BIOCHEMICAL CHARACTERIZATION OF IN VITRO SALT TOLERANT CELL LINES AND REGENERATED PLANTS OF POTATO (SOLANUM TUBEROSUM L.)

Abstract

The present investigation deals with the establishment of an efficient in vitro selection strategy to produce salt-tolerant cell lines and subsequent regeneration protocols in potato (cvs. Cardinal and Desiree). The activities of antioxidant enzymes (peroxidase, catalase and superoxide dismutase) and total soluble protein contents of various tissues under stress were evaluated to understand their possible role in salinity tolerance. Exogenous application of ascorbic acid and salicylic acid were also tested for salt stress alleviation. In order to proceed with these objectives, the initial focus was to establish protocols for micropropagation, callus induction and maintenance, plant regeneration, establishment of cell suspension cultures and ex vitro acclimatization of regenerated plants. Three different concentrations of TDZ (10-8, 10-9, or 10-10 M) in MS medium were tested for the purpose of in vitro clonal propagation. MS basal medium fairly supported micropropagation of both the tested potato cultivars followed closely by MS medium supplemented with TDZ (10-10 M). For callus induction and proliferation in dark, internodal segments proved to be a good explant source whereas MS medium fortified with 2, 4-D (18.09 μM) was the best medium composition equally effective for both the potato cultivars. A combination of NAA (2.64 μM) and TDZ (1.00 μM) supplemented to MS medium was the best choice for shoot initiation from callus cultures after 20 and 21 days in Cardinal and Desiree, respectively. Rooting of regenerated shoots was achieved on MS medium supplemented with 8.87 μM BAP, 2.64 μM NAA and 0.123 μM IBA. Cell suspension cultures using friable calluses were developed successfully using MS2 medium for the two cultivars. The best supporting medium for ex-vitro transplantation of potato plants was vermiculite. It was observed in this study that different in vitro growth parameters, i.e., shoot/root length and numbers of roots decreased while number of shoots increased with an increase in NaCl (20-140 mM) concentration in the medium. In Desiree, rosette-type of shoot development initiated at 100 mM whereas in Cardinal it was evident at 120 mM NaCl level. During this investigation, a direct recurrent selection procedure was employed to select salt- tolerant cell lines in potato (Cvs. Cardinal and Desiree) on the basis of sub-lethal concentration of salt. Results have shown more than 50% reduction in relative fresh weight in both the cultivars above 100 mM NaCl. Callus morphology correspondingly changed from off-white to blackish-brown above 100 mM to acutely-necrotic at 140 mM NaCl. Regeneration potential of recurrently-selected callus cultures (100 mM NaCl-treated) on salt- free medium was more pronounced in Desiree as compared to Cardinal. When well- acclimatized recurrently-selected plants were treated with 100 mM NaCl and compared with control plants to check their acquired salinity tolerance, it was observed that recurrently- selected plants showed higher fresh/dry weight and number of tubers in both the cultivars. A slight decrease in protein contents of in vitro Cardinal cultures was observed as the concentration of NaCl (20-140 mM) gradually increased in the media. However, there was an increase in protein contents in Desiree plants when subjected to increasing salt concentrations. In case of in vitro recurrently-selected plants, protein contents were higher as compared to control (non-selected ones) in both the cultivars. The peroxidase activity exhibited a slightly decreasing trend in Cardinal though an increasing one was observed in Desiree with an increasing NaCl level in the medium. In the present investigation, recurrently-selected plants had higher POD, CAT and SOD activities as compared to the control ones in both the cultivars.