Sukumar Gupta

Effects of salt and osmotic shocks on unadapted and adapted callus lines of tobacco

Growth, viability and proline content of adapted and unadapted calluses of Nicotiana tabacum L. var. Jayasri, affected due to osmotic stresses and particularly to stress-shocks treated with different osmotica like NaCl (ionic-penetrating), mannitol (non-ionic-penetrating) and polyethylene glycol, (PEG) (non-ionic-non penetrating) were studied to evaluate the physiological differences of stress effects. The tissues adapted to a low concentration of NaCl (85 mM) showed low growth with high proline content compared to the tissues adapted to a low concentration of mannitol (165 mM). Proline content was similar in tissues adapted to high concentrations of NaCl (171 mM) and mannitol (329 mM) but growth in the latter case was relatively low. Growth and viability were subsequently correlated with the pattern of retention in or diffusion of proline out of the tissues after shock-treatments. The loss of tissue viability of the adapted calluses was comparatively less than the unadapted callus even after shock-treatments with 1282 mM NaCl and 823 mM mannitol. The former calluses retained the capability of regrowth though at a slow rate. Such adapted tissues also retained more proline. The mannitol-adapted tissues, when shocked with PEG (200 g l-1), showed low viability with more diffusion and a very little retention of proline while, in the unadapted tissue, all the proline was leached out. The results indicated that the effects of different osmotica on plant tissue varied depending upon the physico-chemical nature of the compounds used as stress-inducing-agents, and retention and diffusion of proline was altered when the tissues were shocked with high concentrations of all these compounds.

Micropropagation of Tectona grandis: Assessment of Genetic Fidelity

Random amplified polymorphic DNA (RAPD) markers were used to analyze genetic fidelity of micropropagated teak (Tectona grandis L.) clones with respect to subcultural passage. Of the twenty primers screened, no variation in RAPD profiles was noticed in the in vitro clones of fifth, tenth, fifteenth and twentieth passage in comparison to the in vivo mother plants. Only one micropropagated plant of twenty-fifth subcultural passage, however, differed from the in vivo ones. It revealed the appearance of a new polymorphic DNA fragment (molecular mass 379 kb) in case of primer OPB-08. This primer, manifesting detectable variation, may be utilized as a diagnostic marker for assessing genetic fidelity of micropropagted teak plants.

Plant regeneration of salt adapted callus of indica rice (var. Basmati 370) in saline conditions

NaCl adapted callus of a salt sensitive scented indica variety of rice (Oryza sativa var. Basmati 370) showed 55% regeneration in culture medium supplemented with IAA and kinetin. Regeneration was low in 85 mM NaCl but a concentration of 128 mM was inhibitory to regeneration. SEM study revealed organogenesis and somatic embryogenesis from the same callus. The rate of survival and endogenous free proline content of the plants regenerated from the NaCl adapted callus was significantly higher than for those obtained from unadapted callus in liquid maintenance media supplemented with NaCl.

In vitro selection and physiological characterization of NaCl- and mannitol-adapted callus lines in Brassica juncea

Na Cl (salt)- and mannitol (drought)- tolerant callue lines of Brassica juncea (L) Czern. Var. RW-85-59 were isolated by a plated cell suspension culture technique against 43 mM NaCl and 165 mM mannitol, respectively. Callus lines, adapted to a high concentration of Na Cl (171 mM) and mannitol (329 mM) were then established bv direct adaptation procedures. In the initial passages, the calluses showed severe reduction in tissue growth when grown on NaCl/mannitol-containing media but growth of adapted calluses recovered and was sustainable in the subsequent passages. Adapted calluses showed considerable accumulation of free proline in NaCl-/mannitol- containing media compared to the control callus grown on stress-free medium. A significant increase of intensity of one particular acid phosphatase isozymic band in the adapted calluses, irrespective of NaCl or mannitol stress, indicated that it may be used as an osmotic stress-marker in this system. Short-term salt/osmotic-shock-treatment with high concentrations of osmotica revealed that only the adapted lines retained the maximum amount of free proline within the cells for osmoregulation. This response probably helped the cells to restore their normal growth when the stress was withdrawn.

Callus culture and plantlet production in Carica papaya (Var. Honey Dew)

SummaryIn order to develop techniques for efficient callus production and regeneration in Carica papaya (Var. Honey Dew), lamina, petiole, stem and root explants from in vitro plantlets were cultured in media supplemented with 2.0 mg/1 IBA and 0.5 mg/1 BAP. Use of in vitro-grown plantlets as an explant source helped to avoid contamination common in papaya tissue culture. Callusing was maximum in root explants cultured in a modified MS (half-strength) medium. Shoot reganeration was maxium in root-derived callus grown in full-strength modified MS medium supplemented with 0.5 mg/1 IBA and 1 to 2 mg/1 kinetin. A histological study indicated that shoot buds originated from peripheral cell layers of the callus. Each shoot regenerated from callus was subcultured using a multiplication medium. Root formation was induced in all shoots treated in half-strength of modified MS medium containing 2 mg/1 IBA and rooted shoots were transferred successfully to the field.

In vitro propagation of shoot buds of Carica papaya L. (caricaceae) var. Honey Dew.

Shoot buds from the saplings and the fruit bearing plants of Carica papaya L.. var. Honey Dew (papaya) initially treated with Gentamycin were cultured in modified MS media, each with a different hormonal combination, for the establishment of cultures and multiplication and rooting of plants. About 43% of explants from fruit bearing plants and 69% of those from saplings remained free of contamination and retained regeneration capacity when treated in 500 mg/l Gentamycin. For the establishment of the explants a medium containing 1 mg/l GA3 and 2 mg/l kinetin was necessary. When established buds were transferred to medium containing 1 mg/l NAA and 3 mg/l kinetin, calli were initiated at cut ends of shoot buds; multiplication started on transfer to NAA (0.1 mg/l) and BAP (0.5 mg/l) medium. Cultures have been maintained for the last twenty months without any loss in multiplication rate. Rooting was induced in medium with reduced salt concentration containing 2 mg/l IBA. Shoot elongation was induced after prolonged culture in the same rooting medium.