Indirect shoot organogenesis from leaf explants of Adhatoda vasica Nees

Abstract

A novel protocol for indirect shoot organogenesis of Adhatoda vasica was developed using petiole explants derived from mature shrubby plants. Media with concentrations of cytokinins in combination with auxins were used to induce callus formation in two explants types: petiole and leaf segment. The frequency of callus formation from petiole and leaf segment explants on Murashige and Skoog (MS) basal medium supplemented with 0.25 mg l(-1) thidiazuron (TDZ) and 0.25 mg l(-1) α-naphthaleneacetic acid (NAA) was 100 ± 0.0 and 83.70 ± 0.52% respectively, while on this medium supplemented with 0.25 mg l(-1) 6-(γ-γ, dimethylallyamino purine) (2iP) and 0.25 mg l(-1) NAA, the callus frequency was 100 ± 0.0 and 96.70 ± 0.67% respectively. The highest shoot regeneration (90.60 ± 0.52%) response and the maximum shoots (8.10 ± 0.28) per callus were achieved from petiole explants on MS medium containing 0.25 mg l(-1) TDZ and 0.25 mg l(-1) NAA. On the contrary, on Schenk & Hildebrandt (SH) basal medium supplemented with 0.25 mg l(-1) TDZ and 0.25 mg l(-1) NAA, the frequency of callus formation from petiole and leaf segment explants was 100 ± 0.0 and 90.50 ± 0.89% respectively while the callus frequency on this medium containing 0.25 mg l(-1) 2iP and 0.25 mg l(-1) NAA was 100 ± 0.0 and 89.90 ± 0.72% respectively. The shoot regeneration frequency for petiole explants was 89.90 ± 0.46% producing 6.00 ± 0.21 shoots per callus on SH basal medium supplemented with 0.25 mg l(-1) TDZ and 0.25 mg l(-1) NAA. Whereas petiole explants could induce 83.70 ± 0.50% shoot regeneration and 7.3 ± 1.05 shoots per callus on SH medium containing 0.25 mg l(-1) indole-3-butyric acid (IBA), 0.5 mg l(-1) 6-benzyladenine (BA) and 0.5 mg l(-1) 2iP. Elongation of regenerated shoot was obtained on MS basal medium supplemented with 0.25 mg l(-1) TDZ. All regenerated shoots developed adventitious roots within 4 weeks when transferred to rooting medium containing SH medium supplemented with 0.5 mg l(-1) IBA. Total nine rooted plantlets were transferred from in vitro to in vivo conditions and eight plants survived and successfully acclimatized in the shaded greenhouse 12 weeks after transplanting.

Keywords: Adhatoda vasica; Adventitious shoots; Callus induction; Organogenesis; Shoot regeneration.

Figure 1

Mean % of callus induction of leaf segment and petiole explants of Adhatoda vasica on MS medium containing plant growth regulators after 4 weeks. Different letter(s) indicate a significant difference between treatments at P ≤0.05 according to Tukey test.

Figure 2

Mean % of callus induction of leaf segment and petiole explants of Adhatoda vasica on SH medium containing plant growth regulators after 4 weeks. Different letter(s) indicate a significant difference between treatments at P ≤0.05 according to Tukey test.

Figure 3

a-g Adventitious shoot regeneration of Adhatoda vasica . a Friable callus formation from petiole explants after 3 weeks of culture on MS medium supplemented with 0.25 mg l ⁻¹ 2,4-D and 0.25 mg l ⁻¹ TDZ. b Compact callus formation from petiole explants on MS medium supplemented with 0.25 mg l ⁻¹ NAA and 0.25 mg l ⁻¹ TDZ after 3 weeks of culture on the same medium. c Onset of organogenesis on MS medium supplemented with 0.25 mg l ⁻¹ NAA and 0.25 mg l ⁻¹ TDZ 4 weeks after culture initiation. d Adventitious multiple shoots on the same medium after 8 weeks of culture initiation. e Elongation growth of shoots on MS medium supplemented with 0.25 mg l ⁻¹ TDZ after 2 weeks of culture. f Rooting of in vitro shoot on MS medium supplemented with 0.5 mg l⁻¹ IBA after 4 weeks of culture. g Acclimatized plant after 8 weeks of transfer to garden soil. Bars =1 cm (a and b), 3 cm (c), 1 cm (d and e), 3 cm (f), and 1 cm (g).

Figure 4

a-c Organogenic responses of petiole explants of Adhatoda vasica on MS and SH medium supplemented with plant growth regulators after 4 weeks. a) Mean % regenerating calli. b) Mean number of shoots per callus. c) Mean shoot length (cm). Different letter(s) indicate a significant difference between treatments at P ≤0.05 according to Tukey test.