Plant Cell Reports

Plant Cell Reports (1996) 15:704-706

9 Springer-Verlag 1996

Micropropagation of Sterculia urens Roxb. - an endangered tree species S . D . Purohit and Ashish Dave Post Box No. 100, Plant Biotechnology Laboratory, Department of Botany, M.L. Sukhadia University, Udaipur-313001, India Received t4 June 1995/Revised version received 26 October 1995 - Communicated by F. Constabel

A b s t r a c t . A n in vitro p r o c e d u r e for large scale multiplication o f S t e r c u l i a urens Roxb. ( G u m Kadaya Tree) has b e e n developed using cotyledonary node segments. A n average o f 4.0 shoots per node were obtained on Murashige and S k o o g ' s (MS) m e d i u m containing 2.0 mg1-1 6-benzyl a m i n o - p u r i n e ( B A P ) w i t h i n 21 days of initial culture. U p o n s u b s e q u e n t subculture 16 shoots/node could be harvested every three weeks and upto three times. Sixty per cent of the shoots w e r e s u c c e s s f u l l y rooted. Rooted plantlets were transferred to plastic pots c o n t a i n i n g soil under mist house conditions before they were finally exposed to an external e n v i r o n m e n t . Fifty seven per cent o f the plantlets survived in n u r s e r y sheds. BAP, 6-benzyl amino-purine; Kn, Kinetin; IBA, indole-3-butyrie acid; IAA, indole-3-acetic acid; NAA, 1-naphthalene acetic acid; 2,4-D, 2,4-dichlorophenoxy acetic acid; MS; Murasbige and Skoog (1962) medium.

Introduction Sterculia urens Roxb. (Sterculiaceae) c o m m o n l y called " G u m K a d a y a Tree" is valued for its g u m k n o w n as "Indian T r a g a c a n t h " . O n c e a c o m m o n p l a n t of A r a v a l l i s in Rajasthan, it has b e e n indiscriminately exploited and is n o w listed as a n " e n d a n g e r e d plant species" in the r e g i o n ( S h a r m a 1993). C o n v e n t i o n a l m e t h o d s o f multiplication such as cutting, b u d d i n g , grafting, and air layering are not available in this species a n d seed germination is very poor. Therefore, a m e t h o d to multiply this plant using m o d e r n m e t h o d s is needed.

Tissue culture methods have been successfully e m p l o y e d for large scale m u l t i p l i c a t i o n of a n u m b e r o f w o o d y plants (Mascarenhas and Muralidharan 1989; Ahuja 1991, T h o r p e et al. 1991; Aitken-Christie and Connett 1992; Correspondence to: S. D. Purohit

Rathore e t al. 1993). T h i s paper d e s c r i b e s a n in vitro procedure for propagation of S. urens using cotyledonary node segments.

Material and Methods Malure dry follicles of Sterculia urens Roxb. were collected from superior trees growing in the forest areas of Aravallis in Rajasthan (India). Seeds were surface sterilized with 0.1% HgC12 for 15 rain, washed thoroughly with sterilized dist. water for 4 times and placed aseptically on 0.8% water-agar. Cotyledonary nodes, epicotyledonary nodes and hypocotyl segments obtained from 20-day 01d in vitro raised seedlings were explanted onto various nutrient media [Murashige and Skoog (MS) (1962), Sehenk and Hildebrandt (SH) (1972), Llyod and McCown, (WP) (1980), 135 (Gamborg et aL, 1968), Broad leaved tree medium (BTM) (Chalupa, 1981) and White (Wh) (1963)]. Different concentrations of BAP and kinetin (Kn) (0.1-5.0 mgl-1) were used individually and in combination for shoot proliferation from the cotyledonary nodes. Also various auxins (NAA, IAA, 2,4-D) were combined with the optimum BAP concentration. Explants were cultured in conical flasks (100 ml) covered with non-absorbent cotton plugs and kept in controlled conditions of temperature (28 _+2~ light (45 ~ttool m2s"I for 16 h aday by fluorescent tubes) and 60-70% relative humidity. Experiments were performed with a minimum of 5 replicates and repeated 3 times. Observations were recorded after an interval of 3 weeks. Explants were'subcultured and shoots harvested every 21 days. For rooting of shoots, auxins tNAA, IAA and IBA) were applied in various concentrations (0.1-5.0 mgl" ) individually in full and in quarter strength MS medium. Also the cut ends of shoots were dipped in various concentrations (50-1500 mgl"t) Of preautoclaved IBA solution for different durations (5-15 rain) followed by their implantation onto 1/4 MS medium. Initially, the cultures were maintained either in darkness or the lower part of culture vessel was kept wrapped with black paper for 5-7 days. Thirty day old rooted plantlets were transferred to 400 ml screw cap glass bottles one-fourth filled with SoilriteTM (Karnataka Explosives Ltd., Bangalore, India) and irrigated with 40 ml MS inorganic salt solution (major salts reduced to one-fourth strength) for in vitro hardeni~,~ Such plants were subsequently shifted to pots containing soil: Soilrite : sand (1:1:1) mixture and kept under lm x lm x lm po lythene covered tent which was spray-mlsted with water to maintain relative humidity of more than 70 %. Both, the media (pH 5.8) and SoilriteTM had been steam sterilized at 1.06 kg cm"2 for 15 rain.

705 Results

a n d

Discussion

On MS medium containing 2.0 mg1-1 BAP (Table - 1) hypocotyl segments produced callus, while epicotyledonary nodes produced one single shoot and cotyledonary nodes generated multiple shoots per explant.

Table 3. Effectof differentcytokininson multipleshoot proliferationfrom cotyledonary nodes of S. urens on MS medium (Observationsrecorded after 21 days) MS + Concentrationof cytokininsin mgld

1. Shoot proliferationfrom different explantsof S. urens on MS medium + 2.0 mgld BAP (Observationsrecorded after 21 days)

Number of shoots • SD

Lengthof shoots • SD (era)

1.3• 1.3• 1.3• 1.3• 2.0• 1.3• 1.6• 1.6• 2.0• 4.3• 1.0• 1.0 • 0.00 2.0 • 0.20 2.6 • 1.15 4.0• 2.6•

0.3• 4.0• 3.1~0.28 3.5• 3.6• 4.1• 2.3• 3.1• 3.1• 3.3• 1.2• 3.2 • 0.28 3.3 • 0.50 3.4 • 0.28 3.6• 2.0•

Table

Explants Hypocotyl Epicotyledonarynode Cotyledonarynode

Mean numberof shoots • SD

Length of shoots • SD (cm)

1.0 • 0.00 4.3 • 0.57

3.2 • 1.22 3.3 • 0.12

Initiation o f s h o o t cultures

I n o c u l a t i o n of cotyledonary nodes on MS m e d i u m without growth regulator generally resulted in a single shoot (0.3 cm). Incorporation of BAP (0.1-5.0 mg1-1) t o the m e d i u m improved shoot proliferation. The number of shoots per explant was highest on MS containing 2.0 mg1-1 BAP (4.3) followed by SH, B5, 3/4 MS, WP, BTM and Wh media (Table-2). Consequently, in all subsequent Table 2. Effectof differentmediaon shoot proliferationfromcotyledonary nodes of S. urens on nutrient medium + 2.0 mg1-1BAP (Observations recorded after 21 days) Type of medium 3NMS MS SH WP B5 BTM Wh

Number of shoots • SD 3.6• 4.3• 4.0• 3.3• 3.8• 2.3• 2.0•

Length of Shoots ~ SD (era) 2.1• 3.3• 2.4• 1.8• 2.1• 4.2• 3.0•

experiments, only MS medium was used. On medium with BAP (2.0 mgl -l) over 4 shoots (3.3 cm) were generated within 21 da_ylsof culture. Kinetin at different concentrations (0.1-5.0 mgl- ) did not improve the number of proliferating shoots except that average shoot length was increased considerably. Combined cytokinins (BAP and Kinetin) and auxins (NAA, I A A , 2,4-D) in combination with BAP (2.0 mg1-1) failed to improve shoot multiplication and caused undesirable callusing (Table 3 & 4). Rooting

Explants with proliferated shoots were subcultured every 21 days on MS medium containing 2.0 mg1-1 BAP. Sixteen shoots per explant (Fig.l) were harvested during each of three subcultures and used for rooting. After that the mother explant ceased to produce new shoots (Cf Aitken-Christie and Jones, 1987; Sharma and Chaturvedi, 1988). Shoots rooted best (40%) on MS medium with IBA. They produced about 2.0 roots/shoot (1.0 cm) and much less callus than IAA and NAA. A pulse treatment of shoots with different concentrations and durations of IBA improved the rooting

Kinetin

BAP

~ntrol 0.1 0.5 1.0 2.0 5.0 0.05 0.25 0.5 1.0 2.5

-

0.1 0.5 1.0 2.0 5.0 0.05 0.25 0.5 1.0 2.5

Table4. Effectof auxinscombinedwith BAP(2.0 mgl"1)on multipleshoot proliferation from cotyledonary nodes of S. urens on MS medium (Observationsrecorded ;after21 days) MS+BAP2.0 mgl1 + Auxin concentrationsin mgl"1 NAA 0.1 0.5

IAA

2,4-D

-

-

Mean Mean length numberof of shoots • shoots • SD SD (cm)

-

-

2.3 • 1.52 2.0 • 0.95 2.0 _ 0.10

-

01

-

2.6

-

0.5

-

2.3 • 0.83

-

1.0

-

-

_

-

-

01

-

-

-

-

0.5

-

-

-

-

1.0

-

-

1.0

•

1.15

1.7 • 0.25 0.7 • 0.10 0.4 • 0 . 1 1 2.4 • 0 . 1 0 2.2 • 0.18

response and minimized the callusing (Table-5). Maximum root induction (60%) was observed w h e n shoots were treated with 500 mg1-1 I B A for 10 minutes prior to implanting them on MS semi-solid medium. The best rooting was observed when the salt concentration of MS was reduced to one quarter (1/4). On this medium shoots usually developed 3.0 roots from the basal end within 15 days (Fig.2). Similar method of rooting of shoots by a dip treatment of auxin has been applied by Harry and Thorpe (1991). The promotory effect of reducing the salt concentration of MS on in vitro rooting of shoots has been described in several reports (Constantine 1978; Skirvin e1 al. 1980). We have found that in S. urens that reducing MS salt strength to 1/4 not only enhanced rooting frequency but also reduced callusing.

706 Table 5. Effect of pulse treatment (5-15 minutes) of different IBA concentrations on shoots of S. urens implanted on 1/4 MS medium (Observations recorded after 30 days)

1/4MS + IBA concentration (in mgl"1)

Time (in minutes)

Per cent rooting

.

50

5 10 15

10 20

1.0 • 0.20 1;0 • 0.50

3.5 • 0.02 3.0 • 0.14

100

5 10 15

25 25 25

1.0 -+0.65 1.0 • 0.10 1.0 +--0.20

2.5 • 0.45 2.8 • 0.37 2.5 • 0.10

500

5 10 15

50 60 50

1.3 • 0.64 3.0 + 0.00 1.0 • 0.10

1.5 • 0.61 2.7 • 0.53 2.4 • 0.49

1000

5 10

-

-

-

-

-

-

15

10

1.0 • 0.20

0.6 • 0,50

5 10 15

10 -

1,0 • 0.50 -

0.5 • 0.35 -

Acknowledgements.

.

Mean length of roots • SD (era)

Control (1/4MS)

1500

.

Mean number of roots • SD

Authors

are

.

thankful

to

Head.

D e p a r t m e n t o f Botany, M . L . S u k h a d i a U n i v e r s i t y , tJdaipur for facilities. References Ahuja MR (1991) Plant Research and Development 55 : 106-120 Aitken-Christie J, Jones C (1987) Plant Cell Tiss Org Cult 8 : 185-196 Aitken-Christie J, Connett M (1992) In : Kurata K, Kozai T (eds) Transplant Production Systems, Kluwer Academic Publishers, Netherlands, pp 163-194 Chalupa V (1981) Commun Inst For Czech 12 : 255-271 Constantine D (1978) Round Table Conference, Gembloux, Belgium pp 134 Gamborg OL, Miller RA, Ojima K(1968) Exp Ceil IRes50 : 151-158

Figure Legends Multiple shoot proliferation from cotyldeonary node of S. urens on MS medium containing 2.0 rag["1 BAP (After 63 days of culture) Fig.2 Rooting of in vitro produced shoot of S. urens implanted on 1/4 MS medium after a pulse treatment of 500 mgl-I IBA for 10 minutes. Fig.1

Transplantation P l a n t l e t s d i r e c t l y t r a n s f e r r e d f r o m r o o t i n g m e d i u m to p o t s s u c c u m b e d due to l e a f d e s i c c a t i o n . T h e survival p e r c e n t a g e c o u l d b e i n c r e a s e d b y a d o p t i n g t h e in vitro h a r d e n i n g p r o c e d u r e d e s c r i b e d in M a t e r i a l and M e t h o d s . D u r i n g in vitro h a r d e n i n g t h e s h o o t s w e r e e l o n g a t e d , leaves turned g r e e n e r a n d e x p a n d e d . S u c h plants w e r e r e m o v e d f r o m the c u l t u r e b o t t l e s a n d w e r e further a c c l i m a t i z e d u n d e r m i s t h o u s e c o n d i t i o n s . H e r e the p l a n t s w e r e k e p t in p o t t i n g m i x t u r e c o n t a i n i n g soil: S o i l r i t e q ~ : sand (1:1:1). By using the a b o v e p r o c e d u r e 5 7 o u t o f 100 plants survived.

Harry IS, Thorpe TA (1991) In : Bajaj YPS (ed) Biotechnology in Agrlc-ulture and Forestry, Vol 16. Springer-Voting, Berlin, Heidelberg pp 408-422 Lloyd GG, McCown BH (1980) International Propagators Society Combined Proceedings 30 : 421-427 Mascarenhas AF, Muralidharan EM (1989) Curr Science 58 : 606-613 Murashige T, Skoog F (1962) Physiol Plant 15 : 473-497 Rathore TS, Deora NS, Shekhawat NS, Singh RP (1993) Biologla Plantarum 35 : 381-386 Schenk RU, Hildebrandt AC (1972) Can J Bot 50 : 199-204 Sharma AK, Chaturvedi HC (1988) Ind J Exp Bio126 : 285-288 Sharma S (1993) In : Jain NC (ed) Proc of Workshop on Tree Improvement and Provenance Research, Dept of Forest, Govt of Rajasthan, Jaipur, pp 120-128 Skirvin RM, Chu MC, Rukan H (1980) Proc III State Hortie Soc 113 pp 30-38 Thorpe TA, Harry IS, Kumar PP (1991) In : Debergh PC, Zimmermann RH (eds) Micropropagation Technology and Application Kluwer Academic Publishers, Dordrecht, pp 311-336 White PR (1963) (Ed) The Cultivation of Animal and Plant Cells, 2nd ed Ronald Press, New York