Mutation Breeding workshop


- Project Review
- Introduction of the Project Leaders
- Mutation Breeding Database
- Mutation Breeding Publication Database
- Mutation Breeding Manual
- Sorghum & Soybean
- Insect Resistance in Orchid
- Disease Resistance in Banana
- Composition or Quality in Rice
- Rice for Sustainable Agriculture
- Papers for Project Outcome

  Banana Meeting 2008
  Orchid Meeting 2007
  Banana Meeting 2006
  Orchid Meeting 2005



Center for Reseach and Development of Isotope
and Radiation Technology
National Nuclear Energy Agency (BATAN)

Conventional breeding techniques are difficult to apply in most vegetatively propagated plant species. Induced mutation is therefore very important in varietal improvement of these plant species , but practical results have been difficult to obtain due to the problems in recovering the mutated cells in the plant. In Indonesia , varietal improvement of vegetatively propagated crops by mutation techniques has mainly been carried out at BATAN and in collaboration with several research institutes under the Ministry of Agriculture and some Universities. The importance of biotechnological approach such as the advantage of developing various tissue culture techniques has been incorporated in mutation breeding program. Mutation breeding in vegetatively propagated crops has unfortunately not yet resulted any officially released mutant variety. Most of the mutants are now being evaluated for official release preparation such those in some ornamental crops , banana , and ginger as reported in this paper.
A large number of plant species are asexually or vegetatively propagated in their reproductive system. These vegetatively propagated crops include root and tuber crops , ornamental crops , woody perenial and forest trees , fruit crops , and other crops such as peppermint , sugarcane , tea , and grasses.

Cross breeding is often limited by specific problems in most vegetatively propagated crops. Many vegetatively propagated crops have a rather long vegetative phase before going into sexual reproduction. In addition , these plants are generally highly heterozygous , which causes complicated segregations and makes the detection of a useful recombinant very difficult. This problem is further enhanced by frequently polyploidi in such plants. All these factors make cross breeding very difficult and time consuming. Furthermore , incompatibility and other cross barriers , apomixis and sterility exist quite often and hinder the plant breeder in making use of conventional cross breeding. It is , therefore , quite clear that improvement of vegetatively propagated crops by mutaion techniques becomes an attractive alternative in breeding these crops. The most promising aspect of induced mutation in vegetatively propagated crops compared to cross breeding method is the ability to change only a few characters of an otherwise good cultivar without altering significantly the original well established genotype. Induced mutation must , therefore , be considered as the obvious means to improve the leading cultivars and as a possible shortcut for inducing desired genetic alterations in outstanding cultivars. Obviously , mutations are the only mean for producing variability in sterile vegetatively propagated crops. Furthermore , mutations can be useful to break apomixis , to overcome self-sterility and cross barriers as well as to uncover rearrange chimeras. In spite of these many advantages of breeding by mutation techniques in vegetatively propagated crops , the results have been rather small compared to the seed propagated crops such as rice , barley , wheat and many other crops (Maluszynski et al. , 2000). The main problem has been the recovery of mutants in vegetatively propagated crops. Normally , a shoot or any other multicellular organ is treated for mutation induction and the desired mutation has occurred in one cell. The chances of such mutated cell growing into a sector or layer and be able to manifest itself will depend on its position within the apex as well as its growth rate as compared to the surrounding cells or tissue layers. It is obvious that mutated cells will be easily hidden and not be visible in such multicellular tissues and a special method is required to unciver such mutated cells (Mikaelsen , 1985). The first requirement is , therefore , that mutagen treated material has to be propagated to permit the formation of periclinal layers before selection can be applied. Very often repeated propagations are necessary for enhancing large sectors of mutated tissue from which a maximum number of mutants can be recovered. In fruit trees , for example , the best condition is found in the auxillary meristems of the basal leaf primordia of the dormant buds. The primordia to be used for mutagenic treatments should consist of as few cells as possible. Experience shows that the buds derived from leaves approximately 4-8 of the primary shoot are those exhibiting the higest frequency of relatively broad mutated sectors in the second vegetative generation. Measures by pruning have to be taken to force these buds into growth. Several authors have reported better results after irradiating buds that have just started growth than with buds in deep dormancy (Broertjes and Van Harten , 1978). If growing plants are irradiated , decapitation of the main shoot will force new buds to develop through regeneration and thereby increase the chance for recovering a large number of mutants. Many of the complications described above could be eliminated if chimera formation could be avoided. One of the method available is the adventitious bud techniques which was developed by Broertjes et. al. (1968). This technique is based on the phenomenon that the apex of the adventitious buds , such as that may be formed the base of the petiole of detached leaves , originates from only one epidermal cell. Consequently , adventitous plantlets will produce complete and solid mutants. In other words , chimera formation does not take place. This adventitious bud technique offers a great advantage for practical crop improvement by induced mutations and has given good results particulary in ornamental plants (Broertjes et. al. , 1968).

A number of 350 plant species can be propagated by adventitious bud techniques (Broertjes et. al. , 1968). Many species economically important plant families have not yet tested for developing the adventitious bud techniques and may not always develop adventitious buds from only one cell. In addition , the condition of the mather plant , the age of leaves , environmental conditions during and after rooting and the auxin-cytokinin balance appear to influence the formation and differentiation of adventitious buds.

Tissue Culture Techniques in Vegetatively Propagated Crops
The modern development of culturing somatic plant cells should also be investigated for use in vegetatively propagated crops. In this respect , tissue culture technology holds much promise in speeding up breeding for crop improvements. Recent advances in the field of plant protoplast , cell , tissue , and organ (embryo , anther) cultures have transferred this area from fundamental research to the one that is dynamic and promising also for obtaining further advances in crop improvement programs. Since breeding vegetatively propagated crops is difficult to conduct by cross breeding , thus , the potential of this technology can become a powerfull tool in crop improvements.

During the last few years , the culture of ovule , ovary , and embryo has been employed to overcome sterility , incompatability , dormancy , to induce polyembriony , and to succesfully hybridize various crops (Mikaelsen , 1985). By meristem culture , large numbers of important horticultural crops have been commercially propagated with good quality and free from pathogens. These established techniques have played an important role in wide hybridization and clonal propagation programs and will , no doubt , continue to contribute to future demands. However , some of the recent advances in plant biotechnology including protoplast , cell , tissue , and organ cultures have attracted international attention because of their significance in and far-reaching implications for agricultural research and crop improvement programs.

Varietal improvement of vegetatively propagated crops by using mutation techniques is expected to get more desirable results by combining the biotechnological approaches into plant breeding program. These approaches include (1) wide hybridization through in vitro pollination and fertilization , (2) production of haploid and homozygous plant from exised anthers , isolated pollen and by chromosome elimination , (3) somatic hybridization and genetic engineering through protoplast fusion and transfer of DNA , (4) induction of genetic variability in crops by mutations.

Mutation Breeding of Vegetatively Propagated Crops in Indonesia
Mutation breeding in Indonesia has mainly been very successful for the seed propagated crop species such as rice , soybean , and mungbean. Up till now , Indonesia has officially released 12 mutant varieties including 8 varieties of rice , 3 varieties of soybean , and 1 variety of mungbean (Soeranto et. al. , 2001). These released mutant varieties have widely been grown by farmers in several provinces in Indonesia. Mutation breeding in vegetatively propagated crops , unfortunately , has not resulted any officially released mutant variety yet. The main problem might be due to the occurance of mutant recovery which commonly appeared in vegetatively propagated crops as it was noted by Mikaelsen (1985). This phenomenon is relevant to general result of the overall mutation breeding program as reported in the IAEA database (Maluszynski et al. , 2000) which showed that the number of mutant varieties of vegetatively propagated crops is less than that of seed propagated crop species. Nevetheless , varietal improvement of these crop species keeps continuing such as that in ornamental crops (orchids , chrysanthemum , jasmine) , banana , ginger , shallot , garlic , and so on. Plant breeding by using mutation techniques has mainly been carried out at BATAN and in collaboration with several institutions including the Department of Agriculture and the Universities. Research results of some investigated crops are presented below.
Mutation Breeding in Ornamental Crops
Floriculture farmings in Indonesia have developed fast to be a commercial business applying industrial principles with agrobusiness orientation. The use of principle of industrial system is that to produce high quality and uniformity of yields as demanded by the consumers. As a consequence of using this principle , the demand for exellent cultivars is remarkably increasing. Up till now the exellent cultivars used by growers are mostly originated from import. This situation is certainly not profitable because high value royalty and expensive transportation will cause high market prices and low competitiveness of the products. To solve this problem , procurement of new superior cultivars must be judged as an important priority.

Research Institute of Ornamental Plants (RIOP) has intensively conducted plant breeding program for the major ornamental crops such as orchid , rose , chrysanthemum , gladiolus etc. as presented in RIOP Report 2000. The breeding objective is to improve genotypes which are more resistant to some insects and diseases , more productive , and more adaptive to the Indonesian climate. Conventionally varietal improvement is done by crossing among cultivars and selecting the F1 plants which have preffered trait combinations. With the rapid progress in biotechnology , new superior cultivars may be created by trasferring the interested gene(s) into the old cultivars. This attempt can theoritically shorten the breeding time but it is more costly compared to the conventional breeding. Other breeding alternative for ornamental crops is through mutation techniques. Research on mutation breeding in ornamental crops in Indonesia mostly conducted through colaboration between BATAN , RIOP and some universities. Some results of mutation breeding in ornamental crops have been documented , especially for orchid , rose , and chrysanthemum as presented below.

Rahayu (1985) did mutation breeding on orchid Dendrobium kahaloa Beauty by irradiating the protocorm with Gamma rays with the dose of 32.5 Gy (the D50 value). The results indicated that Gamma irradiation caused some changes in the plant and its flower characters as presented in Table 1.

Table 1. The effects of Gamma irradiation on plant/flower characters of orchid Dendrobium kahaloa Beauty.
Plant/Flower Characters
Gamma irradiation
(32.5 Gy
No irradiation
Plant height (cm)
Number of flowers
Petal width (cm)
Sepal width (cm)
Petal length (cm)
Sepal length (cm)
Flower diameter (cm)
Other mutation breeding in orchid Dendrobium and Vanda was reported in the RIOP Report 2000. In this research , the protocorms were irradiated with Gamma rays with the dose levels of up to 100 Gy , and then cultured in Vacin and Went media. The results found that mutations both in Dendrobim and Vanda were detected in the Gamma irradiation treatment with the dose of 30 Gy. These mutations have been confirmed by RAPD analysis which indicated different genetic changes found in the mutants compared to the original varieties. These mutants are now being investigated further through in-vitro techniques to learn the stability of the mutations. Phenotypic performance of the mutants showed more rapid growth and more leaves if compared to the original varieties.

Mutation breeding in rose was also reported in the RIOP Report 2000. The rose variety used as the original variety was “Black Magic”. Two-bud cuttings were treated with Gamma irradiation with the dose levels of up to 100 Gy. The irradiated buds were then oculated to two-month old root stocks originated from Multic variety. The research results found that the dose levels of 60-100 Gy caused lethality in the buds. Some chlorophyll mutations were observed in the Gamma irradiation treatment with the dose of 20 Gy , showing variegata on the young leaves. However , the mutations seemed to recover to normal plant again as the leaves grew older. At the dose of 40 Gy , more than 50% of the buds were unable to grow. Since this research was just started in 2000 , the evaluation of mutations has not completed yet and it still continuing up till now.

In the RIOP Report 2000 also noted about mutation breeding in chrysanthemum. Explants were irradiated with Gamma rays with the dose levels of up to 50 Gy , and then grown in modified Linsmaier Skoog (LS) media in in-vitro culture. Observations were done after regenerating the plants to the other new media. The research results found that the irradiation doses of 10-20 Gy inhibited callus growth and differentiation. Gamma irradiation caused abnormality in the growth pattern as it was shown by morphological changes in the plants. No callus growth and differentiation is found in the treatment of Gamma irradiation with the dose of 25 Gy or higher.

Mutation Breeding in Ginger
Ginger plant (Zinger officinale Rosc.) is an important spice crop which demand is always increasing year by year. In Indonesia , ginger is generally used in various medicinal and culinary preparation in most local communities. Ginger is vegetatively propagated through the underground rhizomes but , unfortunately , its multiplication rate is very low. Hosoki et.al. (1977) reported heavy losses in ginger production in some plantations due to some disease attacks caused by bacterial wilt (Pseudomonas solanacearum) and soft rot (Pythium aphanidermatum). Because the diseases are mainly transmitted by rhizomes propagated every year , a production of disease-free clones are necessitated in order to get a successful ginger cultivation. Micro propagation by using tissue culture techniques can be a proper alternative to produce disease-free clones of ginger plant. Problems faced in ginger breeding has so far been the very low genetic variation in ginger plant. This is because ginger is vegetatively propagated crop and hybridization is not effective since its flower biology has not been properly observed yet. Wide genetic variation is needed in plant breeding in order to search ideal plant types during the process of selection (Simmonds , 1986). In a breeding for disease resistance , the narrow genetic variation will slow down the selection process due to the risk of susceptibility to the disease. A choice to increase plant genetic variability can be through somaclonal variation derived from tissue culture techniques , or through induced mutation techniques. Larkin (1981) stated that the somaclonal variation was a genetic variation produced from a tissue or cell cultures. Meanwhile , Reisch (1983) mentioned that somaclonal variation happened in differentiation stage of tissue or cell cultures can be increased by applying either chemical or physical mutagen. A research on induced mutation in ginger plant combined with tissue culture techniques has been conducted at BATAN. The explants used were the shoot tips sized 0.4 - 0.5 cm long taken from the rhizomes. The explants were exposed to gamma rays emitted from Cobalt-60 source with different level of doses. Gamma chamber is available at BATAN with initial activity of about 10980 curries. The irradiated explants were then sterilized with 70 % alcohol and 0.2 % HgCl2 , rinsed 5 times with distilled water , plated in the modified MS basal media , and subcultured every 4 weeks. After 8 weeks , observations were done for the variable survival growth rate , number of shoot emerged , shoot height , and abnormality. Results of the observations were presented in Table 2. As shown at Table 2 , the best growth performance of ginger explants was for those irradiated with Gamma rays with the dose of 9 Gy. It seemed that dose inhibited bacterial and fungal contamination and stimulated explant growth. Lower doses as to 3 and 6 Gy did not show any better growth performance and it might be due to bacterial and fungal contamination. The Gamma ray doses higher than 9 Gy seemed to cause significant abnormalities as shown by dwarf plant with pale leaves. Best fitting models found relationship between Gamma irradiation doses and survival rate follows the Sinusoidal equation with coefficient of regression r = 0.921 (Soeranto et al. , 2002).
Table 2. The effects of Gamma rays on ginger tissue culture after 2nd subcultured.
Gamma rays doses (Gy) Survival rate (%)Number of Shoots Number of Shoots Shoot height (cm) Abnormality
0 85.0 2-7 1.5-8.0 None
3 62.5 4-8 1.5-8.0 None
6 43.8 2-8 2.0-8.0 None
9 87.5 2-16 1.5-9.0 None
10 85.0 2-6 1.5-6.0 Dwarf , pale leaf
12 25.0 2-3 1.5-5.0 Dwarf , pale leaf
15 50.5 2-5 1.0-5.0 Dwarf , pale leaf
20 37.5 1-2 1.0-2.0 Dwarf , yellow leaf
Modification in MS basal media with thidiazuron (TDZ) hormone significantly improved explant growth as shown in Table 3 (Ismiyati et. al. , 2000). Following the next subcultures , plantlets were transplanted to the field for screening against bacterial wilt (Pseudomonas solanacearum) and soft rot (Pythium aphanidermatum) diseases.
Table 3. Growth performance of ginger explant culture in modified MS basal media with TDZ hormone.
Gamma ray (doses)
Explant growth (%)
No. of plantlets/ explant
No. of leaves/ plantlet
Plant height (cm)
0 100 3.3 3.0 5.9
6 80 2.6 3.0 6.9
8 100 2.5 2.7 6.9
10 97 2.8 3.5 6.7
15 92 3.7 3.1 7.0
20 86 2.1 3.4 6.4
Mutation Breeding in Banana
Banana is an important fruit crops , ranked as second most important fruit in the world after citrus (Swennen & Rosales , 1994). The world production of banana in 1993 reaches 74 million tons (FAO , 1993). Banana production is concentrated in Central and South America , Africa , and Asia-Pacific with their contribution to the world production of 36 , 35 , and 29 % , respectively.

In Indonesia , the majority of edible banana is cultivated around the backyard with high soil fertility. In a country with densely increased population , like Indonesia , it is difficult to maintain banana cultivation around the backyard. Shifting its cultivation to another large-scale arable land may also compete with annual food crops or with other horticultural crops. Thus , banana production in the arable land is low and insignificant. Banana is traditionally propagated by separating the suckers from their mother plant , which is then planted individually to establish a new mother plant. Because of that nature of propagation system , and the fact that banana flowers are mostly sterile , breeding banana by mean of hybridization is difficult to be conducted. Induced mutation combined with tissue culture techniques can be used as an alternative to increase plant genetic variation in a breeding program. Subsequent breeding procedures through selection processes can theoretically shorten breeding time of banana to reach the desired objectives.

Almost banana cultivars grown in Indonesia are susceptible to a plant disease caused by Fusarium oxysporum Cubense (FOC). Mutation breeding for this disease resistance has been conducted in combination with tissue culture techniques (Ismiyati et. al. , 1998). A local banana variety Ambon Kuning (cavendish type) was used as the plant material. Its plantlets with height of about 5 cm were irradiated with Gamma rays emitted from Cobalt-60 source with the dose levels of 5-35 Gy. The irradiated plantlets were grown in MS basal media modified with adding growth regulators 4 ppm BAP and 2 ppm IAA. The grown plantlets were then subcultured up to M1V5 generation , acclimatized , and transferred to a hot spot field for Fusarium disease. Observations were done for plantlet performances during in-vitro stage and severity of Fusarium attacks in the field. Results of the observations were presented in Table 4 , 5 , and 6.

Table 4. Number of survival plantlets at different plant age after Gamma rays irradiation treatments.
Gamma ray (doses)
Number of survival plantlets
3 months
6 month
9 month
12 month
0 312 423 511 554
5 324 414 482 538
10 272 347 392 468
15 275 351 412 471
20 94 0 0 0
25 96 0 0 0
30 78 0 0 0
35 45 0 0 0
Table 5. Plant performances in the field at 3 months after transplanting.
Gamma ray
doses (Gy)
Plant height
No. of leaves
No. of suckers
0 69.17 12.3 1.56
5 88 13.82 2.65
10 87.51 12.61 2.39
15 88.75 11.4 1.82
Table 6. Number of healthy and infected plants at 6 , 7 and 8 months after transplanting in the field.
Gamma ray
doses (Gy)
6 months
7 months 8 months
Infected Healthy Infected
0 19 31 16 34 8 42
5 12 38 9 41 7 43
10 25 25 22 28 15 35
15 35 15 34 16 28 22

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Swennen , R.; Rosales , F.E. (1994). Bananas. In Encyclopedia of Agricultural Science Vol. 1 , A-D. Ed. Arntzen , C.J. and Ritter , E.M. 1994. Academic Press. p215-232. ISBN 0-12-226671-4.

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