Basic Operation Flow of Tissue Culture and Seedling Raising of Walnut

   tissue culture has high propagation efficiency, is not affected by season and disastrous climate, can propagate every year, and the material can multiply geometrically and propagate quickly. The training technology is simple and easy, the management is convenient, and the training conditions can be artificially controlled, which is conducive to automatic management and realize factory production. The way of asexual reproduction can maintain the excellent characters of the mother, uniform and good quality of seedlings. It can also solve the problem of rapid propagation which can not be propagated by seeds in some plants.

   1. Material selection and treatment. Excellent walnut varieties were selected, and the mother plants with strong growth and less pollution were used as the source of explants. Generally speaking, the materials in the field are dyed more, and it is not easy to obtain aseptic lines, so they can be cultivated in the greenhouse to reduce pollution. A small amount of materials can also be obtained by hydroponic culture.

During the culture of   , the shoots of the same year were cut, the compound leaves were cut off and cut into 3-4 cm stem segments with axillary buds. First rinse with tap water for 3 hours, sterilize with 75% alcohol for 30 seconds under sterile conditions, then soak with 0.01% liter of mercury for 3 minutes for 8 minutes, and finally rinse with sterile water for 3 times for inoculation.

   2. Training steps

The most commonly used stem segment culture medium for    (1) is DKW medium. The sterilized stem segment was cut off the 1 mm end face at both ends and implanted in human culture medium. The culture conditions were as follows: temperature 25 ℃, light intensity 1 000 ~ 3 000 lux, photoperiod 14 ~ 16 hours, dark 8 hours.

   (2) subculture is the main step of stem culture. One method is to promote the rapid growth of axillary buds, the other is to induce the formation of a large number of adventitious buds. The excellent characteristics of the variety can be maintained by the method of axillary bud proliferation, the probability of variation is small, and the proliferation rate is fast. theoretically, more than 100000 buds can be proliferated in one year. The induction of adventitious buds will produce a high probability of variation. After multiplication, the clustered seedlings or buds were subcultured in a new medium and subcultured once every 4-8 weeks. One bud can proliferate 5 ~ 25 seedlings and subculture for many times to meet the demand of production.

   (3) rooting culture the shoots propagated in subculture have no roots and generally need to be induced to take root in rooting medium. The basic culture medium used for rooting is MS medium, but it is necessary to reduce the concentration of inorganic salt, generally use the amount of 1 to 2 or 1 to 4, and reduce or remove cytokinin and increase the concentration of auxin. In the rooting stage, supplemented by dark conditions, the rooting effect is better.

   also has a method of rooting by cutting shoots. First, the shoots were rapidly soaked in auxin or cultured in a medium containing relatively high liquid auxin for 5 ~ 10 days, and then planted in the culture medium in greenhouse (perlite: frogstone = 2:1), often sprayed, and could take root by themselves after a few days.

   because the problem of rooting of walnut tissue culture seedlings has not been well solved, so it is only necessary to provide grafting without rooting, bud grafting and no rooting culture.

   3. Seedling refining and transplanting is a process from heterotrophic to autotrophic. Transplanting test-tube plantlets is not only an important part of tissue culture, but also the most labor-intensive link. If this link is not done well, the previous efforts of tissue culture will be wasted.

   (1) the common transplanting substrates for transplanting and containers are perlite, vermiculite, sand and so on. The containers for transplanting tissue culture seedlings are often 6cm x 6cm plastic bowls, or seedling trays can be used.

   (2) before transplanting, the test-tube plantlets should be exposed to strong light for 2-3 days without opening in the culture container for 2-3 days, and then open for 1-2 days to undergo low temperature training to gradually adapt to the external environment.

   (3) transplanting and seedling management when transplanting test-tube plantlets, the culture medium attached to the roots should be washed off first to avoid microbial propagation pollution and cause seedling death. Keep high humidity and shade after transplanting. After that, gradually reduce the humidity and increase the light until it is the same as the external environment.

   can be transplanted to the field after transplanting survival seedlings in greenhouse for a period of time, and after growing new roots and leaves.