Colour is one of the most important traits in orchids and has created great interest in breeding programmes. Gamma irradiation is an alternative way for generation of somaclonal variation for new flower colours. Phenotypic changes are usually observed during screening and selection of mutants. Understanding of targeted gene expression level and evaluation of the changes facilitate in the development of functional markers for selection of desired flower colour mutants. Four Dendrobium orchid sequences (NCBI accessions: AM490639, AY41319, FM209429 and DQ462460) were selected to design gene specific primers based on information for chalcone synthase (CHS) from NCBI database. Quantitative real-time PCR (qPCR) was used to understand flower colour expression quantitatively derived from the CHS gene activities in different flower tissues (petal and sepal) from control Dendrobium Sonia (red purple), mutant DS 35-1/M (purple pink) and mutant DS 35-WhiteA. It was found that expression of CHS gene was highest in sepals of white flowers and lowest in both sepals and petals of purple pink flowers. Genomic DNA was amplified and PCR products were sequenced, aligned and compared. Sequence variations of CHS partial gene in Dendrobium Sonia mutants with different flower colour showed that two protein positions have been changed as compared to the control. These non-synonymous mutations may have contributed to the colour alterations in the white and purple pink mutants. This paper describes important procedures to quantify gene expression such as RNA isolation (quantity and quality), cDNA synthesis and primer design steps for CHS genes.
The research on radiation induced mutation has been conducted as one of the promising method of plant breeding in Malaysia since 1980s. Nuclear Malaysia is leading research institute inMalaysia conducting plant mutationbreeding research. Gamma Greenhousefacility located in Nuclear Malaysiais one of the irradiation facilitiesto serve as a chronic irradiation facility for inducing mutation in various organisms including plants, fungi and microbes.Chronic irradiation refers to the exposure of materials at a lower dose rate over a long period of time. Previous studies have shown that this type of irradiation can minimize radiation damages to living materials and produces a wider mutation spectrum, therefore is very useful for trait improvements in irradiated organisms. Experiments on induce mutation using Gamma Greenhouse facility for crop improvement program have been conducted since its first operation in 2009. Various plant species including ornamental and herbal plants, food crops and industrial crops have been irradiated to improve their traits such as higher yield and biomass, pest and disease tolerance, higher bioactive compounds, longer bloom time and many others. Most of these crop improvement programs were done through collaborations with other agencies in Malaysia such as universities, research institutes and government departments. A number of publications on crop improvement using Gamma Greenhouse have been published inlocal and international journals as well as seminar presentations at national and international levels. The outputs from induced mutation via chronic radiation using Gamma Greenhouse could be of great interest for plant breeders dealing with improvement and development of new cultivars. This paper discusses the activities and achievement in plant breeding and improvement using Gamma Greenhouse Facility in Malaysia.