| Summary: | N-nitroso-N-ethyleurea (NEU) and N-nitroso-N-methyleurea (NMU) induced mutants and control plants had been maintained in in-vitro condition for 3 years by continuous sub-culturing and screened 2 generations for resistant mutants selection. In this study highly resistant mutants were regenerated and assessed by leaf discs assay for drought, salt and frost resistance to confirm the persistence of mutation over generations of subculture. Assessment was carried out using mannitol (drought stress), NaCl (salt stress) and freezing (frost stress). Cold-acclimated and non-acclimated leaves were assessed for frost resistance. Results confirmed the persistence of mutations in clones with enhanced tolerance levels to stresses over control plants. Response of individual mutants was different for each of the stresses, some mutants were resistant to two stresses whilst others demonstrated multiple resistance and no one mutant was resistant to a single stress. Acclimation at 4 °C appeared good enough to increase frost resistance compared to non-acclimation. Acclimation also tended to emphasis the difference between mutants and some mutants (K18 & K19) showed highly significant increase in frost resistance at -6 °C compared to control. Responses of in-vitro and in-vivo plants within a clone were correlated. Molecular and biochemical analysis was carried out with objectives (1) To investigate the presence of CBF/DREB1 and COR15 genes in cauliflower (2) To investigate whether the induced resistance can be attributed to the expression of these genes and proline level. The clones (mutants and control) were analyzed under cold acclimation (4 °C) and non-acclimation (22 °C). Total RNA was isolated after 3 h, 6 h, 24 h and 14 d acclimation. Proteins and free proline were isolated after 14 d acclimation. Under non-acclimation, RNA, protein and proline isolated once at end of experiment. cDNA was produced using RT-PCR, with specific primers the gene was detected only in acclimated clones and no PCR product appeared under non-acclimation. The PCR product was isolated, sequenced, and compared the nucleotides and deduced amino acid sequences with other plants. Very high resemblance (- 91 %) with Brassica species (BnCBF5/DREB1, BrDREB1 and BjDREB1B) were found and confirmed the first reporting of the transcription factor BoCBF/DREB1 in cauliflower. This resemblance was reduced to 67% when compared to other plants, confirms that this sequence is conserved in Brassica. The transcript level increased up to 24 h acclimation and then declined. The response of the mutants was different, some showed PCR product at 3 h while others only after 6 h and 24 h acclimation. Through SDS-PAGE and Western blotting, the COR15a protein was detected with specific antibodies obtained from MSU (USA), and the blots appeared in all clones under cold acclimation correlated with frost resistance but under non-acclimation the COR15a constitutively expressed only in 3 mutants with increased frost resistance that confirms the persistence of mutation. The genotypes showed positive correlation between BoCBF/DREB1 expression and frost resistance and this correlation was significant after 24 h and 14 d cold acclimation. The highest R² value was found between BoCBF/DREB1 expression at 14 d and EC% at -6 °C (93.43% of variation accounted for) followed by BoCBF/DREB1 expression at 24 h and EC% at -6 °C (82.57%). The proline level under acclimation increased about 8 times compared to non-acclimation and demonstrated positive and significant correlation with BoCBF/DREB1 expression. Proline also showed positive and significant correlation with frost resistance under cold acclimation but very weak under non-acclimation. The effect of cold acclimation on proline and total protein was evaluated and negative correlation was found to be non significant between free proline and total protein content in clones.
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