DEVELOPMENT OF STRESS TOLERANT POTATO (SOLANUM TUBEROSUM L.) THROUGH GENETIC TRANSFORMATION

Abstract

Potato (Solanum tuberosum L.) is a herbaceous and tuberous plant belongs to the family Solanaceae. It is one of the most commonly cultivated vegetable crops in Bangladesh as well as all over the world. Potato is energy rich, nutritious and easy to grow on small pots and ready to cook without any expensive processing. Improvement of potato production faces several constraints; various biotic and abiotic stresses limit the production and quality of potato. Abiotic stresses include drought, salinity, extreme temperature, chemical toxicity and oxidative stress that are serious threats to agriculture including potato. The severity of such issue goes big when it comes as obstacle to ensure an optimum agricultural production for a country like Bangladesh. Even if a gene for drought or salt tolerance is found in a wild relative of potato, it is difficult to integrate the gene into the cultivated potato varieties due to the reproductive barriers. Moreover, in Bangladesh true potato breeding is hampered due to absence of required day length for potato flowering The present investigation was carried out for the establishment of in vitro regeneration and Agrobacterium- mediated genetic transformation in potato (Solanum tuberosum L.). Four potato varieties cultivated in Bangladesh, namely, Asterix, Diamant, Granola and Lady Rosseta were used to develop a suitable in vitro regeneration and efficient genetic transformation protocol for potato using marker as well as salinity and drought tolerant genes. Nodal segments, microtuber discs, leaf and intermodal segment explants were used for direct and indirect regeneration of shoots. The best responses obtained when the explants of nodal segments and microtuber discs were cultured on MS medium supplemented with 4.0 mg/l BAP and 1.0 mg/l IAA. In case of indirect shoot regeneration leaf and internodal segment explants were cultured on MS supplemented with 4.0 mg/l BAP, 1.0 mg/l IAA and 0.5 mg/l GA . Among the explants, the best response towards multiple shoot regeneration was obtained from nodal segments on MS medium supplemented with 4.0 mg/l BAP and 1.0 mg/l IAA. Hormone free MS medium was found to be most effective for healthy root induction from the in vitro raised excised shoots. Following root induction, in vitro regenerated plantlets were successfully transplanted and established to soil. 3 Transformation experiments were performed using Agrobacterium strain LBA4404/pBI121 harboring GUS and nptII and EHA105/pCAMBIA 1301-PDH45 harboring PDH45 and hptII genes. Agrobacterium tumefaciens strain LBA4404/pBI121 showed maximum transformation efficiency in nodal segment with bacterial suspension having an optical density of 0.6 at 600 nm in Asterix. Moreover, 30 min incubation period followed by 72 hrs of co-cultivation was found the most effective for transformation as determined by transient GUS histochemical assay. Transformed shoots were selected using MS medium supplemented with 4.0 mg/l BAP, 1.0 mg/l IAA, 0.5 mg/l GA , 300 mg/l carbenicillin and 200 mg/l kanamycin. In case of strain EHA105/pCAMBIA 1301PDH45,

maximum transformation efficiency was observed in nodal segment with bacterial suspension having an optical density 0.5 at 600 nm, 15 min incubation and 48 hrs co-cultivation period. In this case transformed shoots were selected using 20 mg/l hygromycin. Stable integration of GUS, nptII, PDH45 and hptII genes were confirmed through PCR analysis using the genomic DNA isolated from transformed shoots. Transgenic tuber generation (TG ) and control plants were subjected to salt and drought stress. After 15 days of treatment, plant tissues were used to evaluate different physiological parameters i.e. chlorophyll, carotenoid content, proline content, H 2

content and water loss, etc. Transgenic plants challenged to NaCl stress showed better performance towards growth, chlorophyll and carotenoid content than wild type (WT) control. Similarly, less amount of water loss was observed in transgenic lines. Transgenic lines (TG 1 and TG generations of PDH45-potato) performed well under stress condition were maintained in the greenhouse. Molecular characterization and phenotypic analysis of TG 1 2 generation were performed. Amplification of 1200 bp band for PDH45 gene and 750 bp band for hptII gene in transformed lines confirmed presence of PDH45 and hptII gene.