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A.G. Lapade, A.M.S. Veluz, L.J. Marbella, A.C. Barrida and
M.G. Rama. Philippine Nuclear Research Institute.


This paper summarizes the status of mutation breeding in vegetatively propagated crops in the Philippines. The use of gamma radiation coupled with in-vitro culture techniques and related biotechnology have resulted in the development of crop varieties with desirable traits in fruit crops (pineapple and banana) and ornamentals (cutflower and foliage). In pineapple Queen variety, two mutants have been induced: Chlorophyll mutant that look-like “ornamental bromeliads” and a mutant with reduced thorns along the leaf margins. A putative mutant that is resistant to bunchy top virus (BTV) has been induced in banana cv. Lakatan.

Likewise, mutation breeding in ornamentals has lead to the genetic improvement of cutflower and foliage ornamentals. Putative mutants for earliness to bear flowers were induced in Chrysanthemum morifolium, color variation in mussaenda, shortening of thorns in Freycinetia and chlorophyll mutations in Dracaena sp. and Cordyline.

An important breakthrough this year in the Mutation Breeding Program of the Philippine Nuclear Research Institute is the registration of the Dracaena chlorophyll mutant (Dracaena ‘Marea’) with the National Seed Industry Council of the Department of Agriculture.

1/ Paper to be presented in the 2002 FNCA Workshop on Mutation Breeding
August 20-23, 2002. Beijing, China.
1. Introduction

The Philippines major thrust in agriculture is to attain efficiency in crop production for sustainable development and global competitiveness. Crop improvement thru the collaboration with different agencies and scientists of various disciplines plays a key role in achieving the goal.

Vegetatively propagated crops such as banana and plantain, pineapple, sweet potato, yam, cassava and sugarcane represent important sources of food in developing countries. These crops contribute significantly to the gross national product as cash crops (ornamentals, timber and fibers) and as food (fruits and rootcrops). For instance, global production of banana is about 68.5 million tons and approximately 7.0 million tons go to the world export trade. The Philippines is one of the world’s top producers of some vegetatively propagated crops (VPC). The country supplies 80% of the global fiber needs (abaca) and contribute about 30.0% of banana in the world market. Likewise, the Philippines leads in the pineapple world export trade (Philippine Almanac, 2000).

Plant breeding requires genetic variation of useful traits for crop improvement. Often, however, desired variation is lacking. Mutagenic agents such as ionizing radiation and certain chemicals can be used to induce mutations and generate genetic variations from which desired mutants may be selected.

Based on the FAO/IAEA mutant varieties database, more than 1,800 mutant varieties have been officially released. Of this total, 1,237 are seed propagated crops (FAO/IAEA, 1997).These data indicate that varietal improvement of vegetatively propagated crops lags behind seed propagated crops. Hence, there is a need to focus breeding efforts on the induction of mutations for genetic improvement of these economically important crops.
This paper summarizes the status of mutation breeding of vegetatively propagated crops in the Philippines.

The Philippine Nuclear Research Institute (PNRI) DOST is the lead agency in radiation-induced mutation breeding for crop improvement. The Mutation Breeding Program is presently being undertaken by the Agricultural Research Section of the Atomic Research Division, PNRI.

In recent years, induced mutations have played an increasing role in the breeding programs of sweet potato (Ipomea batatas L. Poir), pineapple (Ananas comosus (L.) Merr) and foliage ornamentals (Dracaena sanderiana D. godseffiana, Cordyline terminales, Freycinetia ). Several improvement varieties of VPC with desirable traits were successfully developed through induced mutation breeding at our Institute. Likewise, the Institute of Plant Breeding, of the University of the Philippines in collaboration with PNRI has induced mutants of banana and mussaenda through gamma irradiation. Research studies on the radiosensitivities of other vegetatively propagated
crops to ionizing radiation have been undertaken by the academe and other research institutions (Table 1).

A. Pineapple
Pineapple (Ananas comosus L. Merr.) is one of the most important export crops of the Philippines. The pineapple Queen variety is the most desirable for fresh consumption because of its sweetness, low fiber content and crispiness of the flesh. However, one objection to this variety is the presence of troublesome hard spines which interferes with weeding and harvesting.
Mutation breeding through the use of ionizing radiation (gamma rays) coupled with in-vitro culture techniques is being undertaken for the development of improved pineapple Queen Variety with the following objectives.

1. To eliminate or reduce the thorns along the leaf margins
2. To induce chlorophyll mutation which can be the bases of ornamental types
3. To establish protocol for tissue culture techniques for the Queen Variety of pineapple
4. To make use in-vitro culture techniques as a tool for mutation induction

In vegetatively propagated crops that do not produce some seeds like pineapple, the mutation breeding technique may be considered an advantage over the conventional method because of the difficulty of producing seeds and/or making crosses in this crop and that mutation breeding should be used when it is most important to retain a complex trait such as good eating quality in the variety that needs improvement for a simple-inherited characters. Likewise, it is of utmost importance to develop a method whereby shoots or plants are obtained which originate from only one cell. Irradiation of axillary buds has been reported to be a successful technique for inducing mutations (Lapins, 1971). Buds consist of a few cells which provide larger mutated sectors in the growing shoot (Lapins, 1971). Hence, the use of the in-vivo approach (through irradiation of axillary buds) and the in-vitro technology (through tissue culture techniques) were the methods used to attain the above-mentioned objectives.

Crown axillary buds of pineapple variety Queen were irradiated with 5, 10, 20, 30,40 and 50 Gy of gamma radiation.

In the MV1 generation the percentage emergence, height of the plantlets, length of the roots and survival in the MV1 generation decreased with the increasing dose of gamma radiation.

The morphological changes induced by gamma radiation were: plantlets with tumorous roots, compact plantlets with thick leaves, and plantlets with leaf abnormalities such as twisted, narrow or reduced spines, or inward curved, striated or fused leaves. Generally, the frequency of these morphological changes increased with the increasing dose of radiation.

The chlorophyll mutations obtained in the MV2 plantlets ranged from viridis, chlorina, striata and xantha to albina. An increase in anthocyanin pigmentation was obtained with doses ranging from 5 to 40 Gy of gamma radiation.

Desirable mutations with reduced spines and chlorophyll mutants were selected in MV2, MV3 and later generations. These mutants were propagated asexually using the axillary bud technique. Further selections and evaluation of usefulness of these mutants were undertaken.

Recurrent irradiation was also performed to increase the frequency of mutations. Axillary buds that had previously been irradiated with 5-50 Gy were recurrently irradiated with 30 and 40 Gy.

Chlorophyll mutants that look-like ‘ornamental bromeliads’ were selected from the recurrently irradiated population. These mutants were grown in pots under greenhouse conditions and later on transplanted in the field, where it developed normal fruit with a crown, exhibiting the same morphology as the leaves of the plant. Both the chlorophyll mutant and the plants with reduced spines were propagated using the axillary bud techniques. Once a sufficient number (100 plants) is attained this chlorophyll mutant will be submitted to the National Seed Industry Council for registration as an improved variety.

Purification of mutant plants with reduced thorns along the leaf margins were continued. These mutants will be multiplied and grown for further evaluation.
Putative mutants with bigger fruits were isolated. This mutant will be multiplied through the use of suckers for further verification.

Tissue culture is a potential tool for induced mutation breeding. However, before one can make use of this technique for mutation induction purposes, there is a need to establish the protocol for plantlet regeneration.

In pineapple Queen Variety, callus tissues were induced from crown axillary sections inoculated in Murashige and Skoogs Medium (MS) with benzyl adenine (BA) and naphthalene acetic acid (NAA). These callus tissues were successfully regenerated into plantlets using the above culture medium.

Callus tissue were irradiated with different doses of gamma radiation ranging from 5 to 50 Gy. After irradiation, the control as well as irradiated calli were inoculated in MS medium with 2 ppm BA + 2 ppm NAA since the best formulation for callus induction and shoot regeneration was observed in this medium. Calli irradiated with 20 Gy produced the most number of plantlets (90%) cultured in these. Callus tissues were further subcultured in the same medium formulation every 2-3 weeks. Vigorous plant growth was noted at 20 Gy.

Regenerated plantlets with well-developed roots were compotted and acclimatized before planting. Somaclonal variants with reduced spines were obtained from in-vitro culture technique.
Based on the results of these studies, the use of gamma radiation coupled with tisssue culture techniques have resulted in the development of the following mutants in pineapple Queen variety:


1. Chlorophyll mutants that look like ornamental bromeliads
2. Plant with reduced thorns
3. Putative mutants with increase size of the fruits
B. Banana

Banana (Musa sp.) is a major export of the Philippines. It is planted to 333,430 hectares with 3.2 million metric tons export valued at P109 billion (approximately US$2.18 billion).

Bunchy top remains to be the most destructive virus disease of banana (Musa sp.) in the Philippines. While rapid propagation of disease-free planting materials is a viable disease management option, its effectivity is limited where residual or alternative inoculum sources are present. Thus, built-in resistance remains to be the most effective disease control measure. There is no known resistance to brunchy top virus (BTV) in the banana germplasm.

Thus, the use of gamma radiation in combination with in-vitro related biotechnology is being used in the development of banana that is resistant/tolerant to BTV.

Results of this study indicated that the LD50 of shoot explants of banana var Lakatan was established to be 20-25 Gy. The procedure from initial culture to multiplication of shoots, irradiation and subsequent multiplication of shoots was optimized and standardized. A total of 3,099 and 4,466 plantlets were regenerated from radiosensitivity experiments and from explants irradiated at the optimum dose, respectively. Out of 4,042 artificially inoculated plants, 216 were putative BTV resistant. As of Dec. 2000, putative BTV resistant banana plants in the field numbered: 92 from irradiated materials and 6 from in vitro somaclonal variation experiment (no irradiation). Initial PCR-based diagnosis of BTV in putative BTV resistant materials showed 18 plants to be consistently negative out of 25 tested. ELISA tests will follow. Evaluation for field BTV resistance and horticultural traits of the banana clones is being continued (Mendoza et. al, 2001).


Ornamentals have become an important commodity, a promising dollar-earning crop in the country. Cutflowers such as chrysanthemum and foliage plants such as Dracaena sp., Cordyline terminales and Freycinetia sp. have big market potential both locally and abroad. Some of the problems facing the Ornamental Horticulture Industry are: (1) lack of tested varieties for cutflower production, (2) poor quality of cutflowers which fail to meet international standards, (3) lack of new and improved ornamental plants to satisfy changing consumer demands and (4) inadequate supply of planting materials with superior quality.

Chrysanthemum morifolium is a hexaploid, outcrossing and vegetatively propagated plant. With this breeding system, heterozygosity is the rule, which makes this crop amenable to mutation breeding. In vegetatively propagated crops such as chrysanthemum, genetic improvement is through induced mutation breeding with the objective of developing dwarf mutants with longer vase life and earliness to bear flowers.

In foliage plants such as Dracaena sp., Cordyline terminals and Freycinetia sp., ionizing radiation is used to induce variation in form and color of leaves, plant size and growth habit (dwarfism).

Ionizing radiation complemented by tissue culture is being undertaken to accelerate genetic improvement of ornamental crops mentioned above.

A. Cutflower Ornamentals

Stem cuttings of Chrysanthemum morifolium were irradiated with 10 and 20 Gy gamma rays. In the M1V3 and M1V4 generations morphological changes induced by gamma radiation were shortening of the internode, stem bifurcation and chlorophyll mutations. Suckers were taken from this generation and planted as M1V5 generation. Putative mutants for earliness to bear flowers were selected.

Tissue culture studies in chrysanthemum is being done as a tool for mutation induction and as a means of micropropagation. The effects of different doses of gamma radiation on callus induction from nodal sections of chrysanthemum grown in Murashige and Skoog’s (MS) with naphthalene acetic acid (NAA) and benzyl adenine (BA) were studied.

Best results for callus induction using nodal section as explants was observed at 5Gy gamma rays and grown in MS +2 ppm BA and 2 ppm NAA; and 20 Gy cultured in MS +6 ppm NAA. The biggest calli were developed in those levels of gamma radiation and media formulation. For the regeneration of shoots, the best treatments were 5 and 10 Gy in MS basal medium, 0 and 5 Gy sub-cultured in MS +6 ppm NAA and dose level 5 and 10 Gy in MS +2 ppm BA +2 ppm NAA.

Micropropagation of the irradiated and unirradiated chrysanthemum using MS basal medium is presently being done. Research studies showed that stem sections irradiated with 10 and 20 Gy dose gamma rays showed vigorous growth of plantlets as compared to 30-50 Gy doses. From these results, chrysanthemum stem sections were irradiated with 10 and 20 Gy gamma rays and micropropagated aseptically in full and half strength MS basal medium.

Root formation was observed in all the stem sections cultured but multiple root formation was noted in 10 Gy dose at the M1V2 generation. Cultures were further subcultured in the same medium and grown up to M1V5 generation.

Whorling and changes in leaf color were observed at 10 Gy dose and doubling of leaf growth at the node at 20 Gy of the 3rd vegetative generation. Morphological changes observed in the 4th generation was multiple branching per node in the 10 Gy dose as well as in the 20 Gy. From the 5th vegetative generation, plantlets with well-developed roots were transferred and grown in seedling boxes for 10-12 days for acclimatization and later grown in greenhouse.

B. Foliage Ornamentals

IDracaena sanderiana ‘virescens’ belongs to Lily Family. It is slender, more or less succulent plant with unbranched stems. Leaves are distant, alternate, lanceolate, acuminate, dark green with occasional faint lines of pale green.

Stem cuttings of D. sanderiana were exposed to 10 and 20 Gy dose of gamma rays. Immediately after irradiation, these were planted in seedbed and grown under greenhouse conditions as M1V1 generation. When these plants are big enough, cuttings were obtained from M1V1 and were planted in pots as M1V2 generation. Chlorophyll mutations and other morphological changes were induced. Continuous selection of desirable mutants was done from M1V3 to M1V5 generations. Dracaena Chlorophyll Mutants were propagated through cuttings and are grown up to M1V7 generation.

In the M1V7 generation, D. sanderiana chlorophyll mutant was purified. It is a robust plant with variegation of the leaves (the center is silver green with white stripes and the broad margins in deep green). The Dracaena chlorophyll mutant registered with the National Seed Industry Council of the Department of Agriculture as improved variety. The registered named for this mutant is Dracaena ‘Marea’. This mutant was highlighted and test-marketed during this year’s Atomic Energy Week Celebration. At present, there are 180 full-grown plants available for sale to interested clients.

Cuttings of D. godseffiana were exposed to 60Co gamma radiation and grown to first vegetative generation. When plants attain maturity, cuttings were obtained and planted in pots as second vegetative generation. Morphological changes observed were reduction in leaf size and chlorophyll mutations.

Cordyline terminales is an erect, unbranching shrub with recurved, strap-shaped leaves, tinged with orange and red near the tip of the stem. Stem cuttings were irradiated with 10 and 20 Gy that resulted in the selection of chlorophyll mutants.

Freycinetia multiflora Merr is characterized by several slender shoots arising from the base of the plant, forming a clump. Leaves are spirally arranged, forming three distinct row. It has bright orange bracts of inflorescences that are very attractive. Irradiation of stem cuttings with gamma rays resulted in the selection of putative mutants with reduced thorns and plants with short internode and reduced leaf area.

C. Mussaenda
Studies on the gamma irradiation of rooted cuttings of Mussaenda at 30 Gy resulted in the induction of two desirable mutants: one with patches of white on the peach petaloids of otherwise solid peach petaloids of non-irradiated Doña Hilaria; and the other mutant has open and thicker petaloids compared to non-irradiated Doña Aurora. These mutants are now being stabilized and subjected to cytological and horticultural evaluation. Recurrent irradiation of rooted cuttings which did not show any mutation in flower morphology is being presently done (Mendoza et. al, 2001).


Research studies on the induction of mutations using ionizing radiation in vegetatively propagated crops (i.e. sampaguita and black pepper) were undertaken by academic institution (De La Salle-Araneta University Foundation) in collaboration with Philippine Nuclear Research Institute. Basic studies on the determination of the radiosensitivity of the different propagules of these two crops were completed. Results of these studies are presented below.

A. Sampaguita (Jasminium sambac L. Ait)
This is viny ornamental known for its profuse, white and fragrant flowers. Casyao (1991) studied the effects of gamma radiation using different doses ranging from 5 to 100 Gy on three popagules: leaf cuttings, unrooted and rooted stem cuttings or whole plant. Gamma radiation increased the number of flowers per plant, number of petals and stimulated the fragrance of fully opened blossoms.
B. Black Pepper (Piper Nigrum)
Determination of the radiosensitivity of the stem cuttings of black pepper was studied by Baluyot (1993). The results indicated that the stem cuttings can tolerate radiation dose to 5 to 10 Gy, while seeds can tolerate up to 100 Gy. The effects of gamma radiation ranged from increase leaf size, leaf streaking and early flowering.

Induced mutation holds promise in the genetic improvement of plants. In crops that do not produce any seed, mutation breeding is the only way to improve the selected variety, or when the desired character is not present in the existing population within the reach of the plant breeding in a situation wherein the desired character is controlled by a single gene and a desirable complex character such as good eating quality has to be retained.

Radiation-induced mutagenesis, in combination with tissue culture techniques greatly enhances the efficiency of mutations. There are several situations which call for the use of mutation breeding. These are when the objectives are: (1) reduction in height or dwarfiness (2) chlorophyll mutation (3) diseases resistance controlled by a recessive gene (4) photo-period insensitivity and (5) earliness.


Radiation-induced mutagenesis coupled with in-vitro culture techniques and related biotechnology will be applied in the International Atomic Energy Agency (IAEA) Technical Cooperation Project “Enhancing Agricultural Productivity in Mindanao through Radiation Technology: Component II. Mutation Breeding in Fruit Crops.”

Utilization of gene amplification by polymerase chain reaction (PCR)/micro-array will facilitate screening of polymorphism among a wide variety of crops. Amplified DNA from PCR could be used in the characterization of mutants induced by gamma radiation.


The authors wish to thank the IAEA Technical Cooperation Project, Department of Science and Technology (DOST) Grants-in-Aid Program and the Philippine Nuclear Research Institute for the financial support and Irradiation Services for irradiating our samples.


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