The Mini-juvenile-type Bud Stick Were Cultured In Vitro And The Method Of Seed Seedling Bud Grafting Of Hevea Rasiliensis

In the course of rubber seed seedling grafting process, the diameter of young green bud stick is about 0.5-1.2 cm. But the diameter of seed seedling is only about 0.2-0.3 cm. It's difficult to graft the above big bud and small seed seedling together. Because the big bud need to be cut very small to fit for the seed seedling, which depresses the surviving efficient of grafting seed seedlings. And only squama bud and leaf bud can be used in existence. So the grafting efficient is very low. In this paper, the technique for cultivating the mini-juvenile-type bud stick by tissue culture has been studied. The result shows that the somatic cell plantlets obtained from anther or stamen cultured in vitro pass the process of dedifferentiation and redifferentiation, and their developmental stage returns from mature type to the juvenile-type, extremely similar to that of zygote embryo. The bud stick from micro propagated in vitro is named mini-juvenile-type bud stick., which diameter is about 0.2-0.3cm. We also studied the seed seedling grafting with the mini-juvenile-type bud stick. The result shows that the method can solve the problem of nonsymmetrical stock and bud stick (stock is too thin, and bud stick is too thick) in seed seedling grafting process.The propagation of the mini-juvenile-type bud stick is carried on in the laboratory. The germfree axillary buds and apical buds of the young anther somatic plantlet cut as explant are inoculated on the improved Ms medium with 6-BA 2-3mg/l,NAA 0.1-0.2mg/l and sucrose50-60g/l. Side buds will come up in 7-10 days on the medium. The new buds will come into being mini-juvenile-type bud stick high about 5-10 cm in 40-50 days. The axillary buds and apical buds of mini-juvenile-type bud stick can continue to be used in bud proliferating. The multiplication coefficient every year is above 1000 times. However, the multiplication coefficient of ordinary bud stick in field is about 20 times, and the coefficient of green lateral branch bud stick after cutting the apical bud many times in field is most 200 times.The thickness of sucrose in the medium affects the proliferation of mini-juvenile-type bud stick obviously. When the thickness of sucrose increases from 30g/L to 60g/L, the proliferating coefficient of mini-juvenile-type bud stick increase from 1.50 to 2.98.Another important factor affecting the height and proliferating coefficient of mini-juvenile-type bud stick is the cultivating temperature. When the temperature increases from 26℃to 30℃, the height of mini-juvenile-type bud stick increases from 5.2 cm to 9.1 cm and the proliferating coefficient increases from 2.6 to 4.6. The difference between 26℃and 28℃is terribly marked(|u|=11.71>2.58). The difference between 26℃and 30℃is also terribly marked(|u|=12.03>2.58). But the difference between 28℃and 30℃is not marked(| u|=0.24< 1.96).The bud grafting speed is quickened when the seed seedlings graft with the mini-juvenile-type bud. Because the bud sticks are small. It means that the bud-patch and the budplap small too. The bud patch need not trim. It can be inserted directly into the budplap without striping off the xylem. And the binding is easy. Therefore the bud grafting is easy to operate. A skilled worker can graft about 250-300 within 1day. The number is 2.5 times higher than green lateral branch bud stick grafting in field. The survival rate is high when the seed seedling grafting with mini-juvenile-type bud. The seed seedling grafting with the phenophase suitable miniature bud, even in winter (after bud grafting the plantlets are kept in green house), the survival rate can achieve 80-90%. However, the survival rate is only 70-80% even the optimum green field lateral branch being used.On mini-juvenile-type bud stick, the axillary buds before germination or on exactly germinating stage, and the apical buds in jarless phenophase or in unstable phenophase were all used to graft with seed seedlings, when the seed seedling's first pair of bronze-coloured leaves. The results show that the axillary buds before germination have the highest survival coefficient, the survival coefficient of axillary buds on exactly germinating stage is lower than it, and the apical buds in unstable phenophase have the lowest survival coefficient. Comparing the axillary buds before germination and on exactly germinating stage, and the apical buds in jarless phenophase with the apical buds in unstable phenophase, the difference is terribly marked(prob=0.001<0.01,prob=0.001<0.01,prob=0.002<0.01). It shows that we should avoid the grafting between the apical buds in unstable phenophase and the seed seedlings.The different phenophase of seed seedling affects the grafting survival rate of the seed seedling. The seed seedlings before the stage of first pair of leaves, and the first pair of leaves on bronze-coloured stage, the first pair of leaves on spread stage, the first pair of leaves on mature stage were all choosed to graft with mini-juvenile-type bud. The result shows that the survival rate of seed seedlings before the stage of first pair of leaves is 83%, the survival rate of seed seedlings the first pair of leaves on bronze-coloured stage is 100%, the survival rate of seed seedlings the first pair of leaves on spread stage is 93%, but the survival rate of seed seedlings the first pair of leaves in jarless phenophase is only 20%.For the propagation of Hevea planting mateiral by seed seedling bud grafting is denseness, A lot of seed seedling bud grafting plantlets can be obtained in a small land. The management including watering, weeding, fertilizing, and plant disease controlling and so on are convenient.The way of different stock cuts will affect the bourgeoning speed of the seed seedling bud grafting plantlet. Not only the bourgeoning is slowly when the terminal bud is removed and the first pair of leaves retained, but also it is a troublesome work for excision stock bud 1-2 times each week. Taking stock cuts underneath the first pair of leaves, can make the bud bourgeon early and consistent, even the seedling size consistent. The difference between the above two methods is terribly marked(prob=0.001<0.01).The latter method is better than the former in promoting grafting buds bourgeoning and availability of seed seedlings.The bud grafting can carry on in the winter with the mini-juvenile-type bud stick. In North Hainan, although cold time is not long in winter, but the young rubber seedling sometimes encounters the cold hurting. Moreover, the seed seedling bud grafting survival rate is low when we use the field cultivation bud stick in winter. Therefore no one carries on the bud grafting in winter. For the mini-juvenile-type bud sticks of rubber tree cultured in the plant tissue culture room, they will be not affected by season. A batch of seeds in Hainan Province can be obtained in November, and in west of Guangdong province it happens in January or February. They can be sowed in the green house, and we can plant the seed seedling bud grafting plantlets in the green house too. Each square meter seedbed may plant 150-200 seedlings. The Bud grafting survival rate is above 80%. When (in March ) the weather turns warm, they are moved outside. In this period, often no serious cold hurting will happen.The plant material from mini-juvenile-type bud stick seed seedling bud grafting is high yield and fast-growing. Using the anther somatic plantlets as the explant., the mini-juvenile-type bud stick were cultured with method of micro-propagation in vitro. The kind of bud stick possesses of the donor tree's fine characters and the excellence of seed tree, such as fast-growing and the resistance.At present, no report about the propagation of rubber tree via mini-juvenile-type bud stick through tissue culture techniques and grafting with seed seedlings. This technology will become a new method to obtain high yield and fast-growing rubber plant material.