IN VITRO PROPAGATION OF WADAKAHA (ACORUS CALAMUS L.)

Wadakaha (Acorus calamus L.) is a medicinal plant of great commercial value and a potential export crop. The practice of propagating this plant based on rhizomes is inadequate to provide the planting material required for large-scale cultivation. This paper describes a tissue culture based method for mass production of Wadakaha propagules. Apical shoot meristems were cultured on MS (Murashige & Skoog, 1962) medium supplemented with BAP or kinetin(0.1-2.0 mgl-') along with IAA, IBAandNAA(0.01-1.0 mgl-l)forculture initiation. Well developed shoots were transferred to solid or liquid MS medium with BAP or kinetin (0.5-5.0 mgl-l)forshootmultiplication. Forculture initiation the medium containing BAP (0.5 mgl-l) and NAA(0.2 mgl-') was the best. Liquid media containing BAP 1.0 mgl-I and 2.0 mgl-I both gave the highest number of shoots (26 shoots/explant). The results show that BAP produced significantly more shoots than kinetin. Liquid media were more promising than solid media.


INTRODUCTION
Wadakaha (Sinhala); Vashambu (Tamil); Bach (Hindi) [family Araceael is an aromatic herb of marshy habitats with a stout creeping and branching rhizome.It occurs throughout India, Sri Lanka, Philippines, temperate regions of Asia, Europe and North America, China, Japan and southern Russia.'In Sri Lanka, the plant is cultivated as a medicinal herb.The rhizome of Wadakaha contains alkaloids, mainly choline, the bitter glucosides acorin and calamine A, an essential oil calamol, a resin, gum, starch and tannin.The essential oil is said to contain asarone, palmitic and heptoic acids.Calamus oil is used for preparation of aromatic cordials and liquors, flavouring beer and making perfumes.In ayurveda the rhizome ofthis herb is used for treating diseases such as dyspepsia, flatulence, choleraic diarrhoea in children, cough, fever, piles and asthma.In the USA the rootstock is often eaten raw for relief from indigestion.The powdered rhizome is an insecticide and is used in the preparation of toilet powder.I t is also an antidote for several poison^.^Due to its varied uses, there has been a growing demand for the plant.The herb rarely produces seeds and is only propagated by vegetative means.1t is not known to flower in Sri Lanka and has possibly been introduced to the country.Conventional propagation techniques are inadequate to provide the planting material required for large scale cultivation.The present study describes an effective method for rapid multiplication of Wadakaha.

METHODS AND MATERIALS
Rhizomes of Wadakaha were collected from field-grown plants from the Matale area.They were washed thoroughly using a soap solution (SunlightTM) and rinsed under running tap water for about 30 min.Then shoot-meristems of approximately 5 mm in length were dissected out from the rhizome and were sterilized with 70% ethanol (vlv) for 1 min.and 20% (vlv) commercial clorox (bleach containing 5.2% wlv NaOCl) with Teepol (2-3 drops1100 ml) for 10 min.followed by 0.1% HgC1, for 3 min under constant stirring.After rinsing three times with sterilized distilled water the final explants (about 2 mm) were prepared by removing the outermost layers.The shoot-meristems were cultured on agar-solidified (0.8% wlv) MS (Murashige & Skoog, 1962)3 medium containing either 6-benzylaminopurine (BAP) or kinetin 0.1-2.0mgl-l along with indoleacetic acid (IAA), indolebutyric acid (IBA) and naphthaleneacetic acid.(NAA) a t a rate of 0.01 -1.0 mgl-'.The cultures were maintained under fluorescent light (55.5 ~E s -l m -~, 16 h) a t 26 + 1°C.After 8 wk, growing shoot meristems in the medium containing BAF' (0.5 mgl-') and NAA (0.2 mgl-l) were transferred to 20 ml of solid or liquid MS media containing BAJ? or kinetin a t 0.5, 1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5 and 5.0 mgl-l in flasks for shoot multiplication.Four replicates per treatment were used.The liquid cultures were agitated (60 rpm) on a shaker to facilitate shoot multiplication.
Well-grown shoots were taken out from the shoot-multiplication media (shoot proliferation also occurred in the media), isolated, m d cultured on MS solid medium without any growth regulators for root induction.Subsequently the rooted plantlets were transferred to a soil mixture in pots (5 cm diameter) of sand: top soil: compost 1:l:l and maintained in the glasshouse for 2-3 wk.Finally the plantlets were established in the field.Data analysis: Analysis of variance was done using MINITAB data analysis software package for a three factorial randomized complete block design and treatments were compared using least' significant values.

RESULTS
Cultures were established in the MS media containing BAP or kinetin (0.5 -1.0 mgl-l) along with IAA, IBA and NAA (0.01-0.5 mgl-') within 8-10 wk of culture.Of these culture media, the medium supplemented with BAP (0.5 mgl-l) and NAA (0.2 mgl-l) produced what appeared to be the most vigorous shoots.However, IAA, IBA or NAA a t higher levels (over 0.5 mgl-') induced calli a t the base of the shoot-meristems, suppressing shoot growth.When cytokinin (BAF', kinetin) levels were above 1.0 mgl-l, shoot elongation was less, and callus formation was observed at the base of the shoot-meristems.
When single shoots taken from the culture medium with BAP (0.5 mgl-I) and NAA (0.2 mgl-I) were transferred to shoot-multiplication media (solid and liquid), shoot proliferation [Fig. 2 --These results show that the mean number of shoots that formed on solid media was 7.0 k 0.2 per explant.Shoot multiplication was significantly enhanced (19.0 + 0.5 mean number of shoots/explant) in liquid cultures (Table 2).Also the results show that BAP was significantly more effective than kinetin for shoot-proliferation (Table 1).At higher levels (> 2.0 mgl-I), particularly BAP in liquid media inhibit shoot proliferation.
When cytokinin (BAP, kinetin) levels were above 2.0 mgl-l, calli formed at the base of the newly formed shoots, both in the solid and the liquid media.When the cytokinin levels were increased further (above 4.0 mgl-l) these shoots did not show elongation.
When these single shoots were isolated and transferred to root induction medium of MS without any growthregulators, within 4 5 wk ofculture, 6-8 roots were visible.When these plantlets were established in soil [Fig.2(b)] the survival rate was approximately 75%.

DISCUSSION
An initial problem encountered was the elimination of microorganisms from the explants as they were taken from rhizomes growing in soil.However, the problem could be overcome when the rhizomes were washed thoroughly using a soap solution (SunlightTM) and rinsed under running tap water for about 30 min followed by chemical sterilization.Although HgCl, is highly toxic and its use for sterilization is not recommended, it was found that out of the chemicals used in the present study soil microorganisms could be eliminated only by the use of HgC1,.The sterilization procedure adopted in this study gave about 85-90% survival.The contaminants were mostly fungal and rarely bacterial.The developed methodology has also been successful in the elimination of microorganisms from explants of ginger which also harbour soil microorganism^.^Thus this method could be utilized for explants taken from soil.The preseqt resdlts show that in the proliferation phase, when the cytokinin level was above 2.0 mgl-l, callus formation occurred both in the solidified and liquid media.Hence, it is not advisable to use cytokinin over 2.0 mgl-l fur shoot multiplication of these plants, as the formation of callus can induce genetic ~a r i a t i o n .~ In liquid cultures both kinetin and BAP enhanced shoot multiplication, but BAP was more effective than kinetin.BAP coAcentrations of 1.0 mgl-' and 2.0 mgl-I gave the highest amount of shoot proliferation (mean number of shoots/explant 26).Since there was no difference between them, BAP a t 1.0 mgl-l was selected as the optimum concentration for the proliferation phase.Almost similar results have been reported for shoot multiplication in ginger.4 In this study MS medium without any growth regulators performed well for ' ,, \ root induction.Therefore the authors did not examine the effectiveness of any other growth regulators for root induction.Earlier studies with komarika indicated that root formation could easily be induced in similar media without much diffi~ulty.~It is interesting to note that the medium used for root induction of Wadakaha could also be used to conserve the i n vitro cultures for a period of approximately one year without subculturing at a reduced temperature of 20°C (unpublished data).Therefore the established method can be used to conserve this plant in the i n vitro collection.If materials like sorbitol or mannitol are added and at further reduced temperatures the conservation period (1 112 years) is said to be p r ~l o n g e d .~ Establishment of i n vitro grown plantlets in soil'is one of the major difficulties encountered in a micropropagation procedure.The plantlet survival rate on transfer to soil is reported to be extremely low in woody p l a n k g The present study showed the survival rate of Wadakaha was about 75% which is satisfactory.In conclusion, the results suggest that i n vitro culture ofwadakaha, a useful technique for rapid multiplication, is feasible.

1 . 2 Figure 1 :
Figure 1: Effect of BAP and Kinetin on shoot proliferation in solid and liquid media.

Figure 2 :
Figure 2: In vitro culture of Acorus calamus L. (Wadakaha).( a ) Shoot multiplication (b) In vitro derived plant after transferring to soil

Table 1 : Analysis of variance on the effect of hormones (BAP & kinetin) on shoot proliferation of Wadakaha.
= degrees of freedom; ss = sums of squares; ms = mean square; f = F value; * = p < 0.05 df

Table 2 : Analysis of variance on the effect of status (solid or liquid) of media on shoot proliferation.
df = degrees of freedom; ss = sums of squares; ms = mean square; f = F value; ** = p < 0.01