Genetic transformation of indica rice varieties involving Am-SOD gene for improved abiotic stress tolerance

Agrobacterium mediated genetic transformation has become an important tool in crops for molecular breeding. Am-SOD quality containing transgenic plants were created from embryogenic calli of Sambha mahsuri and cotton sannalu by Agrobacterium tumifaciens co-development. The superoxide desmutase quality was housed responsible for CaMV 355 promoter and Nos polyadenylation motion in double vector pCAMBIA 1301. Good change productivity was gotten. Mix of quality at genome level in the plants was exhibited by PCR examination and Southern smear, and furthermore delineated by a few physiological studies.     

Ectopic over-expression of peroxisomal ascorbate peroxidase (SbpAPX) gene confers salt stress tolerance in transgenic peanut (Arachis hypogaea)

Peroxisomal ascorbate peroxidase gene (SbpAPX) of an extreme halophyte Salicornia brachiata imparts abiotic stress endurance and plays a key role in the protection against oxidative stress. The cloned SbpAPX gene was transformed to local variety of peanut and about 100 transgenic plants were developed using optimized in vitro regeneration and Agrobacterium mediated genetic transformation method. The T₀ transgenic plants were confirmed for the gene integration; grown under controlled condition in containment green house facility; seeds were harvested and T₁ plants were raised. Transgenic plants (T₁) were further confirmed by PCR using gene specific primers and histochemical GUS assay. About 40 transgenic plants (T₁) were selected randomly and subjected for salt stress tolerance study. Transgenic plants remained green however non-transgenic plants showed bleaching and yellowish leaves under salt stress conditions. Under stress condition, transgenic plants continued normal growth and completed their life cycle. Transgenic peanut plants exhibited adequate tolerance under salt stress condition and thus could be explored for the cultivation in salt affected areas for the sustainable agriculture.     

Use of doubled haploid technology for development of stable drought tolerant bread wheat (Triticum aestivum L.) transgenics

Anther culture–derived haploid embryos were used as explants for Agrobacterium‐mediated genetic transformation of bread wheat (Triticum aestivum L. cv CPAN1676) using barley HVA1 gene for drought tolerance. Regenerated plantlets were checked for transgene integration in T₀ generation, and positive transgenic haploid plants were doubled by colchicine treatment. Stable transgenic doubled haploid plants were obtained, and transgene expression was monitored till T₄ generation, and no transgene silencing was observed over the generations. Doubled haploid transgenic plants have faster seed germination and seedling establishment and show better drought tolerance in comparison with nontransgenic, doubled haploid plants, as measured by per cent germination, seedling growth and biomass accumulation. Physiological evaluation for abiotic stress by assessing nitrate reductase enzyme activity and plant yield under post‐anthesis water limitation revealed a better tolerance of the transgenics over the wild type. This is the first report on the production of double haploid transgenic wheat through anther culture technique in a commercial cultivar for a desirable trait. This method would also be useful in functional genomics of wheat and other allopolyploids of agronomic importance.     

Increased Tolerance to Salinity and Drought in Transgenic Indica Rice by Mannitol Accumulation

The osmolytes, including mannitol have been shown to be very important in abiotic stress tolerance. Thus, the present study was undertaken with the aim to enhance abiotic stress tolerance in basmati indica rice by introduction of the E. coli mannitol-1-phospho dehydrogenase (mt/D) gene, which is involved in mannitol synthesis in plants. Several putative transgenic rice plants were generated by Agrobacterium-mediated transformation. The presence of the transgene in the primary transformants was confirmed by PCR using hygromycin phosphotransferase (hpt) and mt/D gene specific primers. Southern hybridization also revealed the integration of the transgene. Transgenic lines exhibited mannitol accumulation, which was correlated to the increased tolerance of the transgenics against salinity and drought stress. The T₁ transgenic seed germination and seedlings growth showed better performance than that of wild type during abiotic stresses under in vitro and in vivo growth conditions.     

Arabidopsis galactinol synthase AtGolS2 improves drought tolerance in the monocot model Brachypodium distachyon

Brachypodium distachyon (purple false brome) is a herbaceous species belonging to the grass subfamily Pooideae, which also includes major crops like wheat, barley, oat and rye. The species has been established as experimental model organism for understanding and improving cereal crops and temperate grasses. The complete genome of Bd21, the community standard line of B. distachyon, has been sequenced and protocols for Agrobacterium-mediated transformation have been published. Further improvements to the experimental platform including better evaluation systems for transgenic plants are still needed. Here we describe the growth conditions for Bd21 plants yielding highly responsive immature embryos that can generate embryogenic calli for transformation. A prolonged 20-h photoperiod produced seeds with superior immature embryos. In addition, osmotic treatment of embryogenic calli enhanced the efficiency of transfection by particle bombardment. We generated transgenic plants expressing Arabidopsis thaliana galactinol synthase 2 (AtGolS2) in these experiments. AtGolS2-expressing transgenics displayed significantly improved drought tolerance, increasing with increased expression of AtGolS2. These results demonstrate that AtGolS2 can confer drought tolerance to monocots and confirm that Brachypodium is a useful model to further explore ways to understand and improve major monocot crop species.     

The TaDREB3 transgene transferred by conventional crossings to different genetic backgrounds of bread wheat improves drought tolerance

Drought tolerance of the wheat cultivar Bobwhite was previously enhanced by transformation with a construct containing the wheat DREB3 gene driven by the stress‐inducible maize Rab17 promoter. Progeny of a single T₂ transgenic line were used as pollinators in crosses with four elite bread wheat cultivars from Western Australia: Bonnie Rock, IGW‐2971, Magenta and Wyalkatchem, with the aim of evaluating transgene performance in different genetic backgrounds. The selected pollinator line, BW8‐9‐10‐3, contained multiple transgene copies, had significantly improved drought tolerance compared with wild‐type plants and showed no growth and development penalties or abnormalities. A single hybrid plant was selected from each cross‐combination for three rounds of backcrossing with the corresponding maternal wheat cultivar. The transgene was detected in all four F₁BC₃ combinations, but stress‐inducible transgene expression was found in only three of the four combinations. Under well‐watered conditions, the phenotypes and grain yield components of the F₂BC₃ transgene‐expressing lines were similar to those of corresponding recurrent parents and null‐segregants. Under severe drought conditions, the backcross lines demonstrated 12–18% higher survival rates than the corresponding control plants. Two from four F₃BC₃ transgenic lines showed significantly higher yield (18.9% and 21.5%) than control plants under limited water conditions. There was no induction of transgene expression under cold stress, and therefore, no improvement of frost tolerance observed in the progenies of drought‐tolerant F₃BC₃ lines.     

Heterologous expression of the AtDREB1A gene in chrysanthemum increases drought and salt stress tolerance

DNA cassette containing an AtDREB1A cDNA and a nos terminator, driven by a cauliflower mosaic 35S promoter, or a stress-inducible rd29A promoter, was transformed into the ground cover chrysanthemum (Dendranthema grandiflorum) ‘Fall Color’ genome. Compared with wild type plants, severe growth retardation was observed in 35S:DREB1A plants, but not in rd29A:DREB1A plants. RT-PCR analysis revealed that, under stress conditions, the DREB1A gene was over-expressed constitutively in 35S:DREB1A plants, but was over-expressed inductively in rd29A:DREB1A plants. The transgenic plants exhibited tolerance to drought and salt stress, and the tolerance was significantly stronger in rd29A:DREB1A plants than in 35S:DREB1A plants. Proline content and SOD activity were increased inductively in rd29A:DREB1A plants than in 35S:DREB1A plants under stress conditions. These results indicate that heterologous AtDREB1A can confer drought and salt tolerance in transgenic chrysanthemum, and improvement of the stress tolerance may be related to enhancement of proline content and SOD activity.     

Abiotic stress tolerance in transgenic eggplant (Solanum melongena L.) by introduction of bacterial mannitol phosphodehydrogenase gene

In the present work, the bacterial mannitol-1-phosphodehydrogenase(mtlD) gene was introduced into eggplant(Solanummelongena L.) by Agrobacteriumtumefaciens-mediated transformation. Several transformants weregenerated and the transgene integration was confirmed by PCR, dot blot andSouthern blot analysis. Transgenic lines of T₀ and T₁generations were examined for tolerance to NaCl-induced salt stress,polyethylene glycol-mediated drought and chilling stress under bothinvitro and in vivo growth conditions. Aconsiderable proportions of transgenic seeds germinated and seedlings grew wellon 200 mM salt-amended MS basal medium, whereas seeds ofuntransformed control plants failed to germinate. Further, leaf explants fromthe transgenics could grow and showed signs of shoot regeneration onsalt-amended MS regeneration medium, whereas wild type did not respond, and infact the explants showed necrosis and loss of chlorophyll after about one week.The transgenic leaves could also withstand desiccation, and transgenics couldgrow well under chilling stress, and hydroponic conditions with salt stress ascompared to wild type plants. Thus, the transgenic lines were found to betolerant against osmotic stress induced by salt, drought and chilling stress.The morphology of the transgenic plants was normal as controls, but thechlorophyll content was higher in some of the lines. These observations suggestthat mtlD gene can impart abiotic stress tolerance ineggplant.     

Genetic modification of Gossypium arboreum universal stress protein (GUSP1) improves drought tolerance in transgenic cotton (Gossypium hirsutum)

Cotton crop suffers shortage of irrigation water at reproductive stage which reduces the yield and fibre quality. Universal stress proteins belong to Pfam00582 which enables several plants to cope with multiple stresses via ATP binding. GUSP1 (Gossypium arboreum USP) is one of such proteins; its amino acids were mutated after in silico simulations including homology modeling and molecular docking analysis. Transgenic cotton plants were developed through Agrobacterium mediated genetic transformation by using mutated pmGP1 and non mutated pGP1 constructs under CaMV35S promoter. PCR and semi-quantitative PCR analyses confirmed the amplification and expression of transgene in transgenic plants. It was revealed that leaf relative water content, total chlorophyll content, CO₂ assimilation as net photosynthesis, stomatal conductance, total soluble sugars and proline content was significantly increased at P ≤ 0.0001 and P ≤ 0.001 in both the pmGP1 and pGP1 transgenic plants as compared to non transgenic control plants. Moreover, relative membrane permeability and the transpiration rate were reduced significantly at P ≤ 0.0001 and P ≤ 0.001 respectively in transgenic plants under drought stress. Furthermore, the T1 transgenic seedlings containing pmGP1 mutated construct showed longer roots under desiccation stress imposed by 5% PEG. Transgene inheritance into the T1 progeny plants was confirmed by amplification through PCR and integration through Southern blot. Hence, our results pave the way to utilize the mutagenized known genes for increasing endurance of plants under drought stress. This will help to increase our understanding of drought tolerance/ sensitivity in cotton plants at the molecular level.Supplementary Information The online version contains supplementary material available at 10.1007/s12298-021-01048-5.