A commercially viable protocol for mass propagation of Vanilla tahitensis, a cultivated species of vanilla, was standardized. A multiplication ratio of 1 : 4.7 was observed over a culture period of 60-70 days on benzylaminopurine (1 mgl-1) and a-naphthaleneacetic acid (O.1 mgl-1).
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R Lakshmanan, K J Madhusoodanan, K Mary Mathew, Y S Rao, Gilu Lizy George

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of Spices and Aromatic Crops 9 (2) : 171-173 (2000) Invitro propagation of Vanilla tahitensis Moore K Mary Mathew, Y S Rao, Gilu Lizy George, R Lakshmanan & K J Madhusoodanan , . Indian Cardamom Research Institute Kochi - 682 025, India. Received 10 November 1999; Revised 17 February 2000; Accepted 3 March 2000. Abstract. A commercially viable protocol for mass propagation of Vanilla tahitensis, a cultivated species of vanilla, was standardized. A multiplication ratio of 1 : 4.7 was observed over a culture period of 60-70 days on benzylaminopurine (ImgP) and a-naphthaleneacetic acid (O.lmgl'I). Key words: micropropagation, plantlets, Vanilla tahitensis. Abbreviations: BAP : 6-Benzy laminopurine IAA : Indole-3-butyric acid KN : Kinetin MS : Murashige & Skoog medium NAA : a-Naphthaleneacetic acid Vanilla planifolia Andrews is the most commonly cultivated species of vanilla of commerce apart from V. pompona Schiede and V. tahitensis Moore. V. tahitensis commonly called 'Tahiti vanilla', indigenous to Tahiti Island is of recent introduc-tion to India and no data on its field performance is available. Hence, en masse production of plant-ing materials is a pre-requisite for establishment of plantations. In India, little variation is observed in V. planifolia since it has a narrow genetic base (Ravindran et al. 1996). Introduction of plant material which is predominantly vegetatively propagated is hardly likely to exhibit any variability (Purseglove et al. 1988). This would probably be one of the reasons for the limited work done on vanilla improvement by breeding. This paper presents a micropropagation protocol for V. tahitensis that may enable mass production of plantlets for establishment of plantations and their utilization in breeding programmes of vanilla. Nodal segments were excised from vines of V. tahitensis raised at the Germplasm Repository of Indian Cardamom Research Institute, Spices Board, Kerala, India. Disinfection and surface steriliza-tion of explants was performed as described earlier (Mary et al. 1999). MS basal salts (Murashige & Skoog 1962) supplemented with BAP, IAA or NAA was used for culture experiments. Sucrose was added at 3% and agar at 0.65% and the pH was adjusted to 5.8. Cultures were incubated at 25 ± 2°e and 16 h photoperiod at an intensity of 2500 lux. Proliferated buds from established primary cul-tures were subcultured on the same medium for multiplication. For further shoot multiplication, well developed shoot buds were transferred to elongation and rooting media. MS basal salts were supplemented with KN and or NAA as growth regulators. The micropropagated plants were maintained under intermittent mist for 2-3 weeks in a glasshouse prior to transfer to the greenhouse. For greenhouse planting, a mixture of garden soil and sand (1:1) and soilrite mixture was used. Data for 172 Mathew et al. Fig 1 Micropropagation of Vanilla tahitensis a. In vitro multiple shoot formation h. Rooted plantlets c. Hardened plant shoot multiplication and percentages of rooting and hardening were recorded. Primary cultures of V. tahitensis were established within S-<'i weeks of culture initiation. Among the various growth regulators tested, MS with BAP at 1mgl-' and NAA at 0.1 mgl-' proved most suitable for shoot multiplication and for further growth and development of shoots (Table 1). lAA was the most suitable auxin for in vitro multiplication of V. planifolia (Mary et al. 1999; Rao et al. 1999) whe-Table 1. Multiple shoot regeneration in Vanilla tahitensis reas NAA was best suited for V. tahitensis. NAA when used in V. planifolia cultures promote exces-sive aerial root formation in multiplication sub-culture cycles, whereas in V. lahitensis this was not observed. Repeated sub culture on the afore-said medium resulted in production of a large nu-mber of buds with an average multiplication ratio of 1:4.7 at each cycle (Fig 1 a) over a culture period of 60-70 days (Table 1). The shoots were separated from multiple shoot clumps and cultured on Growth No. of multiple Shoot No. of days . in culture regulator shoots/explant morphology (mgl-') BAP 0.5 + NAA 0.2 BAP 1.0 + NAA 0.1 BAP 1.0 + KN 1.0 + NAA 0.1 BAP 3.0 Basal medium : MS 2.0 4.7 4.8 Thin stemmed; rooting profuse Healthy, vigorously growing Clustered, short shoots Yellowish callus; nodular protocorm like growth without much differentiation SO--UO 60-70 60-70 70-90 ;~ :-~ Mi,:ro}?rOjpa8:ati,on in Vanilla tahitensis for elongation and rooting. It was nCltic,ea that simultaneous elongation and rooting oc,cw"~'~ in 95% shoots on medium with reduced 'levels of BA (0.1 mgl") and NAA, (0.2 mgJ-l) (Fig 1b). In V. planijolia (Mary et al. 1999), KN (0.2 mgl") was effeclive for shoot elongation and , rooting. Plantlets attained a height of 6-7 cm with healthy leaves, aerial roots and one or two basal roots, within 65-70 days. subsequently, the plantlets were removed from the cultures, washed free of agar, and transferred to pots containing soil/ soilrite mixture. They were transferred tei a mist chamber with 80-90% relative humidity for 2 to 3 weeks and thereafter hardened in the greenhouse for 3D-45 days (Fig 1c) and subsequently trans-planted in the field under partial shade. The survival percentage was 70-80% showing that micropropagation technique can be effeclively used for mass propagating V. tahitensis. The technique used is simple, efficient and reproduc-ible and 500 plantlets were produced using the protocol described. 173 References Mary Mathew K, Rao Y S, Pradip Kumar K, Madhusoodhanan K J & Potty S N 1999 In vitro culture systems in Vanilla In : Kavi Kishor P B (Ed.) Plant Tissue Culture and Biotechnology -Emerging Trends (pp. 171-179). University Press (India) Ltd., Hyderabad. Murashige T & Skoog F 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 15 : 473-497. Purseglove j W, Brown E G, Green C L & Robbins S Rj 1988. Vanilla. In: Spices Vol. 2 (pp. 657-{;58). Longmani UK. Ravindran P N, Peter K V, Nirmal Babu K, Rema j, Samsudeen KI Minod D, Geetha S P, Sajina A, Mini P M, Manjula C & john C Z. 1996. Biotechnological approaches in spice crops -present scenario and future prospects. In : Biotechnology for Development. A Compen-dium of Essays (pp. 179-180). State Committee on Science, Technology and Environment, Kerala. Rao Y S, Mary Mathew K, Pradip Kumar K, Madhusoodhanan K j, Potty S N & George C K 1999 A Manual for Micropropagation of Se-lected Spices. Indian Cardamom Research Insti-tute, Spices Board, Kochi. 

In vitro propagation of Vanilla tahitensis Moore

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In vitro propagation of Vanilla tahitensis Moore

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