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

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 (Sorghum bicolor). However, concurrent carotenoid and mineral enhancement has not been thoroughly assessed and antagonism between these chemical classes has been reported. This work evaluated two genetically engineered constructs containing a suite of heterologous genes to increase carotenoid stability and pathway flux, as well as phytase to catabolize phytate and increase mineral bioaccessibility. Model porridges made from transgenic events were evaluated for carotenoid and mineral content as well as bioaccessibility. Transgenic events produced markedly higher amounts of carotenoids (26.4 µg g-1 DW) compared to null segregants (4.2 µg g-1 DW) and wild-type control (Tx430; 3.7 µg g-1 DW). Phytase activation by pre-steeping flour resulted in significant phytate reduction (9.4 to 4.2 mg g-1 DW), altered the profile of inositol phosphate catabolites, and reduced molar ratios of phytate to iron (16.0 to 4.1), and zinc (19.0 to 4.9) in engineered material, suggesting improved mineral bioaccessibility. Improved phytate : mineral ratios did not significantly affect micellarization and bioaccessible provitamin A carotenoids were over 23 times greater in transgenic events compared to corresponding null segregants and wild-type controls. A 200 g serving of porridge made with these transgenic events provide an estimated 53.7% of a 4-8-year-old child's vitamin A estimated average requirement. These data suggest that combinatorial approaches to enhance micronutrient content and bioaccessibility are feasible and warrant further assessment in human studies.

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 (ß-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. Once these nutritionally enhanced sorghum lines have undergone biosafety examination and have been deregulated, they will be ready for incorporation into sorghum varieties that will benefit Africa and other areas that rely upon sorghum as a staple food.

Evaluation of Agronomic Performance of ß-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-ß-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. The studies described in this chapter have laid the groundwork for development of the next generation of ß-carotene elevated sorghum as a food product.

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. We report regeneration frequencies ranging from 6% to 29% in Tx430 using different selectable markers and single copy, backbone free 'quality events' ranging from 45% to 66% of the total events produced. Furthermore, we successfully applied this ternary system to develop transformation protocols for popular but recalcitrant African varieties including Macia, Malisor 84-7 and Tegemeo. In addition, we report the use of this technology to develop the first stable CRISPR/Cas9-mediated gene knockouts in Tx430.

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. Two different selection marker genes, moPAT and PMI, were used in this protocol.

Elevated vitamin E content improves all-trans ß-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 (ß-carotene) and in the bioavailability of iron and zinc. Biofortification is a process to improve crops for one or more micronutrient deficiencies. We have developed sorghum with increased ß-carotene accumulation that will alleviate vitamin A deficiency among people who rely on sorghum as their dietary staple. However, subsequent ß-carotene instability during storage negatively affects the full utilization of this essential micronutrient. We determined that oxidation is the main factor causing ß-carotene degradation under ambient conditions. We further demonstrated that coexpression of homogentisate geranylgeranyl transferase (HGGT), stacked with carotenoid biosynthesis genes, can mitigate ß-carotene oxidative degradation, resulting in increased ß-carotene accumulation and stability. A kinetic study of ß-carotene degradation showed that the half-life of ß-carotene is extended from less than 4 wk to 10 wk on average with HGGT coexpression.

Morphogenic Regulators Baby boom and Wuschel 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 (Zea mays) Baby boom (Bbm) and maize Wuschel2 (Wus2) genes, which produced high transformation frequencies in numerous previously nontransformable maize inbred lines. For example, the Pioneer inbred PHH5G is recalcitrant to biolistic and Agrobacterium tumefaciens transformation. However, when Bbm and Wus2 were expressed, transgenic calli were recovered from over 40% of the starting explants, with most producing healthy, fertile plants. Another limitation for many monocots is the intensive labor and greenhouse space required to supply immature embryos for transformation. This problem could be alleviated using alternative target tissues that could be supplied consistently with automated preparation. As a major step toward this objective, we transformed Bbm and Wus2 directly into either embryo slices from mature seed or leaf segments from seedlings in a variety of Pioneer inbred lines, routinely recovering healthy, fertile T0 plants. Finally, we demonstrated that the maize Bbm and Wus2 genes stimulate transformation in sorghum (Sorghum bicolor) immature embryos, sugarcane (Saccharum officinarum) callus, and indica rice (Oryza sativa ssp indica) callus.

Evaluation of fitness in F2 generations of Africa Biofortified Sorghum event 188 and weedy Sorghum bicolor ssp. drummondii

Background: Introgression of transgenes from crops to their wild species may enhance the adaptive advantage and therefore the invasiveness of and weedy forms. The study evaluated the effect of Africa Biofortified Sorghum (ABS) genes from ABS event 188 on the vegetative and reproductive features of the F2 populations derived from crosses with Sorghum bicolor subsp. drummondii. Results: F1 populations were obtained from reciprocal crosses involving ABS event 188 and its null segregant with inbred weedy parents from S. bicolor subsp. drummondii. Four F2 populations and four parental populations were raised in RCBD with 4 replications in a confined field plot for two seasons. Vegetative and reproductive traits were evaluated. The vigour shown in the F2 populations from the reciprocal crosses involving ABS event 188 and S. bicolor subsp. drummondii was similar to that in the crosses involving the null segregant and S. bicolor subsp. drummondii. Differences in vegetative and reproductive parameters were observed between the parental controls and the F2 populations. Examination of the above and below ground vegetative biomass showed lack of novel weedy related features like rhizomes. Conclusions: Therefore, release of crops with ABS 188 transgenes into cropping systems is not likely to pose a risk of conferring additional adaptive advantage in the introgressing populations. The interaction of ABS genes in weedy backgrounds will also not have an effect towards enhancing the weedy features in these populations.

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. ABS 203 events are so far the most advanced and well-characterised lead events with about 12 µg ß-carotene/g tissue which would supply about 40-50 % of the daily recommended vitamin A at harvest. Through gene expression optimisation other events with higher amounts of pro-vitamin A, including ABS 214, ABS 235, ABS 239 with 25, 30-40, 40-50 µg ß-carotene/g tissue, respectively, have been developed. ABS 239 would provide twice recommended pro-vitamin A at harvest, 50-90 % after 3 months storage and 13-45 % after 6 months storage for children. Preliminary results of introgression of ABS pro-vitamin A traits into local sorghum varieties in target countries Nigeria and Kenya show stable introgression of ABS vitamin A into local farmer-preferred sorghums varieties. ABS gene Intellectual Property Rights and Freedom to Operate have been donated for use royalty free for Africa. Prior to the focus on the current target countries, the project was implemented by fourteen institutions in Africa and the USA. For the next 5 years, the project will complete ABS product development, complete regulatory science data package and apply for product deregulation in target African countries.

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

KEY MESSAGE: 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 strain AGL0 gave a higher transformation frequency, but a reduced QE frequency than LBA4404 due to a higher number of vector backbone insertions. Higher binary plasmid copy number positively correlated with transformation frequency and usable event recovery. The above findings can be exploited to develop high-throughput transformation protocols, improve the quality of transgenic events in maize and other plants.

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

KEY MESSAGE: 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. The use of green regenerative tissue medium components, high copper and 6-benzylaminopurine, in resting and selection media dramatically increased the transformation frequency. The use of glucose in resting medium further increased transformation frequency by improving the tissue induction rate, tissue survival and tissue proliferation from immature embryos. Consequently, an optimal combination of glucose, copper and cytokinin in the co-cultivation, resting and selection media resulted in significant improvement from 2.6 % up to tenfold at the T0 plant level using Agrobacterium strain LBA4404 in transformation of PHR03. Furthermore, we evaluated four different Agrobacterium strains, LBA4404, AGL1, EHA105, and GV3101 for transformation frequency and event quality. AGL1 had the highest transformation frequency with up to 57.1 % at the T0 plant level. However, AGL1 resulted in lower quality events (defined as single copy for transgenes without Agrobacterium T-DNA backbone) when compared to LBA4404 (30.1 vs 25.6 %). We propose that these improvements can be applied to other recalcitrant commercial maize inbreds.

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

Agrobacterium-mediated sorghum transformation frequency has been enhanced significantly via 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 Agrobacterium 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. With Agrobacterium strain AGL1, the transformation frequencies were as high as 33%. Using quantitative PCR analyses of 1,182 T0 transgenic plants representing 675 independent transgenic events, data was collected for T-DNA copy number, intact or truncated T-DNA integration, and vector backbone integration into the sorghum genome. A comparison of the transformation frequencies and molecular data characterizing T-DNA integration patterns in the transgenic plants derived from LBA4404 versus AGL1 transformation revealed that twice as many transgenic high-quality events were generated when AGL1 was used compared to LBA4404. This is the first report providing molecular data for T-DNA integration patterns in a large number of independent transgenic plants in sorghum.

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. The germplasm background and transgenic sorghum contained 1.0-1.5 and 3.3-14.0 µg/g ß-carotene equivalents on a dry weight basis (DW), respectively. Test porridges made from milled transgenic sorghum contained up to 250 µg of ß-carotene equivalents per 100 g of porridge on a fresh weight basis (FW). Micellarization efficiency of all-trans-ß-carotene was lower (p < 0.05) from transgenic sorghum (1-5%) than from null/nontransgenic sorghum (6-11%) but not different between vector constructs. Carotenoid bioaccessibility was significantly improved (p < 0.05) by increasing the amount of coformulated lipid in test porridges from 5% w/w to 10% w/w. Transgenic sorghum event Homo188-A contained the greatest bioaccessible ß-carotene content, with a 4-8-fold increase from null/nontransgenic sorghum. While the bioavailability and bioconversion of provitamin A carotenoids from these grains must be confirmed in vivo, these data support the notion that biofortification of sorghum can enhance total and bioaccessible provitamin A carotenoid levels.

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. Agrobacterium co-transformation vectors, one T-DNA containing the selectable marker gene and another T-DNA containing the trait gene(s), were also introduced in sorghum transformation for eliminating the selectable marker gene in the resulting transgenic plants. This chapter provides recommendations for analysis of the transgenic plants to confirm T-DNA integration into the sorghum genome and segregation of the selectable marker gene from the trait gene(s).

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. Recommendations for molecular confirmation and evaluation of transgenic tissue/plants are also provided.

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. Bialaphos resistant calli were generated from 52 to 59% of inoculated immature embryos depending on treatment. A total of 93.4% of the bialaphos selected calli from the 2 T-DNA vector treatment exhibited GUS activity compared to 11.7% for the mixed strain treatment and 98.2% for the cis control vector treatment. For the 2 T-DNA vector treatment, 86.7% of the bialaphos resistant/GUS active calli produced R0 plants exhibiting both transgenic phenotypes compared to 10% for the mixed strain treatment and 99% for the single T-DNA control vector treatment. A total of 87 Liberty herbicide (contains bialaphos as the active ingredient) resistant/GUS active R0 events from the 2 T-DNA binary vector treatment were evaluated for phenotypic segregation of these traits in the R1 generation. Of these R0 events, 71.4% exhibited segregation of Liberty resistance and GUS activity in the R1 generation. A total of 64.4% of the R0 2 T-DNA vector events produced Liberty sensitive/GUS active (indicating selectable-marker-free) R1 progeny. A high frequency of phenotypic segregation was also observed using the mixed strain approach, but a low frequency of calli producing R0 plants displaying both transgenic phenotypes makes this method less efficient. Molecular analyses were then used to confirm that the observed segregation of R1 phenotypes were highly correlated to genetic segregation of the bar and GUS genes. A high efficiency system to segregate transgenes in co-transformed maize plants has now been demonstrated.