Effects Of Exogenous Methyl Jasmonate And Gibberellin On Floral Development And Its Implication In The Utilization Of Heterosis In Oilseed Rape (Brassica Napus L.)

Oilseed rape oil is one of the most important vegetable oils for edible consumption in the world, and the meal with high protein content and well balanced amino acids is a valuable supplement in animal feed mixtures. The economic significance of the crop demands intensive studies on its physiological processes, including its transition from the vegetative to the reproductive stage. Delayed flowering time, insufficient flowering period, or abnormal floral organs including male sterility may result in a significant decline of yield in oilseed rape production. However, male sterility is a crucial element in a heterosis utilization system guarantying a yield promotion. Flower development involves a large group of genes that respond to environmental stimuli and endogenous cues. The role of jasmonates and gibberellins in flowering remains largely not understood. The advent of molecular biology era provides us a new perspective in comprehending the mechanism of floral organ development and male sterility. In this paper we investigated the effect of exogenous methyl jasmonate (MeJA) and gibberellins (GA3) on the development of floral development and its implication in the utilization of heterosis in oilseed rape (Brassica napus L.).In Chapter 3.1 and 3.2, effects of exogenous MeJA and GA3 on the flowering time, floral organ morphology, and transcript levels of a group of genes implicated in floral development are demonstrated. Through controlled greenhouse experiments, we found that the effect of MeJA depended on both plant genotype and jasmonate dosage. MeJA promoted maximum flowering when it was applied to the cultivars of early-flowering types of oilseed rape. In addition, a concentration of 100μM resulted in the most number of early open flowers, in comparison with the results obtained for the concentrations of 50μM and 80μM, indicating a dosage effect. Moreover, the application of high concentrations of MeJA (100μM) also produced various kinds of abnormal flowers. Molecular approaches showed that the combined actions of the floral identity genes, specifically BnAP1, BnAP2, BnAP3, BnAG1, and BnPI3, as reflected by their respective relative transcript levels, were responsible for causing the different kinds of flower abnormalities previously un-described. On the other hand, the application of 50μM GA3 gave rise to the shift of flowering time varied from 0.6 to 2.6 days among six winter rapeseed cultivars. Some cultivars did not respond to GA3 in terms of flowering time, whereas others (Xinyang-7833, S-205, Mei-Jian and Fu-You 4) started to flower significantly earlier after the treatment of GA3 than their respective untreated control. In addition, the 50μM GA3 had a clear effect on flower opening and the elongation of filament epidermis cells. Analysis of relative transcript levels of a group of functionally identified genes indicated that GA3 regulated the expression of the floral homeotic genes such as BnAP3, BnAGl, BnPI3 and the genes on the GA signal transduction pathway like BnRGA2, BnGAI, as well as the genes belonging to MYB family as BnMYB21, BnMYB24 and BnMYB28.As the disequilibrium of jasmonates or gibberellins results in a variety of abnormal flowers caused by the combined action of a group of identified genes, the genome-wide transcriptome changes and the unidentified GA3-and MeJA responsive genes were further interested. In Chapter 3.3, we employed cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis to identify genes that exhibited modulated expression following the application of MeJA and GA3 to flower buds. By using 64 primer pair combinations, about 2787 cDNA fragments were counted and all bands longer than 50 bp in size were compared among the four treatments, viz. the water control, the 50μM MeJA, the 50μM GA3, and the 100μM MeJA. A total of 168 transcript derived fragments (TDFs) were differentially displayed among the treatments. The expression pattern of several of these genes was confirmed by RT-PCR. A total of 106 of the differentially displayed TDFs were cloned and sequenced, and were identified as homologues to the Arabidopsis genes classified into twelve categories, viz. transcription factors, stress responsive genes, genes regulating fatty acid metabolism, and genes relating to signal transduction and so on.34 and 39 of them were GA-and MeJA-responsive, respectively. A total of 24 TDFs were both GA3-and MeJA-responsive, suggesting a cross-talk of the two plant growth regulators in modulating the development of oilseed rape flower.Male sterility is interested in the utilization of heterosis that means an efficient method to promote oilseed rape production. Various kind of heterosis utilization systems, including the cytoplasmic male sterility (CMS) system (such as pol-CMS, Shan2A-CMS, ogu-CMS, tour-CMS, nap-CMS), the nuclear gene male sterility (GMS) system, the self incompatibility (SI) and the chemical induced male sterility (CIMS), have been established. Each system has its advantageous and its clear inapplicability. In Chapter 3.4 and 3.5, we made valuable efforts to establish a new system of heterosis utilization in oilseed rape, which is based on the discovery of the male sterilities that are maintainable by the application of exogenous GA or MeJA. Such male sterilities were sought through both genetic engineering approach (Chapter 3.4) and traditional EMS mutagenesis (Chapter 3.5). BnDAD1, a putative Brassica napus orthologue to Arabidopsis DAD1, was isolated and cloned. Molecular cassettes containing the promoter of BnDAD1 and antisense orientation of the BnDAD1 coding region was constructed in a binary vector pCAMBIA1300, which is used to transform Agrobacteria and rapeseed cultivar'Zhe-You 758'. On the other hand, EMS mutagenesis resulted in a total of 75 M1 individual plants that could be maintained by the exogenous stimulations like thermal water, and MeJA or GA3. The male sterility of the M1 lines was maintained by crossing with fertile plants. The male sterile descends were further characterized in the M3 generation for their anther and pollen development under microscope and the ability to set seeds under the applications of various concentrations of MeJA or GA3 growth regulators.Taken together, we described the flowering time, floral organ morphology that were affected by exogeneous MeJA and GA3 in oilseed rape, and characterized the molecular mechanism underlying the phenotypic appearance. Our results could be an enriching addition to the body of work that attempts to understand the signaling function of MeJA and GA in the floral inductive pathway in oilseed rape. The start-up work to screen for the MeJA-or GA3-maintainable male sterile lines laid a solid foundation for the attractive prospect to establish an efficient new system of heterosis utilization in oilseed rape.