Paramvir Singh Ahuja

Saponin production in callus and cell suspension cultures of Panax quinquefolium

Abstract Callus and cell suspension cultures of American ginseng (Panax quinquefolium) were compared for growth and in vitro ginsenoside production over a 35-day culture cycle on modified Murashige and Skoogs medium. A time course study at five day intervals revealed that biomass yield in suspension and callus cultures was maximal on the 25th and 30th day of growth, respectively. Both types of cultures were able to produce ginsenosides in amounts and quality comparable to the cultivated plants. TLC-densitometry and HPLC analyses of the crude ginsenosides revealed that yield and relative distribution of different fractions belonging to the Rb and Rg groups of ginsenosides were greatly influenced by culture age. For the Rb group components, 25-day-old callus or suspension cultures were the best source of these compounds, while for the Rg group fractions 30–35-day-old cell cultures gave the maximum yield. Appreciable amounts of ginsenosides, particularly Rg1, were found to leach out in the culture medium of 30–35-day-old suspension cultures.

Somatic embryogenesis and plant regeneration from callus cultures of Aconitum heterophyllum Wall

Plants were obtained via somatic embryogenesis in callus derived from in vitro raised leaf and petiole explants of Aconitum heterophyllum Wall. Callus was induced on a Murashige-Skoog medium supplemented with either 2,4-dichlorophenoxy acetic acid (2,4-d 1 mg l-1) and kinetin (KN 0.5 mg l-1) with coconut water (CW 10% v/v) or naphthalene acetic acid (NAA 5 mg l-1) and benzylaminopurine (BAP 1 mg l-1). Somatic embryos appeared after 2–3 months or 2 subculture passages when 2,4-d or NAA induced source of the callus was transferred to a MS medium containing BAP (1 mg l-1) and NAA (0.1 mg l-1). For successful plantlet formation, the somatic embryos were transferred to a medium containing 1/4 strength MS nutrient with indole-3-butyric acid (IBA 1 mg l-1). Alternatively, the somatic embryos were dipped in a concentrated solution of IBA for 5 min and placed on a hormone free medium. Complete plantlets were formed after 4 weeks and were transferred successfully to soil.

Screening and evaluation of agronomically useful somaclonal variations in Japanese mint (Mentha arvensis L.)

SummaryA procedure has been standardized for high frequency plant regeneration response from nodal explant cultures of Mentha arvensis Linn. var. piperascens Holmes. Murashige and Skoogs medium supplemented with IAA or NAA (0.5–2.0 mgl−1) alone, supported axillary shoot elongation while BAP (2.0–3.0 mgl−1) alongwith IAA (1.0 mgl−1) supported multiple shoot production. In vitro-derived shoots readily developed roots when cultured on NAA (1.0 mgl−1) fortified MS medium. Regenerated plantlets were successfully transferred to glasshouse (90–95% survival rate) and ultimately to the field. Among 280 plants transferred to the field a wide range of variation was observed for various agronomic traits i.e. plant height (32.0–92.0 cm), leaf-stem weight ratio (0.53–2.32), herb yield (105.0–870.0 g), oil content (0.32–1.10%) and oil yield (0.66–5.22 ml/plant). In addition, variations were also recorded for four major constituents of the essential oil i.e. menthol (65.2–94.77%), menthone (1.40–20.89%), isomenthone (0.96–5.14%) and menthyl acetate (0.75–8.52%). A positive correlation is found for oil yield with plant height and herb yield, whereas a negative correlation exists between herb yield and oil content. Based on the initial agronomic assessments on individual plant basis, 27 somaclones were selected and further evaluated in a replicated plant to row trial with parent plant CIMAP/Hy-77 as standard check. Somaclones Sc 59 and Sc 179, selected on the basis of higher herb yield in the initial screening, recorded 55.8% and 64.3% increase in oil yield over the control, respectively. Somaclones Sc 93, Sc 114, Sc 121 and Sc 124 that were selected for their better oil content exhibited 47.2%, 50.6%, 57.5% and 48.2% increase in oil yield over the parent variety, respectively. The performance of these clones in evaluation trials is discussed in relation to the possibility of genetic improvement of mints through somaclonal breeding.

Propagation of Indian rhubarh (Rheum emodi Wall.) using shoot-tip and leaf explant culture

Shoot-tip explants of Rheum emodi Wall. (Polygonaceae) gave rise to multiple shoots when cultured on a Murashige and Skoog (1962) medium (MS) with 2.0 mg/l 6-benzylaminopurine (BAP) and 1.0 mg/l indole-3-butyric acid (IBA). Also, shoot buds developed from leaf explants using MS medium with 2.0 mg/l BAP and 0.25 to 1.0 mg/l indole-3-acetic acid (IAA) or IBA. Roots were induced when the resulting shoots were placed on MS medium with 1.0 mg/l IBA. Both regeneration procedures gave rise to healthy plantlets that were established in soil under glasshouse conditions at 80% frequency after hardening phase of two weeks. Regenerated plants showed a constant chromosome number of 2n=2x=22, same as the parent plant. The use of liquid shake cultures minimized the time and culture medium requirements for propagation. This procedure can be applied for the conservation and utilization of elite clones of R. emodi.

Morphogenetic potential of foliar explants in Duboisia myoporoides R.Br. (Solanaceae)

The effects of serial combinations of either indole-3-acetic acid, indole-3-butyric acid or α-naphthaleneacetic acid (0.5–10.0 mg/l) with either kinetin, 6-benzyl-amino-purine, zeatin or 6-methylaminopurine (0.5–5.0 mg/l) have been investigated to assess the morphogenetic potential of foliar explants of Duboisia myoporoides. Shoot buds developed either directly or via a callus interphase. Combinations involving indole-3-acetic acid with any of the cytokinins were more effective in inducing shoot bud formation compared to those containing indole-3-butyric acid or α-napthalenacetic acid as an auxin. Among cytokinins, zeatin, kinetin and 6-benzylamino-purine were equally effective for shoot formation. However, optimum response with zeatin could be achieved at low concentrations (0.5–2.0 mg/l), while kinetin and 6-benzylamino-purine exhibited comparable efficacy at higher levels (3.0–5.0 mg/l). 6-Methylaminopurine proved least effective in all concentrations and combinations tested. Rooting of the differentiated shoots was readily achieved with α-naphthaleneacetic acid alone (0.5 mg/l) after changing the physical form of the medium from gel to static liquid. Regenerated plantlets were transferred to pots and grown to maturity in the field with a high rate of survival (80–90%).

Assessment of liquid culture procedures for in vitro propagation of Rheum emodi

Micropropagation of an endangered Indian medicinal plant, Rheum emodi Wall., was achieved on Murashige and Skoogs medium using different liquid culture procedures. Liquid static (submerged, semi-submerged and with filter paper bridge) and shake (80 and 120 rpm) culture procedures were assessed for their effects on growth and multiplication rates. Best results were obtained using liquid shake cultures, which resulted in 50% reduction in medium requirement, 37.5% reduction in time and 1.5–2.2 fold increase in growth and multiplication rate. Liquid culture-raised plantlets facilitated easy transplantation and 90–95% survived transfer to potting mix in glasshouse.

Establishment and multiplication of colchi-autotetraploids of Rauvolfia serpentina L. Benth. ex Kurz. through tissue culture

A tissue culture procedure was developed for the establishment and propagation of a colchi-autotetraploid of Rauvolfia serpentina for possible commercial exploitation. Multiplication of autotetraploid shoots was obtained either through axillary bud elongation on Murashige and Skoog [1] medium (MS) containing 2.65 μM (0.5 mgl−1) α-naphthaleneacetic acid and 0.33 μM (0.05 mgl−1) kinetin, or via multiple shoot formation on MS medium supplemented with 4.44 μM (1.0 mgl−1) 6-benzylaminopurine and 0.53 μM (0.1 mgl−1) α-naphthaleneacetic acid. Rooting could be induced by transferring the shoots to MS medium containing 7.95 μM (1.5 mgl−1) α-naphthaleneacetic acid alone. The plantlets, thus formed, were tetraploid in nature by cytological observations of the root tips. They exhibited 80–90% success in establishment under glass house and field conditions.

Fertile somatic hybrid between sexually incompatible Hyoscyamus muticus and Hyoscyamus albus

SummarySomatic hybrid plants have been regenerated from fused protoplasts of a chlorophyll deficient mutant of H. muticus (2n=28) with wild type protoplasts of H. albus (2n=68). The inability of protoplasts of H. albus to regenerate was utilized in complementation with achlorophyllous, but regenerating, protoplasts of H. muticus for the selection of green somatic hybrid colonies and plants. The somatic hybrid plants showed intermediate morphological characters, and possessed 82–120 chromosomes, with a modal number of 96 which is also the amphidiploid complement of the two species. The isozyme patterns indicated the presence and expression of genes from both parents. The hybrid plants produced 33–78% viable pollen and set viable seeds upon selfing and backcrossing in a directional manner.