Results

Explants source

In present communications one year old potted plantlets and in-vitro germinated seedlings were used as explants. For in-vitro germination, seeds were grown in half-strength MS and WPM medium. The effect of basal medium and the various cytokinins on shoot initiation was studied.

Effect of basal medium on shoot initiation

For shoot initiation, two basal media were used MS and WPM. When WPM was used as basal medium, at 8.87 μM BAP concentration the shoot initiation response was 66.66 % which was increase to 80 % when the concentration of BAP was increased to13.31 μM Where as when MS was used as basal medium at 8.87 μM BAP the shoot initiation response was only 46.66 % which was decreased to 33.33 % at 13.31 μM BAP (Table 1).

Table 1: Effects of different concentrations of BAP and KN in MS on shoot regeneration from in vitro grown seedling explants of M. ferrea Data (Mean ± S.D) were recorded after 3 subculture.

There was also difference in mean number of shoots arising from the explants. In WPM medium at 13.31 μM BAP the mean number of shoots was 2.77 and at the same concentration of BAP when MS was used as basal medium, the mean number per explants was 1.2. Similar observation was made when shoots were inoculated in KN. In WPM medium at 9.29 μM KN the main number of shoot initiation was 2.11, where as in MS medium at same concentration it was 1.12. Hence for further experiments WPM was chosen as basal medium.

Effect of various cytokinin concentrations of shoot regeneration

Initially in-vitro grown seedlings were used as explants source. WPM medium containing a range of BAP (4.43μM to 17.75μM) and Kinetin (KN) (4.64μM to 18.58μM) were selected for shoot regeneration (Figure 1a). At lower concentration of KN from 4.64μM to 9.29μM, shoot initiated from apical or axillary meristem only elongated (Figure 1b), But at higher KN concentrations (13.93μM to 18.58μM) the initiated shoot shriveled up and from the base of this shoot 2-3 new shoot initials developed (Figure 1c). At 18.58μM KN concentration the mean number of shoot initials at the end of 3 month was maximum i.e. 3.4 initials. At 13.93μM KN comparatively produced less shoot initials the mean number was 2.8 (Figure 1d).

Figure 1: Micropropagation of M. ferrea. from in-vitro seedling explants. a) In-vitro seed germination. b) Shoot development from apical and lateral meristem on WPM basal medium + 4.46 μM KN. c) Multiple shoots development on WPM medium + 18.58 μM KN d) Shoot development on WPM medium + 8.87 μM BAP. e) Shoot proliferation and greening on WPM medium + 13.31 μM BAP f) Greening of the nodal explant and multiple shoot development on WPM medium + 13.31 μM BAP g) Root initiation on WPM Half-strength + 9800 μM IBA.

BAP comparatively gave better shoot initiation response. At BAP concentration (13.31μM to 17.75μM) the axillary shoot initiated shriveled up and basal tissue proliferated, underwent greening (Figure 1e) and produced number of shoot initials from the explant. At the end of 3rd subculture average number and shoot initials were 4 which increased to 5 to 7 initials at the end of 5th sub-culture (Figure 1f).

Effect of explant source in shoot initiation

When Axillary and apical buds from potted plantlets were used as explants, the shoot regeneration response was comparatively slow. For KN (4.64μM) the number of days required for shoot initial was 30 days. For higher concentration of KN (18.58μM) the number of days reduced to 27 days, contrary to this when in-vitro germinated seedlings were used for KN (18.58μM) the number of days required for shoot regeneration was less (22 days) (Table 2 & 3).

Table 2: Effects of different concentrations of BAP and KN in WPM on shoot regeneration from in vitro grown seedling explants of M. ferrea Data (Mean ± S.D) were recorded after 3 subculture.

Table 3: Effects of different concentrations of BAP and KN in WPM on shoot regeneration from one year potted plantlet explants of M. ferrea Data (Mean ± S.D) were recorded after 3 subculture.

The percentage of shoot regeneration response was also comparatively more for in-vitro plantlets. For potted plantlet at(13.93μM) KN the percentage of shoot regeneration was 66% where as for in-vitro germinated seedling at same concentration of KN (13.93μM) the shoot response was as high as 90%. For (13.31μM) BAP for potted plantlets the shoot initial was 60% and for in-vitro germinated plantlets the shoot regeneration was 80%.

In in-vitro plantlets, the highest number of shoot initiation obtained at 13.31 μM BAP was 2.7. And the same concentration of BAP in potted plantlets the mean number of shoot initials was 1.6. Whereas, potted plantlets at this concentration of BAP the explants never proliferated or shown greening to give rise to multiple shoots.

Elongation of in-vitro shoots

KN 13.93 μM showed higher number of shoot initials. These shoots were separated from the mother plant and then transferred to lower concentrations of KN for elongation (2.3 and 4.6 μM). The best shoot elongation was achieved in 4.6 μM KN. Similarly from BAP 13.31 μM the shoots were transferred to 2.3 μM and 4.3 μM BAP for shoot elongation.

Rooting of regenerated shoots

For the root induction in-vitro elongated shoots were subjected to pulse treatment of different concentration of IBA (1225, 2440, 4900, 9800 μM). Within 27 days, the initiation of roots was observed in medium containing 9800 μM IBA (Figure 1g). However, no root induction was observed on the medium supplemented with lower concentrations of IBA (1225 μM to 4900 μM). The elongated shoots incorporated in half strength WPM medium supplemented with IBA (4.92 μM and 9.84 μM) concentration did not show any root initiation. Effort for hardening of rooted shoots will be undertaken.