Comprehensive Identification and Expression Analysis of CRY Gene Family in Gossypium

Background: The cryptochromes (CRY) comprise a specific blue light receptor for plants and animals, which play crucial roles in physiological processes of plant growth, development, and stress tolerance. Results: In the present work, a systematical analysis of CRY gene family from five allotetraploid cotton species, G. hirsutum, G. barbadense, G. tomentosum, G. mustelinum and G. darwinii together with seven diploid species. There were 18, 17, 17, 17, and 17 CRYs identified in G. hirsutum, G. barbadense, G. tomentosum, G. mustelinum and G. darwinii, respectively, whereas five to nine CRY genes were identified in the diploid species. Phylogenetic analysis of the protein-coding sequences revealed that CRY genes from the allotetraploids G. hirsutum and G. barbadense, three diploid cotton species (G. raimondii, G. herbaceum, and G. arboreum), and Arabidopsis thaliana could be classified into seven clades. Synteny analysis suggested that the homoeolog of G. hirsutum Gh_A02G0384 has undergone an evolutionary loss event in the other four allotetraploid cotton species. Cis-element analysis predicated the possible functions of CRY genes in G. hirsutum. Public RNA-seq data were investigated to analyze the expression patterns of G. hirsutum CRY genes in various tissues as well as gene expressions under abiotic stress treatments. Conclusion: These results indicated the possible functions of G. hirsutum CRY genes in differential tissues as well as in response to abiotic stress during the cotton plants life cycle.

Genetic Transformation for Pod Borer Resistance in Dolichos Bean [Lablab purpureus (L.) Sweet]

Background: Agrobacterium mediated genetic transformation experiments were carried out in Dolichos bean Cv. (Konkan Bhushan) showing better regenerability. Methods: Three cry genes viz. cry1Aabc, cry1Fa1 and cry2Aa were used for the transformation each of which were linked to CaMV35S promoter and nptII gene under control of nos promoter and terminator. A vector system consisting of the disarmed hyper virulent Agrobacterium tumefaciens strain EHA-105 harboring pBinAR or BinBt3 was used. Mature embryo axis with single cotyledon was used as explant. Kanamycin as well as PCR screening was carried out to assess the transformation frequency. Progeny analysis using PCR was also carried out to assess the transgene segregation and stable transformation.Result: Kanamycin concentration of 500 mg/l was found as optimum for selection of a transgenic turning leaf blades albino. Among five methods of colonization used, the method employing mild injury to explant with dipping in Agrobacterium culture for 20 minutes followed by co-cultivation for 48 hours, cefotaxime washing and sowing in soil resulted in maximum survival (74.80%) associated with maximum transformation frequency through PCR analysis (2.13%). Among three cry genes, the gene cry2Aa was found the most effective in transforming Dolichos bean. The progeny analysis of transformants has shown the 3:1 mendelian segregation ratio confirming stable transformation of transgene.

Pyramiding of Cry Toxins and Methanol Producing Genes to Increase Insect Resistance in Cotton

The idea of enhanced methanol production from cell wall by pectin methyl esterase enzymes (PME) combined with expression of cry genes from Bacillus thuringiensis as a strategy to improve pests control in cotton is presented. We constructed a cassette containing two cry genes (cry1Fa and Cry32Aa) and two pme genes, one from Arabidopsis thaliana (AtPME), and other from Aspergillus niger (AnPME) in pCAMBIA1301 plant expression vector using CAMV-35S promoter. This construction was transformed in Eagle-2 cotton variety using shoot apex-cut Agrobacterium-mediated transformation. The expression of cry genes and pme genes was confirmed by qPCR. Methanol production was measured in control and in the cry and pme transformed plants showing methanol production only in transformed plants, then the non-transgenic cotton plants. Finally, insect bioassays performed with transgenic plants expressing cry and pme genes, showed 100 % mortality for Helicoverpa armigera (cotton bollworm) larvae, 70% mortality for pectinophore gossypiella (pink bollworm) larvae and 95% mortality of Earias fabia, (spotted bollworm) larvae, that was higher than the transgenic plants expressing only cry genes that showed 84%, 49% and 79% mortality, respectively. These results demonstrate that Bt. cry-genes coupled with pme genes is an effective strategy to improve the control of different insect pests.

Optimization of genetic transformation method for an indica rice (Oryza sativa L.) variety Ratnagiri-711

A study for method optimization of Agrobacterium-mediated genetic transformation for insect resistance was carried out for a rice variety Ratnagiri-711 showing better regenerability. Three different cry genes viz. cry1Aabc, cry1Fa1 and cry2Aa with a vector system consisting of the disarmed hyper virulent Agrobacterium tumefaciens strain EHA-105 harboring pBinAR or BinBt3 were used. The respective genes were linked to CaMV35S promoter and nptII gene under control of nos promoter and terminator. Scutellum-derived callus bits and embryonic shoot apical meristem of germinating seeds were used as target tissues for callus-mediated and in planta transformation, respectively. Kanamycin screening and PCR analysis was employed for confirmation of presence of transgene. Among five methods of colonization and co-cultivation tried with three cry genes, a callus-mediated transformation method consisting of 20 minutes colonization and 3 days co-cultivation with cry2Aa gene recorded highest transformation frequency (13.79%) but minimum survival (5.27%). On the contrary, considerable transformation frequency (6.35%) with highest survival (79.42%) was observed in an in planta method employing mild injury to embryonic shoot apical meristem of germinating seeds followed by injection of Agrobacterium having cry2Aa gene followed by 15 minutes colonization and then directly sowing in pots. Among three cry genes used, the gene cry2Aa was found most effective showing more transformation frequency.