Identification, Morphologic Observation And Primary Genetic Analysis Of Three Rice T-DNA Insert Floral Organ Mutants

Spike of rice belongs to panicle anthotaxy, composed of rachilla, primary branch, secondary branch and spikelet, and is the reproductive organ of rice. Among the composing elements of rice panicle, the spikelet is an important organ of sink, in which the economic yield of rice will be formed. It has a very important role on revealing the morphological construction, revolving the mechanism in development and regulation of rice panicle and directing the practice of rice breeding and cultivation to get kinds of reproductive organ mutation of rice and resolve the function of the relative genes and the interaction among them.In the nomal seasons of 2003 and 2004, during the period from heading to filling stages, we observed 4,416 T-DNA inserted label lines japonica rice cultivars, zhonghua11 and zhonghua15. Inheritable spikelet mutants of over 20 label lines, involved in more than 10 types of mutants, were identified, such as no panicle differentiation mutants (only nutritional growth), degradated or abnormal glume mutants, multi-glume mutants, no florets or extremely sparse florets mutants, erect panicle mutants, degraded top spikelets mutants and so on. And Some mutants appeared the whole line to be fake-homozygous mutations in T1 generation, other areas showed the separation of targeted characters in one line. It implied that different mutants of rice floral organ were generated by different reasons,some of which might be caused by T-DNA insertion, some might belong to the mutation in tissue cultivation and the others were formed by other unknown factors. These mutants provides a basis to exploit systematically the regulation mechanism of rice spike development. In order to further analyze the phase and position of mutation trait occurrence, three mutants, degeneration hull mutation, degeneration floret mutation and multi-glume mutation, were selected for further research.The basic characteristics of these three mutants were detailed as follow. (1) The typical characteristic of the degenerated hull 1 mutant (dh1 mutant) was the serious degradation of glume (including palea and lemmas), and the bareness of internal floral organ. When the panicle the mutant emerged, it looked like to be blooming and its stamens and pistil dried up soon. Furthermore, the number of its stamens and pistil was also abnormal. A few of spikelets generated two or three florets on the same rachilla. And the transparent tumor tissue existed in some florets. (2) The typical characteristic of the degradation floret mutant (df mutant) was that, at the rachilla, only primary branch and secondary branch were differentiated, the length of branches was obviously shortened, and the spike of rice looked like a stick, of which few number of florets and some degradated traces of florets could be seen. (3) The typical characteristic of multi-glume mutant (mg) was that its glume number of almost every florets increased obviously. The most florets appeared two or more paleas and lemmas, and others lacked paleas but had more lemmas. Additionally, most of florets had no lodicule and abnormal stamens and pistil in the mg mutant, which resulted in male sterility.We watched the microstructure of these three mutants by the paraffin sections and scanning electron microscope (SEM) and found the differences of the floral development process between wild type and mutants. (1)The phenotype of the DH1 mutant occurred early in the glume primordium differentiation stage. By the SEM, we could found that when stamens primordium of the mutant was forming, the primordium convex of palea and lemma could not observed clearly or the length of the primordium was less than half of the wild type plant florets. Furthermore, the different stamen primordia in the same floret were formed without synchronization and the feather stigma in mutant divided often unevenly;(2)The phenotype of the df mutant occurred in the stage of primary and second branch primordium differentiation. Most mutants only formed primary branch and second branch primordium, can hardly see differentiated floral primordium, so stamen and pistil primordia were almost absent;(3) The morphological change of mg mutant appeared after the glume (palea and lemmas) primordium differentiation. After palea and lemmas formed of mg mutant, its stamen and pistil primordia had not developed immediately, while extra sevarel glume-like tissue was formed. Moreover, the differentiation of its stamens and pistils primordia was un-synchronous and without orderliness.After genetic analyses for these three mutants in two continuous generations, it was found that these three mutants were controlled by different single genes. The correlative analysis between mutant phenotypes and T-DNA taggings (PCR marker) showed the results of co-segregation as follows;(1) The dh1 and mg mutants all belonged to the function-loss mutation generated by different single copy T-DNA insertion; (2) The df mutant might be caused by one of the two non-tandem T-DNA insert segments, which was needed to be verified further.In summary, (1)for the dh1 mutant, its degenerated glume did not transform homologically into inner ring organ. So we speculated, the gene controlling this mutation phenotype was possibly different from some MADS-box genes of"ABC"model and may be another kind of function gene interrelated with rice floret growth;(2)For the df mutant, its development course of panicle may be restrained during the period from branch differentiation to florets forming, in which its inflorescence meristem could not switch to floral meristematic tissue. So, even if the blossom induction signal existed, its development programme from branch differentiation to frolet formation could not be activated. Consequently, its inflorescence meristem could not switch to flowered meristematic tissue and there was no any floret in mutant to open;(3)As for mg mutant, its mutation phenotype under microscope and electron microscope appeared behind the formation of palea and lemmas primordium and except a pair of normal palea/lemma, formed some structure like palea/lemma. Furthermore, most florets of the mg mutant were lack of lodicule. So, we speculated that some of the lodicules might be convered into glume-like organ. The mg mutation might be the result of the loss-of-function of a gene belonging to E type of MADS-box family.The phenotypes of these three mutants were quite different though the stage of mutation emergence was similar. It might give new insight into elucidating the mechanism of morphogenesis of floral organ in rice to further investigate the function and relationship of the genes controlling the mutation phenotypes.