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.
Date of Award | 2010 |
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Original language | English |
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Awarding Institution | |
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Sponsors | Government of the Islamic Republic of Pakistan |
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The Physiological and Molecular Characteristics of Chemically Induced Abiotic Stress Resistant Mutants of Cauliflower (Brassica oleracea var. botrytis)
Hadi, F. (Author). 2010
Student thesis: PhD