Regeneration of Stevia Plant Through Callus Culture

Stevia rebaudiana Bertoni that conventionally propagated by seed or by cuttings or clump division which has a limitation of quality and quantity seed material. In present study, callus culture technique was tried to achieve rapid plant multiplication for quality seed material. Callus induction and multiplication medium was standardized from nodal as well as leaf sagments. It is possible to maintain callus on Murashige and Skoog medium supplemented with 6-benzyl amino purine and naphthalene acetic acid. Maximum callus induction was obtained on Murashige and Skoog medium incorporated with 6-benzyl amino purine (2.0-3.0 mg/l) and naphthalene acetic acid (2.0 mg/l) treatments. However, Murashige and Skoog medium containing 2.0 mg/l 6-benzyl amino purine+2.0 mg/l naphthalene acetic acid was found to be the best for callus induction. Higher regeneration frequency was noticed with Murashige and Skoog medium supplemented with 2.0 mg/l 6-benzyl amino purine+0.2 mg/l naphthalene acetic acid. Regenerated plants were rooted better on ¼ Murashige and Skoog strength supplemented with 0.1 mg/l indole-3-butyric acid. The rooted plantlets were hardened successfully in tera care medium with 63 per cent survival rate. The developed protocol can be utilized for mass production of true to type planting material on large scale independent of season, i.e. external environmental conditions.

a limitation on account of the low number of new plants, simultaneusly, from a donor plant. Stevia can be grown throughout India in subtroprical climatic region. Therefore, considering its scope and future need of planting material, this experiment was conducted to standardise protocol for rapid multiplication of Stevia through callus culture method.
Nodal segments and leaves were collected as source of explants from Stevia plant. Nodal segments of about 1.5 cm length and leaves were isolated from collected explants. The explants were washed in running tap water for about 30 min and then, treated with 10 per cent solution of detergent for 5 min. The traces of detergent were removed by wasing thoroughly with double glass distilled water. The explants were then, surface sterilized by using 0.1% mercuric chloride solution for 3 minutes under aseptic conditions in a laminar air-flow cabinet followed by rinsing four times with sterilized double glass distilled water. The size of sterilized nodes were further reduced to 0.8 cm by trimming both the ends. While in case of leaf explants, sterilized leaves were cut into 0.5 cm² size pieces for inoculation on the medium.
Murashige and Skoog (MS) medium [6] supplemented with 6-benzyl amino purine (BAP-1, 2 and 3 mg/l) in combination with naphthalene acetic acid (NAA-0.2 and 2.0 mg/l). The cultures were inoculated at 26±2° temperature in air-conditioned culture room and provided with 1000 lux light intensity from fluorescence cool tube light. The callus obtained on different treatments were recultured to S 3 (MS supplemented with 2.0 mg/l BAP and 0.2 mg/l NAA) for regenerarion of shoots. The shoots obtained on regeneration medium were excised and transferred to rooting medium for in vitro rooting. MS liquid medium with quarter and half strength were tried. Each medium was supplemented with different levels of indole-3-butyric acid (IBA levels: 0.05, 0.1, 0.2, 0.5, and 1.0 mg/l). Observations on establishment and proliferation were recorded after three and four weeks of incubation of cultures, respectively, while in case of rooting, it was recorded after three weeks.
The rooted plantlets were transferrred in polyethylene bags filled with four types of potting mixtures i.e. tera care; soil:tera care; soil:leaf mould and soil:leaf mould:tera care each in equal proportions. Survival % of plantlets were recorded after three weeks.
The data on culture establishment of both types of explants i.e. leaf piece and nodal segment on each treatment are presented in Table 1. Among both the explants, establishment of nodal segments was found significantly superior than leaf pieces. The establishment of explants was statistically infuenced by treatments. The maximum establishment was recorded on MS medium supplemented with 1.0 mg/l BAP+0.2 mg/l NAA treatment. Nodal segments established 100% on MS medium supplemented with 1.0-2.0 mg/l BAP+0.2-2.0 mg/l NAA. While, piece of leaf explants, showed better establishment (58.58%) on S 2 (MS supplemented with 1.0 mg/l BAP+2.0 mg/l NAA) medium than other treatments. It is apparently seen from the data ( Table 1) that in both types of explants, callus was induced on all the treatments. However, S 2 (MS supplemented with 1.0 mg/l BAP+2.0 mg/l NAA) induced better callus among all the treatments. The colour of callus ranged from green to white green to brown yellow in different treatments. At the end of 1st subculture, the intensity of callus was increased and maximum callus was noticed on S 2 treatment in both the explants (Table 2). However, higher intensity was observed in nodal segments than leaf pieces. Normally, nodal segments produced globular callus.
The treatment S 3 (MS supplemented with 2.0 mg/l BAP+0.2 mg/l NAA) registered maximum regeneration of shoots. (Table 1). Therefore, calli developed on other media combinations were also transferred to S 3 treatment for regeneration of shoots from callus. The shoots were regenerated through organogenesis from callus. The regeneration frequency was significantly lower in case of S 1 and S 6 calli. Maximum number of shoots were regenerated, when callus produced on S 2 treatment was transferred to S 3 treatment, whereas, length of shoot was noticed significantly higher on S 1 followed by S 2 and S 3 ( Table 3).
The data on rooting response to different levels of IBA supplemented in half strength MS and one fourth strength MS medium are presented in   The survival rate of plantlets was infuenced by potting mixtures. Maximum number of survival of plantlets (63 per cent) was found in tera care potting medium ( Table 5). Addition of leaf mould or soil in tera care reduced the survival rate of plantlets.
For the culture of a number of callus tissues, auxin is the essential supplement which is needed to be added to the basal medium supplying inorganic ions and sugars [7] . Navarro et al. [8] also noted that auxin was essential supplement for induction of callus. The addition of cytokinin in the auxin medium has an additive effect on tissue growth has been demonstrated by other workers in a number of plant species [6] . Incorporation of BAP was also found to have beneficial effect on growth of callus in the present investigation.
The role of cytokinin in shoot organogenesis is well established [9][10][11] . In the present investigation, MS medium supplemented with high ratio of cytokinin (BAP) and auxin (NAA) responsed favourably for regeneration of shoots. However, in other studies of micropropagation [4] , maximum number of shoots were reported on MS medium supplemented with 0.6 mg/l BA. The difference may be due to source of explant and hormonal levels used in callus culture.
Addition of Auxin in the MS medium at adequate levels enhanced root formation. In our studies better response of rooting were observed on ¼ strength MS medium supplemented with 0.1 mg/l IBA. The present investigation is in agreement with findings of Ferreira and Handro [12] who also reported optimum concenrtation for root formation at 0.1 mg/l IBA. On the other hand, better rooting response was reported by Tadhani et al. [4] on MS medium supplementd with 1 mg/l IBA in case of Stevia. The variation  may be due to the source of explant, the change in microclimate and may vary with the strength of rooting MS medium, which was observed in the present study.
In vitro plants need to be hardened before being transplanted in the field. In the present study, potting mixture tera care performed well with better survival of the plantlets. The potting mixture used may help in giving better grip for the rooting and ample aeration. The survival of in vitro plants also depends upon plant species and the potting mixture used for raising in vitro plants under greenhouse conditions. The other reports on potting mixtures such as peat:perlite [13] , sand:soil:vermiculite [11] , etc. were reported for establishment of in vitro plants under greenhouse conditions. The technique developed may be utilized efficiently for producing true type and disease free planting materials on large scale during any time of the year.