Taiwan Banana Research Institute
P.O. Box 18
This paper discusses the micropropagation of banana plantlets in Taiwan. The main purpose is to produce healthy plants that are free of virus diseases, particularly banana bunchy top and banana mosaic. The plantlets also have the advantages that they are more uniform than suckers, and the adult plants can be harvested over a shorter period. The paper describes the protocol of micropropagation, and also the protocol used for virus indexing of mother plants and a random sample of tissue culture plantlets.
Banana is one of the most important fruit crops grown for export in Taiwan, as well as for local consumption. The flavor and texture of Cavendish bananas grown in a subtropical climate are superior to those of bananas grown in tropical regions. Thus, Taiwan bananas have maintained their popularity in the Japanese market over the last three decades, in spite of keen competition from the Philippines and Latin American countries.
The triploid Cavendish banana is traditionally propagated vegetatively by suckers. Most banana growers in the southern lowlands of Taiwan practice annual cropping. Replanting the orchard between March and May ensures that high-quality fruit can be harvested within the export season, without danger of typhoon damage.
Fusarial wilt appeared in southern Taiwan in 1967, and spread rapidly in suckers used as planting materials. It soon became a major threat to Taiwan’s banana industry. The most effective way of stopping the spread of this soil-borne pathogen is to provide disease-free planting materials. The traditional method of producing seed corms is slow and expensive.
Thus, the urgent need for a large amount of disease-free planting material triggered the development of the banana meristem culture technique (first reported by Ma and Shii 1972, 1974) to mass propagate tissue culture plantlets for commercial planting. These tissue culture plantlets first became available in 1983 (Hwang et al. 1984) and are now widely used by banana growers in Taiwan.
Banana tissue culture plantlets have several advantages. They have a higher survival rate than suckers, reduce the cost of disease and pest control, show uniform and vigorous growth, and have a shorter harvesting period. From 1983 to 2000, a total of 33.3 million plantlets have been produced for commercial planting to check the spread of fusarial wilt in Taiwan. They have enabled the banana industry in Taiwan to achieve stable levels of production.
Meristem culture can eliminate a number of pests and diseases. However, it is possible for viruses to be transmitted through micropropagated materials. Thus, a virus indexing system (ELISA test) has been established to prevent the spread of virus diseases such as banana bunchy top (caused by Banana Bunchy Top Virus (BBTV)) and banana mosaic (caused by Cucumber Mosaic Virus (CMV)) in infected tissue culture plantlets.
Somaclonal variation is common in tissue culture plantlets. In the present system, the variants amounted to about 3%. The annual planting of two to three million plantlets over the last fifteen years has provided an excellent opportunity for the selection and improvement of banana cultivars through somaclonal variation for resistance to fusarial wilt, and other agronomic traits such as plant height, bunch size and yield (Hwang and Ko 1988, 1989; Tang and Hwang 1998).
The micropropagation of banana plantlets consists of five stages: the preparative stage the establishment of the culture the multiplication of shoots the regeneration of plantlets and acclimatization of the plantlets in the nursery.
The Preparative Stage
Young suckers selected from healthy and true-to-type mother plants are checked for symptoms of fusarial wilt. They are then indexed (using the ELISA test) to check for Banana Bunchy Top Virus and Cucumber Mosaic Virus.
The outer layers of leaves and the corm tissue of the sucker are removed. What remains is a piece 10 cm long and 6-8 cm in diameter. The piece is wiped clean with 75% alcohol. Under aseptic conditions, the sheaths and bases of the leaves are trimmed to expose the meristematic region.
The shoot tip is decapitated, and a block of tissue measuring 1.5 x 1.5 x 1.0 cm is excised and inoculated onto multiplication medium (MS salts supplemented with thiamine CH1 0.4 mg/L, myo-inositol and L-tyrosine both at 100 mg/L, sugar 30 g/L, agar 5 g/L, growth regulators including BA 4 mg/L, IAA 1.6 mg/L and adenine sulfate 80 mg/L.
The pH is adjusted to 5.8 before autoclaving). The explant is incubated at 26-28oC with a 12 hour light/dark cycle. After four or five weeks, 15 to 25 buds can be induced from each explant.
The adventitious buds induced from the meristematic tissue can be divided into smaller pieces and subcultured onto fresh multiplication medium. The multiplication rate of buds depends on the cultivar, the concentration of cytokinin and the number of subcultures. The number of subcultures should be limited to six or seven, in order to reduce the incidence of off-types arising through somaclonal variation.
Elongation and rooting of adventitious buds is accomplished by adding liquid regeneration medium to the established shoot/bud culture. The regeneration medium consists of MS salts, sugar (20 g/L) and activated charcoal (1.6 g/L). Plantlets in culture vessels are hardened under 50% shade for two or three weeks before they are transplanted into a screened nursery.
Acclimatization in the Nursery
After plantlets have been removed from the culture vessel, the clusters are washed to remove the agar, separated into individual plants, and sorted according to size (large: more than 5 cm high; medium: 3 to 5 cm and small: less than 3 cm). They are then transplanted into boxes of soil.
Planting density is high, with 126 plantlets in a box measuring 58 x 36 x 12.5 cm. After planting, the box is covered with a clear plastic cover for seven to ten days, to conserve moisture.
The plastic is then removed, and two or three weeks later, the rooted plantlets are removed from the box and delivered to the screened nurseries of the Fruit Cooperatives in banana-growing areas of Taiwan. In the cooperative murseries, the plantlets are transplanted into plastic pots 9 cm high and 10 cm in diameter. They are kept in the screened nursery for six to eight weeks until they reach a height of around 15 cm. At this stage, they are released to growers to plant out in the field.
Diseases caused by Banana Bunchy Top Virus (BBTV) and Banana Mosaic Virus (CMV) are widespread in Taiwan. They affect all the major commercial Cavendish cultivars. Viral diseases caused by BBTV and CMV can be spread in infected vegetative planting materials such as suckers, and can also be transmitted between plants by aphid vectors. Control measures include the destruction of diseased plants, the control of vector populations, and the use of virus-free planting materials.
The direct Double Antibody Sandwich Enzyme Linked Immunosorbent Assay (DAS ELISA) is routinely used for the indexing of BBTV (Wu and Su 1990). The midrib tissue of the first young leaf is used for the extraction of sap, at a rate of 1 to 4 (w/v) using 0.05M Tris buffer containing 0.1% DIECA and 5% sucrose, to which has been added 0.5% sodium sulfite.
Isolates of CMV from banana plants were used to produce monoclonal antibodies at National Taiwan University (Wu 1994). These antibodies are used to detect CMV in banana plants. The pooled sample of the first three leaves is used in sap extraction, at a rate of 1 to 10 (w/v), with a 0.05M citrate buffer containing 0.5mM EDTA, 0.5% thioglycerol, 2% PVP and 0.05% Tween 20 (Thomas 1991).
All suckers used in the micropropagation of tissue culture plantlets are indexed for BBTV and CMV. At the stage of acclimatization in the nursery, random samples of banana plantlets are taken from local nurseries for ELISA testing. This second virus indexing serves to monitor the health of plantlets and the effectiveness of insect control carried out in the local nurseries.
From the time of field planting, growers are responsible for protecting the plantlets by the control of vectors and weeds which serve as hosts, and by avoiding growing the young banana plants near legumes or vegetable crops. The eradication of any diseased plants is also important in checking the spread of disease.
IMPROVING MICROPROPAGATION TECHNIQUES
The banana micropropagation system in Taiwan was established as a service for banana growers who are members of the Taiwan Fruit and Marketing Cooperative. The disease-free plantlets are supplied at a minimal fee, to encourage members to plant them. Within the last decade, rapid industrialization and the aging of the rural population has caused a considerable shortage of farm labor, and a rapid increase in farm wages. Hence, there is constant pressure on the micropropagation system to become more efficient, in order to counterbalance the rapid increase in production costs. Recent improvements made in the micropropagation technique are shown below.
Suckers used in micropropagation are obtained from healthy true-to-type mother plants which are virus indexed and kept in the stock nursery (an insect-proof net-house). In view of the large number of suckers required each growing season, true-to-type stock plants of different cultivars are also maintained in the stock nurseries under strict field management. All suckers are virus indexed for BBTV and CMV.
At this stage, dividing the piece of meristematic tissue (explant) into halves or quarters can increase the total number and quality of adventitious buds induced. The use of gelrite in culture initiation means that the medium is less likely to blacken (from the oxidation of phenolic compounds) than if agar is used.
Multiplication of Adventitious Buds
In the multiplication of adventitious buds, adjusting the level of growth regulators can improve the quality of plantlets by reducing the proportion of small plantlets (i.e. those less than 3 cm. high). The level of growth regulators should be 4 mg/L BA and 1.6 mg/L IAA in MS medium during subcultures. At the last subculture before the regeneration of plantlets, the concentration of BA should be reduced to 3 mg/L and IAA 1.2 mg/L. Larger plantlets are more easily and quickly acclimatized in the nursery than very small ones.
At the early stage of multiplying adventitious buds, if gelrite is used as the gelling agent in the multiplication medium, the shoot cluster obtained is larger than if agar is used. After one to two further subcultures, there are fewer shoots and the buds become greatly enlarged. However, when 2.5 g/L agar mixed with 1.0 g/L gelrite was used as the gelling agent, the adventitious buds induced were superior to those induced by the use of agar or gelrite alone.
The use of gelrite in place of agar in the final subculture before liquid is added slightly reduced the number of plantlets regenerated. In a study of the effect of temperature on the multiplication of adventitious buds, incubation at 27oC was found to be better than 22oC or 32oC. The use of table sugar instead of sucrose, and locally manufactured agar instead of tissue culture grade agar, has reduced the cost of production considerably.
Regeneration of Plantlets
The regeneration of plantlets is achieved by the elongation of buds into shoots, and the rooting of these shoots. A high concentration of cytokinin in the multiplication medium at the stage when adventitious buds are forming may inhibit shoot elongation and rooting. For this reason, a different regeneration medium is required. Plantlets can be regenerated by dividing the cluster of adventitious shoots into groups of two or three buds. These are then transferred to agar regeneration medium which does not include any growth regulators.
However, a much more efficient and labor-saving procedure is to reduce the concentration of BA to 3.0 mg/L at the last multiplication stage, and add liquid regeneration medium directly to the established culture of shoots. It was found that liquid medium containing MS macro and micro salts, 20 g/L sugar and 1.6 g/L activated charcoal produced healthier plantlets and reduced the number of small plantlets (less than 3 cm high), compared to the use of liquid media containing 3 g/L or 5 g/L Hyponex No. 1 (Lee 1993).
Adding growth retardant (Paclobutrazol) to the liquid regeneration medium improved the development of the root system and increased the number of new leaves and the fresh weight of the plantlets. When plantlets were transplanted into potting mix, the higher tolerance to wilting of plantlets cultured in a medium with Paclobutrazol increased their survival rate in the nursery.
Acclimatization in a Screened Nursery
In our search for a better potting medium, we found that the addition of organic potting mix consisting of peat moss, carbonized rice hull and sand (1:1:1 v/v), increased the growth rate of plantlets, compared to the traditional potting mix with sand only. However, we also wanted a cheaper soilless potting mix. We found that a mixture of organic fertilizer from the Taiwan Sugar Company (the main component of which was cow manure) and sawdust, at a rate of 1:2.5 by volume, could support the growth of plantlets for two months. The light weight of this mixture also means makes transporting the plantlets easy and convenient.
If the plantlets are removed from the culture vessels and directly transplanted into plastic pots under clear plastic, they show a higher rate of survival. This makes it possible to omit the labor-intensive step of first transplanting them into boxes of soil. However, smaller plantlets (less than 3 cm tall) still require to be first transplanted into soil during the low-temperature months of January and February. The minimum temperature at night during these two months may drop to only 8-10oC.
Somaclonal Variation in the Improvement of Banana Cultivars
The occurrence of off-types has been reported in both bananas and plantains all over the world. Their frequency varies from as high as 50% to as low as 1-3% (Israeli et al. 1995). In Taiwan, the percentage of off-types in 30,000 young tissue culture plantlets was estimated to be 0.37%. The percentage of off-types among 46,260 mature plants growing in the field was 2.43% (Hwang 1986).
In the micropropagation system, the major cultivars for commercial propagation are Pei Chiao, Tai Chiao No.1 (a fusarial wilt resistant variant from Pei Chiao) and Tai Chiao No. 2 (a semi-dwarf cultivar). All three cultivars belong to the Cavendish group. These cultivars are rather stable, and the occurrence of somaclonal variation is less than 5%. When tissue culture plantlets are distributed to farmers, growers are given 5% extra plantlets as compensation for off-types.
Various precautions are taken for the control of somaclonal variation. The stock plants are maintained either in an insect-proof nethouse, or in stock nurseries under strict field management. Suckers for micro-propagation are collected from true-to-type stock plants between January and June. A large number of suckers are used every cycle in the initiation of stock material for micropropagation. In the production of one million plantlets, about 1000 suckers are used. On average, therefore, one to two thousand plantlets are propagated from each sucker.
As mentioned above, the number of subcultures should be limited to six to seven, and the concentration of cytokinin used is gradually reduced towards the final subculture for rooting. During subculturing, any underdeveloped or abnormal buds are discarded. At the stage of deflasking, any very small or abnormal plantlets are discarded. Variants, especially those with variegated leaves or abnormal leaf shape, are eliminated at the nursery stage by trained personnel before the young plants are distributed to growers.
Previous studies of the improvement of banana cultivars in Taiwan have shown that somaclonal variation is an effective way of improving cultivars (Tang and Hwang 1994). In the last decade, about two million plantlets were propagated for commercial planting each year. This system has provided a large number of tissue culture plantlets for the improvement of banana cultivars through the selection of somaclonal variants.
In 1984, a mass screening program of tissue culture plantlets in diseased fields selected ten clones resistant to race 4 of fusarial wilt (Hwang and Ko 1988). After cycles of improvement and field trials, the selected somaclone GCTCV-215-1 was registered and officially released as Tai Chiao No. 1 in 1992. Up to the present, a total of 6 million tissue culture plantlets have been propagated for commercial planting. In 1992, a semi-dwarf mutant (TC1-229) derived from GCTCV-215-1 was found in a farmer’s field. The semi-dwarf plant growth habit is truly inherited, and the line also retains its resistance to fusarial wilt (Tang and Hwang 1998). Application for registration of this cultivar as Tai-Chiao No. 3 was approved in December 2000. Between March and April 2001, 120,180 tissue culture plantlets of Tai-Chiao No. 3 were produced for field planting.
The micropropagation of tissue culture plantlets has played an important role in the successful selection of clones which are resistant to race 4 of fusarial wilt. Somaclonal variation also offers the opportunity for the improvement of other agronomic traits, such as a reduction in plant height, early shooting and increased bunch weight. A semi-dwarf cultivar introduced from Barbados, after many cycles of selection and domestication, was registered in 1993 and released to framers as Tai Chiao No. 2. Up to the present, a total of four million plantlets of Tai Chiao No. 2 have been propagated for commercial planting.
With the support and guidance of the Council of Agriculture and the Department of Agriculture and Forestry, the Taiwan Banana Research Institute (TBRI) and the Taiwan Provincial Fruit and Marketing Cooperative have jointly established a micropropagation system for virus-indexed disease-free banana plantlets for commercial planting. TBRI is responsible for the virus indexing and the in vitro stages of micropropagation. The Fruit Cooperative manages the last stage of acclimatizing the plantlets, in screened nurseries located in banana-growing regions. The Fruit Cooperative surveys market demand, and advises members when to register (six to eight months in advance) for the number of plantlets they will need, the cultivar, and the time of planting. About 70 to 80% of the total supply is needed in February and March, in accordance with the cultural practices of banana growers. The total number of plantlets supplied to growers between 1983 and 2002 was 39 million (see Table 1 (627)).
Monoclonal antibodies specific to BBTV and CMV are produced at National Taiwan University. A virus indexing routine using DAS-ELISA has been established for the micropropagation of disease-free tissue culture plantlets. It is used at two stages: firstly, for the individual suckers which are used as stock material for micropropagation, and secondly, for culture plantlets transplanted into plastic pots in the local nurseries. These are randomly sampled for indexing before plants are released to growers for planting out in the field.
The banana micropropagation system has served growers in Taiwan over the past twenty years through the strong support of the government. Research and development activities will continue to improve the micropropagation system, in an effort to reduce production costs. This unique system has succeeded in providing disease-free and virus-indexed banana plantlets to growers at a minimal cost, to stabilize Taiwan’s banana industry in an increasingly competitive export market.
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- Tang, C.Y. and S.C. Hwang. 1998. Selection and asexual inheritance of a dwarf variant of Cavendish banana resistant to race 4 of Fusarium oxysporum f. sp. cubense. Australian Journal of Experimental Agriculture 38: 189-94.
- Thomas, J.E. 1991. Virus indexing procedures for banana in Australia. In: Banana Diseases in Asia and the Pacific, Valmayor, R.V. B.E. Umali, and C.P. Bejosano (Eds.). INIBAP, Montpellier, France, pp. 144-157.
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- Wu, R.Y. and H.J. Su. 1990. Production of monoclonal antibodies against banana bunchy top virus and their use in enzyme-linked immunosorbent assay. Jour. Phytopathol, 128: 203-208.
- Wu, M.L. 1994. Preparation and Application of Monoclonal Antibodies to Studies on Strains and Infection Ecology of Cucumber Mosaic Virus Causing Banana Mosaic. Unpub. Ph.D. Thesis, National Taiwan University, Taipei, Taiwan, R.O.C.
Index of Images
Figure 1 Diagram of Micropropagation Procedure
Table 1 Number of Banana Plantlets Propagated in Taiwan for Distribution to Growers, 1983-2002.