Tissue Culture technique of Rhododendron

Rhododendron is an evergreen or deciduous shrub of rhododendron family, which has the characteristics of many varieties, early flowering, rich flower color, long flowering period and so on. In recent years, due to the development of cutting propagation technology, the yield of rhododendron in China has been increased. However, due to the small number of female parents and low reproduction coefficient, valuable varieties still can not meet the needs of society. Tissue culture is an effective measure to realize the industrial breeding of rhododendron. Here is a brief introduction as follows:

I. the basic technical links of rhododendron tissue culture

In tissue culture of rhododendron, stem tips, stem nodes and buds are commonly used. Because the differentiation of flower buds is difficult and the cycle is long, and the selection of stem tips is limited, stem nodes are often used as the explants of primary culture in China.

During sterilization treatment, mercuric chloride (mercuric chloride) with strong germicidal ability is easy to cause browning and death of explant materials, so it is not suitable to be used. The experiment shows that the effect of using the supernatant of saturated calcium hypochlorite solution or 5% sodium hypochlorite solution as sterilizer is good, and the treatment time is 15 to 20 minutes. In order to enhance the sterilization effect, 1% carbendan or 0.1% Tween-20 (sorbitol) can be added. The pretreatment of the material before sterilization is usually soaked in 70% alcohol for no more than 30 seconds. In addition, the pretreatment of materials with neutral detergents can also effectively reduce the contamination rate after sterilization. The sterilization process of explants is as follows: soak in detergent solution for 20 minutes, rinse in running water to clear 70% alcohol for 30 seconds, rinse in sterilized water for 1 minute, 5% sodium chlorate for 15 minutes, rinse 3 times, 1 minute each time. In this way, the explant contamination rate can be reduced to less than 15%.

The medium used for tissue culture of rhododendron was Andeson modified MS medium: the amount of ammonium nitrate and potassium nitrate in MS medium was reduced to 1pm 4, potassium iodide was eliminated, and the amount of iron salt was doubled. Because Azaleaceae plants are native to alpine acid soil, the pH value of the culture medium should be 5.05.4. The temperature of the culture room was 25 ℃, the light intensity was 3000 lux, and the light time was 12 to 14 hours.

The hormones used in bud induction culture of rhododendron are different from those of ordinary plants, and cytokinins with strong activity are needed in order to achieve satisfactory results. The results showed that the bud induction effect of ZT was the most significant; the treatment with KT had almost no cluster buds or lateral buds, only the elongation of the original stem tip; while the treatment of 6-BA led to the browning of the culture materials, and the effect was not as good as that of the control. In addition, in the subsequent proliferation culture, the requirement of cytokinin in rhododendron was still better with the addition of ZT.

2ip is often used as exogenous cytokinin in the tissue culture of rhododendron abroad, and the effect is better than that of ZT. However, from an economic point of view, ZT is still recommended.

Second, the primary and subculture of rhododendron.

In the primary bud induction culture of rhododendron, the effect of bud induction was obviously promoted by the increase of the concentration of NAA not more than 0.1 mg. However, when the concentration of ZT is increased, the concentration of NAA should not increase synchronously, and the higher value of ZT/ NAA is better. From the significance of economic culture, the hormone ratio of primary culture is ZT5+NAA0.01 (mg/L). After about one month of culture, the bud induction rate can reach 100%, and the bud induction coefficient is 6.5.

The requirement of cytokinin in the subculture of rhododendron was lower than that in the primary culture. Although the proliferation effect still increased with the increase of the dosage of ZT, it was not as obvious as that in the primary culture. In addition, the proliferation effects of the materials used for proliferation culture were different, and there were significant differences among varieties. Among the tested varieties, the proliferation coefficient of 'Xiao Taohong' was higher when using cluster buds as proliferation culture medium (7.6), while that of 'Hua Chunyu' was the opposite, and the response of bud strips was the best (7.5).

The time from inoculation to bud induction in tissue culture of rhododendron is obviously longer than that of rose and chrysanthemum, so the cycle of subculture is also longer. In the experiment, statistics were carried out with 30 days and 45 days as the culture cycle, respectively. in the treatment of the ratio of hormone levels, the total number of proliferative buds cultured for 45 days was 50% to 80% higher than that cultured for 30 days. Therefore, in the process of rhododendron tissue culture, cutting and switching culture too frequently can not achieve the purpose of rapid propagation.

3. Rooting culture and transplanting of test-tube plantlets

Although the rooting culture of rhododendron is not very difficult, the requirement of culture medium is still different from that of ordinary plants. First, the amount of large elements of inorganic salt in the basic culture can not be halved; second, the amount of sucrose is still 30g/L, the addition amount of auxin NAA is 1.5 to 2.0mg/L, and the rooting rate is about 60% after 45 days of culture.

The test-tube plantlets for rooting and culture are required to be more than 15 cm high, with more than 7 true leaves and strong development. Rooting was not seen until 2 weeks after rooting culture, and the highest rooting rate was reached at 6 weeks. In the experiment, it was found that when KT or 6-BA was used to replace ZT in the process of proliferation culture, although the effect of bud induction and proliferation was poor, the development of bud strips was stronger. Therefore, after the multiplication culture reached a certain number of populations, the rooting rate and transplanting survival rate could be significantly increased by subculture with KT or 6 BA. In addition, the ontogeny of test-tube plantlets could be significantly enhanced by adding 2g/L activated carbon to the rooting medium, and the rooting rate could reach more than 90% after one month.

Foreign research data show that for some plants which are extremely difficult to root by conventional propagation (cutting), it will be much easier for test-tube plantlets to take root after tissue culture. Some can be directly transplanted to the rooting medium without rooting culture, and the rooting rate can reach 100%. After 3 to 4 weeks, the rooting rate can be transferred to the greenhouse for pot cultivation. The rooting medium is a mixture of peat, vermiculite and perlite, and the suitable pH is 4 to 4.5.

The rooting test-tube plantlets were transplanted into the soil, and the medium was 1 ∶ 1 peat + vermiculite mixture. The surface layer is required to be screened to facilitate root adhesion, and the lower layer is covered with coarse grains to facilitate matrix water seepage. When transplanting, tweezers are used to remove the culture medium attached to the roots (do not damage the roots). Make a small hole in the matrix with the tip of the tweezers, put the root system into it, cover the soil, gently compact it, and pour enough water. The key to the survival of test-tube plantlets is to ensure certain temperature and wet conditions. there is a great difference between transplanting environment and test-tube culture conditions, which often cause discomfort or death of transplanted plantlets.

Although the transplanting of test-tube rooting plantlets can be carried out throughout the year, it is more safe and convenient in spring and autumn without rooting chamber.

4. application of tissue culture technique in the breeding of new rhododendron varieties.

At the beginning of the experiment, in view of the difficulty of sterilization of Rhododendron explants, the inoculation population available for the experiment was too small to meet the normal test. In order to select the suitable medium formula and corresponding culture conditions as soon as possible, the author combined with cross breeding, adopted the method of test-tube germination of hybrid seeds, and then used the epicotyl of test-tube seedlings as explants, and achieved twice the result with half the effort. It was found that the hybrid seedlings of rhododendron could not blossom until 3 to 5 years after conventional sowing and propagation, while the hybrid seedlings cultured on epicotyl could blossom in the second year after transplanting, which greatly shortened the juvenile period of hybrid seedlings. It is of great significance to speed up the breeding process of flowering and fruiting plants.

Generally speaking, seed sterilization is much easier than other organs or tissues. The sterilization treatment of rhododendron hybrid seeds is as follows: soak the surface of 70% alcohol for 1 minute, rinse with sterilized water and transfer to 0.1 liter mercury solution for 10 minutes, and rinse with sterilized water for 3 times, more than 1 minute each time. After sterilization, the seeds were cut, inoculated and cultured, and sprouting was not seen until two weeks after inoculation. After emergence, the Hypocotyl was used for bud induction and rapid propagation, and the propagation coefficient was higher. After the seedling is formed, the stem tip or stem node should be re-cultured, and the culture conditions and methods are the same as before. In China, there are few tissue culture related to the selection of new varieties, especially in ornamental horticultural plants. From the point of view of development, tissue culture technology should not leave the breeding of new varieties in order to maintain strong vitality. Therefore, the organic combination of tissue culture technology and new variety breeding of rhododendron is very beneficial to improve the construction level of green plant landscape and promote the industrialization development of rhododendron.