Regeneration of Healthy Banana Plantlets from Banana Bunchy Top Virus-Infected Tissues Cultured at High Temperatures

R.Y. Wu* and H.J. Su
Department of Plant Pathology and Entomology,
National Taiwan University,
1 Roosevelt Rd, Section 4,
Taipei, Taiwan, ROC
*Development Center for Biotechnology,
81 Chang Hsing St., Taipei, Taiwan, ROC, 2004-11-01

Introduction

Banana bunchy top disease, which was first reported from Fiji in 1889 (Stover 1972) and noted in Taiwan in 1900 (Sun 1961), is the most destructive virus disease of banana in the Eastern hemisphere. Banana bunchy top virus (BBTV) is considered to be a luteovirus by Matthews (1982) by virtue of its transmission in the persistent manner by aphids and the phloem damage caused by infection. Research progress on the disease has been very slow in comparison with many other serious virus diseases because of an inability to purify the virus. This is because of the presence of large amounts of latex and phenolic compounds in banana tissue which interfere with virus extraction and purification (Dale et al. 1986).

BBTV was recently purified by a procedure which included pulverizing frozen diseased tissues in liquid nitrogen to reduce the interference by latex and phenolic compounds. The extract obtained was clarified with chloroform-butanol and by differential centrifugation to remove contaminants which would otherwise have prevented the detection of BBTV by electron microscope observation and UV scanning (Wu and Su 1990a). BBTV was shown to be a small luteovirus (20-22 nm in diameter) consisting of single-stranded RNA with a relative molecular mass (Mr) of 2.0 x 106 and coat protein subunit with Mr of 21 000. Subsequently monoclonal antibodies against BBTV have been produced (Wu and Su 1990b).

The Taiwan Banana Research Institute has a large collection of banana varieties planted in the field. As bunchy top is frequently found in banana plantations in Taiwan, it is difficult, if not impossible, to prevent banana plants in the collection from being infected with this disease. A program was, therefore, initiated to develop a method of freeing infected banana plants from BBTV.

Materials and Methods

Tissue Culture

The method of Ma and Shii (1972, 1974) was used to obtain plantlets of Cavendish banana (Musa acuminata). Explants obtained from the decapitated shoot apices of suckers infected with BBTV were cultured at 28oC on the medium of Smith & Murashige (1970) in glass vials (28 x 95 mm) to induce the formation of adventitious buds. When 5-10 adventitious buds were produced on each explant, the explant was cut longitudinally into smaller pieces, each having 1-2 buds. These pieces were then transferred to fresh medium for further proliferation of adventitious buds.

Tissue with adventitious buds was transferred to fresh medium amended with 0.1% (w/v) activated charcoal and incubated under continuous cool white fluorescent light to induce root and shoot development. When plantlets reached a height of 5-8 cm, they were transplanted into a 50% sand and 50% peat-moss growing medium in plastic pots (7 x 10 cm).

Heat Treatment of Diseased Rooted Plantlets

Diseased plantlets c. 35 cm high were exposed in growth chambers to alternating temperatures of 40oC for 16 h during the day and 30oC for 8 h at night. After heat treatment for 3 or 5 weeks, more than 90 banana aphids (Pentalonia nigronervosa) were placed on each treated plantlet and held at 28oC for 1 day. The aphids were then transferred to healthy, 2-month-old banana plantlets for 1 day in an infectivity assay as described previously (Wu and Su 1990c). The tests were repeated 2 weeks later after further incubation at 28oC. Thirty aphids were used to inoculate each plantlet. Inoculated plantlets were incubated at 28oC for symptoms to develop.

Heat Treatment of Diseased Tissues

BBTV-infected tissue from two cultures, each consisting of 4-5 adventitious buds maintained on agar medium in glass vials, was exposed to alternating temperatures as described above. After treatment for 4, 6, 8, 10 or 12 weeks, two vials per treatment were moved to a growth chamber at 28oC for further growth. Treated tissue was transferred to fresh medium for the production and development of adventitious buds. Disease incidence was assessed after two months.

Culture of Diseased Tissues at High Temperatures

BBTV-infected tissues, consisting of 4-5 adventitious buds maintained on the medium in glass vials, were incubated at 35oC in a growth chamber under continuous fluorescent light. The tissues were cut longitudinally and transferred monthly to fresh medium and further incubated at the same temperature. When healthy looking plantlets developed from the tissues, they were separated and transplanted. Disease incidence was assessed after two months.

Detection of BBTV with Monoclonal Antibodies

The monoclonal antibody method of Wu and Su (1990b) was used to detect BBTV in banana extracts by means of direct ELISA. Small pieces (c. 15 x 15 mm) of midribs and petioles of BBTV-infected banana leaves were ground in a mortar with 0.1 M potassium phosphate buffer, pH 7.4, containing 0.2% (v/v) 2-mercaptoethanol and 0.1% (w/v) sodium diethyldithiocarbamate, using 1 g of leaf tissue in 2 ml of buffer. The extracts were clarified by low-speed centrifugation at 7000 r.p.m. for 10 min.

Results and Discussion

Exposure of BBTV-infected rooted banana plantlets to 40oC for 16 h daily for periods of up to 5 weeks did not free the plantlets from BBTV. Aphids still transmitted BBTV from treated plantlets to infect healthy plantlets (Table 1). However, the treatment apparently reduced the concentration of BBTV in the plantlets, as the transmission rate of BBTV from diseased to healthy plantlets fell from 100 to 25% after three weeks' treatment and to 7% after five weeks. Thereafter the concentration of BBTV in the treated plantlets increased rapidly following incubation at 28oC. After two weeks' incubation, the transmission rate increased to c. 67% for three weeks' treatment and 80% for five weeks' treatment (Table 1). These results suggest that heat therapy was ineffective in eliminating BBTV from infected banana plantlets, although heat therapy is effective for many other host-virus combinations (Nyland and Goheen 1969).

Exposure of BBTV-infected tissues in nutrient culture to 40oC for 16 h daily tended to inhibit the growth of tissue and plantlets, but still failed to eliminate BBTV. Even after heat treatment for 12 weeks, all the plantlets produced from treated tissue displayed bunchy-top symptoms following incubation at 28oC.

When BBTV-infected tissue was cultured at 35oC, the adventitious buds grew and multiplied but did so slowly. After three months, some of the buds started to produce roots and developed into healthy looking plantlets (Fig. 1). Five of 11 treated cultures produced one or more healthy looking plantlets in six months (Table 2). When these healthy looking plantlets were transferred from tissue culture to growing medium in pots, all remained healthy looking after three months' further growth at 28oC in a growth chamber.

A monoclonal antibody against BBTV was used to test for BBTV in leaf tissue. The ELISA values of crude extracts from diseased plantlets, healthy-looking plantlets and the healthy plantlets used as controls were 1.543, 0.085, and 0.091, respectively. This indicates that the healthy-looking plantlets were BBTV free.

Mutants resistant to race 4 of Fusarium oxysporum f.sp. cubense have been isolated from plantlets derived from tissue cultures of Cavendish banana (Hwang and Ko 1988). We therefore tested the susceptibility to BBTV of healthy plantlets that had been regenerated from BBTV-infected tissue kept at high temperatures. When the heat-induced healthy plantlets were each inoculated with 30 viruliferous aphids, all developed bunchy-top symptoms within one month, indicating that these plantlets were not resistant to infection with BBTV.

Our results suggest that heat treatment inhibited multiplication of BBTV in banana tissues, and that some primordial cells were freed from BBTV at 35oC, possibly associated with an uneven distribution of the virus. These cells subsequently developed into healthy buds and plantlets.

References

• Dale, J.L., Philips D.A. and Parry J.N. 1986. Double-standard RNA in banana plants with bunchy-top disease. Journal of General Virology 67: 371-375.
• Hwang, S.C. and W.H. Ko. 1988. Mutants of Cavendish banana resistant to race 4 of Fusarium oxysporum f.sp. cubense. Plant Protection Bulletin 30: 386-392.
• Ma, S.S. and C.T. Shii. 1972. In vitro formation of adventitious buds in banana shoot apex following decapitatioin. Journal of Chinese Society of Horticultural Science 18: 135-142.
• Ma, S.S. and C.T. Shii. 1974. Growing banana plantlets from adventitious buds. Journal of Chinese Society of Horticultural Science 20: 6-12.
• Matthews, R.E.F. 1982. Classification and nomenclature of viruses. Intervirology 17: 1-200.
• Nyland, G. and A.C. Goheen. 1969. Heat therapy of virus diseases of perennial plants. Annual Review of Phytopathology 7: 331-354.
• Smith, R. H. and T. Murashige. 1970. In vitro development of the isolated shoot apical meristem of angiosperms. American Journal of Botany 57: 562-568.
• Stover, R.H. 1972. Banana, Plantain and Abaca Diseases. Commonwealth Mycological Institute. Kew, England.
• Sun, S.K. 1961. Studies on the bunchy-top disease of banana. Special Publication of College of Agriculture, National Taiwan University 10: 82-109.
• Wu, R.Y. and H.J. Su. 1990a. Purification and characterization of banana bunchy top virus. Journal of Phytopathology 128: 153-160.
• Wu, R.Y. and H.J. Su. 1990b. Production of monoclonal antibodies against banana bunchy top virus and their use in enzyme-linked immunosorbent assay. Journal of Phytopathology 128: 203-208.
• Wu, R.Y. and H.J. Su. 1990c. Transmission of banana bunchy top virus by aphids to banana plantlets produced by tissue culture. Botanical Bulletin of Academia Sinica 31: 1-4.

Index of Images

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  Figure 1 Diseased (Left) and Healthy Looking Banana Plantlets (Right) Regenerated from BBTV-Infected Tissues Cultured at 35oc

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  Table 1 Effect of Heat Treating Infected Banana Plantlets on Transmission Rate of Banana Bunchy Top Virus

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  Table 2 Disease Incidence of Plantlets Regenerated from Banana Bunchy Top Virus-Infected Tissues Cultured at 35oc

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