and Skoog (MS) media supplemented with 0.1 mg/NAA (á-naphthalene acetic acid) and 15% coconut water. (CW) was found the best formation for seed ...
World Journal of Agricultural Sciences 10 (4): 163-168, 2014 ISSN 1817-3047 © IDOSI Publications, 2014 DOI: 10.5829/idosi.wjas.2014.10.4.1819
Efficient in vitro Cultural Techniques for Seeds Germination of Vanda roxburghii Md. Rafiqul Islam, 1Khandakar Md. Rayhanul Kabir, Md. Sabir Hossain, 3Md. Fuad Hossain and 4Md. Ibrahim Khalil 1
2
Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia 7003, Bangladesh Department of Biochemistry and Molecular Biology, Jahangirnaar University, Savar, Dhaka, Bangladesh 3 Department of Agriculture Biology, University of Ruhuna, Sri Lanka 1
2
4
Human Genome Center, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia
Abstract: Since orchid seeds lack endosperm, their germination in nature is very limited. Moreover, ruthless collections, destruction of habitats by reclamation and deforestation, unauthorized trade have led to reduction in natural population of many orchid species. In the meantime some of the orchid species have been extinct and a large number of species have become rare and endangered. It is therefore important to take step for saving this group of plants from extinction and also to support their quick propagation to meet up our demand for their ornamental, medicinal and environmental need. This study was carried out to develop a suitable protocol for in vitro seed germination of an indigenous orchid, Vanda roxburghii in different nutrient media. Murashige and Skoog (MS) media supplemented with 0.1 mg/NAA (á-naphthalene acetic acid) and 15% coconut water (CW) was found the best formation for seed germination and subsequent plantlet development. This media composition also shortens the germination period from 35 days to 22 days only. In conclusion, this protocol could be useful for biodiversity conservation and commercial application for Vanda seed germination. Key words: Orchid
Vanda roxburghii
Germination
INTRODUCTION
The name Vanda, came from India language, means that people like these plants by their fragrance, color and the shape of flower. The genus was established by Sir W. Jones in 1795 and his type species is Vanda roxburghi. There are about 80 species plants, native to China the Himalayas, Bangladesh, Indonesia and northern Australia. Vanda is monopodial orchids and most are epiphytic. In Bangladesh, it can be found on trunks and branches of trees. Sometimes it grows as lithophyte on rocks. All Vanda enjoy the light and with sufficient sunlight the may bloom two or three times a year. The inflorescences appear from the axis of the leaves. Flowers are long lasting and appear in cluster along the spike. The flower of orchids has great aesthetic value. This genus is one of the five most horticulturally important orchid genera, because it has some of the most magnificent flower to found in entire orchid family. This has contributed much to the work of hybridists producing flowers for cut flower market. Vanda coerulea is one of the few botanical orchids with blue flower (actuall a very bluish purple), a property much appreciated for producing interspecific and inter generic hybrids.
Orchid is one of the richest heritage of natural resources of Bangladesh. The climate of Bangladesh is one of the important vegetational places in the world. Bangladesh lies in the transition of three important vegetational zone of the world. Hooker (1895) first reported about 1250 species from the Bangladesh region of the British India [1]. Orchids are nature’s most extravagant group of flowering plants distributed throughout the world from tropics to high alpine [2]. Orchids belong to the family Orchidaceae is one of the most diversified and largest among the flowering plants. It is an attractive monocot family with its large number of species and natural hybrids. There are various estimates about the number of genera, species and varieties. The number of genera varies from 500-800 and the number of species from 1500035000 according to various repots [3-8]. However, some conservative statement accounts about as many as 750 genera with about 17000 species constituting the family orchidaceae [9, 10].
Corresponding Author: Md. Ibrahim Khalil, Department of Biochemistry and Molecular Biology, Jahangirnaar University, Savar, Dhaka, Bangladesh.
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MATERIALS AND METHODS
Commercial cultivation of orchids both for pot plant and cut flower production has been developed into sizeable industries in many countries as the lovely flower fetch a very high price in the international market and the sale of flower run a million of dollars [11]. The techniques of plant organ, tissue and cell culture are now established in many research laboratories throughout the world and rare being used in different areas of plant science. These techniques are widely used for mass scale propagation of commercially important plant, development of virus free plants, induction of somaclonal variation and also in execution of genetic engineering programmes. The research in the field of tissue culture is still continuing for the development of new protocols, refinement of the media and increasing the efficiencies of the techniques. Orchid seeds usually germinate in symbiotic association with mycorrhizal fungus, which supplies food to the germinating undifferentiated orchid embryos. Therefore, the presence of mycorrhizal fungus is prerequisite for germination of orchid seeds. The distribution of orchids is related to availability of mycorrhizal fungus. As a result, orchid generally propagate in nature very slowly. Now a day, commercial orchid are predominantly propagated by tissue culture technique. Kundson (1922) first succeeded in development of in vitro germination technique of some orchid viz. Cattleya, Laelia and Epidendrum seeds on agar medium containing sugar [12]. From the commercial point of view, rapid multiplication is necessary and accordingly in 1950’s more refined techniques of in vitro germination of some orchid seeds have been developed and established. In 1960’s very rapid in vitro clonal propagation of commercially important orchids such as Cymbidum sp; Phalaenopsis sp; Dendrobium sp. was achieved by meristem, organ and tissue culture [13-15]. Different explants including shoot tip [16-18], leaf stalk [16], leaf segment [19], young leaves [20], inflorescence [21], root tip [22] and rhizome segments [23-24] have been used successfully for commercial propagation of orchids. Based on these techniques in 1980’s a number of commercial farms have been set up in different countries especially in South East Asia. Million of ornamental orchid seedlings are produced in these countries and sold in local and foreign markets. An attempt was taken to develop a suitable in vitro protocol for seed germination of indigenous orchid, Vanda sp. both for their commercial uses and conservation purposes.
Plant Materials: In the present study, one indigenous orchid species viz. Vanda sp. was selected. The plant specimens were collected form Rabindranat Kuti Bari Shalidah, Kumarkali, Kushtia, Bangladesh. Seeds were used as explants of in vitro germination. Culture Media: A culture media is composed of inorganic salts, an iron source, vitamins, amino acids, growth substance (hormones) and carbohydrate supply. In most media, iron may be chelated as ferric- sodium ethyleneamine tetra-acetate (Fe-EDTA) in this medium as it is utilized by the living cells. Vitamins used in culture media are mesoinositol, nicotinic acid, pyridoxine thiamine etc. Carbohydrate is supplied usually as sucrose. The most commonly used amino acid is glycine. In addition, phytohormones (auxins and cytokinins) that are commonly used in culture medium are IAA (indol-3-acetic acid), NAA (á-naphthalene acetic acid, 6-BAP) (6, Benzylaminopurine). Preparation of Stock Solutions for Culture Media: The stock solution of various constituents of Murashige and Skoog (MS) [25] media were prepared for ready use. As different media constituents were required in different concentrations, separate stock solutions of macronutrients, micronutrients, organic compounds (vitamins and amino acids) for MS and growth regulators were prepared. Stock Solution (I-III): Macronutrients: 20 times the weight of each of the major salts required for 1(one) liter of medium was weighed accurately, dissolved once at a time and sequentially in 750 ml of distilled water and then final valium was made up to 1000 ml by further addition of distilled water. The stock solution was filtered through Whatmann 1 (Whatmann Ltd. England) filter paper, to remove all the solid contaminants and solid particles. The stock was then poured in a clean glass container and stored in a refrigerator at 4°C. Stock Solution (IV-VI): Micronutrients: The stock solution of IV and V were prepared at 20x or 100x like as stock solution of macronutrients as described above. Required amount of FeSO4.7H2O and Na2EDTA.2H20 were taken and dissolved separately in 400 ml distilled water by heating and constant stirring to make stock solution IV. 164
World J. Agric. Sci., 10 (4): 163-168, 2014
The two solutions were mixed properly, the pH was adjust to 5.5 and distilled water was added to make up the final volume to 1000 ml and stored in refrigerator at 4°C. This solution must be kept in an amber coloured bottle or covered by black paper. The rest of the micronutirents (solution V) was made at 1000x. All components of solution VI were weighed separately and dissolved in 800 ml of distilled water. Finally the volume of the solution was adjusted to 1000 ml and after filtering stored at 4°C in glass container.
Final volume of the medium was then made 1 (one) liter by adding distilled water. The pH of the media was adjust to 5.8 by a digital pH meter using 0.1 N NaOH or 0.1 N HCl. The whole mixture was then transferred to one liter flask and the agar was melted by micro oven. Medium Dispensing: Requisite volume of the melted medium (while still hot) was then dispensed into culture vessels and were autoclaved. Culture Environment: All cultures were grown in an aircondition culture room, illuminated by 40w white warm fluorescent table fitted at a distance of 30-40 cm from culture racks. Culture room temperature was 25±1C and light intensity ranged from 2000-3000 lux with a photo period of 16 hours light and 8 hours dark. The relative humidity of the culture room was maintained around 60%70% by hygrometer. The culture vessels were observed daily to note the growth response.
Stock Solution (VII): Vitamins and Amino Acids: This stock solution was prepared at 100x, dissolved in distilled water or in appropriate solvent to make the volume 200 ml. After being filtered was poured into clean plastic container and stored at deep freeze. Preparation of Coconut Water (CW): Green coconut were cut and CW wax collected filtered and stored in plastic bottle at deep freeze.
Setting and Designing of Experiments: In order to determine the optimum condition of a specific factor; experiments were conduction with various modification of one factor, keeping the others constant. Most of experiments were conducted following standard tissue culture method. The experiments had 8-15 or 20-30 replicated culture per treatment and they were grow for 1-4 consecutive passages of 2-6 weeks each and data on different parameters were recorded at the end of each passage.
Stock Solution: Plant Growth Regulators (PGR): Stock solution of different PGRs were prepared separately. Details of the method of preparation of stock solution are given in the following table 1: Assembling of the Media Components: The following steps were followed to make one litter of MS medium: At required volume of each of the stock solutions were taken in 1 (one) liter volumetric flask containing 500 ml distilled water and mixed well. Different amount of PGR and supplements as required were added to the solution and thoroughly mixed. PGR concentration was made different by varying the volume of the stock solution as per requirement. Other supplements such as agar 5 gm/l (BDH. high gel strength), sugar 30 gm/l etc. were also added to the medium.
Culture Methods: The capsule of Vanda sp were treated with 0.1% (w/v) HgCl2 for 10 minutes for surface sterilization and thereafter washed 2-3 times with autoclaved distilled water in a laminar airflow cabinet. Sterilization of non- living Articles. The one liter medium routine sterilization procedure of non-living articles such as nutrient medium glass goods, distilled water, instruments (wrapped with brown paper) were sterilized by autoclaving.
Table 1: Different PGRs and their solvents Growth regulator
Amount taken(ml)
Solvent(ml)
Final volume of the stock(ml)
Final concentration of the stock solution(mg/ml)
IAA
10
1N NaOH
100
0.1
NAA
10
1N NaOH
100
0.1
BAP
10
1N NaOH
100
0.1
The solution was then stored at 4°C for up to 12 weeks.
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Seed Culture: In the present study, symbiotic in vitro seed germination and successful plantlet development was achieved for Vanda roxburghii. The seeds were germinated normally and continued to develop green seedlings. Approximately seed germination time was same in these species. It was found that hormone free MS media required maximum time 35 days, which is longer germination time then other hormone, hormone+coconut water (CW) adding media. On the other hand, MS media supplemented with 0.2 mg/l IAA, 0.5 mg/l IAA+BAP and 0.2 mg/l NAA the culture seeds were started to germination in comparatively shorter time 24-26 days. Callus formation was not observed in any of the media formulation another noticeable difference of plant growth regulator supplemented culture media was their effect on tissue growth. Finally, we observed that grater germination rate and the lowest time required (22-24 days) in MS media containing 15% CW+0.1 mg/l NAA than other hormone containing and hormone free MS media.
Table 2: Different culture media formulation used for seed germination. Culture media formulations MS MS+0.5 mg/l BAP MS+0.2 mg/l IAA MS+0.5 mg/l IAA+0.5 mg/l BAP MS+0.2 mg/l NAA MS+0.1 mg/l NAA+15% CW MS=Murashige and Skoog (1962) medium, CW=Coconut water
Capsules opening and Culturing of Seeds were done aseptically under laminar airflow cabinet. The culture vessels with inoculated seeds were incubated in 16:8 h Light:Dark photoperiod at 25±1°C. Subculture of Orchid Seedlings: Sub-cultures were maintained in the same conditions as for in germination. Thus regular sub-culturing was done at every 28 days interval when the masses of seedlings were further thinned. Preparation of Stock Solution for Culture Media: The stock solution of various constituents of MS media were prepared for ready use. Different concentrations, separate stock solution of macronutrients, micronutrients, organic compound (vitamin and amino acids) for Murashige and Skoog (MS) and growth regulators were prepared. The constituents of stock solution of MS media are given in Appendix.
Seed Germination: MS media without any supplementation of growth regulators on coconut water showed seed germination after 35 days to 39 days. Subsequent plantlet development was also observed but the process is very slow. Separate MS media supplement with MS+0.5 mg/l BAP shorten the germination period which was 26 days to 29 days, while MS+0.2 mg/l IAA, MS+0.5 mg/l IAA+ 0.5 mg/l BAP and MS+0.2 mg/l NAA the germination period was more shortened, 24 days to 26 days. The lowest germination period 22 days to 24 days was observed when MS+0.1 mg/l NAA+ 15% CW used (Table 3 and Figure 1).
Seed Culture: The seeds from green capsule Vanda sp. cultured onto six different semisolids culture media the culture media formulation used for seed culture are given in following table
Table 3: Effect of culture media on germination and seedling development
Data Recording: Data were collected on the following parameters for the evaluation of the result of different sets of experiments.
of V. sp seeds. The sterilized seeds were cultured onto semisolid medium and incubated under defused light (8000 Lux) 16 h/day for 5 weeks Time required for seed Morphogenic
Time Taken for Seed Germination: The lag period seed germination was counted in days starting from the day of culture inoculation to the first sign of shoot emergence.
Culture media
RESULT In the present study, capsules were surface sterilized with HgCl2. It was found that 0.1% HgCl2 treatment for 10 minute was suitable for contamination free capsule culture development.
germination (days)
responses
MS
35-39
Green seedlings
MS+ 0.5 mg/l BAP
26-29
Green seedlings
MS+ 0.2 mg/l IAA
24-26
Green seedlings
MS+ 0.5 mg/l IAA+ BAP
24-26
Green seedlings
MS+ 0.2 mg/l NAA
24-26
Green seedlings with multiple shoot
MS+ 0.0.1 mg/l NAA+ 15% CW
166
Green seedlings with 22-24
high germination rate
World J. Agric. Sci., 10 (4): 163-168, 2014
A B C Fig. 1: Germination of Vanda seeds on different media composition. A=MS+0.1 mg/l NAA+ 15% CW, B=MS+ 0.2 mg/l NAA and C=MS media. 2. 3. 4. 5. 6.
Fig. 2: Plantlets of Vanda roxburghii.
7. 8.
DISCUSSION It was studied the seed germination and seedling development of orchid species in five different basic culture media supplemented with 20% CW. They found that seed germination and subsequent growth conditions of the 26 orchid species were varied from species to species and media, which are concomitant with the findings of the present study [26]. It is also reported the beneficial effect of PGR/NAA on seed germination and subsequent PLB growth of Cymbidium aloifolium, Aerides multioflorum, Dendrobium chrysotxy and hybrid of D. pieravdii [27]. Another research supports the similar effect of NAA on seed culture of temperature Cymbidium species. These reports support the result of the present study of in vitro seed culture of Vanda roxburghii [28].
9. 10. 11. 12. 13. 14. 15.
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