Trait stacking simultaneously enhances provitamin A carotenoid and mineral bioaccessibility in biofortified .

Vitamin A, iron, and zinc deficiencies are major nutritional inadequacies in sub-Saharan Africa and disproportionately affect women and children. Biotechnology strategies have been tested to individually improve provitamin A carotenoid or mineral content and/or bioaccessibility in staple crops including sorghum (). However, concurrent carotenoid and mineral enhancement has not been thoroughly assessed and antagonism between these chemical classes has been reported.

Evaluation of Agronomic Performance of β-Carotene Elevated Sorghum in Confined Field Conditions.

To help alleviate vitamin A deficiency in Africa, we have developed nutritionally enhanced sorghum with stabilized high all-trans-β-carotene accumulation. Toward the finalization of this nutritionally enhanced sorghum for food production, confined field trials were conducted to determine the agronomic performance of thirteen independent transgenic events in Iowa and Hawaii. Through these trials, three leading events with no negative impact on agronomic performance were identified.

Nutritionally Enhanced Sorghum for the Arid and Semiarid Tropical Areas of Africa.

To help alleviate malnutrition in Africa, nutritionally enhanced sorghum was developed through genetic transformation to increase pro-vitamin A (β-carotene) accumulation and stability, to improve iron and zinc bioavailability, and to improve protein digestibility. Through many years of efforts, significant achievements have been made for these goals. We generated nutritionally enhanced sorghum lines with enhanced and stabilized pro-vitamin A that provide 20-90% of the Estimated Average Requirement (EAR) for children under age 3, lines with a 90% reduction in phytate that increase iron and zinc bioavailability and provide 40-80% of the EAR for iron and zinc, and lines that show no reduction in protein digestibility after cooking compared with normal levels.

Developing a flexible, high-efficiency Agrobacterium-mediated sorghum transformation system with broad application.

Sorghum is the fifth most widely planted cereal crop in the world and is commonly cultivated in arid and semi-arid regions such as Africa. Despite its importance as a food source, sorghum genetic improvement through transgenic approaches has been limited because of an inefficient transformation system. Here, we report a ternary vector (also known as cohabitating vector) system using a recently described pVIR accessory plasmid that facilitates efficient Agrobacterium-mediated transformation of sorghum.

Agrobacterium-Mediated Sorghum Transformation.

Agrobacterium-mediated plant transformation is commonly used in crop genome modification. An optimized sorghum transformation protocol we developed is described here. Using this protocol, the transformation frequency of sorghum inbred TX430 is over 10% with Agrobacterium strain LBA4404 and 33% with Agrobacterium strain AGL1.

Elevated vitamin E content improves all-trans β-carotene accumulation and stability in biofortified sorghum.

Micronutrient deficiencies are common in locales where people must rely upon sorghum as their staple diet. Sorghum grain is seriously deficient in provitamin A (β-carotene) and in the bioavailability of iron and zinc. Biofortification is a process to improve crops for one or more micronutrient deficiencies.

Morphogenic Regulators and Improve Monocot Transformation.

While transformation of the major monocot crops is currently possible, the process typically remains confined to one or two genotypes per species, often with poor agronomics, and efficiencies that place these methods beyond the reach of most academic laboratories. Here, we report a transformation approach involving overexpression of the maize () () and maize () genes, which produced high transformation frequencies in numerous previously nontransformable maize inbred lines. For example, the Pioneer inbred PHH5G is recalcitrant to biolistic and transformation.

Is there a place for nutrition-sensitive agriculture?

The focus of the review paper is to discuss how biotechnological innovations are opening new frontiers to mitigate nutrition in key agricultural crops with potential for large-scale health impact to people in Africa. The general objective of the Africa Biofortified Sorghum (ABS) project is to develop and deploy sorghum with enhanced pro-vitamin A to farmers and end-users in Africa to alleviate vitamin A-related micronutrient deficiency diseases. To achieve this objective the project technology development team has developed several promising high pro-vitamin A sorghum events.

Effect of Agrobacterium strain and plasmid copy number on transformation frequency, event quality and usable event quality in an elite maize cultivar.

Improving Agrobacterium -mediated transformation frequency and event quality by increasing binary plasmid copy number and appropriate strain selection is reported in an elite maize cultivar. Agrobacterium-mediated maize transformation is a well-established method for gene testing and for introducing useful traits in a commercial biotech product pipeline. To develop a highly efficient maize transformation system, we investigated the effect of two Agrobacterium tumefaciens strains and three different binary plasmid origins of replication (ORI) on transformation frequency, vector backbone insertion, single copy event frequency (percentage of events which are single copy for all transgenes), quality event frequency (percentage of single copy events with no vector backbone insertions among all events generated; QE) and usable event quality frequency (transformation frequency times QE frequency; UE) in an elite maize cultivar PHR03.

Agrobacterium-mediated high-frequency transformation of an elite commercial maize (Zea mays L.) inbred line.

An improved Agrobacterium -mediated transformation protocol is described for a recalcitrant commercial maize elite inbred with optimized media modifications and AGL1. These improvements can be applied to other commercial inbreds. This study describes a significantly improved Agrobacterium-mediated transformation protocol in a recalcitrant commercial maize elite inbred, PHR03, using optimal co-cultivation, resting and selection media.

Optimized -mediated sorghum transformation protocol and molecular data of transgenic sorghum plants.

-mediated sorghum transformation frequency has been enhanced significantly medium optimization using immature embryos from sorghum variety TX430 as the target tissue. The new transformation protocol includes the addition of elevated copper sulfate and 6-benzylaminopurine in the resting and selection media. Using strain LBA4404, the transformation frequency reached over 10% using either of two different selection marker genes, moPAT or PMI, and any of three different vectors in large-scale transformation experiments.

Bioaccessibility of carotenoids from transgenic provitamin A biofortified sorghum.

Biofortified sorghum (Sorghum bicolor (L.) Moench) lines are being developed to target vitamin A deficiency in Sub-Saharan Africa, but the delivery of provitamin A carotenoids from such diverse germplasms has not been evaluated. The purpose of this study was to screen vectors and independent transgenic events for the bioaccessibility of provitamin A carotenoids using an in vitro digestion model.

Sorghum (Sorghum bicolor L.).

This chapter describes a stepwise protocol for Agrobacterium-mediated sorghum genetic transformation. Immature embryos from sorghum plants were used as the target explants. The Agrobacterium strain LBA4404, carrying a "super-binary" vector, was used in this protocol.

Transformation of maize via Agrobacterium tumefaciens using a binary co-integrate vector system.

This chapter describes a stepwise protocol to achieve success in genetic transformation of maize using Agrobacterium tumefaciens as a DNA delivery system. Researchers will be able to effectively transform immature embryos of Hi-II and related genotypes with this protocol. The outcome of the transformation process will be transgenic embryogenic callus tissue, transgenic plants, and transgenic progeny seeds.

High efficiency transgene segregation in co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system.

For regulatory issues and research purposes it would be desirable to have the ability to segregate transgenes in co-transformed maize. We have developed a highly efficient system to segregate transgenes in maize that was co-transformed using an Agrobacterium tumefaciens 2 T-DNA binary system. Three vector treatments were compared in this study; (1) a 2 T-DNA vector, where the selectable marker gene bar (confers resistance to bialaphos) and the beta-glucuronidase (GUS) reporter gene are on two separate T-DNA's contained on a single binary vector; (2) a mixed strain treatment, where bar and GUS are contained on single T-DNA vectors in two separate Agrobacterium strains; (3) and a single T-DNA binary vector containing both bar and GUS as control treatment.