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FNCA 2002 WORKSHOP ON MUTATION BREEDING

Orchid Improvement through Mutation Induction
by Gamma rays

Chitrapan Piluek1, and Siranut Lamseejan 2
1Department of Horticulture, Faculty of Agriculture,
Kasetsart University, Thailand
2Department of Applied Radiation and Isotopes, Kasetsart University,
Thailand


 
Abstract
Tropical orchids are the major commercial ornamental plants grow for cut flower and pot plant production in Thailand. New varieties of them are selected from breeding and propagated by tissue culture technique. Many somaclonal variation clones are selected for cut flower or pot plant production or as parent plant for produce extinct hybrids. Introduce mutagenesis by gamma rays irradiation at protocorms stage in vitro will have more chance to derive new flower colors or plant with variegated leaves.
 
Key words: orchids, acute irradiation, cronic irradiation, gamma rays, in vitro culture
 
1. Introduction

Thailand is the major producer of the tropical orchids. There were about 6,800 acres of growing area under cultivation by 2,300 farmers. The cut flower orchid area are mainly in Metropolitan Bangkok, Nakorn Pathom, Samuthsakorn, and Nontaburi, a nearby provinces.
Export of tropical orchids flower from Thailand is around 1965. And the export of cut flower reach 10,000 tons to all over the world in 1990. The dominating cut flower is genus Dendrobium. Dendrobium hybrids account for 70-80 % of tropical orchid plant and flower trade. Dendrobium hybrids are easy to grow, fast growing, floriferous and suitable to flower arrangeent due to its various colors and forms. Other major tropical orchids include Oncidium, Aranda, Mokara, Vanda and Ascocenda hybrids.
The exported quantity of orchid cut flower in 2001 was 13,940.8 metrictons earning of 40 million US$. The major imported countries were Japan, America, Italy, Hong Kong and Taiwan. The exported quantity of orchid plants in 2001 was 24,750.3 metrictons earning of 6.73 million US$. The major imported countries were Japan.

 
2. Orchid Breeding

New varieties of orchid for cut flower or pot plant derive from breeding and select the best clone for multiply by tissue culture technique. Dendrobium Pompadour was the first clone to be grown for commercial scale. Later on, other Dendrobium as well as orchids of other genera began to share a substantial portion. However Dendrobium hybrids is still holding the first rank.
The characteristics of cut flower are attractive flower and sprays, long with erect or arching spray, high yields, year-round flowering, low bud drop (under 5 %), long vase life, resistance to diseases and insects. For pot plant, the stem must be compacted with beautiful leaves.
Many hybrids are produced by the orchid grower every year. Unfortunately, only few crosses can use for commercial cut flowers and pot plants.

3. MUTATION BREEDING IN ORNAMENTALS

Tissue culture methods were use for clonal propagation of orchids. In Thailand there are more than 30 commercial laboratories serve for tissue culture and seed germination which produce ten millions seedlings per year. Usually every mericlones must exhibit exactly the same characteristics. When the population become very large, more variation among its individuals is to be expected. Mutation are more likely to occurse during asexual phases than during sexual ones. However, recessive mutations may remain hidden and become apparent after recombination in sexual reproductions. Some of variation can be transmitted to off spring.
Mutation from tissue culture that found in Thailand are

1. Change in chromosome number from diploid to tetraploid e.g. Dendrobium, vanda, Mokara. The flowers are larger than diploid plant.
2. Leaf variegation. Leaf variegation is a chimera that usually in values chlorophyll production. The amount of chlorophyll varies from area to area, resulting in mottling or striping. This variegation has been found in several orchid genera e.g. Dendrobium, Vanda, Mokara. The variegation of one Dendrobium hybid is not limit to leaves but extend to the flower which are green with white outline.
3. Variation in size of flower part. Vajrabhaya and Vajrahaya (1974) showed statistical analysis of petals and labella of 20 selected mericlones of Dendrobium Pompadour that show clearly the difference in length and width of petal and labella were genuine variations.
4. Color variations. Flower color of some orchid hybrids are change to new color e.g. from red purple to blue purple in Dendrobium ; from red purple with dark sports to white with dark sports, in Mokara Walter Oumae ; from light violet blue to orange and red in Mokara Wan Chark Kuan.
5. Color variegation. Unusual color distribution on their floret parts. The orchid flowers are often incredibly beautiful, exotic with bold stripes and splashes e.g. color green with white outline (Dendrobium hybrid), serrated edging, some has a lighter color at the edge of petals while other exhibited a darker color, splash petals (D. Ekapal ‘King Drogon’) or crown like yellow stripes on white petals (D. Kasem White)
 
4. Mutation Breeding

Somaclonal variation happened in differentiation stage of tissue or cell culture can be increased by apply in either chemical or physical mutagen. Many techniques are available for increasing the number of variation, but rarely been applied to orchids. Since orchids are slow–growing plants which produce relatively few shoot per year, experiment along these line are very difficult. Recent investigation have contributed much to the induction of mutation in vitro (Nickell, 1973). And the results with orchids are promising (Sanguthai and Sagawa, 1973, Sanguthai, Sanguthai and Sagawa, 1973),
Tolerance of tissues to radiation varies greatly depending on organism and stage of growth. Dosages of gamma rays ranging from 2000 to 4000 R at a rate of 900 R/hour seem to be optimum dose for the purpose inducing mutation in Cymbidium protocomes. Young protocorms are more sensitive than older one (Harn, 1970). Vajrabhaya (1977) stated that Dendrobium mericlones 2 to 2.5 cm high can tolerate gamma radiated. Application with higher dosages. Some of the irradiated Dendrobium Pompadour produced flowers with modifications in shape or color or both.

5. Mutation breeding of Dendrobium hybrids
Angamnuasiri (2001) a researcher of the C.P. Company, reported about the company work in improve of cut flower of Dendrobium hybrids. One method was induced mutation by gamma rays irradiation. Protocorm-like Bodies of 5 varieties were irradiated in vitro. The irradiated protocorms were induced to be plantlets then grew in the nursery until they flowered. The mutant showed wide range of purple shade in flower color. The different color variations are expressed as whole or gradual distribution of intensity form pale to darker shade in the petals and sepals. The mutant flowers show difference in size and shape.
6. Effect of gamma irradiation on protocorm - like Bodies of Cattleya Alliances.
Thammasiri (1996) reported that induced mutations by gamma irradiation can be one of the promising methods for orchid breeding. Protocorm-like bodies (PLBs) of Brassolaeliocattleya (Blc.) Alma Kee and Blc. Greenwich were established from in vitro apical and axillary buds of young shoots in Vacin–Went (1949) liquid medium supplemented with 150 ml/L coconut water. PLBs were irradiated with acute gamma rays at 0, 20, 60, 80, 110, and 130 Gray (Gy). Doses between 80-110 Gy were suitable to apply for induced mutation in both cultivars. Another experiment with more samples was conducted. Gamma rays were applied at 0, 70, 100, and 130 Gy. The result showed that irradiated PLBs of both cultivars had slow growth rate and some PLBs turned brown and died. A dose of 70 Gy was suitable for induced mutation. More severe effects were observed when higher doses had been applied.
7. Effect of gamma irradiation on survival rate of orchid protocorms
Seeds of orchids of species were germinated on solid medium of modified
Vacin – Went in vitro protocorms with 2 leaves of 1) Dendrobium chrysotoxum,
2) Dendrobiunm albosanguineum , 3) Dendrobium albosanguineum (variegated leaf), 4) Dendrobium cruentum, 5) Grammatophyllum speciosum, 6) Rhynchostylis gigantea 7) Phalaenopsis violacea and 8) Calanthe rubens were irradiated with acute gamma rays of 20, 40, 60, 80 Gy and with cronic gamma rays of 100, 150, and 200 Gy. After the protocorms were subculture to new medium for one month, growth and % survival of all experimental unit were recorded. Protocorms of orchids radiated with acute gamma rays of 20 and 40 Gy had more survival rate than those that irradiated with 60 and 80 Gy. Protocorms of Grammatophyllum specisum are tolerance to all doses of irradiation. They could survive and develop to be normal plantlets. While all of the treated Calanthe rubens protocorms were complete dead. Protocorms that irradiated with cronic gamma rays showed normal growth and development could developed to be plantlet in every treatments. (Table 1) Protocorms of Grammatophyllum speciosum could survive and develop to normal plantlets in all treatment. While protocorms of Calanthe rubens were completey dead.Protocorms that irradiated with cronic gamma rays showed normal growth and development. (Table 1)
 
Table 1 Survival rate of orchid protocorms after in vitro acute and cronic irradiation
with gamma rays
 
Species Acute (Gy) Cronic (Gy)
0 20 40 60 80 107 150 200
1.Dendrobium chrysotoxum
100 79.96 61.73 42.07 43.06 100 100 200
2. D. albosanguineu
100 91.12 96.04 48.59 26.67 100 100 100
3. D. albosanguineum
(variegated leaf)
100 61.43 83.62 68.06 51.85 100 100 100
4. D.cruentum
100 50.37 17.42 13.55 17.76 100 100 100
5. Gram. speciosum
100 100 100 100 98.53 100 100 100
6. Rhyn. gigantea
100 100 61.18 59.67 48.17 100 95.00 97.33
7. Phal. violacea
100 100 58.97 13.38 0 96.11 94.78 58.97
8. Calanthe rubens 100 0 0 0 0 - - -
8. Effect of gamma irradiation on growth of Dendrobium chrysotoxum
Irradiated protocorms of D. chrysotoxum and Grammatophyllum speciosum were subculture for seedling formation. Seedlings of D. chrysotoxum irradiated with 20, 40, and 60 Gy showed normal growth. Dwarf and abnormal growth were found in 80 Gy treatments. (Table 2) While irradiated Grammatophyllum speciosum seedling showed shorter stem height and leaf length. The normal plant at mature stage is about 1.5–2.5 m. high. The dwarf size derived from irradiated protocorms will be a beautiful pot plant. (Table 3)
 
Table 2 Effects of gamma rays on seedlings growth of Dendrobium chrysotoxum
derived from in vitro acute irradiation
 
Radiation dose
(Gray)
Fresh weight (g) Height of pseudobulb
(cm)
Leaf length
(cm)
0
2.73 2.73 4.37
20
3.14 2.73 4.39
40
2.43 2.26 3.45
60
1.93 2.11 3.15
80
1.81 2.02 2.67
 
Table 3 Effects of gamma rays on seedlings growth of Grammatophyllum speciosum
derived from in vitro acute irradiation
 
Radiation dose
(Gray)
Fresh weight (g) Height of pseudobulb
(cm)
Leaf length
(cm)
0
2.25 17.9 11.64
20
1.98 13.60 8.95
40
2.13 10.29 7.08
60
2.62 12.84 9.64
80
1.71 10.85 7.03
9. Effects of gamma irradiation on seed and protocorms survival and seedling development in Rhynchostylis gigentea
Seeds and protocorms (20 days from germination) of Rhynchostylis gigentea were irradiated with acute gamma rays of 0, 20, 40, 60, and 80 Gy. After spray on in vitro solid modified Vacin-Went medium.
Two months after radiated, seeds and protocorms were subculture to new medium and observed for growth and development. Seeds treated with acute 20 and 40 Gy could germinated and developed to seedling. More radiated treated the seedlings show slow growth. While the protocorms that treated with 20 Gy were survived and developed to be seedlings. Protocorms treated with 40, 60, and 80 Gy could survived in a small number, slow growth and all of them died in 4 months after treated. The treated seeds with 627.5 Gy gamma rays can germinate as same as untreated seeds
 
Table 4 Effects of gamma irradiation on seeds and protocorms survival and seedling
development in Rhynchostylis gigantea
 
Dose
In
Gy
Number of Seedlings (%)
Germinated
seedling
Growth Index
Protocorms
2-3 mm
Small seedling
Large seedling
Seed  
Acute  
0
 
20
   
40
   
60
   
80
0
0
1.67
100.00
100.00
0
50.00
36.66
0
0
100.00
50.00
0
0
0
60
50
50
3
5
300.0
200.0
264.4
100.0
100.0
Protocorms
Acute  
20
 
40
   
60
   
80
8.47
100.00
100.00
100.00
57.63
0
0
0
33.9
0
0
0
59
9
12
8
225.4
100.0
100.0
100.0
Cronic
 
627.5
 
16.95
 
27.12
 
55.93
 
59
 
238.9

 

10. Workshop and Trainings
Workshop and Trainings were organized by Gamma Irradiation Service and Nuclear Technology Research Center (GISC), Kasetsart University During 2001-2002
 
Year 1999
1. Hosting the Regional Meeting of the FAO/ IAEA on “Project Formulation Meeting on Mutational Enhancement for Genetic Diversity in Rice“ during 8-12 February 1999. (15 participants from 11 countries in the region.)
2. Workshop on “Use of Induced Mutation Technique for Genetic Diversity and Economic Crop Improvement” during 16-20 August 1999. (35 participants from different organizations and institutions.)
3. Workshops on “ Ornamental Improvement through Gamma rays Induced Mutation” .Two workshops were held on 20 July and 6 August 1999. Workshops were sponsored by DOAE for 90 ornamental growers from Bangkok and Nontaburi provinces.
Year 2000
1. Training on “ Production of Exotic Ornamentals through Gamma rays Induced Mutation ”on 26 May 2000. For 20 extension officers from Department of Agriculture Extension (DOAE)
2. Training on “ Production of Exotic Ornamentals through Gamma rays Induced Mutation”
Six training were held during 2000 for 240 participants selected from ornamental growers in Thailand. Training were sponsored by DOAE
3. Workshops on “In Vitro Mutagenesis and Molecular Marker Analysis of Ornamental Plants” during 18-22 December 2000. For 25 researchers and instructors from different organizations and institutions in Thailand. This workshops was supported by IAEA under THA/5/045.
Year 2001
1. Training on “The Use of Gamma rays for Crop Improvement” during 12-14 September 2001. For 20 extension officer from Department of Agriculture Extension, Ministry of Agriculture.
2. Training on “Production of Exotic Ornamentals through Gamma rays Induced Mutation “ Training organized under the Technology Transfer Project supported by the Ministry of University Affairs.
Six training were held in 2001 for 240 ornamental growers and interested persons. Many participants take their orchids to irradiate and grow in the nurseries. They want to select the mutant clone for cut flower.
Year 2002
1. Workshop on “Induced Mutation Technique for Genetic Diversity and Economic Crop Improvement – II” during 23-26 April 2002. For 16 researchers and instructors from different institutions and organizations in Thailand.
11. Acknowledgement
The authors appreciate the research grant from Kasetsart University Research and Development Institute (KURDI) and the expert service from Gamma Irradiation service and Nuclear Technology Research Center, Kasetsart University
12. Literature Cited
Angamnuasiri, K. 2001. Orchid breeding in The C.P. Company. Report in the seminar
Thai Orchid production for Export. Jan. 20-21, 2001. Surat Thani, Thailand.
p. 15-24.
Nickell, L. G. 1973. Test- tube approaches to bypass sex. Hawaiian Planters’ Record 58
: 293-314.
Sanguthai, S. and Y. Sagawa. 1973. Induction of polyploidy in Vanda by colchicine
treatment. Hawaii Orchid J. 2(2) : 17-19.

Sanguthai, O., S. Sanguthai and H. Kamemoto. 1973. Chlomosome doubling of a Dendrobium Hybrid with colchicine in meristem culture. Hawaii Orchid J. 2(2) :
12-16.
Thammasirii, K. 1996. Effect of gamma Irradiation on Protocorm – like Bodies of Cattleya
Alliances. Proceeding of the 15 th World Orchid Conference. p. 403-409.
Vajrabhaya, T. 1977. Variation in Clonal Propagation in Orchid Biology, Reviews
and Perspectives, I.


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