Naseem Ahmad

Shoot multiplication in Rauvolfia tetraphylla L. using thidiazuron

The effect of thidiazuron (TDZ) was investigated on in vitro shoot proliferation from nodal explants of Rauvolfia tetraphylla. Murashige and Skoog (MS) medium containing TDZ (0.5–10μM) was effective in inducing shoot buds and maintaining high rates of shoot multiplication on hormone free medium. The highest shoot regeneration frequency (90%) and mean number (18.50 ± 1.25) of shoots per explant were achieved from nodal segments cultured on MS medium supplemented with 5μM TDZ for 4 weeks prior to transfer to MS medium without TDZ for 8 weeks. The regenerated shoots rooted best on MS medium containing 0.5μM indole-3-butyric acid (IBA). Micropropagated plantlets were hardened to survive ex vitro conditions and were then established into soil.

Improved plant regeneration in Capsicum annuum L. from nodal segments

Multiple shoots were induced by culturing nodal explants excised from 1-month-old aseptic seedlings of red pepper (Capsicum annuum L. cv. Pusa Jwala) on Murashige and Skoog (MS) medium supplemented with (0.1–10 μM) thidiazuron (TDZ). The rate of multiple shoot induction per explant was maximum (14.4 ± 0.06) on MS medium supplemented with 1.0 μM TDZ. Regenerated shoots were elongated well on growth regulator free MS medium. Adventitious roots were induced two weeks after transfer of elongated shoots to MS medium supplemented with auxins (IAA, IBA or NAA) in different concentrations. Optimum root formation frequency was obtained in medium containing 1.0 μM IBA. Ex-vitro rooting was also achieved by pulse treatment with 300 μM IBA for 10 min. Rooted shoots were transplanted in plastic pots containing garden soil (with 90 % survival rate), where they grew well and attained maturity. Regenerated plants were phenotypically and cytologically normal.

Direct plant regeneration from encapsulated nodal segments of Vitex negundo

An efficient protocol for encapsulation of nodal segments of Vitex negundo L. has been developed for the production of non-embryogenic synthetic seeds. The encapsulations of nodal segments were significantly affected by the concentrations of sodium alginate and calcium chloride. A 3 % Na2-alginate with 100 mM CaCl2 has been found to be optimum concentration for the production of uniform synthetic seed. For germination, the synseeds were cultured on Murashige and Skoog (MS) basal medium supplemented with kinetin (KIN) and α-naphthalene acetic acid (NAA) either singly or in various combinations. MS medium containing 2.5 μM KIN in combination with 1.0 μM NAA was found to be the optimum for maximum (92.6 ± 3.71 %) plantlet conversion frequency. Well developed regenerated plantlets were hardened, acclimatized and established in field, where they grew well without any detectable variation.

Assessment of Genetic Fidelity in Rauvolfia serpentina Plantlets Grown from Synthetic (Encapsulated) Seeds Following in Vitro Storage at 4 °C

An efficient method was developed for plant regeneration and establishment from alginate encapsulated synthetic seeds of Rauvolfia serpentina. Synthetic seeds were produced using in vitro proliferated microshoots upon complexation of 3% sodium alginate prepared in Llyod and McCown woody plant medium (WPM) and 100 mM calcium chloride. Re-growth ability of encapsulated nodal segments was evaluated after storage at 4 °C for 0, 1, 2, 4, 6 and 8 weeks and compared with non-encapsulated buds. Effects of different media viz; Murashige and Skoog medium; Lloyd and McCown woody Plant medium, Gamborg’s B5 medium and Schenk and Hildebrandt medium was also investigated for conversion into plantlets. The maximum frequency of conversion into plantlets from encapsulated nodal segments stored at 4 °C for 4 weeks was achieved on woody plant medium supplement with 5.0 μM BA and 1.0 μM NAA. Rooting in plantlets was achieved in half-strength Murashige and Skoog liquid medium containing 0.5 μM indole-3-acetic acid (IAA) on filter paper bridges. Plantlets obtained from stored synseeds were hardened, established successfully ex vitro and were morphologically similar to each other as well as their mother plant. The genetic fidelity of Rauvolfia clones raised from synthetic seeds following four weeks of storage at 4 °C were assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. All the RAPD and ISSR profiles from generated plantlets were monomorphic and comparable to the mother plant, which confirms the genetic stability among the clones. This synseed protocol could be useful for establishing a particular system for conservation, short-term storage and production of genetically identical and stable plants before it is released for commercial purposes.

Enhanced in vitro regeneration and change in photosynthetic pigments, biomass and proline content in Withania somnifera L. (Dunal) induced by copper and zinc ions.

In the present study the effect of inorganic nutrients (CuSO₄ & ZnSO₄) on morphogenic and biochemical responses from nodal explants in Withania somnifera L. was investigated. Incorporation of either Copper sulphate (25-200 μM) or Zinc sulphate (50-500 μM) in the optimized Murashige and Skoog (MS) medium highly influenced the shoot bud formation and subsequent elongation, which induced maximum percentage (95%) regeneration, number (61.7 ± 0.25) of shoots with shoot length (5.46 ± 0.16 cm) on CuSO₄ (100 μM) and maximum percentage regeneration (100%), number of shoots (66.1 ± 0.96) with shoot length (6.24 ± 0.21 cm) on ZnSO₄ (300 μM) after 12 weeks of culture. Healthy growing in vitro microshoots rooted efficiently on ½ MS medium supplemented with NAA (0.5 μM), which induced (16.2 ± 0.12) roots with root length (3.30 ± 0.12 cm) after 4 weeks. Pigment content increased with increasing concentration of Cu and Zn and the maximum Chl. a (0.47), (0.41); Chl. b (0.52), (0.42); total Chl. (0.99), (0.83) and Carotenoid (0.16), (0.16) mg/g FW contents in regenerants were found on CuSO₄ (100 μM) and ZnSO₄ (300 μM), respectively. Maximum proline content (0.17), (0.16) μg/g FW was observed on high concentrations of CuSO₄ (200 μM) and ZnSO₄ (500 μM) respectively, in the basal medium. Regenerated plantlets were acclimatized successfully in soilrite with a survival rate of 95%. No morphological variations were detected among the micropropagated plants when compared with seedling raised plants of the same age.

In vitro production of true-to-type plants of Vitex negundo L. from nodal explants

Summary An efficient micropropagation protocol, involving axillary bud multiplication, was established to clone Vitex negundo L. plants. An optimum regeneration frequency (97%) and a desirable organogenic response, in the form of multiple shoots, was obtained on Murashige and Skoog (MS) medium augmented with 5.0 µM 6-benzyladenine (BA) in combination with 0.5 µM 1-naphthaleneacetic acid (NAA). Healthy, growing in vitro-raised microshoots rooted efficiently on MS medium supplemented with 1.0 µM indole-3-butyric acid (IBA). Following standard hardening procedures, rooted shoots were transferred to the field with ≥ 90% survival. No morphological variations were detected among the micropropagated plants when compared with the mother plant. Inter-simple sequence repeat (ISSR) markers were used to evaluate the genetic stability of both the in vitro and in vivo propagated plants. All ISSR profiles from micropropagated plants were monomorphic, and similar to those of field-grown control plants. These results suggest that the culture conditions used for axillary bud proliferation are appropriate for clonal propagation of this medicinally important plant as they do not appear to interfere with the genetic integrity of in vitro-regenerated plants (i.e., no somaclonal variation).

Improved shoot regeneration system through leaf derived callus and nodule culture of Sansevieria cylindrica

Long-term culture establishment and efficient in vitro regeneration protocol for Sansevieria cylindrica Bojer ex Hook was developed using leaf derived callus and nodule culture. Profuse callus induction on leaf discs was achieved on Murashige and Skoog (MS) medium supplemented with 10 µM indole-3-butyric acid (IBA), while a high frequency of nodulation was induced on 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) containing media. Shoot regeneration ability from cultured tissues occurred at varying degrees on all media. Through callus culture a maximum of 17.6 ± 0.14 shoots per culture was formed on medium containing 5µM 6-benzyladenine (BA) and 2 µM α-naphthaleneacetic acid (NAA). Among nodule cultures, the 2,4-D generated nodules were more proliferative and regenerative as compared to 2,4,5-T induced nodules and a maximum of 25 ± 0.16 shoots per culture was produced on a medium containing 5 µM BA plus 1 µM NAA. The regenerated shoots were successfully rooted on a semi-solid half strength MS medium containing 5 µM IBA with an average root number 3.5 ± 0.18 and root length 6.5 ± 0.14 cm. The regenerative ability of callus tissues was steady upto one year, while the nodules retained the totipotency to regenerate on optimal medium even after 3 years of subculturing. The histological sections of nodules confirm the typical anatomy exhibiting the vascular elements in bundles with well demarcated cortex and epidermal covering.

An efficient system for in vitro multiplication of Ocimum basilicum through node culture

An efficient in vitro micropropagation system was developed for direct shoot growth of Ocimum basilicum , an important medicinal plant, using nodal explants. The excised nodes were cultured on Murashige and Skoog (MS) medium containing two plant growth regulators (6-benzyladenine and 2isopentanyl adenine) with various combinations and concentrations for the study of shoot induction. Addition of L-glutamine was essential to induce sprouting of axillary buds. The best condition for shoot growth was with 6-benzyladenine (BA) 10.0 µM + L-glutamine 30 mg/L in MS medium. The optimum shoot formation frequency was 100% and about 13.4 ± 1.80 shoots were obtained from each explant after 8 weeks of culture. Shoots (more than 4 cm) were rooted most effectively in 5.0 µM indole-3-butyric acid (IBA) supplemented with half-strength MS medium. The plantlets thus obtained hardened off and were transferred to natural soil, where they grew well and attained sexual maturity.

Effect of PGRs in adventitious root culture in vitro: present scenario and future prospects

Plants, a source of undeviating therapeutic agents, are used as a model system for new synthetic compounds and a taxonomic marker for discovery of novel compounds. The expanding awareness of the health hazards and toxicity associated with the indiscriminate use of synthetic drugs has enhanced the interest in the use of plants and plant-based drugs. The evolving commercial significance of secondary metabolites in recent years has resulted in a great interest in secondary metabolism, particularly in the prospect of varying the production of bioactive plant metabolites by means of tissue culture technology. Adventitious root culture system (AR) provides an alternative approach for the improvement and enhancement of plant-based pharmaceutical compounds. AR serves as a base for conservation of important plant species by reducing pressure on its natural populations. The present review focuses on the application of plant growth regulators (particularly auxins and cytokinins) for the initiation and establishment of adventitious root cultures through various explants in vitro. The production of root biomass could provide bioactive compounds of interest in laboratory conditions. In addition, the root biomass production, accumulation and quantification of secondary metabolites studies in different medicinal plant species have been discussed in the light of different PGRs.

Two-way germination system of encapsulated clonal propagules of Vitex trifolia L.: an important medicinal plant

ABSTRACT In the present study we have developed an efficient and effective method of synthetic seed production and its two-way germination system of Vitex trifolia, for easy transport of the propagules and efficient utilization of its in vitro regeneration system. Nodal segments harvested from 8-week-old in vitro cultures were encapsulated in calcium alginate beads. Three percent (w/v) Na-alginate polymerized in 100 mmol/L CaCl2.2H2O for 30 min produced clear and uniform beads. Germination of encapsulated beads with shoot and roots was achieved on Murashige and Skoog (MS) medium augmented with 6-furfurylaminopurine (KN, 2.5 µmol/L) + α-naphthalene acetic acid (NAA, 1.0 µmol/L). For multiple shoot production, synseeds were incubated on 6-benzyladenine (BA, 5.0 µmol/L) + NAA (0.5 µmol/L) augmented MS medium followed by in vitro rooting on MS + indole-3-butyric acid (IBA, 1.0 µmol/L). The synseeds produced retained about 90% regeneration potential even after 4 weeks of storage at 4°C. Genetic stability of the regenerated plants was evaluated using 13 inter simple sequence repeats (ISSR) primers. The study thus provides an efficient system for production of synthetic seeds, their storage and subsequent conversion into genetically identical plants.