Independent Transgenic Events Research Articles

Host-induced RNA interference targeting the neuromotor gene FMRFamide-like peptide-14 (Mi-flp14) perturbs Meloidogyne incognita parasitic success in eggplant.

The study demonstrates the successful management of Meloidogyne incognita in eggplant using Mi-flp14 RNA interference, showing reduced nematode penetration and reproduction without off-target effects across multiple generations. Root-knot nematode, Meloidogyne incognita, causes huge yield losses worldwide. Neuromotor function in M. incognita governed by 19 neuropeptides is vital for parasitism and parasite biology. The present study establishes the utility of Mi-flp14 for managing M. incognita in eggplant in continuation of our earlier proof of concept in tobacco (US patent US2015/0361445A1). Mi-flp14 hairpin RNA construct was used for generating 19 independent transgenic eggplant events. PCR and Southern hybridization analysis confirmed transgene integration and its orientation, while RT-qPCR and Northern hybridization established the generation of dsRNA and siRNA of Mi-flp14. In vitro and in vivo bio-efficacy analysis of single-copy events against M. incognita showed reduced nematode penetration and development at various intervals that negatively impacted reproduction. Interestingly, M. incognita preferred wild-type plants over the transgenics even when unbiased equal opportunity was provided for the infection. A significant reduction in disease parameters was observed in transgenic plants viz., galls (40-48%), females (40-50%), egg masses (35-40%), eggs/egg mass (50-55%), and derived multiplication factor (60-65%) compared to wild type. A unique demonstration of perturbed expression of Mi-flp14 in partially penetrated juveniles and female nematodes established successful host-mediated RNAi both at the time of penetration even before the nematodes started withdrawing plant nutrients and later stage, respectively. The absence of off-target effects in transgenic plants was supported by the normal growth phenotype of the plants and T-DNA integration loci. Stability in the bio-efficacy against M. incognita across T1- to T4-generation transgenic plants established the utility of silencing Mi-flp14 for nematode management. This study demonstrates the significance of targeting Mi-flp14 in eggplant for nematode management, particularly to address global agricultural challenges posed by M. incognita.

Efficient genetic transformation of papaya using RNAi CP gene against papaya ringspot virus

Papaya Ring Spot Virus disease is wide spread across papaya growing countries of the world and is one of the major impediments in successful papaya cultivation. Genetically engineered papaya varieties viz., SunUp and Rainbow have already been developed and commercialized in USA using coat protein mediated resistance. However, transgenic papaya conferring resistance to papaya ringspot virus has not been developed in India till date due to lack of suitable genetic transformation protocol for Indian papaya varieties and unavailability of coat protein gene construct for harbouring broad-spectrum resistance. The current study reports an efficient gene delivery mechanism in papaya. Young globular embryos infected for 30 minutes with Agrobacterium tumefaciens strain LBA 4404 harbouring hairpin loop of truncated coat protein gene and subsequently co-cultivated in presence of 100 pM acetosyringone and 1mM spermidine in dark for 72 hours gave rise to independent transgenic events characterized by PCR, dot blot hybridisation and RT-PCR.

Expression of Arabidopsis NPR1 (AtNPR1) in Brassica juncea var Varuna confers significant resistance against Sclerotinia sclerotiorum and Alternaria brassicae

The productivity of Brassica juncea, a major oilseed crop grown widely in South Asia, is limited by two major fungal pathogens: Sclerotinia sclerotiorum, and Alternaria brassicae. Currently, no effective resistance against these pathogens has been identified in the B. juncea germplasm. To improve the resistance against these fungal pathogens, Non-expressor of PR1 (NPR1) from Arabidopsis was expressed in the mega variety Varuna via Agrobacterium-mediated transformation. A total of 129, independent transgenic events were generated. Segregation analysis for Basta resistance and subsequent Southern hybridization identified six single-copy transgenic lines (PN23, 42, 60, 72, HN30, and HN32) which were taken forward for disease assays over two generations. Transgenic lines challenged with S. sclerotiorum exhibited a significant reduction in stem lesions ranging from 13 to 42% in the T3 generation and 4 to 31% in the T4 generation. We also observed a reduced infection against A. brassicae in the transgenic lines which developed smaller and fewer necrotic lesions as compared to untransformed lines. A correlation between the upregulation of defense marker genes and enhanced tolerance was observed in the transgenic lines upon S. sclerotiorum infection.

Enhanced seedling growth by 3-n-pentadecylphenolethanolamide is mediated by fatty acid amide hydrolases in upland cotton (Gossypium hirsutum L.).

Fatty acid amide hydrolase (FAAH) is a conserved amidase that is known to modulate the levels of endogenous N‐acylethanolamines (NAEs) in both plants and animals. The activity of FAAH is enhanced in vitro by synthetic phenoxyacylethanolamides resulting in greater hydrolysis of NAEs. Previously, 3‐n‐pentadecylphenolethanolamide (PDP‐EA) was shown to exert positive effects on the development of Arabidopsis seedlings by enhancing Arabidopsis FAAH (AtFAAH) activity. However, there is little information regarding FAAH activity and the impact of PDP‐EA in the development of seedlings of other plant species. Here, we examined the effects of PDP‐EA on growth of upland cotton ( Gossypium hirsutum L. cv Coker 312) seedlings including two lines of transgenic seedlings overexpressing AtFAAH. Independent transgenic events showed accelerated true‐leaf emergence compared with non‐transgenic controls. Exogenous applications of PDP‐EA led to increases in overall seedling growth in AtFAAH transgenic lines. These enhanced‐growth phenotypes coincided with elevated FAAH activities toward NAEs and NAE oxylipins. Conversely, the endogenous contents of NAEs and NAE‐oxylipin species, especially linoleoylethanolamide and 9‐hydroxy linoleoylethanolamide, were lower in PDP‐EA treated seedlings than in controls. Further, transcripts for endogenous cotton FAAH genes were increased following PDP‐EA exposure. Collectively, our data corroborate that the enhancement of FAAH enzyme activity by PDP‐EA stimulates NAE‐hydrolysis and that this results in enhanced growth in seedlings of a perennial crop species, extending the role of NAE metabolism in seedling development beyond the model annual plant species, Arabidopsis thaliana .

CRISPR/Cas9-mediated saturated mutagenesis of the cotton MIR482 family for dissecting the functionality of individual members in disease response.

Many genes encoding nucleotide‐binding leucine‐rich repeat receptors (NLRs) are regulated and fine‐tuned by miR482 to balance the trade‐off between disease resistance and growth. Dicotyledonous plants, including cotton, usually have multiple miR482 isoforms. Each miR482 isoform can regulate several NLRs that in turn can be regulated by several different miR482 isoforms. Dissecting the functionality of individual miR482 isoforms in disease response and in balancing the disease resistance and growth trade‐off demands a collection of mutants mutated in individual miR482 members (single or multiple). In this study, we generated such a collection of cotton miR482 mutants using CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) genome editing and transformation of pooled guide RNAs (gRNAs). In total, 84 T0 plants representing 40 independent transgenic events and harboring mutation in each of the 10 miR482 isoforms were generated. The average editing efficiency of the 18 transformed gRNAs is 75%, ranging from 0 (3 gRNAs) to 100% (8 gRNAs). Most miR482 isoforms have a diverse range of mutations, including small indels (1–44 bp) and substitutions, which are expected to impair biogenesis of miR482. All nine mutant populations used in Verticillium dahliae infection experiments showed a disease index lower than the control, with four being significantly lower. The disease assay also suggests a different role of different miR482 isoforms in disease response and a potential dosage effect of miR482l. The study demonstrates the feasibility of saturation mutagenesis of plant miRNA families with dozens of genetic loci using CRISPR/Cas9 and provides the cotton community a valuable resource for uncovering the miR482‐NLR module(s) underlying the interaction between cotton and different pathogens.

Production of Conjoined Transgenic and Edited Barley and Wheat Plants for Nud Genes Using the CRISPR/SpCas9 System.

The Nudum (Nud) gene controls the caryopsis type of cereal crops by regulating lipid biosynthetic pathways. Based on the HvNud sequence and its homologous gene sequences in wheat, a conserved sgRNA was designed to obtain the mutants from the barley variety “Vlamingh” and the wheat variety “Fielder” via Agrobacterium-mediated transformation. A total of 19 and 118 transgenic plants were obtained, and 11 and 61 mutant plants were identified in T0 transgenic plants in barley and wheat after PCR-RE detection, and the editing efficiencies of the targeted gene were 57.9 and 51.7% in barley and wheat, respectively. The grain shape of the barley mutants was naked. Five different combinations of mutations for wheat TaNud genes were identified in the T0 generation, and their homozygous-edited plants were obtained in the T1 generation. Interestingly, the conjoined plants in which one plant has different genotypes were first identified. The different tillers in an individual T0 plant showed independent transgenic or mutant events in both barley and wheat, and the different genotypes can stably inherit into T1 generation, indicating that the T0 transgenic plants were the conjoined type. In addition, we did not find any off-target mutations in both barley and wheat. A candidate method for detecting putative-edited wheat plants was suggested to avoid losing mutations in this investigation. This study provides not only materials for studying the function of the Nud gene in barley and wheat but also a system for detecting the mutants in wheat.

Clean gene technology to develop selectable marker-free pod borer-resistant transgenic pigeon pea events involving the constitutive expression of Cry1Ac.

The most crucial yield constraint of pigeon pea is susceptibility to the pod borer Helicoverpa armigera, which causes extensive damage and severe economic losses every year. The Agrobacterium-mediated plumular meristem transformation technique was applied for the development of cry1Ac transgenic pigeon pea. Bioactivity of the cry1Ac gene was compared based on integration and expression driven by two promoters, the constitutive CaMV35S promoter and the green-tissue-specific ats1A promoter, in those transgenic events. The transgenic events also contained the selectable marker gene nptII flanked by loxP sites. Independent transgenic events expressing the Cre recombinase gene along with a linked bar selection marker were also developed. Integration and expression patterns of both cry1Ac and cre were confirmed through Southern and western blot analysis of T1 events. The constitutive expression of the Cry1Ac protein was found to be more effective for conferring resistant activity against H. armigera larvae in comparison to green-tissue-specific expression. Constitutively expressing Cry1Ac T1 events were crossed with Cre recombinase expressing T1 events. The crossing-based Cre/lox-mediated marker gene elimination strategy was demonstrated to generate nptII-free Cry1Ac-expressing T2 events. These events were subsequently analyzed in the T3 generation for the segregation of cre and bar genes. Five Cry1Ac-expressing T3 transgenic pigeon pea events were devoid of the nptII marker as well as cre-bar genes. H. armigera larval mortality in those marker-free T3 events was found to be 80-100%. The development of such nptII selectable marker-free Cry1Ac-expressing pigeon pea transgenics for the first time would greatly support the sustainable biotechnological breeding program for pod borer resistance in pigeon pea. KEY POINTS: • Constitutive expression of Cry1Ac conferred complete resistance against Helicoverpa armigera • Green-tissue-specific expression of Cry1Ac conferred partial pest resistance • Cre/lox-mediated nptII elimination was successful in constitutively expressing Cry1Ac transgenic pigeon pea events.

Transformation of Teosinte (Zea mays ssp. parviglumis) via Biolistic Bombardment of Seedling-Derived Callus Tissues.

Modern maize exhibits a significantly different phenotype than its wild progenitor teosinte despite many genetic similarities. Of the many subspecies of Zea mays identified as teosinte, Zea mays ssp. parviglumis is the most closely related to domesticated maize. Understanding teosinte genes and their regulations can provide great insights into the maize domestication process and facilitate breeding for future crop improvement. However, a protocol of genetic transformation, which is essential for gene functional analyses, is not available in teosinte. In this study, we report the establishment of a robust callus induction and regeneration protocol using whorl segments of seedlings germinated from mature seeds of Zea parviglumis. We also report, for the first time, the production of fertile, transgenic teosinte plants using the particle bombardment. Using herbicide resistance genes such as mutant acetolactate synthase (Als) or bialaphos resistance (bar) as selectable markers, we achieved an average transformation frequency of 4.17% (percentage of independent transgenic events in total bombarded explants that produced callus). Expression of visual marker genes of red fluorescent protein tdTomato and β-glucuronidase (gus) could be detected in bombarded callus culture and in T1 and T2 progeny plants. The protocol established in this work provides a major enabling technology for research toward the understanding of this important plant in crop domestication.

Stress-induced expression of IPT gene in transgenic wheat reduces grain yield penalty under drought

BackgroundThe heterologous expression of isopentenyl transferase (IPT) under the transcriptional control of the senescence-associated receptor-like kinase (SARK) promoter delayed cellular senescence and, through it, increased drought tolerance in plants. To evaluate the effect of pSARK::IPT expression in bread wheat, six independent transgenic events were obtained through the biolistic method and evaluated transgene expression, phenology, grain yield and physiological biomass components in plants grown under both drought and well-irrigating conditions. Experiments were performed at different levels: (i) pots and (ii) microplots inside a biosafety greenhouse, as well as under (iii) field conditions. ResultsTwo transgenic events, called TR1 and TR4, outperformed the wild-type control under drought conditions. Transgenic plants showed higher yield under both greenhouse and field conditions, which was positively correlated to grain number (given by more spikes and grains per spike) than wild type. Interestingly, this yield advantage of the transgenic events was observed under both drought and well-watered conditions. ConclusionsThe results obtained allow us to conclude that the SARK promoter-regulated expression of the IPT gene in bread wheat not only reduced the yield penalty produced by water stress but also led to improved productivity under well-watered conditions.

Resistant performance of T10 Rojolele transgenic rice events harboring cry1B::cry1Aa fusion genes against the rice yellow stem borer Scirpophaga incertulas Wlk

Rice yellow stem borer (Scirpophaga incertulas Wlk) (rice YSB) is one of the most important insect pests in rice (Oryza sativa L.). Plant damage caused by the pest attack in all growth stages could significantly reduce yield. Cry toxins from Bacillus thuringiensis are known to be effective against rice YSB, therefore transgenic rice events cv Rojolele harbouring the cry1B::cry1Aa fusion genes driven by the maize ubiquitin promoter have been developed to improve rice resistance. To determine that the resistant traits has been stably inherited and expressed, in-planta and in-vitro bioassays of the T10 generation were performed on 6 independent transgenic rice events using the 1st instar larvae of rice YSB. In-planta bioassay was performed on 5-week-old rice plants grown in pots in a transgenic glasshouse, in 7 replicates. Each plant was invested with 20 1st instar larvae of rice YSB. The in-vitro bioassay was performed using the stems of the transgenic rice events against 10 1st instar larvae of rice YSB, in aerated Petri dishes in a culture room under room temperature, in three replicates. Results showed that the T10 generation of the transgenic rice events stably maintained the transgene integration and their resistance against the rice YSB.

Efficient Gene Stacking in Rice Using the GAANTRY System

Genetic engineering of rice provides a means for improving rice grain quality and yield, and the introduction and expression of multiple genes can produce new traits that would otherwise be difficult to obtain through conventional breeding. GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) was previously shown to be a precise and robust system to stably stack ten genes (28 kilobases (kb)) within an Agrobacterium virulence plasmid Transfer-DNA (T-DNA) and obtain high-quality Arabidopsis and potato transgenic events. To determine whether the GAANTRY system can be used to engineer a monocotyledonous crop, two new T-DNA constructs, carrying five (16.9 kb) or eleven (37.4 kb) cargo sequences were assembled and transformed into rice. Characterization of 53 independent transgenic events demonstrated that more than 50% of the plants carried all of the desired cargo sequences and exhibited the introduced traits. Additionally, more than 18% of the lines were high-quality events containing a single copy of the introduced transgenes and were free of sequences from outside of the T-DNA. Therefore, GAANTRY provides a simple, precise and versatile tool for transgene stacking in rice and potentially other cereal grain crops.

RNAi of AGAMOUS genes in sweetgum alters reproductive organ identity and decreases fruit persistence.

Sweetgums (Liquidambar), members of the family Altingiaceae (Altingiales), have inflorescences and floral organs that are distinctive in structure compared with other angiosperms in which the roles of floral homeotic genes have been studied. To begin to understand the role of AGAMOUS (AG)—a floral homeotic gene that has a major role in stamen and carpel development—in development of the monosexual flowers of sweetgum, we used RNAi to reduce the expression of two members of the AG subfamily. Because AG suppression should induce floral sterility, RNAi might also provide a tool to mitigate the risks of invasiveness—and to reduce the production of its nuisance fruits or allergenic pollen—when sweetgum is used as an exotic shade or forest tree. We tested 33 independent transgenic events and non‐transgenic controls during 10 years in the field. The RNAi‐AG sweetgum trees maintained normal growth, phenology, and vivid fall coloration during the 10 years of study, but 8 insertion events had highly modified inflorescence and floral morphology. The modified flowers had anthers and carpels that were converted to flat leaf‐like structures lacking pollen grains and ovules, respectively. The female inflorescences developed into dry papery structures that failed to produce seeds. These infructescences were smaller than control infructescences, and lost a greater percentage of biomass in a controlled decay assay. RNAi against AG genes was highly effective at impairing fertility and modifying reproductive development without significant vegetative effects in sweetgum and gave phenotypes distinct from, but similar to, that of AG loss of function in other angiosperms.

Enhanced exudation of malate in the rhizosphere due to AtALMT1 overexpression in blackgram (Vigna mungo L.) confers increased aluminium tolerance.

Worldwide, 50% of soil is acidic, which induces aluminium (Al) toxicity in plants, as the phyto-availability of Al3+ increases in acidic soil. Plants responds to Al3+ toxicity by exuding organic acids into the rhizosphere. The organic acid responsible for Al3+ stress response varies from species to species, which in the case of blackgram (Vigna mungo L.) is citrate. In blackgram, an Arabidopsis malate transporter, AtALMT1, was overexpressed with the motive of inducing enhanced exudation of malate. Transgenics were generated using cotyledon node explants through Agrobacterium tumefaciens-mediated transformation. The putative transgenics were initially screened by AtALMT1-specific genomic DNA PCR, followed by quantitative PCR. Two independent transgenic events were identified and functionally characterized in the T3 generation. The transgenic lines, Line 1 and 2, showed better root growth, relative water content and chlorophyll content under Al3+ stress. Both lines also accounted for less oxidative damage, due to reduced accumulation of ROS molecules. Photosynthetic efficiency, as measured in terms of Fv /Fm , NPQ and Y(II), increased when compared to the wild type (WT). Relative expression of genes (VmSTOP1, VmALS3, VmMATE) responsible for Al3+ stress response in blackgram showed that overexpression of a malate transporter did not have any effect on their expression. Malate exudation increased whereas citrate exudation did not show any divergence from the WT. A pot stress assay found that the transgenics showed better adaptation to acidic soil. This report demonstrates that the overexpression of a malate transporter in a non-malate exuding species improves adaptation to Al3+ toxicity in acidic soil without effecting its stress response mechanism.

An Efficient Stevia rebaudiana Transformation System and In vitro Enzyme Assays Reveal Novel Insights into UGT76G1 Function

Stevia rebaudiana (Bertoni) is one of a very few plant species that produce zero calorie, sweet compounds known as steviol glycosides (SG). SGs differ in their sweetness and organoleptic properties depending on the number and positioning of sugar groups on the core steviol backbone. There is great interest of modulating the SG profiles of the Stevia plant to enhance the flavor profile for a given application in the food and beverage industries. Here, we report a highly efficient Agrobacterium-mediated stable transformation system using axillary shoots as the initial explant. Using this system, we generated over 200 transgenic Stevia plants overexpressing a specific isoform of UGT76G1. By comparing the SG profiles among independent transgenic events, we demonstrated that altering UGT76G1 expression can change the ratios of specific SG species. Furthermore, using recombinant proteins produced in E. coli, we show that two closely related UGT76G1 isoforms differ in their substrate specificities, providing new insights into mechanisms underlying the diversity of SG profiles that are observed across Stevia germplasm. Finally, we found evidence suggesting that alternative and/or aberrant splicing may serve to influence the ability of the plant to produce functional UGT76G1 transcripts, and possibly produce enzyme variants within the plant.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes.

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

Transgenic Wheat Harboring an RNAi Element Confers Dual Resistance Against Synergistically Interacting Wheat Streak Mosaic Virus and Triticum Mosaic Virus.

Wheat streak mosaic virus (WSMV) and triticum mosaic virus (TriMV) are economically important viruses of wheat (Triticum aestivum L.), causing significant yield losses in the Great Plains region of the United States. These two viruses are transmitted by wheat curl mites, which often leads to mixed infections with synergistic interaction in grower fields that exacerbates yield losses. Development of dual-resistant wheat lines would provide effective control of these two viruses. In this study, a genetic resistance strategy employing an RNA interference (RNAi) approach was implemented by assembling a hairpin element composed of a 202-bp (404-bp in total) stem sequence of the NIb (replicase) gene from each of WSMV and TriMV in tandem and of an intron sequence in the loop. The derived RNAi element was cloned into a binary vector and was used to transform spring wheat genotype CB037. Phenotyping of T1 lineages across eight independent transgenic events for resistance revealed that i) two of the transgenic events provided resistance to WSMV and TriMV, ii) four events provided resistance to either WSMV or TriMV, and iii) no resistance was found in two other events. T2 populations derived from the two events classified as dual-resistant were subsequently monitored for stability of the resistance phenotype through the T4 generation. The resistance phenotype in these events was temperature-dependent, with a complete dual resistance at temperatures ≥25°C and an increasingly susceptible response at temperatures below 25°C. Northern blot hybridization of total RNA from transgenic wheat revealed that virus-specific small RNAs (vsRNAs) accumulated progressively with an increase in temperature, with no detectable levels of vsRNA accumulation at 20°C. Thus, the resistance phenotype of wheat harboring an RNAi element was correlated with accumulation of vsRNAs, and the generation of vsRNAs can be used as a molecular marker for the prediction of resistant phenotypes of transgenic plants at a specific temperature.

Improved Wheat Growth and Yield by Delayed Leaf Senescence Using Developmentally Regulated Expression of a Cytokinin Biosynthesis Gene.

Delaying leaf senescence in plants, especially under water stress conditions, can help to maintain the remobilization of stored nutrients in source–sink relationships, thus leading to improved crop yields. Leaf senescence can be delayed by plant hormones such as cytokinin. Here, the Isopentenyl transferase (IPT) gene, encoding a cytokinin biosynthesis enzyme, driven by a modified AtMYB32xs promoter was transformed into wheat. Transgenic wheat plants exhibited delayed leaf senescence, retaining chlorophyll for longer under controlled environment conditions. Selected independent transgenic events and their corresponding nulls were grown under field conditions for two consecutive years under well-watered and water stress treatments using automated rainout shelters. Three independent transgenic events had improved canopy green cover, lower canopy temperatures, and higher leaf water potential than their respective non-transgenic nulls, with no abnormality in morphology and phenology. Increased grain yield was observed in transgenic events under both water treatments, with the yield increase more pronounced under water stress (26–42%). These results have shown that delayed leaf senescence using the chimeric transgene AtMYB32xs-p::IPT can be a useful strategy to achieve grain yield gains in wheat and potentially other crops for sustainable food production.

Effect of plating density, amino acid and osmoticum on transformation efficiency of papaya (Carica papaya L.)

Papaya suffers colossal losses due to Papaya Ring Spot Virus (PRSV) disease. PRSV is responsible for about 70% loss in papaya production worldwide. This disease is present all over the world wherever papaya is grown. Transgenic papaya varieties namely SunUp and Rainbow have been developed and commercialized in the USA using Pathogen Derived Resistance (PDR). The present investigation reports a successful gene delivery system in an Indian papaya variety employing PDR. Agro-infected immature young globular embryos of papaya cv. Pusa Delicious were co-cultivated with A. tumefaciens harboring a hairpin loop construct of a truncated PRSV coat protein gene. The Agro-infected explants were plated at a density of 2–3, in the presence of 100 mg L−1 L-cysteine in dark for 72 h. Transformed plants maturation under the influence of 45 mg L−1 PEG produced 4 independent transgenic events which were characterized by PCR and data sequencing.

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

A modified in planta method of Agrobacterium-mediated transformation enhances the transformation efficiency in safflower (Carthamus tinctorius L.)

Plant transformation has emerged as an important tool to integrate foreign genes in the plant genome to modify the plants for desired traits. Though many techniques of plant transformation are available; getting single copy transgenic events and cost associated remains a big challenge. Thus Agrobacterium-mediated transformation remains the method of choice due to multiple advantages. In the present work a tissue culture free protocol of Agrobacterium-mediated transformation was optimized in safflower, an oil seed crop recalcitrant to transformation. As a proof of concept we selected pCAMBIA2300 gene cassette containing Arabidopsis specific delta 15 desaturase (FAD3) downstream to truncated seed specific promoter beta-conglycinin and optimized tissue culture free protocol of Agrobacterium-mediated transformation using embryos as explants. Addition of silwet L-77, sonication treatment, vacuum infiltration in infection medium and use of paper wicks in co-cultivation period increased the transformation efficiency to 19.3%. Further, success in transformation was confirmed via product accumulation in 21 independent transgenic events wherein oil in transformed seeds showed significant accumulation of alpha-linolenic acid (ALA; 18:3; n3) which is generated from linoleic acid (LA; 18:2; n3) in a FAD3 catalyzed reaction. The present protocol can be utilized to produce transgenic safflower with different desired characters.