Rapid In Vitro Plant Regeneration of Black Gram (Vigna mungo L. Hepper) Var. Sarala,an Important Legume Crop

Efficient in vitro regeneration of black gram (Vigna mungo L. Hepper) var. Sarala was achieved through organogenesis using cotyledonary explants excised from 4 days old seedlings on MS medium supplemented with 2.0 mg/l BAP. Organogenic calli were developed from cotyledonary tissues within 4–6 weeks of culture on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BAP) along with 2.0 mg/l 1-napthaleneacetic acid (NAA). Shoot bud regeneration was achieved on MS medium supplemented with 2.0 mg/l BAP within 3–4 weeks of subculture. The number of shoots per culture varied from 1.12 to 8.75 in different growth media. The cultures incubated initially on dark photoperiod for 2 weeks and subsequently transferred to 16 h photoperiod showed higher number of shoot bud regeneration. The proliferated shoots were further sub-cultured on similar medium for higher rate of shoot bud regeneration. The elongated shoots were rooted on ½ strength MS medium fortified with 0.1–0.5 mg/l NAA or indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) with 2 % (w/v) sucrose within 2–3 weeks of culture. The higher percentage of rooting was obtained on 0.1–0.25 mg/l NAA as compared with IBA or IAA. The rooted plantlets were transferred to soil mixture (soil: sand: vermi-compost, 1:1:1 ratio) and kept in the greenhouse with 85 % humidity. The regenerated plantlets were successfully grown with 75 % survival rate. This protocol can be used for genetic improvement of black gram.     

Assessment of Genetic Fidelity Using Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats Markers of <Emphasis Type="Italic">Lawsonia inermis</Emphasis> L. Plants Regenerated by Axillary Shoot Proliferation

An efficient, reliable and reproducible plant regeneration protocol was developed for Lawsonia inermis L. using mature nodal explants. Shoot proliferation (81.6 %) with 7.8 shoots/explant was achieved on Murashige and Skoog’s (MS) medium supplemented with 1.0 mg l^?1 6-benzyladenine. Shoot numbers were up-scaled by inducing multiple shoots from axenic nodal segments derived from the primary shoots on the shoot regeneration medium. Thus the authors could achieve ca. 129–134 shoots from single nodal explant. Ninety-three percent rooting of in vitro regenerated shoots was achieved on growth regulator free half-strength MS medium. Regenerated plantlets were successfully transferred to soil with 85 % survival rate. Genetic stability analyses of the in vitro regenerated plants using random amplified polymorphic DNA and inter simple sequence repeat markers revealed a homogeneous amplification profile for all micropropagated plants. This is the first report that evaluates the use of molecular markers to establish genetic fidelity of micropropagated L. inermis for the rapid clonal multiplication and true-to-type production of plant for attaining the ever increasing demand in pharmaceutical industries.     

In Vitro Callus Induction and Plantlet Regeneration of <Emphasis Type="Italic">Saussurea lappa</Emphasis> (Clarke.) from Ladakh Region of India

In vitro propagation protocol was developed for Saussurea lappa (Clarke.) species threatened by over exploitation due to medicinal importance and habitat destruction in Ladakh region of India. The aim of the present study was to examine the main aspects of in vitro callus induction (CI) and plantlet regeneration of S. lappa. Explants were cultured on Murashige and Skoog (MS) basal medium supplemented with various concentrations and combinations of auxins and cytokinins for in vitro CI and plantlets regeneration. Callus initiation and induction was observed within eight and fifteen days after inoculation. 3 mg/L 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 5 mg/L Kinetin (Kin) was found to be more effective for CI frequencies in all the explants as compared to other combinations. Root (79.6 ± 0.8) and stem (76.4 ± 0.6) explants were found with better callus frequencies and early response as compared to other explants. Maximum number of shoots (11.8 ± 0.7), highest shoot length (9.2 ± 0.6 cm), maximum root number (6.2 ± 0.7) and highest root length (6.6 ± 0.8 cm) were found on MS medium with 2 mg/L Kinetin (Kin) and 2 mg/L Indole-3-butyric-acid in root derived callus explant. Plantlets with 3–5 shoots were transferred to potting mixture containing sand: soil: perlite (1:1:1) for acclimatization to field conditions and further multiplication. For the first time, protocol has been developed in S. lappa for in vitro CI and plantlet regeneration that holds robust potential for metabolite production and large-scale propagation.     

In Vitro Plantlet Regeneration from Alysicarpus monilifer,a Source of Hepatoprotective Drug

A plantlet regeneration protocol has been developed for Alysicarpus monilifer, a medicinal plant that is a source of hepato-protective drugs. Callus was induced from mature cotyledonary leaves from 4 to 5 days old seedling on Murashige and Skoog (MS) medium supplemented with 1.0 mg l^?1 2,4-dichlorophenoxyacetic acid (2, 4-D). Proliferation of cultures occurred on MS medium with 1.0 mg l^?1 each of 2,4-D, 6-benzylaminopurine (BA) and kinetin (Kin). Shoot regeneration from proliferated callus was influenced by a number of factors namely plant growth regulators (PGRs), gelling agents, culture vessels and carbohydrate source. The highest (85.6 %) shoot regeneration was recorded in 250 ml culture flasks on agar gelled MS medium + 0.1 mg l^?1 α-naphthalene acetic acid (NAA) + 1.0 mg l^?1 each of BA, Kin and 2-isopentenyladenine (2iP) + 1 % glucose and 2 % maltose in addition to the usual 3 % sucrose. The shoots differentiated on PGRs, free MS medium, were stronger and longer than the shoots developed on MS medium containing PGRs (0.1 mg l^?1 NAA + 1.0 mg l^?1 each of BA, Kin and 2iP) with different leaf morphology and were easy to root. Rooting of the regenerated shoots was achieved both in vitro and ex vitro. About 80.4 % of the shoots rooted in vitro on half strength MS medium containing 1.0 mg l^?1 indole-3-butyric acid (IBA), while 84.9 % of the shoots rooted under the ex vitro condition when treated with 250 mg l^?1 IBA for 5 min. The plants were hardened in the green house and showed 85 % survival rate.     

Sustained Shoot Multiplication and Method for Overcoming In Vitro Browning in Medicinally Important Plant, <Emphasis Type="Italic">Piper chaba</Emphasis> Hunt

The current study evaluated for the first time plant regeneration in P. chaba. Adventitious shoot regeneration was achieved in Murashige and Skoog (MS) medium supplemented with three concentrations of 6-benzyladenine (BA; 0.25, 0.5 and 1 mg/l) and 40 mg/l ascorbic acid. Maximum number of 18 adventitious shoot buds was formed in 0.5 mg/l BA but it was accompanied by severe callusing. Therefore, for subsequent multiplication, shoots were subcultured in MS with 0.25 mg/l BA to prevent excessive callusing, though the number of shoots per culture was reduced to 12. A major problem with cultures of this plant was the excessive browning of the shoots and medium due to phenolic exudation. To control browning of tissue and medium two anti-browning agents, ascorbic acid (AA, 10, 40 and 100 mg/l) and activated charcoal (AC, 5 and 10 mg/l) were used individually or in combination with the multiplication medium. Univariate analysis (Kruskal–Wallis test) and principal component analysis plot were performed to assess impact of anti-browning agents on reducing browning but still maintaining consistent rates of proliferation (16.5 ± 0.42) indicated that AC (5 mg/l) was significantly better than either AA alone or any of the combinations of AC and AA. Single shoots were rooted in 0.25 mg/l indole butyric acid and successfully acclimatized under net-house conditions.     

In Vitro Culture of <Emphasis Type="Italic">Rheum emodi</Emphasis> Wall: An Endangered Medicinal Plant of Northwestern Himalaya

Rheum emodi Wall is a well-known medicinal plant found on high altitudes. The plant is under tremendous anthropogenic pressure due to its over exploitation. In this context, biotechnological intervention particularly tissue culture is the need of hours for its conservation. In the present study, a successful and reproducible protocol was standardized for the micropropagation of various R. emodi using various explants and treatments. The explants used were seeds, leaves, shoots and rhizomes which were cultured on Murashige and Skoog (MS) medium, supplemented with different concentrations and combinations of phytohormones i.e. 6-benzylaminopurine (BAP), kinetin, indole-3-acetic acid, napthalene acetic acid, indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid, Zeatin and Thidiazuron. The most effective concentration and combination for the plantlet formation was MS + (15 µM) BAP + (15 µM) IBA.     

In Vitro Propagation of <Emphasis Type="Italic">Ceropegia anjanerica</Emphasis> Malpure et al.: A Rare Endemic Plant from Maharashtra

An in vitro micropropagation system has been developed for the Ceropegia anjanerica Malpure et al., a rare endemic plant species having ornamental as well as medicinal potential but a limited reproductive capacity. Axillary bud explants were cultured on Murashige and Skoog medium supplemented with 6-Benzylaminopurine, Thidiazuron and 6-Furfurylaminopurine along with combination of Indole-3-Butyric Acid. Although all concentrations showed multiple shoot formation, the best results (8.0 ± 0.5) were achieved with MS + 6-Benzylaminopurine (8.87 µM) + 6-Furfurylaminopurine (18.58 µM). Half strength Murashige and Skoog medium fortified with Indole-3-Acetic Acid (8.56 µM) showed good rooting response (4.0 ± 0.7). Well grown plantlets hardened, acclimatized and established in greenhouse exhibited 78 % survival.     

A Liquid Culture System for Improved Micropropagation of Mature Acacia nilotica (L.) Del. ssp. indica and Ex Vitro Rooting

A micropropagation method using liquid culture medium has been developed for mature Acacia nilotica (L.) Del. ssp. indica. Nodal segments obtained from 15 to 20 years old mature trees were used as explants and cultured on 0.8 % agar-gelled Murashige and Skoog medium containing 6-benzylaminopurine for shoot bud induction. Once culture got established, explants were transferred to Murashige and Skoog liquid medium containing 6-benzylaminopurine or kinetin for shoot multiplication. Shoot multiplication was influenced by plant growth regulators, size of vessels, amount of medium in culture vessels and repeated transfer of mother explants. Murashige and Skoog liquid medium containing 4.4 μM 6-benzylaminopurine was found to be the best for shoot multiplication. The performance of liquid and agar-gelled medium for shoot multiplication was compared. About ten times increase in shoot number in liquid culture medium was achieved. Micropropagated shoots were rooted ex vitro. Shoots treated with 2.46 mM indole-3-butyric acid solution for 1 h followed by 1.41 mM chlorogenic acid for 5 min exhibited highest percent of rooting in the green house. This process of micropropagation of A. nilotica, can be utilized for plant production on a large-scale.     

Micropropagation of <Emphasis Type="Italic">Acacia leucophloea</Emphasis> (Roxb.) Willd.: A Multi-Purpose Legume Tree

An in vitro micropropagation system has been developed for Acacia leucophloea (Roxb.), an important legume tree of Aravallis in Rajasthan (India). Cotyledonary node segments proved to be the most suitable explants to induce axillary bud proliferation on Murashige and Skoog’s medium containing growth regulators. Nodal segments obtained from 20 to 25 days old in vitro raised seedlings produced a maximum of 4.5 shoots/explant during initiation when medium were supplemented with 1.0 mg l^?1 6-benzylaminopurine. For shoot proliferation combination of 2.0 mg l^?1 kinetin, 0.5 mg l^?1 BAP and 0.05 mg l^?1 indole-3-acetic acid produced maximum of 14 shoots/explant. On subsequent culture of shoots on the same fresh medium along with mother explant, an average of 14 shoots/node could be obtained. Rooting could be induced in more than 80% shoots inoculated on MS media containing 1.0 mg l^?1 indole-3-butyric acid. The protocol proved to be highly reproducible.     

In Vitro Propagation and Phytochemical Assessment of Aconitum ferox Wall: A Threatened Medicinal Plant of Sikkim Himalaya

This is the first report on in vitro propagation and phytochemical assessment of Aconitum ferox (Ranunculaceae), a threatened medicinal plant of Sikkim Himalaya. A simple and efficient in vitro propagation protocol through indirect shoot organogenesis has been established for A. ferox using root tip explants. Murashige and Skoog (MS) medium supplemented with 2.26 µM 2, 4-dichlorophenoxyacetic acid (2, 4-D) was found to be the best medium to induce and maintain the callus. Different concentrations and combinations of plant growth regulators (PGRs) were tested for in vitro shoot proliferation. Adventitious shoots were produced from callus when it was transferred to MS medium supplemented with N⁶-benzylaminopurine (BAP). The synergistic incorporation of 3 µM indole-3-acetic acid (IAA) in 6 µM BAP containing medium-induced plantlets with well-developed roots and shoots. However, the best rooting responses were observed in shoots placed on paper bridge in liquid MS medium supplemented with 3 µM IAA?+?6 µM BAP. The plantlets were successfully acclimatized in ex vitro conditions with 70% survival rate. Additionally, root extract of in vitro-raised plants showed antioxidant activity closer to wild plants. The study thus signifies the effectiveness of in vitro technique for A. ferox propagation and provides a method for sustainable utilization of this high-value medicinal plant at commercial scale in pharmaceutical industries.