WO2003079768A1

WO2003079768A1 - A method for the clonal propagation of vanilla

Info

Publication number: WO2003079768A1
Application number: PCT/IB2002/001149
Authority: WO
Inventors: Neelwarne Bhagyalakshmi; Gokare Aswathanarayana Ravishankar; Puthenpurakkal Sebastian George
Original assignee: Council of Scientific and Industrial Research CSIR
Current assignee: Council of Scientific and Industrial Research CSIR
Priority date: 2002-03-26
Filing date: 2002-03-26
Publication date: 2003-10-02
Anticipated expiration: 2004-09-26
Also published as: AU2002249513A1

Abstract

The present invention provides an improved method for the clonal propagation of vanilla planifolia, and provides specific nutrient medium with growth regulators which help establishment of shoot bud cultures and rapid aseptic multiplication of vanilla shoots which are subsequently hardened to obtain high quality vanilla planting material.

Description

AMETHOD FORTHE CLONAL PROPAGATION OFVANILLA

Technical field:

The present invention relates to an improved medium and a method for the clonal propagation of vanilla.

Background of the invention:

Plant Tissue Culture (PTC) is a major area biotechnology which has direct impact on present day agriculture. There is a need for enormous supply of desired type of planting / sowing material obtained by applying tissue culture methods. At present, several laboratories in the world are producing over 500 million plants annually.

There are various advantages of tissue culture (TC) plants over the conventional methods of plant propagation. As TC plants are produced in completely controlled environment, the chances of carrying systemic diseases are rare. The plants are induced to multiply at a tremendous rate by developing specific medium formulation for each plant material. Maximum number of plants can be produced using minimum space, time and nutrients. Similarly, storage and transportation can be handled with lesser cost than conventional plants. It facilitates the availability of planting material throughout the year. Vanilla planifolia is one of the most economically important members of Orchidacea (Childers et al, 1959, In: Orchids, a scientific survey, ed by Wither, C. L. Ronald Press, New York, pp. 477-508). This family includes most expensive ornamentals consumed both as pot plants and cut flowers. Vanilla plants are of high commercial value at all stages of its life cycle. Due to its monopodial growth style with thick beautiful long lasting foliages, it has ornamental value also. The flowers of vanilla also make expensive ornamental having demand in cut flower market throughout the world. Finally, the fruits, (the vanilla beans) are of high market value due to its application for the production of vanilla flavour. The world consumption of vanilla beans is over 1800 tonnes annually. Vanilla beans cost US $ 70-75 per kilogram and the natural vanillin (the flavour extract) is priced at US $ 3200-3500 per kg. The specific agro-climatic requirements, slow growth of the plant, tedious artificial pollination, time consuming and labour intensive curing procedures, make vanillin very expensive (H&R, 1993, Vanilla the Queen of spices, H&R Contact, 59: 15-19). Vanillin is the major constituent of vanilla flavour, which in combination with volatile oils, alcohols, aromatic aldehydes, ketones and esters impart the characteristic aroma and flavour. Vanilla extract has been reported to possess anti-oxidant properties. Owing to the high economic importance, there is a continuous demand for the planting material, and hence the rapid production of plantlets by tissue culture means has tremendous scope.

Trials have been made by earlier workers to apply TC techniques to multiply vanilla plant material. Reference may be made to Cervera and Madrigal, 1981 (Env. Exptl. Bot. 21 : 441-443) wherein shoot-tip explants were induced to grow in the tissue culture conditions wherein the drawback was the poor multiplication rate of only 2 - 3 shoots per each bud culture. Reference may also be made to Jerret and Fernandez (1984 Plant Genetic Resources Newsletter 57: 25-27) who demonstrated germplasm preservation of vanilla material wherein the drawbacks are that the method aimed mainly at the long-term preservation of plant material.

Reference may also be made to Konowicz and Janick (1984, HortScience 19: 58-59) reporting tissue culture method for the establishment of rooted plantlets wherein the drawback is that the method emphasized mainly on the hardening (meaning the acclimatization) of tissue culture plants.

The drawbacks associated with the methods followed hitherto is that the conventional method of propagation by cutting requires bulky (60-120 cm-long) plant material which is often associated with the sacrificing of the selected high yielding mother plant (Singhal, 1995, Hand Book of IARI, New Delhi, Vikas Publishing House, pp. 369-375). Conventional method of propagation transmits systemic infection present in the cuttings acquired from one generation to the next leading to diseased planting material. The rate of multiplication is extremely slow due to the presence of only one growth meristem resulting in sympodial growth, which is extremely slow.

Accordingly, the present invention provides improved medium formulation for the clonal propagation of vanilla, wherein the development of specific nutrient medium with growth regulators help establishment of shoot bud cultures and rapid further aseptic multiplication of vanilla shoots which may subsequently be hardened to obtain high quality vanilla planting material. The invention uses axillary buds for the tissue culture initiation and further multiplication of shoot clusters. This method has been observed to be the most reliable method since genetic stability is ensured (Boxus 1987, Nestle Research News 1986/87, Nestle Ltd., Switzerland 72-79; Vasil 1994, Plant Cell Tissue and Organ Culture, 39: 105-108). Objects of the invention:

The main object of the present invention is to develop an improved medium formulation for the clonal propagation of vanilla, which obviates the drawback as detailed above.

Another object of the present invention is to provide an improved nutrient medium formulation for the establishment of aseptic shoot cultures of vanilla using both terminal and lateral shoot buds as explants and using tissue culture methods.

Yet another object of the present invention is to experiment and find out the most suitable nutrient culture medium as well as growth regulators composition for maximum multiplication of shoot cultures. Still yet another object of the present invention is to test the right combination of light and temperature conditions for maximizing the multiplication of the shoot cultures.

Still yet another object of the present invention is to explore the nutrient requirements for the development of roots from shoot cultures to facilitate easy establishment of vanilla plantlets when transferred to soil. Still yet another object of the present invention is to provide an improved vanilla plantlet production process for the exchange and conservation of disease-free vanilla germplasm.

Description of the invention:

Accordingly, the invention provides an improved method for the clonal propagation of vanilla, which comprises of Murashige and Skoog's medium supplement with 2-10% sucrose 0.1-5.0 mg/lt. naphthalene acetic acid; 0.2-10 mg/lt of benzylamino purine; 75-

125% Murashige and Skoog's micro and macro salts; 0.5-1.2%) agar at pH level of 5.4-6.2 and sterilized by autoclaving, where the surface sterilized shoot buds grow to form shoot cultures.

In an embodiment, the shoot cultures of vanilla are multiplied on improved Murashige and Skoog's liquid medium comprising 85-110% macro and micro salts of Murashige and

Skoog; 2-10%) sucrose; 0.1-5.0 mg/lt. of naphthalene acetic acid; 0.2-6.0 mg/lt. of benzyl aminopurine at pH levels of 5.4-6.2 and sterilized by autoclaving wherein the shoot cluster culturing is on a gyratory shaker of 80-130 rotations per minute at 20-35 degree C in the presence of cool white light for a period of 3-6 weeks.

In yet another embodiment, the said multiple shoot bud cultures are transferred to a similar solid medium as referred above and containing 8-12 gr/ It. of agar; autoclaved; the shoot clusters from liquid medium are incubated at 20-35 degree C in the presence of cool white light for a period of 3-6 weeks where further multiplication and elongation takes place. In another embodiment, the said multiple shoot bud cultures are transferred to a solid medium of Murashige and Skoog comprising 25-75% macro and micro salts of Murashige and Skoog; 1-5%) sucrose; 10-15% volume by volume of coconut water, 8-12gr/lt. of agar at pH levels of 5.4-6.2 and sterilized by autoclaving wherein the elongated shoots where cultured at 20-35 degree C in the presence of cool white light for a period of 3-6 weeks. In still another embodiment, the multiplication of shoots of vanilla comprises of a) cutting one or more shoot buds of vanilla, b) removing contaminants from the surface of buds, c) cutting the decontaminated shoot buds to obtain small buds/meristems, d) cutting the meristems / small shoot buds from step (iii) where in culturing is at 20-

35 degree C in dark in a medium capable of producing organised shoot buds / shoots, e) continuing culture of said meristems / small shoot buds until organized shoot buds/ shoots are formed, f) re-culturing the shoots obtained in steps (v) in a medium capable of inducing multiple shoots, g) transferring the shoots for rooting and partial hardening to an improved medium capable of supporting root formation, h) transferring to soil of the rooted / hardened vanilla shoots for further growth. The present invention is meant to provide an improved medium formulation for the clonal propagation of vanilla, which comprises of nutrient medium with macro and microelements, similar to those of mineral salts (Murashige and Skoog's medium available in literature, Physiol. Plantarum 15: 473-497, 1962, generally known as MS medium) and the growth regulators to induce rapid multiplication of shoot cultures, without forming callus and other abnormal outgrowths, which involves identifying healthy vanilla mother plants from which shoot bud material has to be collected and the shoot cuttings with lateral/terminal buds are washed using tap water to get rid of soil and surface debris, which are later appropriately trimmed, surface sterilized under aseptic conditions using standard sterilant (0.1 % w/v aqueous mercuric chloride solution), followed by several rinses with sterile distilled water and dissection of the shoot bud to obtain healthy smaller shoot bud/meristem under aseptic conditions, planting the shoot budVmeristem material on nutrient medium and incubation of cultures and finding out the right growth regulator combination for the development of aseptic shoot buds, changing the nutrient composition and growth regulators to induce rapid multiplication of shoot cultures, transferring the shoots of 3-4 cm to a fresh medium for root growth, transferring the small shoots back to fresh medium for further multiplication and the rooted shoots to soil for hardening and transferring to garden.

In an embodiment of the present invention explants from vanilla shoot cuttings are surface sterilized, aseptically dissected in laminar air-flow and shoot buds are isolated for transfer to improved Murashige and Skoog's tissue culture medium with 3%> w/v of sucrose as the carbon source and various combinations of growth regulators for shoot bud establishment.

In another embodiment of the present invention the cultured shoot buds are subjected for shoot multiplication using improved Murashige and Skoog's nutrient medium with changes in the concentrations of growth regulators to induce multiple shoot cultures.

In yet another embodiment of the present invention, the multiple shoots may aseptically be transferred to yet another improved Murashige and Skoog's medium with reduced levels of nutrients to induce roots helpful for subsequent transfer of the plantlets to soil.

The following examples are given by way of illustration of the present invention and therefore, should not be considered to limit the scope of the invention.

Example 1 Establishment of aseptic cultures:

Shoot buds of healthy vanilla plants are collected and the 2-3 cm long shoot cuttings with lateral/terminal shoot buds are treated to remove bacterial/fungal (contaminants) by dipping the shoot cuttings in 0.1 % w/v of freshly prepared aqueous mercuric chloride solution for 6 minutes followed by several rinses with sterilised distilled water. The shoot buds are aseptically removed by cutting with sterile scalpel under laminar flow and cultured on a first nutrient medium, namely Murashige and Skoog's medium. This first culture medium is prepared by dissolving in water the following nutrients:

ed to homogeneity, dispensed to culture bottles and the screw-capped culture bottles with medium are autoclaved at 121 degree C, 15 lb/cm² for 20 minutes. The surface sterilized shoot cuttings are then placed, one at a time using sterile forceps, in a sterile petri-dish in a laminar flow, the meristem and small shoot-bud explants are separated from the shoot cutting explants with the help of sharp scalpel blades, further appropriately trimmed to obtain small shoot buds and meristem tips and placed on sterile medium contained in the culture bottles. The shoot buds and meristems placed on the sterile nutrient medium are incubated in dark at 25±2 degree C until tiny shoot cultures are established which may take 4-6 weeks period. In addition to forming tiny shoot buds, there may be a six-fold multiplication in the number of shoot buds during this treatment. The response of initial explants to form shoot buds and tiny shoots during this treatment may be upto 40%. The various other levels and combinations of naphthalene acetic acid and benzyl amino purine and other growth regulators such as 2,4-dichlorophenoxy acetic acid, indole acetic acid, indole butyric acid, added to the nutrient medium, may result in poor response or lesser percentage of response to form shoot cultures.

The medium without any growth regulator or lesser sucrose levels may not support the establishment of shoot cultures from meristem/bud explants. Example 2.

Multiplication of aseptic shoots: The shoot cultures established as in example 1 has to be used as the explant material for the clonal multiplication of vanilla shoots. The shoot buds established as in Example 1 are removed aseptically in a laminar flow chamber and placed on a sterile petri plate. If there is clustered shoot bud formation, each bud is separated by cutting using sterile scalpel. About three to four shoot buds are then placed on a liquid nutrient medium containing the macro-elements, microelements, vitamins and sucrose of Murashige and Skoog medium (MS, 1962, Physiol. Plantarum 15:473-497) are similar to those in Example 1 with the following nutrients in the medium is prepared by dissolving in water the following nutrients:

The pH of the second (liquid) medium is adjusted to 5.8 and boiled to homogeneity, dispensed about 40 millilitre to conical flasks and the medium autoclaved at 121 degree C, 15 lb/cm for 20 minutes. The shoot buds are incubated in the second nutrient liquid medium with 2 mg/L of benzyl amino purine and 1 mg/L of naphthalene acetic acid and kept on rotary shaker with a gyration speed of 90 rpm under a photoperiod of 16h with an illumination of 2000 lux at 25±2 degree C for a period of 7 days and then transferred aseptically, in a laminar flow chamber, to a third fresh agar-gelled medium, the composition of which is exactly the same as for liquid medium with the addition of 10 grams/Litre of agar-agar. The cultures were incubated for 21 days under a photoperiod of 16h with an illumination of 2000 lux at 25±2 degree C, by which time each shoot bud forms nearly six shoots. The shoots are then separated aseptically, the larger ones are transferred to a fourth nutrient medium for rooting as in Example 3, and the smaller ones are transferred to the second (liquid) medium and grown for a period of seven days and then transferred to the third (solid) medium and grown for 21 days, the process is repeated to obtain a large number of shoots. Example 3. The larger shoots multiplied as in Example 2 are transferred to a fourth medium developed for rooting and partial hardening containing the following nutrients in water:

The plantlets were nurtured for 3 weeks where 100% rooting may be encountered which is accompanied by further growth of shoots. The rooted shoots are washed in running tap water, and then transferred to soil compost containing 1 part of red soil and 2 parts of garden humus, and kept in a place with 90% humidity for another three weeks period. The hardened plantlets may be transferred to green house till they are planted in the farm. Advantages: 1. The improved medium formulation invented in the present study improves the level of shoot bud establishment as aseptic shoot bud cultures of Vanilla planifolia. 2. The improved medium formulation invented in the present study for shoot multiplication allows 6-fold increase in the shoot number in 4 weeks period, which is the highest rate of multiplication for Vanilla planifolia plants. 3. By using the improved medium formulation invented here, continuous multiplication of vanilla plants throughout the year is possible. 4. By using the improved medium formulation invented plant material free from saprophytic, fungal and bacterial diseases may be ensured.

Claims

CLAIMS:

1. An improved method for the clonal propagation of vanilla, comprising: a) culturing shoot explants of Vanilla planifolia with lateral or terminal buds in a medium comprising Murashige and Skoog's medium supplemented with 2-10% sucrose, 0.1-5.0 mg/lt. naphthalene acetic acid; 0.2-10 mg/lt of benzylamino purine; 75-125%) Murashige and Skoog's micro and macro salts; 0.5-1.2%) agar at pH level of 5.4-6.2 and sterilized by autoclaving, where the surface sterilized buds grow to form shoot cultures, b) multiplying of shoot cultures of vanilla on improved Murashige and Skoog's liquid medium comprising 85-110%) macro and micro salts of Murashige and Skoog; 2-

10% sucrose; 0.1-5.0 mg/lt. of naphthalene acetic acid; 0.2-6.0 mg/lt. of benzyl aminopurine at pH levels of 5.4-6.2 and sterilized by autoclaving wherein the shoot cluster culturing is on a gyratory shaker of 80-130 rotations per minute at 20-35 degree C in the presence of cool white light for a period of 3-6 weeks, c) the multiple shoot bud cultures of step (b) are transferred to a solid medium similar to used in step (b) and containing 8-12 gr/ It. of agar and are autoclaved, d) the shoot clusters from liquid medium are incubated at 20-35 degree C in the presence of cool white light for a period of 3-6 weeks where further multiplication and elongation takes place, e) the multiple shoot bud cultures are transferred to a solid medium of Murashige and Skoog comprising 25-75%> macro and micro salts of Murashige and Skoog; l-5%> sucrose; 10-15% volume by volume of coconut water, 8-12gr/lt. of agar at pH levels of 5.4-6.2 and sterilized by autoclaving wherein the elongated roots and cultured at 20-35 degree C in the presence of cool white light for a period of 3-6 weeks, f) transferring the shoots for rooting and partial hardening to an improved medium capable of supporting root formation, g) transferring to soil of the rooted / hardened vanilla shoots for further growth.

2. A method as claimed in claim 1 wherein the medium used in step (a) comprises:

3. A metho

5. A method as claimed in claim 1 wherein the medium used in step (f) consists of:

5. An improved medium as claimed in claim 1 wherein the multiplication of shoots of vanilla comprises of : a) cutting one or more shoot buds of vanilla, b) removing contaminants from the surface of buds, c) cutting the decontaminated shoot buds to obtain small buds/ meristems, d) cutting the meristems / small shoot buds from step (iii) where in culturing is at 20- 35 degree C in dark in a medium capable of producing organised shoot buds / shoots, e) continuing culture of said meristems / small shoot buds until organized shoot buds/ shoots are formed, f) re-culturing the shoots obtained in steps (v) in a medium capable of inducing multiple shoots, g) transferring the shoots for rooting and partial hardening to an improved medium capable of supporting root formation, and h) transferring to soil of the rooted / hardened vanilla shoots for further growth.

6. A medium for culturing explants of vanilla planifolia with terminal or lateral buds comprising:

7. A medium for multiplying shoots of vanilla planifolia comprising:

8. A medium for rooting and partial hardening oϊ vanilla planifolia shoots consisting of: