In vitro propagation studies and partial biochemical characterization for drought stress in Jatropha curcas L.

Abstract

Development of rapid and efficient propagation methods for Jatropha curcas are highly desirable since its seed oil can be used as biofuel and hence of high economic value around the world. In this study, tissue culture techniques were employed to resolve conventional propagation issues. In vitro seed germination experiments in soil and on half or full strength MS medium, specifically during the dormant periods by using some pretreatments were performed. Pretreatments included presoaking of seeds in water overnight, scarification, stratification, removal of seed coats (before/after disinfection) and combination of these treatments. It was observed that the orientation of the seeds on the culture media also had significant effect on its germination rate. Disinfection of naked seeds could not support subsequent germination so the seeds were disinfected before removing the seed coats. It was observed that the removal of seed coats only could break the dormancy of seeds to get 100% in vitro germination on full strength MS medium kept in the dark at 25 ± 2˚C in the months of December to January. Such seedlings were shifted in light conditions (16 h photoperiod) after the root emergence at the same temperature to support chlorophyll development. Seedlings were successfully acclimatized by shifting to the soil containing a mixture of peat, clay and silt (1:1:1 v/v) in greenhouse. Efficient callus-mediated regeneration system was developed using various explants of Jatropha curcas like young/mature/cotyledonary leaf and hypocotyl. Different growth regulators including TDZ, Kin, BAP, NAA, IAA, 2, 4-D were supplemented in MS medium either singly or in combinations of different concentrations for callus induction and its proliferation. Cultures were kept in either darkness or 16/8 h photoperiod. It was observed that 22.17 µM BAP + 5.35 µM NAA supplemented in medium gave 100% embryogenic callus induction with all the explants used except mature leaf, when kept in 16/8 h photoperiod. Cultures kept in complete darkness also give good callus induction frequency (90%) but calluses were white friable and non-embryogenic. Developed calluses were shifted to shoot bud induction medium. Shoot bud induction medium was also MS medium supplemented with different plant growth regulators both auxins and cytokinins (BAP, NAA, GA3, TDZ, Kin, IBA) in combinations of two or three. Calluses developed on medium containing 22.17 µM BAP + 5.35 µM NAA, shifted to same combination of growth regulators have shown maximum number of shoot buds per culture vessel (17). However, frequency of shoot bud induction was low. Addition of GA3 or Kin in the medium having BAP and NAA have enhanced the frequency of shoot bud induction. However, when both GA3 and Kin were used together, they did not show any significant effect on shoot bud induction frequency. TDZ supplemented in the medium having BAP and NAA, have shown negative effect on regeneration potential. Maximum shoot bud induction frequency (37%) was achieved on MS medium with 6.65 µM BAP + 2.45 µM IBA added. Direct shoot regeneration from young leaf explant of Jatropha currcas was also achieved on MS medium supplemented with 6.65 µM BAP + 2.45 µM IBA. Developed and elongated shoots of average 2 cm length were shifted to another medium for root development. Maximum root induction frequency was achieved on MS medium supplemented with 4.9 µM IBA. Rooting was not very successful in recent experiments because of the callus formation at the base of shoots shifted to the rooting medium. Effect of water/osmotic stress (synonymously referred as drought stress in literature) on morphological and biochemical activities of Jatropha curcas plants were elucidated in the present experiments. The experiments were performed both under in vitro (seed germination, early growth of seedlings and callus cultures) and field conditions (pot-grown 5-month old plants). Different sorbitol treatment levels (0, 0.05, 0.1, 0.15, 0.20, 0.25, 0.3, 0.35, 0.40, 0.45 and 0.5 M) were supplemented to MS medium in order to increase the osmotic stress for in vitro studies. Five-month-old greenhouse plants were subjected to different field capacities of water (100, 75, 50, 25 and 0%). Results have shown that increased osmotic stress in the medium resulted in decreased germination along with its delayed onset. However above 0.3 M sorbitol concentration, germination process was stopped. Similarly fresh/dry weights and shoot lengths of germinating seedlings were also influenced significantly with increase in osmotic stress. Among the biochemical parameters of germinating seedlings studied, it was observed that there was trend towards significant increase in SOD and peroxidase activities with an increase in osmotic stress. However, the soluble protein contents were not affected significantly. Callus cultures were not influenced physiologically and biochemically with increased osmotic stress however, higher osmotic stress lead to reduction in fresh weight and water content and slight enhancement in soluble protein and peroxidase activity. Five-month-old plants subjected to different field capacities of water for 30 days have not shown any visual symptoms of stress like necrosis or chlorosis. However, minimum fresh weight per unit area of leaves was observed in lowest field capacity (0%). Similarly minimum SOD activity was observed in plants subjected to 50% field capacity and there was trend towards increase in SOD activity both in lower and higher field capacities. Peroxidase activities remained unaffected. However, slight increase in soluble protein contents was observed in 0% field capacity. Hence it can be concluded that germination and early seedling growth are influenced by drought stress to a great extent as compared to mature plants where no remarkable changes were observed in both physical and biochemical activities except in extreme stress condition. Same was the case with callus cultures derived from mature leaf explants. Hence Jatropha curcas plants may be planted in areas of low water availability if irrigated properly at seed germination stage.