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3. Factors affecting axillary shoot proliferation
i. Effects of season on culture establishment
The extent of contamination as well as bud-break is highly dependent on the season. The cultures initiated during spring season (January to April) shows best response not only in terms of the frequency of bud-break but also in the vigor of the shoots with least contamination rate. Since, summer (May-August) is the period that concurs with rainy season in certain regions like India, the cultures are prone to infection. By winter the shoots become old and it is difficult to break the dormant state of the buds.
ii. Effect of carbon source on shoot proliferation
In cultured plant tissues, a continuous supply of carbohydrate from the medium is essential which are needed for growth and organized development of the plant and are necessary as a source of energy and carbon skeletons for biosynthetic process. For shoot induction from axillary buds, three carbon sources, sucrose, glucose and maltose are utilized in maximum plant tissue cultures at a fixed concentration of 30 g l-1 . Of these, sucrose is the most commonly used carbohydrate for plant tissue cultures and most culture media have it as the sole carbohydrate source. It favors higher growth of shoot, number of nodes per shoot and the rate of shoot multiplication compare to maltose and glucose. Sucrose is easily recognized and hydrolyzed by cell wall bound invertase into more efficiently utilizable forms of sugars, glucose and fructose which are incorporated into the cells. Glucose, derived from sucrose hydrolysis, is more accessible to the cultured tissues than glucose derived by maltose hydrolysis, due to a rapid sucrose hydrolysis but a slow maltose hydrolysis in the media.
iii. Effect of growth regulators on shoot proliferation
In general, cytokinins favors shoot proliferation and auxins favors root formation. In S. acmella, nodal explants bearing two opposite axillary buds were when cultured on MS basal medium or basal medium supplemented with BAP, Kinetin or 2-iP at 3 µM concentration, the frequency of bud-break was appreciable in basal medium but incorporation of BAP to the basal medium has further improved the incidence of bud-break and promoted multiple shoot formation (2 shoots/explant). While the least bud-break was observed on Kinetin supplemented medium and 2-iP was noticed to be inhibitory for axillary bud proliferation. The addition of a low concentration of GA3 to the BAP supplemented medium further promoted multiple shoot formation. On the other hand, single shoot with long internodes was developed from axillary buds in cultures when NAA was added to BAP containing medium. The frequency of bud-break varied with the concentration of the BAP and at its optimum level of 5 µM, 10-fold shoot multiplication occurred every 5 weeks.