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FNCA 2002 WORKSHOP ON MUTATION BREEDING |
STATUS OF MUTATION BREEDING
IN VEGETATIVELY PROPAGATED CROPS IN THE PHILIPPINES 1/
A.G. Lapade, A.M.S. Veluz, L.J. Marbella, A.C. Barrida
and
M.G. Rama. Philippine Nuclear Research Institute.
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Abstract |
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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. |
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1/ |
Paper to be presented in the 2002 FNCA Workshop on
Mutation Breeding
August 20-23, 2002. Beijing, China. |
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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 worlds 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).
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2. |
MUTATION BREEDING IN FRUIT CROPS |
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A. Pineapple |
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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.
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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 |
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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:
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1. |
Chlorophyll mutants that look
like ornamental bromeliads |
2. |
Plant with reduced thorns |
3. |
Putative mutants with increase
size of the fruits |
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B. Banana |
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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).
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3. |
MUTATION BREEDING IN ORNAMENTALS |
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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.
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A. Cutflower Ornamentals |
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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 Skoogs (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. |
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B. Foliage Ornamentals |
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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.
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C. Mussaenda |
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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).
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4. |
MUTATION BREEDING IN OTHER VEGETATIVELY
PROPAGATED
CROPS |
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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.
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A. Sampaguita (Jasminium
sambac L. Ait) |
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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. |
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B. Black Pepper (Piper
Nigrum) |
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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. |
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5. |
PROSPECTS OF MUTATION BREEDING |
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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.
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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. |
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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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