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Indirect shoot organogenesis from leaf explants of Adhatoda vasica Nees.

Mandal J, Laxminarayana U - Springerplus (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Education in Science and Mathematics, Regional Institute of Education, National Council of Educational Research and Training, Shyamla Hills, Bhopal, 462013 India.

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.

No MeSH data available.


Related in: MedlinePlus

a-c Organogenic responses of petiole explants ofAdhatoda vasicaon 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.
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Fig4: a-c Organogenic responses of petiole explants ofAdhatoda vasicaon 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.

Mentions: After a 2-subculture, each of 14 days on respective callus inducing medium which contained NAA, IBA or 2,4-D (0.25 or 1.0 mg l−1) in combination with TDZ, BA or 2iP (0.25 or 1.0 mg l−1), the callus derived from petiole and leaf segment explants were cut into pieces (2–3 mm diameter pieces) and transferred onto either MS or SH medium supplemented with 0.25 or 1.0 mg l−1 NAA or IBA in combination with 0.25 or 1.0 mg l−1 TDZ or 2iP or BA for induction of regenerating callus. During the induction stage, callus derived from petiole explants was observed efficiently conversion to regenerating callus either on MS or SH medium containing plant growth regulators while regenerating callus could not be induced from callus derived from leaf segment explants even after a 6-month subcultures. The highest frequency of regenerating callus (90.60 ± 0.52%) response and the maxium number of shoots per callus explants (8.10 ± 0.28) (significantly different P <0.05) with maxium shoot length (4.32 ± 0.05 cm) were achieved on MS medium supplemented with 0.25 mg l−1 NAA and 0.25 mg l−1 TDZ (Figures 3c, d and 4a-c). In comparison, induction of regenerating callus and its conversion to shoots were observed on SH medium supplemented with either NAA in combination with TDZ or NAA and TDZ in combination with 2iP or IBA and BA in combination with 2iP (Figure 4a and b). In contrast, the highest percentage of regenerating callus (89.90 ± 0.46%) from petiole explants induced the maximum number of shoots per callus (6.00 ± 0.21) with an average shoot length of 3.65 ± 0.08 cm on SH medium containing 0.25 mg l−1 NAA and 0.25 mg l−1 TDZ. Besides, the addition of 0.25 mg l−1 2iP to SH culture medium containing 0.25 mg l−1 NAA and 0.25 mg l−1 TDZ stimulated regenerating callus rate (85.20 ± 0.51%) and induced the maximum number of shoots per callus explants (4.10 ± 0.18) with an average shoot length of 3.09 ± 0.05 cm within 4 weeks of culture. The addition of higher concentration of 2iP (0.5 mg l−1) in plant regenerating SH medium containing 0.5 mg l−1 BA and 0.25 mg l−1 IBA induced conversion of regenerating callus (83.70 ± 0.50%) into the maximum number of shoots per callus explant (7.3 ± 1.05) with an average shoot length of 3.27 ± 0.04 cm (Figure 4a-c). The adventitious shoots produced via indirect organogenesis were isolated from callus explants and incubated on MS medium supplemented with 0.25 mg l−1 TDZ for elongation growth (Figure 3e). The shoots with well developed leaves were subcultured on this elongation growth medium in every three weeks.Figure 4


Indirect shoot organogenesis from leaf explants of Adhatoda vasica Nees.

Mandal J, Laxminarayana U - Springerplus (2014)

a-c Organogenic responses of petiole explants ofAdhatoda vasicaon 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230685&req=5

Fig4: a-c Organogenic responses of petiole explants ofAdhatoda vasicaon 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.
Mentions: After a 2-subculture, each of 14 days on respective callus inducing medium which contained NAA, IBA or 2,4-D (0.25 or 1.0 mg l−1) in combination with TDZ, BA or 2iP (0.25 or 1.0 mg l−1), the callus derived from petiole and leaf segment explants were cut into pieces (2–3 mm diameter pieces) and transferred onto either MS or SH medium supplemented with 0.25 or 1.0 mg l−1 NAA or IBA in combination with 0.25 or 1.0 mg l−1 TDZ or 2iP or BA for induction of regenerating callus. During the induction stage, callus derived from petiole explants was observed efficiently conversion to regenerating callus either on MS or SH medium containing plant growth regulators while regenerating callus could not be induced from callus derived from leaf segment explants even after a 6-month subcultures. The highest frequency of regenerating callus (90.60 ± 0.52%) response and the maxium number of shoots per callus explants (8.10 ± 0.28) (significantly different P <0.05) with maxium shoot length (4.32 ± 0.05 cm) were achieved on MS medium supplemented with 0.25 mg l−1 NAA and 0.25 mg l−1 TDZ (Figures 3c, d and 4a-c). In comparison, induction of regenerating callus and its conversion to shoots were observed on SH medium supplemented with either NAA in combination with TDZ or NAA and TDZ in combination with 2iP or IBA and BA in combination with 2iP (Figure 4a and b). In contrast, the highest percentage of regenerating callus (89.90 ± 0.46%) from petiole explants induced the maximum number of shoots per callus (6.00 ± 0.21) with an average shoot length of 3.65 ± 0.08 cm on SH medium containing 0.25 mg l−1 NAA and 0.25 mg l−1 TDZ. Besides, the addition of 0.25 mg l−1 2iP to SH culture medium containing 0.25 mg l−1 NAA and 0.25 mg l−1 TDZ stimulated regenerating callus rate (85.20 ± 0.51%) and induced the maximum number of shoots per callus explants (4.10 ± 0.18) with an average shoot length of 3.09 ± 0.05 cm within 4 weeks of culture. The addition of higher concentration of 2iP (0.5 mg l−1) in plant regenerating SH medium containing 0.5 mg l−1 BA and 0.25 mg l−1 IBA induced conversion of regenerating callus (83.70 ± 0.50%) into the maximum number of shoots per callus explant (7.3 ± 1.05) with an average shoot length of 3.27 ± 0.04 cm (Figure 4a-c). The adventitious shoots produced via indirect organogenesis were isolated from callus explants and incubated on MS medium supplemented with 0.25 mg l−1 TDZ for elongation growth (Figure 3e). The shoots with well developed leaves were subcultured on this elongation growth medium in every three weeks.Figure 4

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Education in Science and Mathematics, Regional Institute of Education, National Council of Educational Research and Training, Shyamla Hills, Bhopal, 462013 India.

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.

No MeSH data available.


Related in: MedlinePlus