Selection and validation of reference genes for measuring gene expression in Toona ciliata under different experimental conditions by quantitative real-time PCR analysis.

BACKGROUND: Before studying gene expression of different organisms, it is important to determine the best reference gene. At present, the most accurate method of detecting gene expression is quantitative real-time PCR (RT-qPCR). With this method, reference genes that are stable in different biological systems and under different conditions can be obtained. Toona ciliata Roem (T. ciliata). is a valuable and fast-growing timber specie. In this study, 20 reference genes were identified using RT-qPCR, as a primary prerequisite for future gene expression analysis. Four different methods, geNorm, NormFinder, BestKeeper, and RankAggreg were used to evaluate the expression stability of the 20 candidate reference genes in various tissues under different conditions. RESULTS: The experimental results showed that TUB-α was the most stably expressed reference gene across all samples and UBC17 was the most stable in leaves and young stems under Hypsipyla robusta (H. robusta) and methyl jasmonate (MeJA) treatments. In addition, PP2C59 and UBC5B were the best-performing genes in leaves under H. robusta treatment, while HIS1 and ACT7 were the best reference genes in young stems. The two best reference genes were 60S-18 and TUB-α after treatment at 4 °C. The expression of HIS6 and MUB1 was the most stable under PEG6000 treatment. The accuracy of the selected reference genes was verified using the transcription factor MYB3 (TcMYB3) gene. CONCLUSIONS: This is the first report to verify the best reference genes for normalizing gene expression in T. ciliata under different conditions, which will facilitate future elucidation of gene regulations in this species.

Development of an efficient regeneration system for the precious and fast-growing timber tree Toona ciliata.

Toona ciliata (Chinese mahogany) is an important timber species and secondary protected plant due to excessive exploitation in China. Here we developed a robust and efficient regeneration system for adventitious shoot induction using hypocotyl explants of T. ciliata. To facilitate plant growth, different regulators were added to Murashige-Skoog (MS) medium (0.5 mg/l 6-BA, 1.0 mg/l KT and 0.1 mg/l IBA). A regeneration frequency of 58.67% with four shoots per explant was achieved by horizontal setting of hypocotyls on MS medium and following a 20-day seeding period. MS medium supplemented with 0.3 mg/l 6-BA and 0.2 mg/l NAA was optimal for shoot multiplication and elongation, with a multiplication coefficient of 3.06. A rooting frequency of 93.33% was achieved using the half-strength MS containing 0.1 mg/l NAA. After acclimatization, plantlets were transplanted to sterilized nutrient soil containing a 2 : 1 ratio of vermiculate with 90% survival frequency. Thus, the regeneration system developed in this study would be useful for genetic transformation and other biotechnology endeavours in T. ciliata.