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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 |
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Abstract |
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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. |
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Key words: |
orchids, acute irradiation, cronic irradiation, gamma
rays, in vitro culture |
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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. |
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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. |
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3. |
MUTATION BREEDING IN ORNAMENTALS |
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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. |
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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. |
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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. |
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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. |
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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) |
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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 slowgrowing 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. |
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5. |
Mutation breeding of Dendrobium hybrids |
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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. |
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6. |
Effect of gamma irradiation on protocorm - like Bodies
of Cattleya Alliances. |
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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 VacinWent (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. |
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7. |
Effect of gamma irradiation on survival rate of orchid
protocorms |
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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) |
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Table 1 |
Survival rate of orchid protocorms after
in vitro acute and cronic irradiation
with gamma rays |
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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 |
- |
- |
- |
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8. |
Effect of gamma irradiation on growth of Dendrobium
chrysotoxum |
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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 1.52.5 m. high. The dwarf size derived
from irradiated protocorms will be a beautiful pot plant. (Table
3) |
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Table 2 |
Effects of gamma rays on seedlings
growth of Dendrobium chrysotoxum
derived from in vitro acute irradiation |
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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 |
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Table 3 |
Effects of gamma rays on seedlings
growth of Grammatophyllum speciosum
derived from in vitro acute irradiation |
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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 |
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9. |
Effects of gamma irradiation on seed and protocorms
survival and seedling development in Rhynchostylis gigentea |
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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 |
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Table 4 |
Effects of gamma irradiation on seeds and protocorms
survival and seedling
development in Rhynchostylis gigantea |
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Dose
In
Gy |
Number of Seedlings (%) |
Germinated
seedling |
Growth Index |
Protocorms
2-3 mm |
Small seedling |
Large seedling |
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300.0 |
200.0 |
264.4 |
100.0 |
100.0 |
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Protocorms |
Acute |
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20 |
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40 |
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60 |
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80 |
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8.47 |
100.00 |
100.00 |
100.00 |
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10. |
Workshop and Trainings |
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Workshop and Trainings were organized
by Gamma Irradiation Service and Nuclear Technology Research Center
(GISC), Kasetsart University During 2001-2002 |
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Year 1999 |
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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.) |
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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.) |
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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. |
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Year 2000 |
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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) |
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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 |
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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. |
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Year 2001 |
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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. |
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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. |
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Year 2002 |
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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. |
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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 |
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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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