In vitro plantlet regeneration in Asparagus racemosus through shoot bud differentiation on nodal segments.<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

Neetu Vijay and Ashwani Kumar

SUMMARY

A highly reproducible in vitro regeneration system in Asparagus racemosus using nodal segments has been developed. Multiple shoots were induced in vitro from nodal segments through adventitious shoot bud regeneration as well as indirectly through callus. The ability of nodal segments to produce shoot buds varies depending upon their position on the main shoot of the field grown plants. Nodal segments obtained from 7^th node to 9^th node was proved to be the most suitable for the induction of bud initials. Explants showed the best proliferation and growth on the medium fortified with BA 3.0 mg.l^-1 and NAA 0.5 mg.l^-1 with an average of 40-45 shoot buds / explant and an average 3-3.5 cm length.  In vitro rooting of regenerated shoots was achieved  on half strength MS medium fortified with NAA. The plants were successfully hardened for about 5 weeks thereafter transferred to the field. The acclimatized plantlets were successfully established in the field with approximately more than 75% survival.

INTRODUCTION

Asparagus racemosus Willd. is multiplied through vegetative propagation in nature. There is always problem of heterogeneity in obtaining uniform seed stock. Previous studies using seeds as means of propagation indicated that plants developed through seeds are poor in vigour. Seeds have been reported to have a low viability and they have a slow rate of growth (Gupta et al., 2002). The natural regeneration of this under shrub is through tuberous roots that have been scarce in nature because of indiscriminate collection of wild material. In vitro cultivation, cryopreservation and micropropagation through tissue culture may also aid the preservation of medicinal plants and provide the complimentary conservation option (Benson et al., 2000).

In vitro propagation of economical and medicinal plants offers not only means for the mass multiplication of existing stocks of germplasm for biomass energy production but also for the conservation of important elite or rare plant species those are threatened with danger of extinction. Clonal propagation of favourable lines through in vitro culture of explants would also facilitate breeding programme (Bajaj, 1986). According to Murashige (1974) there are four areas in which plant tissue culture can be applied possibly either presently or in near future viz., i) production of pharmaceutical and natural products, ii) genetic improvement of plants, iii) recovery of disease free clones and preservation of valuable germplasm and iv) rapid clonal multiplication of selected varieties.

Tissue culture technique has been widely accepted as a tool of biotechnology for vegetative propagation of plants of agricultural, horticultural and forestry importance (Dave and Purohit, 2002). In vitro propagation enables a broad range of species to be cloned under highly controlled conditions (Hussey, 1986). Clonal propagation of plants via tissue culture can be carried out by : i) enhanced branching , ii) axillary branching , iii) somatic embryogenesis and iv) callus organogenesis (Thorpe and Harry,1990). The technique has been successfully used in propagation of wide range of herb and shrub species (Murashige, 1974). Extensive work has been done on several ornamental species of family Liliaceae (Krikorian and Kann, 1986) but very few medicinal plants of this family have been cultured in vitro (Jha et al., 1984).

Recently there has been an upsurge of micropropagation of several medicinal plants viz., Dioscorea zingiberensis (Chen et al., 2003), Artemisia petrosa spp. Eriantha (Pace et al., 2004), Albizia procera Benth. (Swamy et al., 2004), Azadirachta indica A. Juss. (Quraishi et al., 2004), <?xml:namespace prefix = st1 ns = "urn:schemas-microsoft-com:office:smarttags" />Rosa damascena Mill. (Sharma et al., 2004), Achrus sapota L. (Liao et al., 2004), Hagenia abyssinica(Feyissa et al., 2005) and Phellodendron amurense Rupa. (Azad et al., 2005) Micropropagation is being utilized commercially in the mass multiplication of ornamental plants worldwide (Huetteman and Preece, 1993). Nodal segment contain axillary buds, capable of producing a complete plant, have been employed for clonal propagation. With the use of tissue culture technique, rate of shoot multiplication can be increased manifold by culturing the nodal explant on a culture medium having an appropriate cytokinin and auxin-cytokinin. Large number of plants have been produced in diverse group of families using nodal segments as explant, Plumbago zeylanica L. (Rout et al., 1999 and Selvakumar et al., 2001), Centella asiatica L. (Tiwari et al., 2000), Holarrhena antidysenterica Wall. (Raha and Roy, 2001), Accacia catechu Willd. (Sahini and Gupta, 2002), Asparagus maritimus (Stajner et al., 2002a), Decalepis hemiltonii (Gururaj et al., 2004), and Ficus carica L. (Pasqual et al., 2004).

Many attempts in the clonal propagation of Asparagus genus have been made using shoot tip and lateral bud clusters (Yang and Clore, 1974). However, in vitro multiplied shoots of Asparagus often produce thin and weak roots or even no root formation at all and the plantlets thus obtained rarely survive when they are transferred to soil (Shigeta et al., 1996). Little work has been undertaken on in vitro culture in Asparagus racemosus as compared to that of A. officinalis, the horticultural variety extensively used for the preparation of soup and as a vegetable in European countries. A. racemosus is less known to world, but it has many uses in ayurvedic medicines. This has led to its increasing demand in the crude drug market.

  A separate family Asparagaceae has now been created by removing the genus from Liliaceae family (Brumitt, 1992). The genus, Asparagus comprises 150 species (Drost, 1997) and consists of herbs, shrubs and vines which are widespread in the Old World. The genus is classified into three subgenera (Asparagus, Protasparagus and Myrsiphyllum) according to Clifford and Conran (1987). They are grown throughout the world but they originated mainly from Asia, Africa and Europe (Encke, 1958). The species of the first subgenus are dioecious, with unisexual flowers, while the second and the third subgenera include only hermaphrodite flowers. The genus Asparagus represents highly valuable plant species having therapeutical and nutraceutical importance in addition to being consumed as food (Shasnay et al., 2003). Genome size, rDNA ITS polymorphism, genetic variability and relationship of economically important Asparagus led to modification in its classification (Stajer et al., 2002b).