Vanilla planifolia - Springer Link

Plant Cell Reports (1997) 16:490-494

© Springer-Verlag 1997

P. S. George • G. A. Ravishankar

In vitro multiplication of Vanilla planifolia using axillary bud explants

Received: 15 September 1995 / Revision received: 30 September 1996 / Accepted: 16 October 1996

Abstract A clonal p r o p a g a t i o n m e t h o d has been develo p e d for efficient m u l t i p l i c a t i o n o f Vanillaplanifolia. M u l tiple shoots were d e v e l o p e d f r o m axillary bud explants using s e m i - s o l i d M u r a s h i g e and S k o o g (MS) m e d i u m s u p p l e m e n t e d with N 6 - b e n z y l a d e n i n e (BA, 2 m g 1-1) and c~-naphthaleneacetic acid ( N A A , 1 m g 1-1). The m u l t i p l e shoots were transferred to agitated liquid M S m e d i u m with B A at 1 m g 1 1 and N A A at 0.5 m g 1- t for 2 - 3 weeks, and subsequently cultured on s e m i - s o l i d m e d i u m . U s i n g this method, an a v e r a g e of 42 shoots were o b t a i n e d f r o m a single a x i l l a r y bud explant over a p e r i o d o f 134 days. Use o f an intervening liquid m e d i u m has b e e n found to enhance m u l t i p l i c a t i o n o f shoots in V. planifolia.

natural vanilla flavour, alternative m e t h o d s using cell and tissue culture have been e x p l o r e d r e c e n t l y to e n h a n c e production (Stafford 1991; K n o r r et al. 1993). In vitro multip l i c a t i o n o f V. planifolia has b e e n reported, through the culture o f callus m a s s e s (Gu et al. 1987; D a v i d o n i s and K n o r r 1991), p r o t o c o r m s , root tips (Philip and N a i n a r 1986) and stem nodes ( K o n o n o w i c z and Janick 1984). This p a p e r reports an efficient m i c r o p r o p a g a t i o n s y s t e m for V. planifolia, using a x i l l a r y b u d cultures.

K e y w o r d s Vanilla planifoIia • A x i l l a r y bud • L i q u i d m e d i a • M u l t i p l e shoots • Plantlets

Plant material

Abbreviations BA N 6 - b e n z y l a d e n i n e • DMRT D u n c a n ' s m u l t i p l e - r a n g e test • KC K n u d s o n (1946) m e d i u m • KCB KC b a s a l m e d i u m . Kn kinetin • MS M u r a s h i g e and S k o o g (1962) m e d i u m • MSB M S basal m e d i u m • 1/2 MSB halfstrength M S B - MS-D d o u b l e - p h a s e M S m e d i u m • MS-L liquid M S m e d i u m • MS-S s e m i - s o l i d M S m e d i u m • NAA ~ - N a p h t h a l e n e a c e t i c acid

Introduction Vanillaplanifolia, a tropical orchid, is g r o w n for its fruits, which y i e l d v a n i l l a flavour, used in foods and b e v e r a g e s ( G o o d e n o u g h 1982). W i t h an e v e r - i n c r e a s i n g d e m a n d for

Communicated by G. C. Phillips R S. George ~ . G. A. Ravishankar ([]) Plant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore-570013, India

Materials and methods Stem node sections with dormant axillary buds were excised from 1-year-old field-grown plants of V. planifolia from The Nilgiris, Tamil Nadu, India. These sections, measuring 1.5-2.0 cm, each with one dormant bud, were washed with liquid detergent (Tween 20, Gurr's, UK) and then rinsed in running tap water for 10 min. The cleaned explants were surface sterilized with 0.15% (wt/vol) mercuric chloride for 5 min followed by three rinses in sterile distilled water. Multiple shoot initiation For initiation of multiple shoots, Murashige and Skoog (t 962) (MS) medium supplemented with N6-benzyladenine (BA) or kinetin (Kn) at 1, 2, 5 or 10 mg 1-1 in combination with indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or c~-naphthaleneacetic acid (NAA) at 1 mg1-1 was used. The pH of the medium was adjusted to 5.7, 1% (wt/vol) agar (HiMedia, Bombay, India) was added and the medium was autoclaved at 1.2 kg cm-2 for 20 min. The bases of surface-sterilized explants were inserted into the above media and the cultures were maintained at 25+_2°C under continuous illumination of 37.5 ~tE m-2s 1 provided by fluorescent lamps (four fluorescent tubes, 40 W each, Philips, India). Each treatment had ten replicates and data were recorded after 90 days of culture. Significant differences in multiple shoot initiation among the treatments were tested using the ANOVA F-test followed by Duncan's multiple-range test (DMRT) at the 5% level (Duncan 1955). Multiple shoot proliferation

Present address: Department of Botany, St. Joseph's College, Devagiri, Calicut-673008, Kerala, India

MS and Knudson (1946) (KC) media supplemented with BA (1 mg 1-1 ) and NAA (0.5 mg 1-1) were used for multiple shoot pro-

491 liferation. Three types of culture media were used: (a) 40 ml liquid medium; (b) double-phase medium, consisting of 10 ml of liquid medium on top of 30 ml of semi-solid medium gelled with agar (1% wt/vol), or (c) 40 ml of semi-solid medium gelled with 1% (wt/vol) agar. The cultures on liquid medium (medium a) were placed on a rotary shaker at 90 rpm, while those on double-phase medium (medium b) were static. Each treatment had seven replicates of cultures with two to three shoots and the number of multiple shoots formed was recorded every 7 days over a 28-day culture period. Significant differences in shoot proliferation among the treatments were tested using the F-test followed by DMRT at the 5% level. The shoots grown in medium a for 7, 14, 21 and 28 days were separated into groups of two shoots, transferred onto medium c and maintained for another 30 days. Each treatment had seven replicates and increases in shoot number and length were recorded after 30 days of culture on medium. In vitro rooting and hardening Shoots multiplied in vitro measuring 3 - 4 cm were excised and used for rooting. Media tested for rooting were MS basal medium (MSB), 1/2 MSB or KC basal medium (KCB), supplemented with 0.2% (wt/vol) activated charcoal or 15% (vol/vol) coconut milk. Coconut milk was prepared according to Borkird and Sink (1983). All media contained 1% (wt/vol) sucrose. Each treatment had ten replicates and rooting response was recorded after 60 days. Significant differences in rooting among the treatments were tested using the F-test and DMRT at the 5% level. Rooted plantlets were removed from the media, freed of agar by washing in running tap water and planted in a sand-compost mixture (1:2) at about 80% relative humidity under polyethylene hoods in the greenhouse. The plantlets were hardened for 30 days and then transplanted to the field.

Results and discussion M u l t i p l e shoot initiation M u l t i p l e shoots were initiated from a x i l l a r y bud explants after 2 1 - 2 8 days o f culture (Fig. l a). O f the various treatments (Fig. 2) tested in M S m e d i u m , B A (2 m g 1 1) in c o m b i n a t i o n with N A A (1 m g 1-1) (Figs. 1 b, 2a) resulted in an a v e r a g e o f 5.7 shoot buds p e r e x p l a n t after 90 days of culture. The n u m b e r o f shoots o b t a i n e d in this t r e a t m e n t was s i g n i f i c a n t l y different from other treatments at the 5% level. In contrast, shoot e l o n g a t i o n was m o r e p r o n o u n c e d in kinetin treatments (Fig. 2d).

M u l t i p l e shoot p r o l i f e r a t i o n Shoot buds m a i n t a i n e d in liquid M S m e d i u m (Fig. 1 c) s h o w e d m o r e than t w o - f o l d greater i n c r e a s e in shoot n u m ber (Fig. 3, treatment M S - L ) than those p l a c e d in either d o u b l e - p h a s e M S m e d i u m (Fig. 3, treatment M S - D ) or s e m i - s o l i d MS m e d i u m (Fig. 3, treatment M S - S ) . The increase in shoot n u m b e r using M S - L was m o r e than t h r e e - f o l d better, than in the K C m e d i a (Fig. 3, treatments K C - S , K C - D or K C - L ) . The e n h a n c e m e n t in shoot n u m ber in M S - L was s i g n i f i c a n t l y different at the 5% level. Use o f the d o u b l e - p h a s e m e d i u m did not e n h a n c e shoot m u l t i p l i c a t i o n in c o m p a r i s o n to liquid or s e m i - s o l i d m e d i a c o n t a i n i n g the s a m e g r o w t h regulators.

Fig. l a - d

In vitro multiplication of Vanilla planifolia, a Multiple shoot initiation from axillary bud explant on MS medium with BA (2 mg 1 1) and NAA (1 mg 1-1). b Multiple shoot proliferation on semi-solid MS medium with BA (2 mg I 1) and NAA (1 mg 1-1). e Multiple shoot proliferation in liquid MS medium with BA (1 mg 1-1) and NAA (0.5 mg 1-1). d Plantlets in greenhouse during hardening

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T h e shoots o b t a i n e d f r o m c u l t u r e s g r o w n in M S - L m e d i u m for 7, 14, 21 or 28 days (Fig. 3) w e r e t r a n s f e r r e d to M S - S m e d i u m a n d c u l t u r e d for 30 days. T h e best r e s p o n s e in shoot g r o w t h a n d m u l t i p l i c a t i o n o n M S - S after 30 d a y s was in shoots o b t a i n e d f r o m 14 days in M S - L , w h e r e a n a v e r a g e o f s e v e n shoots per i n o c u l u m o f t w o shoots was o b t a i n e d (Fig. 4). T h e shoots o b t a i n e d f r o m 21 or 28 days

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Fig. 5 In vitro rooting response in V. planifoIia. Treatments 1-3: MSB; 4-6:1/2 MSB; 7-9: KCB. Treatments 2, 5 and 8 were supplemented with activated charcoal (0.2% wt/vol) and treatments 3, 6 and 9 with coconut milk (15% vol/vol). All media contained 1% (wt/vol) sucrose. Data were recorded after 60 days Comparison among the treatments for rooting. Means underscored by the same line are not significantly different Treatment Mean

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in MS-L resulted in an average of 3.8 or 3.4 shoots per inoculum, respectively, after 30 days on MS-S. Moreover, the shoots from 21 -day and 28-day treatments showed poor survival (83 and 41%, respectively) during the 30-day culture on MS-S. This is due to the stunted and hyperhydric nature of the shoots obtained after 21 or 28 days in liquid medium. On the other hand, shoots obtained from 14 days treatment in MS-L showed vigorous growth and multiplication during 30 days culture on semi-solid medium. These shoots were comparable in quality to those obtained from cultures maintained in MS-S. Hence, in the propagation cycle, the shoot cultures were maintained in liquid medium for 14 days before they were transferred to the semi-solid medium.

In vitro rooting and hardening The number of roots formed per shoot was significantly different among the treatments (Fig. 5) as shown by the F-test and DMRT at the 5% level, whereas the differences in root length obtained were not significant. Root induction occurred in all the shoots tested in treatments supplemented with 0.2% (wt/vol) activated charcoal. The lowest number of roots per shoot was obtained in KCB (Fig. 5, treatment 7). In media supplemented with activated

charcoal (treatments 2, 5 and 8), the maximum number of roots was obtained in 1/2 MSB medium (treatment 5). The rooting response clearly showed enhanced root induction in media supplemented with activated charcoal. The roots formed in vitro were well developed and the plantlets (Fig. 1 d) hardened for 30 days in the greenhouse thrived after transplantation to the field. This study demonstrated that the multiplication system comprising (a) a semi-solid phase for the initiation of multiple shoots, (b) a liquid phase for proliferation of shoots, followed by (c) a semi-solid phase for subsequent growth resulted in enhanced shoot multiplication in V. planifolia. During 90 days of culture on semi-solid medium, an average of 5.7 shoots per explant was obtained (Fig. 2 a). These shoots, when separated into two clusters of 2 - 3 shoots and transferred to liquid medium for 14 days produced an average of 12 shoots per explant (Fig. 3, treatment MS-L). These shoots were then separated into clusters of 2 shoots each and transferred to the semi-solid medium for 30 days. This resulted in an average of 7 shoots per inoculum (Fig. 4). Using this method, an average of 42 shoots of 3 - 4 cm in length can be obtained from a single axillary bud explant over a period of 134 days. On the other hand, 5.7 shoots per explant obtained after 90 days, when transferred to semi-solid medium for 14 days, yielded an average of 9 shoots per explant (Fig. 3, treatment MS-S). These shoots on subculture onto semi-solid medium for 30 days gave a maximum of 18 shoots. This clearly shows the advantage of using an intervening liquid medium to enhance shoot multiplication. Combination of liquid and semi-solid media in the same culture vessel has been reported to enhance axillary bud and shoot production in many dicots, monocots and ferns (Molnar 1987). In the present study, this experimental design did not favour shoot multiplication in V. planifolia. Establishment of propagation systems employing successive semi-solid and liquid media have been reported for Lilium hybrids (Simmonds and Cumming 1976) and daylilies (Krikorian and Kann 1979). Earlier reports have dealt with the plantlet regeneration of V. planifolia from callus (Gu et al. 1987; Davidonis and Knorr 1991), or root tips (Philip and Nainar 1986). However, regenerants from callus were not desirable in clonal propagation due to the potential for variation. Root-meristem-derived plantlets, as reported, only established the regeneration potential of root tip explants. Kononowicz and Janick (1984) reported an in vitro propagation method using node explants, but the rate of multiplication was lower than that reported in this study. Hence, the present investigation is of importance in demonstrating efficient multiplication and clearly defining the rate of multiplication at various stages of in vitro propagation of V. planifolia.

Acknowledgement RS.G. is grateful to the Council of Scientific and Industrial Research, New Delhi, India, for the award of a Senior Research Fellowship. The authors acknowledge the help of Mr. S. Dhanraj and Mr. R Manilal in carrying out the statistical analysis.

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Kononowicz H, Janick J (1984) In vitro propagation of Vanilla planifolia. Hort Science 19:58-59 Krikorian AD, Kann RP (1979) Clonal propagation of daylilies. In: Sharp WR, Larsen PO, Paddock EF, Raghavan V (eds) Plant cell and tissue culture. Ohio State University Press, Columbus, pp 835-836 Molnar GY (1987) A new patented method for mass propagation of shoot cultures. Acta Hort 212:125-130 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-497 Philip VJ, Nainar SAZ (1986) Clonal propagation of Vanilla plan# folia (Salisb) Ames using tissue culture. J Plant Physiol 122: 211-215 Simmonds JA, Cumming BG (1976) Propagation of Lilium hybrids. II. Production of plantlets from bulb scale callus cultures for increased propagation rates. Sci Hort 5:161-170 Stafford A (1991) The manufacture of food ingredients using plant cell and tissue cultures. Trends Food Sci Technol 2:116-122