Onion is a biennial plant that needs to be vernalized to produce flowering stalk and seeds in the second year. In most onion cultivars, the optimal conditions for vernalization is storage at 9 to 12 degrees Celsius for one month. By flower induction in the plant many physiological changes occur in the shoot apex, when the plant is in the vegetative stage the shoot apex is sunken. After transferring to the reproductive stage it becomes dome-shaped, which indicates flower induction phenomenon. Also, at this stage, the genes that stimulate flowering time are expressed in the terminal meristem causing to develop flowers. Three flowering time stimulating genes (FT, SOC1 and LFY) have been identified in many plants, including edible onion. It has been observed that AcFT2 gene expresses in high amounts in vernalized onions. Before vernalization, AcFT2 is not expressed in the plant, but with the beginning of cold treatments, AcFT2 is expressed at a high level in shoot apex, which means this gene is an important flowering time regulator in onion. Knowledge of controlling factors in initiation and development of inflorescences can be used in flowering inhibition or stimulation techniques for breeding and seed production programs. The present study was conducted with the aim of determining the duration and appropriate temperature of vernalization in two edible Iranian onion cultivars, and also investigating the expression pattern of AcFT2 gene.
Materials and methods
This research was designed as a factorial experiment in the form of a completely randomized design with 3 replications. The first factor was the cultivar type (Sefide Qom and Ghermeze Azarshahr); the second, the storage temperature in three different levels (5, 10 and 15 degrees Celsius); and the third, the duration of cold treatment in four levels (two, four, six and eight weeks). After the required time for vernalization, 20 bulbs from each treatment were selected for microscopic studies of the shoot apex and comparison with the meristem of the control plants (kept at 25 degrees Celsius). In order to extract RNA, sampling was done from the terminal meristem of the red Azarshahr cultivar at two and six weeks after cold application. The relative expression level of the AcFT2 gene was investigated by the Real-Time PCR reaction method. The actin encoding gene was used as a reference gene.
Results and discussion
The results obtained from the combination of all three factors (cultivar, time, and temperature) showed that the meristem swelling rate was low after two weeks. So, it can be concluded that two weeks of low temperature was not enough to induce flowering. Both cultivars showed completely different reactions after four weeks storage in cold. In this regard, the highest vernalization percentage was observed in Sefide Qom cultivar at 10 and 15 and in Germeze Azarshahr cultivar at 5 degrees Celsius, respectively. The difference between cultivars in the sixth and eighth weeks of storage was also quite obvious. So that, in Sefide Qom, the highest percentage of vernalization (90was observed in storage at 10 and 15 degrees Celsius without significant difference. However, in Ghermeze Azarshahr cultivar the highest percentage of vernalization (90%) occured at 5 degrees Celsius, while 10 and 15 degrees Celsius remarkably reduced the percentage of vernalized bulbs The significant amount of vernalized bulb of both onion cultivars after six weeks of storage in cold, indicates that a period of six weeks is sufficient for bulb vernalization of studied cultivars. The relative expression of AcFT2 gene in the shoot apex of Germeze Azarshahr cultivar, increased significantly compared to the control, six weeks after cold treatment. The highest relative expression was observed at 5 degrees Celsius (30.59 times in compare to the control) and the lowest at 15 degrees Celsius (7.48 times in compare to the control). Therefore, with increasing temperature, the amount of expression of AcFT2 gene decreased.
In general, it can be concluded that the optimal duration of vernalization is six weeks for both Ghermeze Azarshahr and Sefide Qom cultivars. Flower induction of the Ghermeze Azarshahr takes place at five and for Sefide Qom at 10 and 15 degrees Celsius. AcFT2 gene was expressed six weeks after cold application in the shoot apex of bulbs of Ghermeze Azarshahr cultivar. Two weeks of chilling did not show a significant difference in AcFT2 gene expression in this cultivar. The maximum amount of AcFT2 gene expression occurred at five degrees Celsius, which seems to be optimum temperature for vernalization of this cultivar. The results of AcFT2 gene expression in Ghermeze Azarshahr were in line with the results of meristem swelling.