TECHNICAL ASPECTS IN UNDERSTANDING EFFECTS OF GAMMA IRRADIATION ON FLOWER COLOUR CHANGES IN DENDROBIUM SONIA
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.