HIGH FREQUENCY MULTIPLE SHOOT REGENERATION FROM TISSUE CULTURES OF ASPARAGUS RACEMOSUS
HIGH FREQUENCY MULTIPLE SHOOT REGENERATION FROM TISSUE CULTURES OF ASPARAGUS RACEMOSUS NEETU VIJAY AND ASHWANI KUMAR* Plant Biotechnology Lab, Department of Botany , University of Rajasthan, Jaipur-302004, ` Email- email@example.com firstname.lastname@example.org * Corresponding author ABSTRACT: Attempts at inducing differentiation in various explants of Asparagus racemosus resulted in the production of abundant shoot buds from the nodal segments , shoot tips and cladodes explants both directly and indirectly (i.e. without and with intervention of the callus formation ) on modified MS medium (1962). Maximum shoot formation was achieved when the media was supplemented with 2.0 mg.l-1 Kn + 0.1 mg.l-1 NAA. In vitro rooting of regenerated shoots was achieved in the same media after a long time of inoculation. Besides this excised shoots were rooted on half strength MS medium in the absence of growth regulators. The regenerated plantlets successfully acclimatized and transferred to soil. About 70% of plantlets survived under ex vitro conditions. Abbreviations: IBA- indole 3-butyric acid, NAA- α-naphthalene acetic acid, BAP- 6-benzyl amino purine, Kn- kinetine. Key words: Asparagus racemosus, Cladodes, Differentiation INTRODUCTION: In recent years cultures of plant cells and tissues has gained considerable importance as a promising tool in improving and multiplying economically/ medicinally important crop plants. But so far work has been restricted to the dicots; the monocots which comprises some well known medicinal as well as ornamental crops of the temperate and sub-tropics have generally been neglected. Asparagus racemosus L. (Liliaceae) consists of tuberous roots has been specially recommended as a glactogogue , antacid, tonic and in cases of threatened abortions. Recently its potent immunomodulatory activity has been reported in animal models (Dahanukar et al., 1986; Thatte et al., 1988). The market sample does not belong to this species but is either of A.adscendens, A.gonocladus or A.officinalis (Khan et al., 1991). It contains several steroidal saponin glycosides (Ravikumar et al., 1987). Shatavarin I-IV are the important glycosides. Shatavarin I, II, IV are derived from a common aglycon moiety, sarsapogenin. The chemical structure of shatavarin-IV has been elucidated (Ravikumar et al., 1987) and is found to contain two glucose and one rhmnose. Shatavarin-I is similar to shatavarin-IV but contains an additional glucose moiety. Chromosomal races have also been reported in A.racemosus containing sapogenin like Diosgenin and Sarsapogenin in diploid, Sarsapogenin in tetraploid and Diosgenin in hexaploid races (Kar, et al., 1984). These sarsapogenins along with kaempferol have been isolated from the woody portion of the roots of A.racemosus (Ahmed et al.,1990). Sarsapogenins belongs to 25β series (Heftmann & Mosetting, 1960). Both aerial and root parts have been observe to posses amylase and lipase activities (Dange, 1969).Asparagamine A, a novel polycyclic alkaloid was reported from A.racemosus possessing antitumour activity (Sekine et al., 1994). Clonal propagation of elite plants is therefore, important to obtain high yield and quality in Asparagus. Genetic engineering techniques could be useful in the creation of new breeding approaches to producing plant varieties with novel characteristics (Mol et al., 1995). However, adventitious shoots or somatic embryo regeneration system are a prerequisite for these techniques and any efficient system has not been reported for regeneration of A.racemosus in tissue culture. In contrast, much of the research on Asparagus has been concentrated on A.densiflorus and A.officinalis. Klein and Edsall (1986) first reported callus formation from primary roots and Hanault (1976) obtained callus cultures from the same explants using Linsmaier and Skoog (1965) basal medium in A.densiflorus. Somatic embryogenesis and plant regeneration from protoplasts of A.officinalis L. was studied by Kuntitake & Mii, 1990. Somatic embryogenesis in A.officinalis has also been reported from hypocotyl segments (Wilmar and Hellendoorn 1968), young spear cross-section (Takatori et al., 1968) and shoot segments (Steward and Mapes 1971; Reuther 1977).These embryogenic calluses could be maintained on a hormone free medium and produced numerous secondary embryos (Dellbreil, 1992). Transgenic plants of A.officinalis using Agrobacterium mediated transformation had been produced by Delbreil et al in 1993. Sexual as well as vegetative propagation methods of A.racemosus are beset with many problems and restrict its multiplication on large scale. Natural seed production is erratic and the available seeds possess short viability. The aim of present study carried out here was to establish an efficient plant regeneration system from nodal explants of A.racemosus so that plants selected with higher medicinal value in roots and leaves could be multiplied for commercial purpose. MATERIAL AND METHODS: Donar Plant : Shoots from plants of A.racemosus grown in the medicinal plant nursery of university campus were excised and cut into 1.5-2.0 cm long nodal segments with one node and surface sterilized with 70% alcohol (1 min) followed by 0.1% mercuric chloride with a few drops of detergent (20 min) followed by four rinse with sterile deionized water. Sterilized shoot tips and nodal segments were transferred to agar solidified Murashige and Skoog’s (1962) medium supplemented with various concentration of BAP, Kn, NAA, IBA (Loba). The pH of the medium adjusted to 5.86 before autoclaving at 121o C and 15 psi for 15min. All cultures were incubated for 30-45 days at 25 ± 2o C under cool white fluorescent light at 30µE m-2 s-1 with a 16 h photoperiod. Initiation of bud initial: Nodal segments of in vivo propagated plants were cut into small pieces of 1.5-2.0 cm size and placed on MS medium containing different hormonal combinations. Cultures were placed under conditions described above. Explants showing sign of regeneration events were sub cultured on same and altered media after 30 days. Induction of bud initials were attempted in two sets of experiments. In the first set BAP (0.5 – 5.0 mg.l-1), Kn (0.5 – 5.0 mg.l-1) were incorporated into MS medium to select the best cytokinin for shoot proliferation. In the second set of experiment, a cytokinin that showed a good response in the previous experiment was tested at wide range of concentrations in combination with NAA to determine the synergistic effect and optimum growth regulator treatment for shoot proliferation. Elongation of shoot and bud initial: Bud and shoot clumps were collected after 7 weeks following culture initiation, bulked and randomly transferred to MS(1962) medium containing same and different concentrations of NAA and Kn. Four different treatments NAA + Kn (0.1mg.l-1 + 2.0mg.l-1) , NAA + Kn (0.5mg.l-1 + 2.0mg.l-1), NAA + Kn (1.0mg.l-1 + 2.0mg.l-1), NAA + Kn (2.0mg.l-1 + 2.0mg.l-1), were employed. Cultured on solid media were grown in 100ml flask containing 30 ml of medium. The entire experiment was replicated three times. Shoots longer than 2.0 cm were counted harvested after 4 weeks at which time the medium was replaced with fresh medium. Shoots were again counted and harvested at 8 weeks and the 8 weeks data were added to the 4 week data for a final tally of shoot production. Rooting of elongated shoots: Cultures of 8 weeks, when the medium was reduced to a higher extent, rooting was observed. Shoots harvested at 8 weeks from all treatments were bulked and randomly taken for rooting experiments. Excised shoots were transferred to test tubes containing 20 ml full and half strength MS media supplemented with 1% agar. In another set of experiment shoots were subjected to pulse treatment of IBA solution before culture to full and half MS strength media. RESULTS AND DISCUSSION: From the various explants tried, nodal segment explants showed highest proliferation of multiple shoots which was followed by shoot apex and cladodes both direct and indirect (Fig1. A,B). During present studies, specificity of explants size was found. Nodal segment with one node of size 1.5 – 2.0 cm was best for bud proliferation. There was also a significant difference in adventitious bud or shoot production among segments from different positions. A nodal segment from the 7th node to 15th node was proved to be the most suitable for the induction of bud initials (data not shown). This shows the morphogenic gradient along the shoot axis. Explants cultured on media containing Kn as the sole hormone reacted with profuse bud initials. Bud initials started to appear 3 weeks after culture in Kn supplemented medium. An increase in the concentration of kinetin up to the highest concentration tested 5.0mg.l-1 gave an inhibitory effect. Kinetin at 2.0mg.l-1 where bud production was most prolific, addition of NAA increased the number of bud initials. NAA had little influence at higher conc. of kinetin (4 -5 mg.l-1) although NAA enhanced shoot production at lower conc. of kinetin. MS basal medium without BAP or Kn did not support the induction of multiple shoots. The percentage of explants forming bud initials was higher with kinetin as compared to BAP. BAP alone or in combination with NAA did not respond satisfactory. Even the number of bud initials was maximum i.e. 13-15 in BA supplemented medium (3.0 mg.l-1) but the strength of these initials and the percentage of explants was poor. Only 48% explants respond in the presence of BAP. Since the differential effect of various concentrations of BA on the stimulation of shoot bud formation from cotyledonary nodes has already been reported for Glycine (Cheng et al., 1980), Pisum (Jackson and Hobbs, 1990) and Phaseolus (McClean and Graffon, 1989). BAP was the most effective phytohormone in all these reports, indicating cytokinin specificity for multiple shoot induction in these tissues. Shoot tips explants have produced very limited number of bud initial (2-4) which was very low when compared to that of nodal segment explants. The number of shoots produced per nodal segment explants has also varied with the combination of cytokinin and NAA (table – 1). The highest number of bud initial i.e. 16 per explants were produced from nodal segment explants in the presence of 2.0 mg.l-1 Kn and 0.1 mg.l-1 NAA after 7 weeks of culture period. On the same medium shoot tip explants have produced 3-5 initials. The growth retardant ancymidol has been used to promote shoot formation from nodal segments (Chin, 1982) or shoot apices (Kohmura et al., 1994) in Asparagus. The shoot initials obtained from cultures grown in above medium for 3, 5, 6, 7, and 8 weeks were transferred to the medium with same conc. of Kn and NAA as well as increased amount of NAA up to 2.0 mg.l-1, the best response in shoot growth and multiplication on new medium after 30 days was in bud initials obtained from 7 weeks in previous medium, where an average of seven shoots per inoculum of two bud initial was obtained. During subculture of initials for elongation the percent response and maximum number of shoots were to be observed in the medium supplemented with 0.5 mg.l-1 NAA and 2.0 mg.l-1 Kn (Fig. 1 C). Higher concentration of NAA i.e. 1.0, 2.0 mgl-1 gave lesser number of shoots, together with some nodulated structures at the base of the shoots (Fig 1. D). For comparing multiplication efficiency among different treatments, the percentage of explants responding is more important than the number of shoots regenerated per nodal explants. Because rhizome formation is very important in Asparagus, the separation of individual shoot without rhizome resulted in the death of shoots. Therefore, in an early developmental stage of regeneration (in 8 week old culture) the whole explants together with the newly formed shoots has to be treated as one propagation unit and the number of shoots per explants cannot be regarded as a multiplication index. Rooting was observed after a long time of inoculation about 3 months, when the media was reduced to a greater extent, and stress conditions were developed (Fig 1. E). Roots were white and small in length and in clusters as of in vivo grown plants. Rooted shoots were then transferred to full and half strength MS medium supplemented with 1% agar in the absence of any growth regulator (fig 1. F). Roots were elongate rapidly in half strength media as compared to full strength. Elongated shoots were also transferred to IBA (2.0 – 12.5 mg.l-1) supplemented media, but no response was seen. Pulse treatment of IBA solution (50-150 mg.l-1) of 24 to 72 hours gave some response but it was not satisfactory. The plantlets were potted in vermiculite and peat moss (1:1 mix) and placed in the incubator to acclimatize for 2 – 3 weeks, which were transferred to the greenhouse. These plants grow vigorously in the soil. About 70 % plantlets were successfully grown in ex vitro conditions. 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Kn (mg.l-1) 1.0 7.33±0.333 0.863±0.053 2.0 15±1 2.09±0.113 3.0 12.33±0.33 1.96±0.033 4.0 8.33±0.33 1.13±0.185 5.0 6.33±0.881 1.43±0.293 BA (mg.l-1) 1.0 5.66±0.666 0.555±0.05 2.0 7.66±0.881 1.13±0.185 3.0 14.66±0.666 2.13±0.185 4.0 15±0.577 1.43±0.033 5.0 12.33±0.333 1.62±0.341 S.E. Standard error (mean is of three replicates) Table – 3 Response of bud initials of A.racemosus in terms of percentage (observation recorded after 45 days) NAA Kn % response mean no. of length of shoots % rooting (mg.l-1) (mg.l-1) shoots±S.E (cm) ±S.E. 0.1 2.0 92% 17.33±2.905 3.53±0.272 50% 0.5 2.0 96% 39±4.509 4.46±0.260 45% 1.0 2.0 90% 13.33±2.185 2.95±0.388 28% 2.0 2.0 90% 11±0.577 2.92±0.145 20% S.E. Standard error (mean is of three replicates) FIGURE LEGEND (Fig 1 A-F) Organogenesis in Asparagus racemosus Fig 1. A direct shoot proliferation from nodal segment explant Fig 1. B indirect shoot proliferation from nodal segment explant Fig 1. C elongation of bud initials Fig 1. D nodulated structures at the base of shoots during subculture Fig 1. E induction of roots Fig 1. F rooted shoots on half strength MS media.