| Wheat (Triticum aestivum) is the most important staple food crop. Continuously increasing food demand for the mushrooming population necessitates further genetic improvement of the crop for enhancing its production from all kinds of available land resources. Genetic transformation has emerged as the most valuable tool for genetic improvement of the crop supplementing the conventional breeding strategies. Precise genetic alterations can be made without affecting the previously accumulated economically important traits of the elite varieties. Now the crops can be genetically engineered to thrive in the abiotic stress conditions. BADH gene encodes for betaine aldehyde dehydrogenase, an important enzyme for the biosynthesis of glycine betaine that plays a definite role in stress tolerance through osmoregulation. Integration and expression of BADH gene can confer salt tolerance to the host plant. Present study was designed and conducted to insert this gene into the genome of wheat crop with the objective to improve its salt tolerance through particle bombardment and Agrobacterium-inoculation of callus. In addition, a preliminary study was also conducted to explore the probability of in planta apical meristem transformation of wheat. The purpose of this study was to find the possibility of eliminating callus phase of gene transformation thus making it genotype independent.Chinese winter wheat variety Shi 4185 was selected for the transformation experiments. The plasmid vector pABH9 containing BADH with bar (encoding for phosphinothricin acetyl transferase) under the control of ubiquitine (ubi) promoter and nopaline synthetase (nos) terminator sequences was used for the bombardment of the callus derived from immature embryos by gene gun (PDS-1000/He). Selection was carried out with 1, 3, and 5mg/l ppt (phosphinothricin) and/or 100 and 150mM NaCI in MS based medium solidified with agar followed by its transfer to regeneration medium for shoot induction. DNA from the leaves of the regenerated seedlings was tested for amplification of BADH gene from the genome using specific primers. Leaf-paint assay was conducted using 3mg/l ppt with 0.1% Tween20 to determine the expression of bar gene. PCR positive plants were exposed to 0.5% NaCI to assess their tolerance to osmotic stress.Out of 3263-bombarded calli 15.99% proliferating calli with good morphology were transferred to regeneration medium. About 9% calli produced seedlings out of which only 5 seedlings were PCR positive for BADH gene indicating high selection-escape rate (about 90%). Results of PPT-leaf painting were consistent with PCR test. PCR positive plants were able to grow in presence of 0.5% NaCI whereas check plants exhibited stunted growth. Net transformation efficiency based on total number of calli bombarded was 0.15%. Regeneration was the major problem encountered in these experiments. Selection system was also not very effective. Transgenic plants that couldtolerate osmotic stress were produced but the method is labor intensive and expensive with low turn out. The efficiency of this method is limited by the tissue culture responsiveness of the genotype.Agrobacterium tumefaciens strain AGL1, harboring BADH and nptll (encoding for neomycin phosphotransferase) genes under the CaMV 35S (cauliflower mosaic virus 35S) promoter and nos terminator sequences in its plasmid pBin438, was used as a biological delivery system for gene transfer. Callus induced from mature and immature embryos was inoculated and co-cultivated with Agrobacterium in presence of 100uM acetosyringone and selected on MS based medium with 100 or 150mM NaCI followed by regeneration in presence of 100mM NaCI. Spermidine was used for inducing shoots from the callus that did not produce shoots after 8-10 weeks. Regenerated plants were tested for integration of BADH gene through PCR amplification. Stress tolerance of plants was determined by exposing the plants to 0.5% NaCI.About 12% good calli were selected from 4901 inoculated calli out of which 4.43% regenerated into plant-lets. Regenerated plant-lets yielded 19.23% PCR positive plants for BADH gene and these were able to tolerate 0.5% level of NaCI. Net transformation efficiency was 0.10% on the basis of total umber of treated calli. Regeneration of the plants and overgrowth of Agrobacterium were the main problems faced in this experiment leading to low transformation efficiency. Gene integration was better than that of gene gun transformation that was manifested by PCR test of the calli from selection medium. The method is relatively cheap in terms of operational cost but it is dependent on the regeneration response of the genotype to tissue culture.Realizing the importance of in planta transformation for wheat to avoid tissue culture based transformation through Agrobacterium and particle bombardment an attempt was made for apical meristem transformation. Three experiments were conducted for finding the probability of apical meristem transformation of wheat. Agrobacterium tumefaciens strain LBA4404, containing pAHCGG plasmid with hph (hygromycin phosphotransferase) and GUS (P-glucuronidase) genes under the control of CaMV 35S promoter, was used for inoculation of apical meristems of seedlings (1 to 11 days post germination) after wounding with fiberglass brush. Transient GUS expression was determined by histochemical assay. In the 2nd experiments 1-2 days old seedlings were used and florets were tested for GUS expression. In the second part of this experiment Agrobacterium tumefaciens strain AGL1, harboring BADH and nptll genes in its plasmid pBin438, was used for inoculation of apical meristems of 2 days old seedlings. Regenerating plants were selected on 100mg/l kanamycin and analyzed through PCR test for BADH amplification. In the 3rd experiment the meristem cells were inoculated with GUS gene before seed germination after 4 or 8 hour soaking and florets were examined for GUS expression.Frequency of transient GUS expression ranged from 1.7% to more than10% and it declined with increasing age of the seedlings. Inoculation of apical meristems of 1-2 days old seedlings yielded 2 to 9% GUS positive inflorescences. X-gluc staining was of variable intensity ranging from light to dark blue. Inoculation of meristem cells before germination produced 1.42% inflorescences with strong X-gluc staining out of 1483 treated seeds. X-gluc staining was never observed in check plants. No PCR positive plant was found in the progeny of GUS positive plants.Sixty-three kanamycin resistant green plants (13.6%) were obtained when apical meristems from two days old seedlings were inoculated with Agrobacterium tumefaciens AGL1 harboring nptll and BADH genes in its plasmid pBin438, followed by selection on kanamycin. Three plants were found PCR positive but only one plant could produce seeds. Albeit successful transformation of wheat through apical meristem conclusive inference cannot be postulated for the reliability of this system based on single transformed plant. It needs further experimental support. This experiment can be regarded as a first successful step towards genotype independent transformation system for wheat for which regeneration of transformed plant-lets is difficult and insufficient. Once a consolidated protocol is developed for in planta apical meristem transformation it will invigorate the genetic transformation not only for wheat but also for other crops.
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