Plant Cell Reports

Plant Cell Reports (1990) 8:521-524

© Springer-Verlag 1990

In vitro clonal multiplication of turmeric (Curcuma spp.) and ginger (Zingiber officinale Rosc.) S. M. Balachandran, S. R. Bhat, and K. P. S. Chandel National Plant Tissue Culture Repository, National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi-110012, india Received March 27, 1989/Revised version received November 14, 1989 - Communicated by G. C. Phillips

ABSTRACT R h i z o m e buds, e x c i s e d from three C u r c u m a spp., and ginger, i n o c u l a t e d a s e p t i c a l l y on MS m e d i u m w i t h v a r y i n g levels of BAP and kinetin, p r o d u c e d m u l t i p l e shoots. For shoot m u l t i p l i c a t i o n , a c o n c e n t r a t i o n of 3.0 m g / l BAP was found to be o p t i m u m for all the species. In v i t r o p l a n t s w e r e s u c c e s s f u l l y e s t a b l i s h e d in the field and w e r e m o r p h o l o gically uniform. A simple m e t h o d to e x t e n d the s u b c u l t u r e i n t e r v a l was used and its rel e v a n c e to g e r m p l a s m c o n s e r v a t i o n is d i s c u s sed. Abbreviations : BAP = 6 - b e n z y l a m i n o p u r i n e : kinetin = 6-furfurylaminopurine; MS = M u r a shige and Skoog (1962)

c a t i o n d e s c r i b e s a m e t h o d for the r a p i d clonal m u l t i p l i c a t i o n and short term c o n s e r v a tion of C u r c u m a spp., and ginger. M A T E R I A L S AND M E T H O D S P r e p a r a t i o n of p laqt m a t e r i a l R h i z o m e s of C. d o m e s t i c a Val. var. 'koova', ~. a e r u g i n o s a Roxb., C. caesia Roxb. and Z. o f f i c i n a l e Rosc., w e r e p l a n t e d in sand to a T l o w s p r o u t i n g of buds. Clean rhizome p i e c e s (ca 3 cm) w i t h s p r o u t e d buds w e r e surface s t e r i l i z e d in 0.1% m e r c u r i c c h l o r i d e sol u t i o n for 15 min., and w e r e t h o r o u g h l y w a s h ed 5-6 times in s t e r i l e d i s t i l l e d water. Outer leaves w e r e r e m o v e d a s e p t i c a l l y and exp l a n t s (ca 1 cm each) w i t h buds w e r e inoculated o n t o the c u l t u r e m e d i u m . Cultures were i n c u b a t e d at 27 + 2°C under 16 h p h o t o p e r i o d .

I NTROD U C T I O N In v i t r o m u l t i p l i c a t i o n T u r m e r i c (Curcuma d o m e s t i c a Val.) and g i n g e r (Zingiber o f f i c i n a l e Rose.) are v e r y i m p o r t a n t spices w h i c h are a l s o u s e d in m e d i cine and c o s m e t i c i n d u s t r i e s . Both turmeric and g i n g e r are v e g e t a t i v e l y p r o p a g a t e d through u n d e r g r o u n d r h i z o m e s e x c l u s i v e l y . Rhizome m u l t i p l i c a t i o n rate in these s p e c i e s is very low (6-10 times) w i t h the y i e l d r a n g i n g b e t w e e n 15 and 25 tons per h e c t a r e . Maintenance of g e r m p l a s m by a n n u a l p l a n t i n g is exp e n s i v e and l a b o u r intensive. B e s i d e s , diseases and pests, p a r t i c u l a r l y soft r o t of g i n ger c a u s e d by piythium, take a h e a v y toll on the g e r m p l a s m . Our i n t e r e s t in in v i t r o p r o p a g a t i o n of these p l a n t s stems from the need to effic i e n t l y m u l t i p l y and c o n s e r v e the r i c h g e r m p l a s m of these s p e c i e s in India. T i s s u e culture m e t h o d s h a v e shown p r o m i s e for the cons e r v a t i o n of p l a n t species, e s p e c i a l l y veget a t i v e l y p r o p a g a t e d p l a n t s (Kartha, 1985). T h e r e is v e r y l i m i t e d i n f o r m a t i o n on in v i t r o m u l t i p l i c a t i o n of t u r m e r i c (Nadgauda et al., 1978) and g i n g e r (Hosoki and Sagawa, 1977). National Plant Tissue Culture Repository, NBPGR, N e w Delhi, has i n i t i a t e d a p r o g r a m m e for d e v e l o p i n g a p p r o p r i a t e in v i t r o m e t h o d s for the c o n s e r v a t i o n of g e r m p l a s m of s e v e r a l p r o b l e m c r o p plants. The p r e s e n t c o m m u n i -

Offprint requests to." S. M. Balachandran

M u r a s h i g e and Skoog (1962) b a s a l m e d i u m w i t h 3% s u c r o s e and 0.8% agar (Difco Bacto) was used, s u p p l e m e n t e d w i t h the c y t o k i n i n BAP (0, 0.2, 1, 3 or 5 mg/l) or a c o m b i n a t i o n of BAP (i.0 mg/l) and k i n e t i n (i.0 mg/l) . The pH of the m e d i u m was a d j u s t e d to 5.8 b e f o r e autoclaving. A b o u t 20 ml of m e d i u m was d i s p e n s e d into each tube and e n c l o s e d with, either c o t t o n plug or P O ½ y p r o p y l e n e cap and a u t o c l a v e d at 1.08 kg/cm for 15 min. After 4 weeks, m u l t i p l e p l a n t l e t s w e r e f o r m e d and these w e r e s e p a r a t e d and s u b c u l t u r e d o n t o fresh m e d i u m . B e f o r e s u b c u l t u r i n g , all the removed. The e x p e r i roots and leaves w e r e m e n t s w e r e r e p e a t e d a t l e a s t three times. D u n c a n ' s m u l t i p l e R a n g e Test (Gomez and Gomez, 1984) was a p p l i e d to test the d i f f e r e n c e b e t w e e n the t r e a t m e n t means. Establishment

of p l a n t s

in soil

In vitro g r o w n plants w e r e t h o r o u g h l y w a s h e d in tap w a t e r to r e m o v e agar from roots. Old leaves w e r e c l i p p e d and plants w e r e imm e r s e d in 0.2% B a v i s t i n (a fungicide) for 1520 min. T r a n s p l a n t i n g was d o n e in trays u s i n g a i:i m i x of a u t o c l a v e d sand and soil. P o l y t h e n e covers w e r e u s e d to e n s u r e high h u m i d i t y a r o u n d plants, and trays w e r e kept

522 at 27°C with 16 h photoperiod. Plants were irrigated 2-3 times during the first week, with half strength MS inorganic salts and s u b s e q u e n t l y with tap water. After i0 days, p o l y t h e n e covers were removed and plants were a c c l i m a t i z e d for another 1 week. Such plants were t r a n s p l a n t e d into pots and kept in a nethouse. RESULTS

AND D I S C U S S I O N

E s t a b l i s h m e n t of c o n t a m i n a t i o n free cultures was difficult, because the explants were taken from u n d e r g r o u n d rhizomes. Nearly 50% of the cultures were found to be contaminated initially. However, once a healthy culture was established, there was no further contamination. R h i z o m e buds of Curcuma spp., (Fig. i) and ginger produced shoots and roots simultaneously. W i t h i n 4 weeks, complete plants were formed in all the t r e a t m e n t s (Fig.2-4) and the m a x i m u m m u l t i p l i c a t i o n of shoots was attained. F u r t h e r i n c u b a t i o n only r e s u l t e d in vigorous g r o w t h of already formed shoots. R e s p o n s e of three Curcuma spp., and ginger to various c o n c e n t r a t i o n s of cytokinins in MS basal m e d i u m is p r e s e n t e d in Table i. There were s i g n i f i c a n t d i f f e r e n c e s between the treatments in the rate of m u l t i p l i cation in all the species except C. caesia. M u l t i p l i c a t i o n rate in the t r e a t m e n t with BAP and k i n e t i n (i.0 mg/l each) was intermediate b e t w e e n treatments with 1.0 and 2.0 mg/l BAP, indicating that BAP alone was adequate. The m u l t i p l i c a t i o n rate was g e n e r a l l y higher in ginger than Curcuma spp. The highest rate of m u l t i p l i c a t i o n was o b s e r v e d at 3.0 mg/l BAP in all the species. In ginger, there was a linear r e l a t i o n s h i p between the rate of m u l t i p l i c a t i o n and the conc e n t r a t i o n of BAP, with the m a x i m u m m u l t i p l i cation value at 3.0 mg/l. The m u l t i p l i c a tion rates recorded in the p r e s e n t study are lower than those r e p o r t e d by Nadgauda et al., (1978), K u r u v i n a s h e t t y et al., (1982) (7-10 times) for turmeric and B h a g y a l a k s h m i and Singh (1988) (16 times) for ginger. In the present study, shoot m u l t i p l i c a t i o n was fourd to occur by d e v e l o p m e n t of axillary buds, whereas it has not been indicated in previous reports. M a i n t a i n i n g g e n e t i c s t a b i l i t y is crucial for tissue culture techniques to be useful in c o n s e r v a t i o n and hence a x i l l a r y bud proliferation, rather than a d v e n t i t i o u s d e v e l o p m e n t is desirable. V a r i e t a l differences or mode of r e g e n e r a t i o n m a y a c c o u n t for the d i f f e r e n c e s b e t w e e n the p r e s e n t results and previous reports. R e g e n e r a t e d plants produced prolific roots in all the treatments r e g a r d l e s s of the c o n c e n t r a t i o n of BAP in t h e medium. These results contrast w i t h previous reports (Nadgauda et al., 1978; B h a g y a l a k s h m i and Singh, 1988), w h e r e i n root i n i t i a t i o n and d e v e l o p m e n t were found to decline with increasing levels of BAP with total i n h i b i t i o n of rooting beyond 4.0 m g / l BAP. Consequently, an a d d i t i o n a l step was n e c e s s a r y for rooting of shoots. However, we have consistently observed rooting in all the accessions (more than 20) of ginger and Curcuma

Figs. 1-5 Stages of in v i t r o m u l t i p l i c a tion of C. domestica. --i] F r e s h l y inoculated rhizome bud; 2) M u l t i p l i c a t i o n after 3 weeks incubation; 3) After 6 weeks incubation; 4) 4 m o n t h s old culture; 5) Plants established in soil. spp., tocol

so far studied, using the standard i.e. MS + 3.0 mg/l BAP.

pro-

In vitro produced plants were easily e s t a b l i s h e d in soil with a l m o s t 100% survial (Fig. 5). Earlier studies (Hosoki and Sagawa, 1977; Nadgauda et al., 1978) r e p o r t ed only 60-70% survival. The in v i t r o m u l t i plied plants were m o r p h o l o g i c a l l y u n i f o r m and i n d i s t i n g u i s h a b l e from their r e s p e c t i v e parent varieties. In C u r c u m a spp., plants grown in vitro did not s--~-~Q~he c h a r a c t e r istic ~-~rpl---~olouration in the m i d r i b region, but on transfer to soil d e v e l o p e d normal pigmentation. P r e l i m i n a r y investi-

523 Table

i.

Effect

of

cytokinin

c o n c e n t r a t i o n on in v i t r o Z. o f f i c i n a l e Average

Cytokinin

number

per

explant

each

of C u r c u m a

and

4 weeks*

BAP

Kinetin

0.0

-

1.74 c

1.41 c

2.26 a

2.13 e

0.2

-

1.33 c

1.60 b c

1.85 a

2.75 d e

1.0

-

2.19 c

1.91 b c

2.21 a

3.24 cd

2.0

-

2.86 a b

2.18 b

2.75 a

3.57 bc

3.0

-

3.43 a

2.73 a

2.81 a

4.05 a

5.0

-

1.75 +

2.50 +

_

4.03ab

1.0

1.0

2.38 bc

2.04 b c

2.27 a

3.71 bc

Treatment means were Any two means having

+

Average

of a s i n g l e

C. d o m e s t i c a

compared a common experiment

within letter and

~. a e r u g i n o s a

conservation

o f in v i t r o

C.

caesia

Z. o f f i c i n a l e

t h e s p e c i e s and n o t b e t w e e n t h e s p e c i e s . a r e n o t s i g n i f i c a n t l y d i f f e r e n t a t 5% level.

not used

g a t i o n s of i s o z y m e p a t t e r n s of in v i t r o r e generated plants and their parental stocks h a v e i n d i c a t e d t h e g e n e t i c p u r i t y of in v i t r o plants. F u r t h e r s t u d i e s a r e in p r o g r e s s to c o n f i r m a n d m o n i t o r t h e g e n e t i c s t a b i l i t y of in v i t r o r a i s e d p l a n t s .

term

spp.,

(mg/l)

*

Short

of s h o o t s

multiplication

for

statistical

comparison.

c o u r a g e m e n t in t h i s w o r k . We also thank Mr. R.P. Y a d a v , for t e c h n i c a l a s s i s t a n c e . T h e f i n a n c i a l s u p p o r t to this w o r k w a s p r o v i d e d b y t h e D e p a r t m e n t of B i o t e c h n o l o g y , M i n i s t r y of S c i e n c e a n d T e c h n o l o g y , G O v t . of India.

plants

T w o d i f f e r e n t e n c l o s u r e s for c u l t u r e tubes, namely, cotton plugs and polyprop y l e n e c a p s w e r e u s e d t o p r o l o n g the i n t e r val b e t w e e n s u b c u l t u r e s . There was no dif f e r e n c e in t h e m u l t i p l i c a t i o n r a t e r e s u l t ing f r o m t h e t y p e of e n c l o s u r e . The plants in t u b e s e n c l o s e d w i t h c o t t o n p l u g s d r i e d u p w i t h i n 2.5 m o n t h s i n c u b a t i o n (Fig. 6A) . In c o n t r a s t , p l a n t s in t u b e s e n c l o s e d w i t h p o l y p r o p y l e n e c a p s r e m a i n e d g r e e n a n d a l i v e for 7 m o n t h s (Fig.6B) . A l l t h e s e p l a n t s , w h e n subcultured onto multiplication medium, produced normal plants with the same multiplic a t i o n r a t e s as o b t a i n e d b e f o r e . The main r e a s o n for p r o l o n g e d s u r v i v a l of plants, in tubes with polypropylene enclosures, appears to b e d u e to s l o w loss of w a t e r f r o m t h e tubes. T h i s m e t h o d h a s b e e n u s e d to p r o l o n g s u b c u l t u r e i n t e r v a l , in s e v e r a l c r o p p l a n t s in o u r l a b o r a t o r y . This may provide an econ o m i c a l m e a n s to g e r m p l a s m c o n s e r v a t i o n , e s p e c i a l l y in d e v e l o p i n g c o u n t r i e s , w h e r e f a c i l i t i e s f o r l o w t e m p e r a t u r e s t o r a g e of g e r m p l a s m a r e i n a d e q u a t e or n o t f e a s i b l e .

Fig. 6 S h o r t t e r m in v i t r o c o n s e r v a t i o n of ~. a e r u g i n o s a c u l t u r e s at ~ 5 ° C . A) A f t e r 2.5 m o n t h s of s t o r a g e w i t h c o t t o n p l u g enclosures; B] A f t e r 7 m o n t h s of s t o r a g e w i t h p o l y p r o pylene enclosures.

REFERENCES

ACKNOWLEDGEMENTS W e a r e g r a t e f u l to Dr. R . S . P a r o d a , D e p u t y D i r e c t o r G e n e r a l (CS), I n d i a n C o u n c i l of A g r i c u l t u r a l R e s e a r c h , N e w D e l h i a n d Dr. R.S. Rana, D i r e c t o r , N B P G R f o r t h e i r e n -

B h a g y a l a k s h m i , Singh, NS (1988) S c i 63 : 3 2 1 - 3 2 7 . Gomez,

J Hort

KA, G o m e z A A (1984) Statistical P r o c e d u r e s for A g r i c u l t u r a l R e s e a r c h , J o h n W i l e y a n d Sons, Inc., S i n g a p o r e , pp 207-215

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KK (1985) C r y o p r e s e r v a t i o n of Plant Cells and Organs, CRC Press, Inc., Boca Raton, Florida, pp 115-134

K u r u v i n a s h e t t y MS, H a r i d a s a n P, Iyer RD (1982) In : MK Nair et al (eds)

:

Proc Natl Seminar on ginger turmeric, CPCRI, Kasaragod, India, pp 39-41 Murashige T, Skoog F 15 : 473-497

(1962)

and Kerala, Physiol

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Nadgauda RS, M a s c a r e n h a s AF, Hendre RR, Jagannathan V (1978) Indian J Exp Biol 16:~ 120-122 /