A Study On The Pollen Germination In Vitro And The Analysis Of Electrophoretic Pattern Of Specific Proteins In The Pollen Tube Of Citrus Grandis Var. Shatinyu Hort

Being one of the important fruit trees in south China, the shaddock (Citrus grandis var.Shatinyu Hort.) tree is the typical gametophytic self-incompatibility (GSI) plant. However, its lower yield has greatly limited the development of the shaddock production. Studying the mechanism of GSI in Citrus grandis var.Shatinyu Hort. has important value in both shaddock production and the scientific research. The pollen of shaddock (Citrus grandis var.Shatinyu Hort.) was used in this study. After studying the influences of preservation temperature, unfreezing ways and moisture content on the pollen germination in vitro and the influences of culture temperature, medium pH, sucrose, polyethylene glycol (PEG) and the different style proteins on the pollen tube growth, we have advanced the culture medium for the pollen germination and pollen growth in vitro. Furthermore, we have analyzed the probable mechanism of GSI in the Citrus grandis var.Shatinyu Hort. after the comparison of electrophoretic pattern of specific proteins in the pollen tube of Citrus grandis var. shatinyu Hort. which were cultured in the different medium with different style proteins. All results are summarized as follows: 1. The influences of different preserving conditions on the pollen germination in vitro. The pollen germination rate would be over 80 per cent if the moisture content of anther range from 11.8 per cent to 15.5 per cent, the activity of pollen in the preserving anther with too high or too low moisture content would be brought down. With the dropping of preserving temperature, the preserving time of the pollen will be prolonged. The pollen preserved under -70℃can have high germination rates over 80 per cent after one year later. The phased unfreezing method will be better than other methods in keeping the pollen activity of shaddock (Citrus grandis var. Shatinyu Hort.). Consequently, the optimum program for preserving pollen were summarized: the anthers should be dried at the temperature of 25℃until their moisture content range from 11.8 per cent to 15.5 per cent, then should be preserved at the temperature of -70℃in the ultra low temperature freezer. 2. The influence of culture environment and medium on the pollen germination and pollen tube growth. The optimum temperature for the culture of pollen range from 28℃to 31℃. The pollen germination would be brought down to lower rates at lower temperatures and would stop in a short time at some higher temperatures. The pollen should be floating upon the medium during the culture process in which the oxygen is needed, and the optimal pH value of the culture medium range from 5.0 to 5.6. When the concentration of sucrose is in the range from 15 per cent to 30 per cent, the pollen germination will be kept in a high level and the pollen tube will reach the length about 230um. Cultured on the medium containing 15 per cent or 30 per cent sucrose, the pollen tube will grow in a similar rate. While cultured on those media in which the concentration of sucrose is lower than 15 per cent or higher than 30 per cent, the pollen germination and the pollen tube growth would be both weakened. Sucrose acts on the growth of pollen tube most probably by way of being the carbon source and adjusting the osmotic pressure of the medium. Used with sucrose, PEG in the concentration of 20 per cent will enhance the length of the pollen tube conspicuously. Cultured on these media, the pollen tube will get an average length about 728.0um which is over tree times the length on the normal medium. The effect of PEG on the pollen germination and pollen tube growth probably acted in four aspects: First, it can maintain the stability of the physical property of the medium. Second, it can promote the growth of pollen tube by keeping the turgor pressure protractedly. Third, it can make the pollen floating upon the medium. Last but not least, it may restrain the growth of the germ. On the basis of these features of the pollen, we should culture them about 24 hours under the temperature from 28℃to 31℃on the improved medium which contains 0.02 per cent MgSO4, 0.03 per cent KNO3, 0.01 per cent H3BO4, 15 per cent sucrose and 20 per cent PEG with the PH value from 5.0 to 5.6. 3. After the self-pollinated style proteins and the cross-pollinated style proteins added separately in the media, the pollen germination was hardly influenced. However, the growth of pollen tube in those media with the self-pollinated style proteins was restrained. Comparing the electrophoretic pattern between the two groups of pollen tube proteins which were classified by the style proteins added during the culture process of pollen tube, we have found some differences. A specific protein band (Rf =0.81) was found in the PAGE pattern of the proteins in pollen tube cultured with the self-pollinated style proteins. Through SDS-PAGE, two specific protein subunits with molecular weights of 35.0kd and 37.5kd were detected among the same pollen tube proteins. And by the method of IEF-PAGE, two specific protein bands with iso-electric point 5.85 and 6.20 were found.. The results of our study offer some experimental evidences to the hypothesis of 'SI controlled by two genes'.