In vitro evaluation and selection of some tomato lines/hybrids for drought- stress tolerance and in vivo evaluation of their physiological and morphological differences

This research was carried out at the General Commission for Scientific Agricultural Research (GCSAR), and the National Commission for Biotechnology (NCBT), Syria during 2017 to 2020 in order to exploit somaclonal variations regenerated from callus culture in improving tolerance of tomato plants to induced drought stress.To reach this aim, the research was conducted by many stages, started by screening eight local tomato lines of 5th generation (Daher-Aljabal, Brieh, Baskanta, Kafer-Selwan, Daraa, Wardyat, Jerdy and Bosfair) for their tolerance to drought stress induced by PEG 6000 at five concentrations (2, 4, 6, 8% and control). The followed parameters were measured: root length and diameter, number of roots, plant height and stem diameter, root-to-stem length ratio, plant dry weight, number of leaves, leaf area, total chlorophyll concentration and proline concentration. Cluster analysis and principal component analysis PCA were then performed in order to separate the studied lines based on their tolerance to drought stress and to identify the most effective parameters in showing the variations among these lines.The second stage was performed in order to develop an efficient protocol for callus induction and plant regeneration of four local tomatoes lines. Cotyledons, hypocotyls and root crown explants were extracted from well-developed seedlings and used as explants to evaluate their capacity for callus induction. Different concentrations and combinations of plant growth regulators (PGRs) were added to MS medium to evaluate their efficiency on callus induction and shoot initiation. The following parameters were measured: Callus induction frequency, callus weight, volume, texture and color, regeneration percentage, days for regeneration, number and length of regenerated shoots.Callus was subjected to five different levels of drought stress induced by PEG 6000 (2%, 4%, 6%, and 8% in addition to the non-treated control). After 8 weeks, tolerant callus lines were selected based on relative growth rate (RGR), cell viability, tolerance index (TI) and proline content. Callus was sub-cultured on medium supplemented with the sub-lethal concentration of PEG determined for each tomato line for two months, then it was transferred to regeneration medium. The regenerated somaclonal variant plants were evaluated for drought tolerance induced by PEG6000 at four concentrations (4, 6, 8% in addition to control) compared to mother plant. Field evaluation was performed firstly to study the morphological and physiological traits of the somaclonal variant plants and their parents. Secondly to evaluate their tolerance to water deficit applied by deficit irrigation treatment (75% of field capacity) compared to full irrigation (100% of field capacity). Finally, the genetic variations among somaclonal variant plants and mother plants were studied by ISSR technique. The results showed a reduction in all growth parameters, except proline, by increasing PEG concentration in medium in all evaluated tomato lines. Darra lineexceeded significantly other lines concerning the least reduction percentage in root length, dry weight, leaf area and number of leaves (26.56, 62.01, 52.41 and 85.30%, respectively). On the other hand, Jerdy line was superior concerning the highest root-to-shoot length ratio (1.60) and the least reduction percentage in total chlorophyll concentrations (62.69%) as compared to control treatment. Brieh line was superior concerning the least reduction percentage in plant height (21.67%), while the highest reduction percentage in plant height (69.12%) was recorded in Wardyat line. Moreover, Bosfair line ranked the worst concerning the highest reduction percentage in root length, plant dry weight, leaf area and number of leaves (85.83, 55.65, 72.89 and 87.61%, respectively) as compared to control, while Kafer-Selwan in chlorophyll a, b an total chlorophyll concentration (90.82, 94.13 and 92.11%, respectively). Concerning proline concentration, the highest percentage of accumulated proline was recorded in Daher-Aljabal (453.79%), while the least values were accumulated in Daraa and Brieh lines (277.81 and 274.29%, resepectevely) as compared to control. No significant differences were recorded among other lines as affected by PEG concerning mean number of roots and stem diameter. Daher-Aljabal, Brieh, Baskanta and Daraa were selected for next stages. The best response for callus induction was observed from root crown cultured on MS medium supplemented with 2 mg/L BAP+0.2 mg/L NAA. Root crown explants gave the highest callogenesis compared to cotyledons and hypocotyls in all studied lines where the mean callus induction frequency were 88.47, 64.47 and 45.73%, respectively. Hard friable, nodular and green callus was obtained from root crown explants with a mean callus fresh weight 1.866g and size 355.85mm3. No significant differences were recorded between tomato lines in respect to callogenesis, even though Baskanta recorded the highest callus induction frequency compared to other lines. Furthermore, the highest percentage of shoot regeneration (76.26%) and the highest number of shoots (4.45 shoots/callus clumps) were recorded on 2mg/L BAP+0.5 mg/L IAA. Daher-Aljabal gave the best response for regeneration in term of percentage (50.32%) and shoot number per calli clump (3.396). While the tallest shoot was 0.373 cm in Brieh with no significant differences between lines. An efficient callus formation and plant regeneration protocol has been developed. Results of selection stage showed that RGR, TI and cell viability decreased while free proline content increased in response to increasing PEG concentration. Based on the studied parameters, Daraa was the most tolerant line at cellular level, followed by Daher-Aljabal, Brieh and Baskanta. A single regenerated plantlet was obtained from Daher-Aljabal line named DH1 and another plantlet from Brieh line named BR1, while two regenerated plantlets were obtained from Daraa line named DA1 and DA2. All of them showed better tolerance to drought stress than mother plants at 8% PEG concentration in terms of root length, plant height,root-shoot ratio and dry weight. In conclusion, a successful callus formation and plant regeneration protocol has been developed under drought stress treatments induced by PEG 6000. Field evaluation showed significant differences between somaclonal variants and their donor plants in both irrigation treatments, although both were affected negatively by water deficit. Somaclonal variants exceeded significantly the mother plants in deficit irrigation treatment by less reduction in the studied parameters as compared to control (full irrigation) except for chlorophyll concentration and relative water content in DA1, where the mother plant was the best concerning these parameters. On the other hand, the mother plants exceeded all somaclonal variants based on the studied parameters in full irrigation treatment. Genetic similarity coefficient between Daraa line (cluster four) and DA1 and DA2 (cluster two) were 0.595 and 0.579, respectively, and 0.541 between Daher-Aljabal (four cluster) and DH1 (first cluster). While it was 0.703 between Brieh (third cluster) and BR1 (second cluster).