RESULTS
Shoot multiplication
Nodal explants of S. acmella bearing two opposite axillary buds were cultured on MS basal medium or basal medium supplemented with BAP, Kinetin or 2-iP. MS + BAP medium proved optimum for shoot multiplication and results into 10-fold shoot multiplication at every 5 weeks (Figure 6.2 A). At the end of the passage each shoot was cut into single node segments and planted on the fresh medium of the same composition (Figure 6.2B). Each node again produced multinodal, multiple shoots after 5 weeks. Shoot multiplication rate in various subculture (S) passages, S1 =10.2, S2 =10.3, S3 =10.4, S4 =10.6, S5 =10.6, S6 =10.5, S7 =10.5, S8 =10.5, S9 =10.6, S10 =10.6, was >10 fold from S1 to S10 on MS + BAP (5 µM). Since every time the explants were taken from freshly formed in vitro shoots, therefore, no significant difference (variation) was observed in the results.
Figure 6.2: Nodal segment culture: invitro clonal propagation of Spilanthes acmella by axillary shoots proliferation
Rooting and Transplantation
Terminal 3-4 cm long portions of shoots after the growth period of 5 weeks are used for rooting. The remaining portions of the shoots can be cut into single node segments and utilized for further multiplication. MS basal medium was tested at full and half (½ MS) strengths of the major inorganic salts and the media are supplemented with 10, 30 and 50 gl-1 sucrose. Half MS was distinctly better than full MS basal medium, in terms of length of the shoot, percent rooting, number of roots per shoot and number of laterals present on the roots. The rooting was positively correlated with the sucrose concentration in the medium. On ½ MS + 50 gl-1 sucrose, shoots formed more than 35 roots directly from the basal cut end of the shoots (Figure 6.3A). On this medium roots appeared after 2 weeks and maximum response was observed after 4 weeks. Some of the roots had developed laterals.
Rooted shoots were transferred out of culture. The plants were acclimatized by covering the pots with polythene bags to maintain high humidity for 6-7 days and irrigated with major salt solution of MS medium. After 7 days, 3-4 small holes were made in the bag and plantlets were irrigated as before but, at frequent intervals. After 25 days, polythene bags were removed and the acclimatized plants were transferred to a shaded area under natural conditions at a temperature range of 20-25°C with photoperiod of 12/12 h (light/dark). The plantlets were acclimatized successfully (Figure 6.3B). During in vitro hardening, shoots elongated, leaves turned greener, and their lamina expanded. Consequently, the plants seemed much healthier and grew more vigorously after in vitro hardening.
Figure 6.3: Nodal segment culture: Rooting and acclimatization