Plant Cell Reports (1991) 10:525-528 plantCell Reports �9 Springer-Verlag 1991

Propagation of Costus speciosus (Koen.) Sm. through in vitro rhizome production

Archana Roy and Amita Pal

Plant Molecular & Cellular Genetics, Unit Bose Institute, Calcutta 700054, India

Received August 20, 1990/Revised version received August 28, 2991 - Communicated by J.M. Widholm

ABSTRAGT

Rhizomes developed in vitro on shoots of Costus speciosus (Koen.) Sin. which were initiated from zygotic embryos. The effect of different hormonal and nutritional additions to Schenk and Hildebrandt ' s (SH) basal nutrient medium on rhizome development was studied. Rhizomes developed on SH basal salts but formed with increased frequency on medium supplemented with adenine sulfate, casamino acids (CA) and various combinations of cytokinins and auxins. Incubation in light was necessary for rhizome formation. Isolated basal as well as nodal (aerial) rhizomes formed and produced new shoots. In basal medium without any growth additives (control) the average number of shoots produced per inoculum was 3.3 -+ 0.73 whereas in the best ]suited medium i.e. supplemented with 250 nag i-" CA the number of shoots obtained was 22.7 +- i.92. There was no dormancy period for rhizomes under the cultural conditions investigated. Rhizome-sprouts were easily transplanted from the in vitro conditions to the field. More than five hundred propagules (i.e. sprouted-rhizomes) were obtained within 5 months an~ it has been estimated that more than 2.4 x 10 propagules could be achieved per year through four subsequent in vitro rhizomes-generation cycles. Thus, a rapid method of propagation has been established.

ABBREVIATIONS

AdS, Adenine sulphate; BA, benzyladenine; CA, casamino acids (vitamin free) ; 2,4-D, 2,4- dichlorophenoxyacetic acid; IAA, indoleacetic acid; IBA, indolebutyric acid ~ Kn, Kinetin; NAA , naphthaleneacet ic acid; SH, Schenk and Hi ldebrandt ' s medium (1972).

INTRODUCTION

Costus speciosus (Koen.) Sin., a member of the Zis~iDeraceae family, is a potential source of diosgenin (Dasgupta and Pandey, 1970). Diosgenin is a starting material for a number of pharmaceutically important steroidal drugs including contraceptives. The rhizome of C. speciosus is the plant organ which accumulates

Offprint requests to: A. Pal

diosgenin, Moreover, the plants are cross pollinated and the resultant seeds are non-viable. Consequently the plants are propagated asexually and the rhizomes are the propagule for dissemination. In culture also rhizome formation in the regenerated plants is desired because of the ease of handling them during field transfer and easy rooting of plants under field conditions.

Normal shoot organogenesis can be modified in culture to generate tubers, bulbils , rhizomes and corms (Uduebo, 1971; Hussey, 1980; Grootaarts et at., 1981; Ammirato, 1982, 1984,1986; Forsyth and Van Staden, 1984; Hussey and Stacey, 1984; Sengupta et al., 1984; Alderson and Rice, 1986; Garner and Blake, 1989; Somani et al., 1989). Uduebo (1971) first reported that nodal cuttings of Dioscorea bulbifera would form tubers on medium without any growth substances. However, much of the published work on the induction of microtubers on potato plants in vitro has focussed on the use of growth regulators for this purpose (Wang and Hu, 1982; Hussey and Stacey, 1984; Abbott and Belcher, 1986). During the present investigation we made an attempt to induce maximal aerial rhizome formation using stem cuttings as explants with different hormonal and nutritional additions to ensure rapid propagation of this medicinal species.

MATERIAL AND METHODS

Costus speciosus population 6, obtained from tb-@--R~ionai Research Laboratory (CSIR), Jammu, India, was clonally propagated th rough rhizome cuttings and maintained in the experimental field. All the experiments were conducted with zygotic embryo raised plantlets, collected from an individual clone of population 6, designated as clone P6. This plantlets were produced in vitro on supplemented with 0. I mg 1 -~H

basal medium IBA and were

grown on the basal medium without any growth adjuvants for 30 d. Stem cuttings, each with ten nodes and approx 6.0 _+ 0.25 cms in length, were excised aseptically from such plantlets and placed into I00 ml. conical flasks containing 30 ml of solid SH medium. The nutritional additives and plant growth hormones used for different treatments were AdS, CA, NAA, IAA, IBA, 2,4~,

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BA and Kn. The c u l t u r e s were maintained under both a z16 hr_photoperiods of fluorescent light (200

2u Em- sec ) or in the dark at 25 -+ I~ Ten replicates were used for each treatment, and all the experiments were repeated twice. The culture vessels containing rhizomatous plants were maintained under ambient temperature (30-32~ for 7 d before transplantation. These plants were washed thoroughly with water and then transferred either to Doly bags or to earthen pots containing sand and soil. These plants were watered at regular intervals and covered with bell jars the first 15 d to avoid excess transpiration. The rhizomes and rhizome sprouts were transferred directly to the soil after thorough washing.

RESULTS AND DISCUSSION

E x p l a n t s when p l a c e d on SH n u t r i e n t medium wi thou t any g rowth r e g u l a t o r s ( con t ro l se t ) could r e g e n e r a t e r h i z o m e s at t h e b a s e . The co lo r of t h e in v i t r o r a i s e d b a s e l r h i z o m e s was g reen in c o n t r a s t to cream c o l o r e d r h i z o m e s of t h e w i l d p o p u l a t i o n s of C_z .. s p e c i o s u s . Aer ia l r h i z o m e s a re of f r e q u e n t o c c u r r e n c e under t h e cu l t u r a l cond i t i on , w h i c h n e v e r f o r m e d unde r t h e in v i v o s i t u a t i o n . These aerial rhizomes (Fi~. I) are formed in acroDetal order at the stem nodes. The aerial rhizomes sprouted to produce new shoots when still attached to the parent plant (Fig.2). New shoots also regenerated from the basal rhizomes (Fi~.3).

However , none of t h e Kn t r e a t m e n t s could p r o d u c e b a s a l r h i z o m e s . BA a l so f a i l e d to induce e i t h e r b a s a l or a e r i a l rh i zome f o r m a t i o n . On the o t h e r hand , aux ins when combined wi th Kn and BA induced b a s a l and a e r i a l r h i zo me fo rma t ion . The number of b a s a l r h i z o m e s was a l w a y s one, w h e n e v e r f o r m e d . The mean number of a e r i a l r h i z o m e s fo rmed and t h e t o t a l number of a d v e n t i t i o u s s h o o t s o b t a i ned p e r e x p l a n t in 60 d in t h e p r e s e n c e of v a r i o u s g rowth r e g u l a t o r s a re r e p r e s e n t e d in Table 1. This t a b l e only e m b o d i e s da ta w h e r e t h e number of r h i z o m e s fo rmed was h i g h e r than t h a t of t h e con t ro l s e t . All of t h e s e hormonal t r e a t m e n t s were conduc ted under photoperiodic ( 16 hr light and 8 hr dark) conditions while another set of cultures was kept in the dark. None of these dark treatments induced rhizome formation so the subsequent experiments were only performed under photoperiodic conditions.

The h i g h e s t number of a e r i a l r h i z o m e s was o b t a i n e d when SH b a s a l medium was s u p p l e m e n t e d

wi th 250 mg 1-1 CA. The mean number of s p r o u t s o b t a i n e d f rom r h i z o m e s was a l so h i g h e s t w i th t h i s t r e a t m e n t (Table 1).

Almost a l l of t h e combina t ions of exogenous ly a p p l i e d Kn wi th aux ins p r o d u c e d a p p r e c i a b l e numbers of a e r i a l r h i z o m e s (Tab le 1). However combina t ions of Kn and 2 ,4-D f a i l e d to induce nodal r h i z o m e s . It shou ld be no ted t h a t t h e p r e s e n c e of

F ig .1 Aer ia l r h i z o m e s (a r ) from t h e nodes of in v i t r o r e g e n e r a t e d s h o o t s a f t e r 21 d in SH medium conta in ing 0.01 mg 1-1 IAA.

Fig. 2 Aerial rhizomes (at) sprouted when still attached to the main shoot after 30 d in SH medium cotaining 250 mg 1 -I CA (ruler marks are in era).

Fig .3 An i s o l a t e d b a s a l r h i z o m e s p r o u t e d in v i t r o on SH b a s a l medium ( r u l e r m a r k s a r e in cm) .

IAA and NAA at 0.01 mg 1-1 concen t r a t ion only could induce r h i z o m e s ( b a s a l and a e r i a l ) . Only b a s a l r h i z o m e s were f o r m e d a t t h e b a s e of t h e s tem cu t t ings w i t h a l l IBA c o n c e n t r a t i o n s , i . e . 0 .01 , 0 .1 , 0.5 and 1,0 mg 1-1. All o t h e r t r e a t m e n t s w i th aux ins f a i l e d to induce any r h i z o m e f o r m a t i o n . Aer ia l r h i z o m e s were f o r m e d s p g r a d i c a l l y when t r e a t e d w i t h 0.01 mg 1 ~ kn .

Kn inhibits basal rhizome formation. Combined t r e a t m e n t s of BA and aux ins f a i l e d to induce a e r i a l r h i z o m e s h i g h e r in number than t h e con t ro l s e t (not shown in t h e t a b l e ) . BA when used at 0.1 mg 1-1 wi th IAA at 0.01 and 0.1 mg 1-1 or IBA at 0.01 mg 1-1 i n d u ced fo rmat ion of h i g h number s of r h i z o m e s (Table 1).

Table I: Total number of aerial rhizomes and adventitious shoots (rhizome-sprouts) from each explant after 60 d of treatment with growth adjuvants.

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Growth ~d_~vant s (mg

Mean No.of rhiz omes / explant *

Mean No.of sprouts / explant*

(Control) 3.4 + 0.92 3.3 + 0.73 IAA 0.01 7.5 -+ 0.75 5.6 +- 1.02 CA 250 15.3 + 1.22 22.7 -'1.92 CA 500 7.3 + 0.86 6.7 -+ 0.60 Ads 15 5.0 +- 0.54 5.1 • 0.62 Kn 0.I + IAA 0.01 6.2 + 0.79 6.3 -+ 1.34 Kn 0.I + IAA 0.I 5.3 + 0.56 5.1 • 0.43 Kn 0.5 + IAA 0.I 5.8 • 0.57 5.1 • 0.54 Kn 0.5 + IAA 0.5 5.1 • 0.86 4.7 + 0.55 Kn 0.I + NAA 0.01 7.6 • 0.7 5.9 • 0.62 Kn 0.i + NAA 0.I 6.3 + 0.01 6.6 • 0.75 Kn 0.5 + NAA 0.5 4.6 • 0.46 4.7 + 0.57 Kn 0.I + IBA 0.01 7.4 • 1.05 5.6 -* 0.87 Kn 0.I + IBA 0.I 4.1 • 0.68 5.6 • 0.66 Kn 0.5 + IBA 0.I 4.0 + 0.64 4.1 • 0.62 Kn 0.5 + IBA 0.5 4.1 • 0.17 Nil BA 0.I + IAA 0.01 7.7 • 0.09 5.8 • 0.45 BA 0.i + IAA 0.I 6.3 • 0.8 5.2 • 0.34 BA 0.I + IBA 0.01 I0.I + 1.2 5.3 • 0.52

* Mean -+ S.E. from ten replicate cultures

Isolated basal and aerial rhizomes when placed on SH basal nutrient medium sprouted within 20 d irrespective of their origin. Unlike wild plants of C. speciosus, no dormancy was observed in basal and aerial rhizomes in culture.

Among the various treatments of growth adjuvants which could induce or enhgnce aerial rhizome formation, CA at 250 mg i-- gave the best result. This treatment gives consistent results in repeat experiments, and a single explant i.e. stem-cutting with ten nodes could regenerate 502 shoots after two vegetative growth cycles as shown in the scheme (Fig. 4). Thus, atthe endofthe third vegetative growth cycle the expected number of ~ropagules (rhizome-sprouts) would be II.0 x I0 from one explant. Hence,the expected number of propagules per year f~om a single explant would be more than 2.4 x I0-.

All these rhizome sprouts were easily established in soil (Figs. 5 and 6) with almost 100% survival. These shoots rooted within 7 days and the basal rhizomes formed in the followibg month. The in vitro multiplied plants were morphologically uniform and indistinguishable from their parent. The yield of rhizomes was 43 to 199 gins in six months after field transfer per plant.

So far in vitro propagation of C?ispeciosus was only successful through shoot tip cultures

VGC "I'EXPLANT MEDIUM DAYS

SH + 250 mg 1 - I CA 23 rhizome sprouts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Each isolated and grown separately

I SH(Control)

Ready to be used as explant(23 in number) ............................. 90

I SH + 250 mg - I CA

"-502 shoots formed (Expected no. of shoots = 23 x 23 = 529,5% mor ta l i ty ) . . . . . . . . . . . . . . . . . . . . . 150

~ I I . 0 x 104 rhizome sprouts (propagules) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

2.4 x 105 rhizome ~sprouts (Propagules) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

Fig,4: Schematic r e p r e s e n t a t i ~ of rhizome sprouting and mul t ip l ica t ion in in v i t ro vegetative growth cycles

(VGC).

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(Chaturvedi et al., 1983). The present study revealed a new, rapid method of propagation for this important medicinal plant. This is the first report of in vitro aerial (nodal) rhizome formation of C__ L. speciosus. Moreover this mode of propagation is much more rapid than the shoot tip multiplication method reported earlier by Chaturvedi et al., (1983).

Aerial rhizomes formed under cultural condition do not have any dormancy period, hence, a rapid rate of in vitro propagation is easily achieved.

Figs.5,6:Plantlets established in poly bag (5) and in pot (6).

ACKNOWLEDGEMENT S

The junior author (AR) is grateful to the Council for Scientific and Industrial Research for financial assistance. We are thankful to Dr.

M. K, Mitra, Farm-in-Charge, Bose Institute for acclimatization and maintenance of the culture regenerants in the field.

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