A Detailed Explanation of the Basic Operational Procedure for Walnut Tissue Culture Propagation
Tissue culture propagation has high reproductive efficiency, is unaffected by seasons and disastrous weather, allows year-round propagation, and the material can multiply in geometric progression, giving a fast rate of seedling multiplication. The culture technique is simple and easy to perform, convenient to manage, and the culture conditions can be controlled artificially, which facilitates automated management and enables factory-scale production. Asexual propagation can maintain the superior traits of the mother plant, giving uniformity and good seedling quality. In some plants it can also solve the problem of rapid propagation when seed propagation cannot be used.
1. Selection and treatment of material Select superior walnut varieties, using robust mother plants with relatively little contamination as the source of explants. Generally, field material is more contaminated and it is not easy to obtain an aseptic line, so it can be cultivated in a greenhouse to reduce contamination. Small amounts of material can also be obtained as young shoots by hydroponic culture.
When carrying out culture, cut off the current year's young shoots, remove the compound leaves, and cut them into 3~4 cm stem segments with axillary buds. First rinse under running tap water for 1~3 hours, then under aseptic conditions sterilize with 75% alcohol for 30~50 seconds, then soak in 0.01% mercuric chloride for 3~8 minutes, and finally rinse with sterile water 3~5 times, ready for inoculation.
2. Culture steps
(1) Shoot tip (segment) culture The most commonly used medium for stem segment culture is DKW medium. Cut off 1 mm from each end of the sterilized stem segment and implant it into the medium. The culture conditions are a temperature of 25°C, a light intensity of 1,000~3,000 lux, and a photoperiod of 14~16 hours light and 8~10 hours dark.
(2) Subculture and multiplication This is the main step of stem segment culture. One method is to promote the rapid growth of axillary buds, and the other is to induce the formation of large numbers of adventitious buds. The method of axillary bud multiplication can maintain the superior characteristics of the variety, has a relatively low probability of variation, and gives a fast multiplication rate; in theory, one bud can multiply into more than 100,000 plants within a year. Inducing adventitious buds produces a relatively high probability of variation. After multiplication, the formed clustered shoots or single shoots are divided and transferred to fresh medium for subculture, with subculturing once every 4~8 weeks. One shoot can multiply into 5~25 small shoots, and multiple rounds of subculture can be carried out to meet production needs.
(3) Rooting culture The shoots from subculture and multiplication have no roots, and generally need to be induced to root in a rooting medium. The basic medium used for rooting is MS medium, but the concentration of inorganic salts needs to be reduced, generally to 1/2 or 1/4 of the amount, and cytokinin should be reduced or removed while the auxin concentration is increased. During the rooting stage, supplementing with dark conditions gives a better rooting effect.
There is also a method of rooting by cuttings of the shoots. First soak the shoots rapidly in auxin or culture them for 5~10 days in a medium containing a relatively high concentration of auxin, then plant them into a culture substrate in the greenhouse (perlite : vermiculite = 2:1), mist frequently, and they will root on their own after a few days.
Because the rooting problem of walnut tissue-cultured shoots has never been well solved, it is only necessary to provide unrooted scions for shoot grafting, without carrying out rooting culture.
3. Hardening off and transplanting Transplanting is a process of transition from heterotrophy to autotrophy. Transplanting test-tube plantlets is an important part of the tissue culture process, and also the most labor-intensive part; if this step is not done well, all the previous work of tissue culture propagation will be wasted.
(1) Substrate and containers for transplanting Commonly used transplanting substrates include perlite, vermiculite, sand, etc., and a single substrate or a composite substrate may be used. The containers for transplanting tissue-cultured plantlets are often 6 cm x 6 cm plastic pots, or seedling trays may be used.
(2) Hardening off before transplanting Before transplanting the test-tube plantlets, first keep the culture container unopened and harden them under natural light for 2~3 days, letting the test-tube plantlets receive strong light, then open the container and harden them for 1~2 days, undergoing low-temperature hardening, so as to gradually adapt to the external environmental conditions.
(3) Transplanting and management of seedlings When transplanting test-tube plantlets, the medium attached to the roots should first be washed off to avoid contamination by microbial growth and the resulting death of the small plantlets. After transplanting, relatively high humidity should be maintained and shading should be provided. Afterwards, gradually reduce the humidity and increase the light until it is the same as the external environment.
After the small plantlets that survive transplanting in the greenhouse have been cultivated for a period of time, once they grow new roots and new leaves they can be transplanted to the field.