DEVELOPMENT OF ABIOTIC STRESS TOLERANT EGGPLANT (SOLANUM MELONGENA L.) BREEDING LINES THROUGH AGROBACTERIUM- MEDIATED GENETIC TRANSFORMATION

Abstract

This study was aimed to develop abiotic stress tolerant eggplant (Solanum melongena L.) lines through Agrobacterium-mediated genetic transformation using a specific abiotic stress tolerant gene. For this purpose a transformation compatible in vitro plant regeneration system as well as a protocol for Agrobacterium-mediated genetic transformation using two locally grown eggplant varieties, namely, BARI Begun-4 (Kazla) and BARI Begun-5 (Nayantara) were established. The best response towards multiple shoot regeneration was obtained from in vitro raised cotyledonary leaf segments on MS medium supplemented with 2.0 mg/l BAP and 0.5 mg/l Kn via organogenesis. Elongation of developing shoots was achieved on MS medium without the addition plant growth regulators. MS with or without IBA were found to be less effective for in vitro root induction from the regenerated as well as transformed shoots. Although 85 to 90% of the regenerated shoots produced roots in MS with 2.0 mg/l IBA but in most of the cases vigorous callus was found to produce at the base of the shoots. Moreover, such shoots became light green in colour and became fragile in nature while maintained in the culture. Under these circumstances, in vitro regenerated as well as the transformed shoots were effectively rooted under ex vitro condition following their treatment with 10 mM IBA for 5 min on autoclaved soil. In ex vitro condition plantlets were hardened and acclimatized within 15 days along with 93 to 97% root formation. Following sufficient development of roots, the ex vitro rooted plantlets were transferred to green house having 100% survival rate. A large number of viable seeds were obtained from the fruits developed on the in vitro raised plants. Transformation protocol was established using Agrobacterium strain LBA4404/pBI121 containing GUS and nptII genes, while abiotic stress tolerance was introduced using Agrobacterium strain EHA105/pCAMBIA 1301-PDH45 harbouring PDH45 and hptII genes. In case of Agrobacterium tumefaciens strain LBA4404/pBI121 maximum transformation efficiency was recorded in cotyledonary leaf explants with a bacterial suspension having an optical density of 0.6 at 600 nm in variety Kazla. Moreover, 30 min incubation period followed by 72 hrs of cocultivation

was found to be the most effective for transformation as determined through transient GUS histochemical assay. Transformed shoots were effectively selected using MS with 2.0 mg/l BAP, 0.5 mg/l Kn, 300 mg/l carbenicillin and 100 mg/l kanamycin. In case of strain EHA105/pCAMBIA 1301-PDH45, maximum transformation efficiency was observed in cotyledonary leaf explants with bacterial suspension having an optical density 0.5 at 600 nm, 20 min incubation and 48 hrs co-cultivation period of both varieties of eggplant (Kazla and Nayantara). In this case transformed shoots were selected using 20.0 mg/l hygromycin. Stable integration of GUS, nptII, PDH45 and hptII genes were confirmed by PCR analysis using the genomic DNA isolated from the leaves of transformed shoots. Transgenic plants of T generation as well as their wild types were subjected to salt and drought stress. After salt and drought stress treatment, plant tissues were used to evaluate various physiological parameters including the contents of chlorophyll, carotenoid, proline, H 2 2 O , EC etc. as well as the water loss. Transgenic plants challenged to NaCl and drought stress showed better performance towards their growth, chlorophyll and carotenoid contents than that of the wild type (WT) plants. Similarly, less amount of water loss and less percentage of electrolyte leakage were observed in transgenic lines. Transgenic lines (T 2 1 and T generations of PDH45- eggplant) performed well under stress condition were maintained in the green house. Molecular characterization and phenotypic analysis of T1 and T generations were performed. Amplification of 1200 bp band for PDH45 gene and 750 bp band for hptII gene in transformed lines confirmed the presence of PDH45 and hptII genes.