Sub-project on "Insect Resistance in Orchid"

The main objective for this project is to produce insect resistant orchids. Three orchid varieties are used in this study namely, Dendrobium Sonia "No. 17 Red" ( Thailand ), D. mirbelliannum ( Malaysia ) and D. jayakarta ( Indonesia ). Protocorm-like bodies (PLBs) of these varieties have already been exchanged among the participating countries early in the project. In addition to these varieties, Thailand also uses D. Sonia "Earsakul" for this project.

Present status:

Indonesia

1. Three to 6 months after gamma irradiation of D. Jayakarta at the doses of 0, 40, 80, 160, 320, 640 and 1280 Gy, the LD50 obtained for the 3 types of explants were as follows;
   A.LD50 for PLBs was 40 Gy (3 months)
   B.LD50 for plantlets was 240 Gy (3 months)
   C.LD50 for young shoots was 60 Gy (6 months)
2. The optimum growth medium for PLBs of D. Sonia "No17 Red" was VW medium + coconut water + activated charcoal.

Malaysia

1. Malaysia uses three approaches to obtain insect resistant orchids, namely; in vitro mutagenesis using both ion beam and gamma irradiations, genetic transformation with Agrobacterium tumefaciens carrying a chitinase gene, and in vitro / in vivo insect screening on irradiated as well as transgenic orchids.
2. Using ion beam irradiation, PLBs of both D. mirbelliannum and D. jayakarta have been successfully regenerated into plantlets and acclimatized in the glasshouse. Plantlets of D. mirbelliannum have been inoculated with mites using an in vitro screening method. Putative tolerant plants have been obtained and planted in the glasshouse for further screening.
3. Using gamma irradiation, explants from all three varieties of Dendrobium have been regenerated into plantlets. D. mirbelliannum and D. jayakarta plantlets have been transferred to the glasshouse for in vivo screening with mites. D. Sonia "No 17 Red" plantlets will be transferred to the glasshouse once enough number of plantlets are obtained.
4. Through genetic transformation, putative transgenic plants have been obtained as confirmed by antibiotic selection, GUS analysis and polymerase chain reaction (PCR). These plantlets have been transferred to glasshouse and further screening will be carried out once the transgenic glasshouse is available.
5. Presently, in vivo (glasshouse) selection for insect resistance was carried out by growing the regenerated plants in the glasshouse without the application of pesticides and fungcides.

Thailand

1. LD50 analysis of D. Sonia "Earsakul" at 3 growth stages; PLBs, young plantlets and back bulbs, were carried out. The optimum doses for gamma irradiation for each growth stage were obtained.
2. Natural infestation of thrips on chronic gamma irradiated seedlings of D. Sonia "No. 17 Red" and D. Sonia "Earsakul" were investigated. Some treated plantlets showed less infestation symptoms by thrips. These plantlets will be further analysed for thrips resistance at the flowering stage.

Future Plan:

The following work will be carried out to complete the project;

Indonesia

1. Mass rearing of thrips for screening of irradiated plants
2. Mass propagation of irradiated plantlets for inoculation with thrips
3. Propagation and maintenance of irradiated plants until the flowering stage for screening of both insect resistance and flower colours.

Malaysia

1. In vitro screening of plantlets of D. jayakarta & D. Sonia "No 17 Red" with mites.
2. In vitro screening of transgenic plantlets
3. In vivo screening of putative mite tolerant plantlets. Challenge-inoculation with mites will be carried out once the contained glasshouse is ready in order to avoid cross-contamination with other plants in the glasshouse.
4. Propagation of irradiated plants until the flowering stage in the glasshouse for screening of flower colours.

Thailand

1. Selection for thrips resistance for irradiated plants at the flowering stage.
2. Selection for other characters such as flower colours, size and morphology.
3. Meristem cloning of selected mutants for large scale propagation and testing to confirm the results for thrips resistance and other characteristics.
4. For thrips resistant mutants, challenge-inoculation with thrips will be conducted in order to select for the most resistant lines using the standard protocol for insect resistance analysis recommended by FNCA orchid project leaders at the FNCA 2006 Mutation Breeding Workshop.

Agreement among participating countries

Following discussion on the project, all participants have agreed on the following points;

1. A standard protocol for insect resistance analysis on the irradiated orchids will be performed in all participating countries.
2. Indonesia will provide the rearing technique of thrips to Thailand .

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