The real time quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including ACT2, ELFA, GAPDH, TUA, UBQ9 (traditional housekeeping genes), ACP, CAC, SNF, TIPS-41, TMD, TSB and ZNF (new candidate reference genes), in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of Brassica juncea has been validated. For the normalization of vegetative stages the ELFA, ACT2, CAC and TIPS-41 combination would be appropriate whereas TIPS-41 along with CAC would be suitable for normalization of reproductive stages. A combination of GAPDH, TUA, TIPS-41 and CAC were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, UBQ9 and TIPS-41 had the most stable expression. Across five cultivars of B. juncea, the expression of CAC and TIPS-41 did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization.

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In this study, changes in growth, chlorophyll pigments, proline, hydrogen peroxide (H 2 O 2) and malondialdehyde (MDA) contents, and antioxidative enzyme activities were investigated in the seedlings of four different cultivars (cvs) of mustard [Brassica juncea (L.) Czern. & Coss.], i.e. Varuna, RH-30, Rohini, and Vaibhave under potassium (K) nutrition-deficient conditions. K deficiency induced a significant decrease in concentrations of photosynthetic pigments in all four cvs, however, this decrease was higher in cvs. Varuna and RH-30. During K deficiency, proline concentration increased in all mustard cvs, but a maximum increase in this parameter was shown by cvs. Varuna and RH-30. The activity of the key proline metabolizing enzyme g-glutamyl kinase increased more in cvs. Varuna and RH-30 compared to cvs. Rohini and Vaibhave. The proline oxidase activity showed greater increase in cvs. Vaibhave and Rohini compared to cvs. Varuna and RH-30. K deficiency increased the concentrations of H 2 O 2 and the activities of anti-oxidative defense system enzymes like superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) in the seedlings of all mustard cvs, but higher activities of these enzymes were observed in cvs. Varuna and RH-30 compared to cvs. Rohini and Vaibhave. A significant lipid peroxidation in terms of MDA contents was also observed in the K-deficient seedlings of all four mustard cvs. This study suggests that K-starvation-induced oxidative stress through the high generation of reactive oxygen species (ROS). All mustard cvs counteracted to some extent the effects of ROS by activation of antioxidant machinery. Overall, the results indicate that of all four mustard cvs, Varuna and RH-30 were tolerant to K deficiency.