Orchid is an important industry in Malaysia with production value of RM 80 mil per year. The orchids produced are mainly exported to nearby countries. In order to sustain and expand the 40% market share of orchids as one of cut flowers export of the country, genetic engineering has been recognised as an approach to overcome the limitations of improvement through conventional breeding. Through genetic engineering, genes for shelf life, flower color and architecture can be directly manipulated to develop new varieties that are tailor made to customers' preferences. Such approaches in flower improvements have been successfully implemented in petunia, roses and carnation.

R&D on genetic engineering of the local orchids was first started in RM7 (October 1996 - September 2001) with funding from NBD under the project title: Improvement of orchids and tulips through genetic engineering. The main objective was to improve flower colour and shelf life characteristics using the genetic engineering approach. Significant achievements have been made in both shelf life and colour aspects of the project which include:
· Cloning of shelf-life gene (ACC oxidase) from Phalaenopsis hybrid.
· Cloning of 4 genes involved in two different colour pathways from Oncidium hybrid namely, CHS, F3H, DFR genes from the anthocyanin pathway and Phytoene synthase gene from the carotenoid pathway.
· Construction of 5 antisense gene cassettes from each of the cloned shelf life and colour genes. All have been used in orchid transformation.
· Development of efficient transformation (gene transfer) systems for various Dendrobium and Oncidium hybrids.
· Production of transgenic Dendrobium orchids transformed with the shelf life genes (antisense ACC oxidase gene)
· Production of transgenic orchids with antisense colour genes.
· Development of techniques for induction of flowering in vitro for early analysis of flower colour in transgenics.
· Scientific publications, manpower trained in plant genetic engineering, PhD and MSc graduates specialised in plant genetic engineering.

For RM8, we intend to fully characterised the orchid transgenics produced in the RM7 to confirm the presence of the engineered characteristics. Furthermore, the knowledge obtained, the genes cloned and the transformation systems developed in RM7 will be applied to other commercially important orchids for improvements of their flower shelf life and colour. New strategies and approaches will also be incorporated to further achieve improvements of the local orchids. This project encompasses three main subprojects. Each subproject title and specific activities are as listed below:

1. Genetic enginnering for improvements of orchid flower shelf life. (Leader: Dr. Umi K. Abu Bakar)
· Conduct detailed molecular and biochemical analysis on the 27 shelf life Dendrobium transgenics produced previously to verify the expression of the engineered flower shelf life characteristics.
· To use the shelf life gene to develop transgenic orchids from other commercial varieties such as Phalaenopsis and Oncidium for improvement of flower shelf life.
· To use the techniques developed for Phalaenopsis to clone the shelf life gene, ACC oxidase, from Dandrobium and Oncidium.
· To identify and clone etr-1 gene from Dendrobium orchid, an alternative shelf life gene that have been proven to more effectively improve flower shelf life.

2. Development of new orchid colour variations in selected orchid hybrids using biotechnology.
(Leader: Dr. Nazir Basiran)
· Transformation of Dendrobium and Oncidium hybrids with antisense of DFR, CHS and F3H genes.
· Analysis of transgenic orchids produced using the colour genes
· Induction of in vitro flowering in transgenics orchids for early analysis of flower colour.
· Cloning of full length F3H for flower colour expression rather than suppression (antisense) that can be achieved using the truncated F3H gene cloned earlier.

EXECUTIVE SUMMARY

Project Title:
IMPROVEMENT OF COMMERCIALLY IMPORTANT ORCHIDS USING BIOTECHNOLOGY

 

 

. Characterisation of cloned full length F3H
· Induction of mutation in selected Dendrobium and Oncidium hybrids and selection of mutants as an alternative method for production of colour variation in orchids.
· Analysis of color gene expression in mutant lines.

3. Genetic alteration of flower architecture of Dendrobium hybrids (leader: Dr. Faridah QamaruzZaman)
Apart from improvement of flower shelf life and colour, improvement in flower shape or architecture has also been proposed. With this technology orchid flower can be genetically engineered to have novel flower shape. Engineering for flower architecture is still a new science. Thus for RM8, the work is more exploratory with the main objective to identify and clone MADS box genes in Dandrobium hybrids. MADS box genes have been implicated in flower architecture. Specifically, this subproject plan to:
· Construct cDNA libraries from selected Dendrobium hybrids
· Clone MADS box gene with degenerate primers by RT-PCR
· Isolate full length genes and characterise by sequencing, Northern blots, Southern blots and in situ hybridization
· Assemble transformation construct by protein engineering to produce MADS box proteins lacking transcription activating function.

It is anticipated that the new project will generate valuable outputs and great advancement in the genetic engineering of the local Malaysian orchids. The major output will be the fully characterised transgenic orchids engineered either with shelf life or colour genes. It will then be known whether the concept has worked. If it has, it will be possible to identify and select the best transgenic lines with improved shelf life or colour and commercialise them as new and improved orchid varieties. Furthermore, the whole technology can then be successfully applied to other orchid hybrids or any ornamental flowers. From flower architecture aspect, the genes cloned, the relevant technologies developed and the valuable information generated will better pave the way to successful engineering of orchid for improved flower architecture.

Period of the Project : 3 Years (2001 -2004)

Achievements

Progress/Achievements for 2002

New product

- Nill

New process
· RNA extraction has been carried out from flowers at each stage of senescence.
· Northern analysis of RNA at different stages of senescence for expression of ETR gene.
· RT PCR Cloning of ACCO gene from pollination induced senescence of Oncidium flower.
· Transformation of Dendrobium Pompadour, Phalaenopsis and Oncidium with antisense ACCO gene construct from Phalaenopsis.
· Modified method of CTAB/NaCl by Chang (1993)
· Modified protocols from Prescott & Martin (1987)
· Qiagen - RNeasy MIDI extraction kit to improve the yields and quality of the RNA extracted

Patents filed - 0

Commercialization - 0

Discovery - The presence of hygromycin resistant gene.

Publication - 0

Human Resource Development - 0