| Limonium bicolor,typical recretohalophyte,is a model plant for studying the salt tolerance mechanism of halophytes. However, the life cycle of L. bicolor is long and needs to cross the cold winter. In order to conveniently and efficiently unravel salt tolerance mechanism of L. biocolor, the most important thing is to understand the vernalization and light period conditions and finally to shorten its life cycle. Feng obtained the early flowering mutants by ion implantation in our laboratory but the reason for the early flowering is unclear (2015). In the first part of this thesis, the vernalization conditions of wild type was explored. The second part examined the related traits of early flowering mutants and the possible reasons. The main results are as follows:1. Study on the vernalization time of the wild-type L. bicolor. Wild type of L.bicolor seeds without imbibition were treated with vernalization temperature at 4?for 0,3, 6, 7, 10, 15 and 20 days respectively. The results showed that the earliest bolting time of the plant was 15 days. As the vernalization time of seeds, the flowering rate showed a gradual upward trend, but on the whole, the time required for bolting and flowering was long. The fastest bolting time was 229 days, and the flowering rate was also low. The highest flowering rate was only 50%.2. Study on the imbibition time and temperature of wild type L. bicolor vernalization. Before the vernalization, 0, 15, 30 hours imbibition were applied respectively (0, 15, 30 hours imbibition represented no imbibition, metabolic active period, late period of germination). Then the imbibition seeds started to be treated with different vernalization tempeatures at 2, 4, 6?. The vernalization time was 30 days. The results showed that the flowering rate under 4 ? vernalization was the highest. The flowing of 15 hours imbibition was the best, flowering rate reached 83.33%, and the earliest bolting time was only 136 days, which was the shortest time for all treatment groups. In conclusion, the optimal condition of the vernalization is 4?and the imbibition time before vernalization is 15 hours.3. Study on the seedling vernalization time of wild type L. bicolor. The seedlings were treated with 10?/7 ? (day/night) for 25 days and 35 days respectively. The results showed that flowering rate reached 100% and the earlist bolting time was 168 days under seedling vernalization for 35 days. While under 25 days vernalization,flowering rate reached 75% and the earlist bolting time was 173 days. So the wild type of L. bicolor at 10? for 35 days can achieve flowering conditions at seedling stage.4. Verification of the early flowering characteristics of L. bicolor mutants. Firstly,the seeds of the WT,B15 and D6 were subjected to seed vernalization at 4? for 30 days. After the seed vernalization was completed, the seeds were planted on the same day. When the L. bicolor seedlings grew to four-leaf stage, seedlings of unvernalized L. bicolor WT, B15 and D6 plants were used for vernalization (day/night 8?/4?)under complete vernalization treatment on the same day. The results showed that the flowering rate of seedlings vernalization was 100%, which indicated that it could meet the needs of the vernalization condition for 10 days during the low temperature of 8?. For the flowering rate of seed-vernalized plants, the flowering rate of mutant B15 reached 80%, D6 60%, the WT was 40%. For the flowering rate of plants without vernalization, mutant B15 was 60%, D6 40%, and WT 0. These results showed that the early flowering phenomenon of the mutant is hereditary. Under the condition of seed vernalization, the trait separation ratio is corresponding with Mendel's law(3 : 1).The proportion of early flower is about 75%, but under the condition of no vernalization, the proportion of early flowering is less than 75%. It needs to cultivate F2 generation for further verification.5. Study on the mechanism of early flowering mutants. Flowering requires the accumulation of energy and material. The results showed that the soluble sugar content and protein content of the early flowering mutant B15 and D6 at the boltingstage were significantly higher than those of wild type. The results also showed that the GA3 content in the leaves of the mutant plants was significantly higher than that in the non-bolting wild plants, while the ABA content was significantly lower than that of the non-bolting wild plants. A large number of studies showed that GA3 could promote the flowering and ABA inhibit flowering. Change of hormones may be one of the causes of early flowering mutants.6. Changes in reproductive development of early flowering mutants. In the determination of hormones, we found that the content of BR in the leaves of mutants was significantly lower than that in wild type. The BR content in wild leaves was 1.6 times that of mutant B15. BR was closely related to the development of plant stamens.It was found that the number of pollen, the proportion of active pollen and seed setting rate were lower than that of wild type. The size and 1000-grain weight of the mutant were significantly lower than those of the wild type. It indicates that the decrease of BR content of mutants influenced the development of pollen. And it led to a decrease in seed setting rate and seed weight.7. Study on the mechanism of the late germination of early flowering mutants.When the seeds were subjected to low temperature for vernalization, we ocassionally found that the germination time of mutant B15 and D6 seeds at low temperature was significantly later than that of wild type. Through the pre-experiment we found that the earlist germination time of wild type seeds under 4? vernalization condition was 14 days. So we measure the hormone levels of wild type and mutant seeds in this period. We found that BR content of the mutant seeds was significantly lower than that of the wild type. The BR content in wild seeds was 2.8 times that of mutants. It was found that BR can promote germination,which may be reasons for the late germination of mutant seeds.8. Effects of exogenous BR on seed germination of mutants. BR was applied to the seeds at 4?. We found that BR could promote the germination of wild type seeds,but had no significant effect on the germination of mutant seeds. However,it was found that BR promoted the germination of wild type and mutant seeds at room temperature (25?), and the promotion effect of wild type seeds was better. It is reported that BR could promote the increase of GA3 content at low temperature,promote the increase of a-amylase activity, stimulate the decomposition of starch into glucose, and finally provide energy for germination and promote germination. GA3 was applied to the seeds at the low temperature germination at 4?. And we found that the germination rate of mutants can be restored to the level of WT. This indicates that the mutant seeds may affect the seed germination in two aspects. On the one hand,the BR content is low and the germination promoting effect is weak. On the other hand, the signal transduction pathway of BR promoting GA3 may be affected.Moreover, effect of BR on seed germination of mutants depends on temperature. The detailed mechanisms need to be further explored.9. Key genes of BR synthesis expression quantity determination. We measured the key genes of BR synthesis content in the seeds that germinate at low temperature(4?) for 14 days. The results showed that the expression of BR synthetic key genes DWF1 and CPD in mutant B15 were significantly lower than those of wild type. It suggest that DWF1 and CPD gene expression have been affected by ion implantation in mutant B15, which affects the synthesis of BR and results in the decrease of BR content finally leading to the trait of late germination. These results explain the causes of BR content decrease and the seed late germination in mutants at the genetic level. |
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