In Vitro Cloning of Achyranthes Aspera: An Important Ayurvedic Medicinal Plant

Abstract

The present investigation was carried out on an important Ayurvedic medicinal plant Achyranthes aspera belonging to family Amaranthaceae. Though almost all of its parts are used in traditional systems of medicines, seeds, roots and shoots are the most important parts which are used medicinally. Different vegetative parts like nodal explant, stem and leaves were excised from field grown healthy mature plant and then were planted on variously supplemented Murashige and Skoog’s (MS) medium for multiple shoot proliferation, callus induction and de novo adventitious root and shoot formation. Leaf and stem segments excised from in vitro raised plants were also used for de novo adventitious shoot formation and callus induction. Achyranthes aspera displayed a high degree of propensity of multiple shoot proliferation from nodal segments. Multiple shoot proliferation was obtained on MS medium supplemented with BAP (8.8- 17.6 µM) or Kin (9.2- 18.4 µM) alone where 23- 27 multiple shoots were formed from single nodal explant after 30 days of culturing. Once the clusters of shoots were formed, small clumps of 2-3 shoots were excised and transferred onto fresh multiplication medium where shoots proliferation continued leading to the formation of 40 – 50 shoots per bottle. The present material exhibited a high efficiency of de novo adventitious root and shoot formation directly from the stem and leaf segments. Prolific root formation occurred when leaf segment taken from field grown healthy mature plant was inoculated on MS medium supplemented with NAA alone (5.37-10.74 µM). Highest number of roots were, however, formed on 10.74 µM NAA where approximately 30 -35 roots were formed after 25 days. Likewise prolific root regeneration was observed from leaf explant taken from in vitro raised plants when cultured on MS medium supplemented with IBA or NAA either alone or in synergism with Kin or BAP. The roots were thin and white with dense root hairs. De novo adventitious shoot formation was not at all observed from the leaf explants taken from field grown mature plants. However, leaf explants taken from in vitro raised plants (through axillary shoot proliferation) exhibited high degree of adventitious shoot formation on MS medium supplemented with NAA either alone (10.74 µM - 21.48 µM) or in conjunction with BAP or Kin forming a maximum of 18-20 shoots. The regenerated shoots from various vegetative parts were rooted on basal MS medium to form complete plantlets. It was noticed that multiple shoots formed on concentrations of cytokinin Kin had already developed roots simultaneously, hence no root induction was required. However, multiple shoots formed on cytokinin BAP were carefully planted upright on Basal MS medium for root initiation. Induction of roots occurred after 20 – 25 days which proliferated further leading to the formation of well-developed roots For the acclimatization, the plantlets were transferred to plastic pots containing sterile potting mixture consisting of soil:sand:vermicompost in the ratio of 1:1:1. These pots were then covered with plastic bags having holes to maintain high internal humidity and kept under culture room conditions for 15 days. The plantlets were regularly monitored and watered at regular intervals. The plantlets were then transferred to polybags containing same potting mixture and were kept in growth room for another 15 days. Attempts are underway to transfer these plantlets to the natural field conditions. Callusing was induced when leaf explants of in vitro raised plantlets were inoculated on MS medium augmented with 8.8 µM-17.6 µM BAP alone. Callus formed after 15 days was compact, hard and was whitish in colour initially but became brownish in colour in later stages. Callusing from leaf explants was also induced on MS medium supplemented with 9.2 µM Kin. Stem explants excised from in vitro grown plantlets and transferred to MS medium supplemented with 4.4 µM BAP and 9.2 µM Kin when used alone initiated callus formation. Better callus induction occurred when 4.4 µM BAP was used in conjunction with NAA (10.74-21.48 µM) forming greenish white callus after 30 days and root differentiation occurred from the callus after 45 days. The callus formed from stem and leaf segments on the medium were more or less identical in morphology. The calli obtained was heterogeneous composed of ovoid, oblong, spheroidal, elongated and of other different shapes and sizes. Tracheids were observed in all the calli. Tracheids occurred singly or in groups and possessed reticulated thickenings on their walls. In the present investigation, only histogenetic differentiation in the form of tracheids and rhizogenesis was observed from the callus. No shoot formation could be effected from the callus on any of the media tested.