementing agencies [165,166]. HarvestPlus is part of the CGIAR Research Program on Agriculture for Nutrition and Health (A4NH). The HarvestPlus program is administered by joint venture of the International Center for Tropical Agriculture and the International Food Policy Research Institute and provides global leadership on biofortification evidence and technology. The UK Government, the Bill and Melinda Gates Foundation, the US Government's Feed the Future initiative, the EU Commission, and donors to A4NH are principal investors [163] https://www.harvestplus.org.

Conventional plant breeding approaches can benefit not only large populations but also people living in relatively remote rural areas who have limited access to commercially marketed fortified foods [19,21,22,167]. This approach requires a one-time investment in plant breeding and can be grown and multiplied across years by farmers at virtually zero marginal cost. Recurrent costs are low, and germplasms can be available internationally without any adverse e ffect on productivity and health; thus, biofortification has widespread public acceptance [20,21,100,168].

Genetic diversity is required in the gene pool to achieve success in biofortification through the plant breeding approach. Several studies have shown substantial variation in the concentration of minerals and vitamins in various crops [93,106]. Parental genotypes with high micronutrient concentration can be identified by screening a wide range of germplasms, and these can be utilized in making crosses, genetic studies, and the development of molecular markers to facilitate marker-assisted selection in breeding. Promising lines can be tested at multiple locations to determine the genotype X environment interaction (G X E) [163]. These can be submitted to national governmen<sup>t</sup> agencies for testing for agronomic performance and release after robust regional testing across multiple locations over multiple seasons [163]. Using the above-mentioned strategy, various studies in pulse crops have identified substantial variation in the available gene pools and recombinant inbred line populations developed from promising parental genotypes. These were utilized for identifying genomic regions and associated markers for their use in marker-assisted selection. Recent research advances in pulse crops for improvement of key micronutrients, including Fe, Zn, Se, I, carotenoids, and folates, through conventional plant breeding approaches, will be discussed in the following section.

### **4. Recent Research Advances for Biofortification of Pulse Crops**
