| Usually, whether a mature seed can germinate or not is affected by two factors, one is its physiological status, the other is ambient environmental contions. And developing seeds can not germinate when they are still attached to the maternal plant. However, pre-harvest sprouting, which means seeds germinated before they were harvested, was reported in maize, sorgrum, wheat and rice etc. Also, precocious germination occurred in muskmelon, chayote, jackfruit, papaya, cucumber, pepper, eggplant, Arabidopsis thaliana and soybean etc. At present, the research of earlier germination of developing seeds is focused on the graminaceous crop. Through the research of this pre-harvest sprouting, a variety of genetic mutants were identified, which flourish the diversity of germplasm resources in breeding work, and enriched the basic theoretical knowledge of seed dormancy and germination. Developing seeds in one accession of tomato rin mutant fruit can germinate precociously, and even young seedlings with viable cotyledons appeared in its fruit. Those non-germinated seeds can also germinate precociously if fruits were harvested and placed in room temperature for a period. This was a special characteristic of plant variety and is affected by the environmental cues.Viviparous germination affect seed collection and storage, elucidating its mechanism is beneficial to application research of prevention of vivipary; also revealing its mechnism, combining with the characteristics and theoretical basis of the germination of recalcitrant seeds, can enrich the fundamental theory of seed dormancy and germination; meanwhile, the investigation to vivipary is of great ecology significance: presence of germination inhibitor is benetifical to seed longevity extension and seed preservation.This is more meaningful for the protection of rare wild plant species.Taking the vivipary in one accession of a tomato rin mutant as a base, some characteristics of the developing tomato seeds were studied, as well as the physiological mechanism associated with vivipary. In addition, the PCR amplification and transformation of transcription factor(Le MADS-rin gene) were conducted. The results of this experiment are as follows:Based on observation for several consecutive years and statistical analysis, precociously-germinated seeds firstly occurred in some fruits of 45 days after pollination(DAP) and proportion of viviparous germination was about 7%. In 50 DAP fruits, approximately 10% of total seeds germinated precociously. Then, precocious germinated% was elevated to about 15% at 55 DAP. More and more seeds germinated precociously in fruit and finally, nearly about 45% had germinated precociously if the fruits remained attaching to maternal plant or harvested and placed in room-temperature for a few weeks after 55 DAP. Also, mucilaginous substance in fruit has depleted and cavity appeared.Some of the developing seeds were able to germinate when they were detached and stripped from the surrounding fruit tissue and were transferred to water in the appropriate temperature. This preliminarily indicated that fruit tissues played an important role in suppression of precocious germination.Germination of developing seeds was inhibited completely when they were stripped from the fruit tissue and then returned to its mucilage gel. This provided a further evidence that seeds germination can be prevented in tomato fruit tissue.Results showed that developing seeds can germinate in low concentrations of ABA, osmoticum and a combination of the two. The higher concentrations of the three solutions, the more effectively are developing seeds are prevented from precocious germination. Tolerance of ABA inhibition, osmotic stress and both increases as the seeds mature in the fruit. In addition, rin seeds exhibited more sensitivity than control seeds to these inhibitors and lower germination percent. The inhibitory effect of the combined ABA and osmoticum was more close to that of mucilage gel on developing seeds germination, and with the stronger influence coming from the osmotic stress.Among the several tomato mutants, seeds of ABA-deficient mutant germinated earlier than the other three tomato line, while that of GA-deficient mutant exhibited slowest germination. There was no drastically difference between germination of control tomato and that of rin mutant, and both took an intermediate position. The results indicated that, ABA deficiency could make seed more able to germinate.For ABA-deficient mutant, there was no dramatically change in seed ABA concentration throughout development and its ABA stayed at lowest level among these four tomato lines. ABA content in other three tomato lines exhibited similar changes: initially increased, peaking at about 50 DAP, then decreased, and ABA concentration of these three tomato lines was higher than that in ABA-deficient mutant. ABA concentration in seed was not necessarily related to viviparous germination in rin accession fruit.Throughout the development, ABA content in mucilage gel of ABA-deficient mutant was lowest among all tomato lines tested. The ABA level in mucilage gel was similar for other three tomato cultivars with a little difference. And the ABA concentration in mucilage gel was not associated with occurrence of viviparous germination.Osmolality in control fruit has been increasing throughout development. On the contrary, osmolality in rin fruit increased gradually at first, then declined and began to be lower than that of control since after 45 DAP stage. Osmolality in rin fruit at later developmental stages was close to maximum threshold value that can permit germination of developing seeds. The results showed that decreasing osmolality in rin fruit contributed to its viviparous germination.In this study, rin gene was amplified and its nucleic acid and amino acid sequence were analyzed using swiss-model software, as well as its protein structure and function. The rin gene encodes 397 amino acids.And it contains two kinds of doomains, which, respectively, is MADS-box domain and K-box domain.Therein, 4 amino acids has changed, the first and the second amino acid was located in the MADS-box domain, whereas the third and the fourth located in the K-box domain. The protein structure was simulated. Based on these works, over-expression vector(p35S::rin) and RNA interference vector(Ubi::rin) were constructed and transformed into tomato. With PCR test, 23 over-expression transgenic positive plants were detected and identified. In two consecutive years, phenotype of developing seeds in PCR-positive plants fruit was investigated and analyzed, there was not yet viviparous seeds occurring in the fruits of over-expression positive plants. |
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