Detailed Explanation of Key Points of Rhododendron Tissue Culture Technology

Rhododendron is an evergreen or deciduous small shrub of the genus Rhododendron in the family Ericaceae, characterized by many varieties, early flowering, rich flower colors, and a long flowering period. In recent years, due to the development of cutting propagation technology, China's rhododendron production has increased. However, precious varieties still cannot meet social demand due to few mother plants and a low propagation coefficient. Tissue culture is precisely an effective measure for achieving factory-scale seedling production of rhododendrons. A brief introduction is given below:

1. Basic technical steps of rhododendron tissue culture

In rhododendron tissue culture, the explants usually used are stem tips, stem nodes, and flower buds, etc. Because flower buds are relatively difficult to differentiate and require a long cycle, and the collection of stem tips is subject to certain restrictions, stem nodes are mostly used in China as explants for primary culture.

During sterilization treatment, mercuric chloride (mercury chloride), which has strong sterilizing ability, easily causes browning and death of explant materials and should not be used. Experiments show that using the supernatant of saturated calcium hypochlorite solution or 5% sodium hypochlorite solution as a sterilizing agent gives better results, with a treatment time of 15 to 20 minutes. To strengthen the sterilization effect, 1% captan or 0.1% Tween-20 (sorbitol) can also be added for combined use. For pretreatment of materials before sterilization, soaking in 70% alcohol is usually used, for no more than 30 seconds. In addition, pretreating materials with a neutral detergent can also effectively reduce the contamination rate after sterilization. The sterilization process for explant materials is: soak in washing powder solution for 20 minutes, rinse under running water until clean, soak in 70% alcohol for 30 seconds, rinse with sterile water for 1 minute, soak in 5% sodium hypochlorite for 15 minutes, rinse with sterile water 3 times, 1 minute each time. In this way, the contamination rate of explants can be reduced to below 15%.

The medium used for rhododendron tissue culture is modified Anderson MS medium: the amounts of ammonium nitrate and potassium nitrate in MS medium are reduced to 1/4 each, the potassium iodide component is eliminated, and the amount of iron salt is doubled. Because plants in the family Ericaceae originate from acidic soils in high mountains, the pH value of the medium should be 5.0 to 5.4. The culture room temperature is 25°C, the light intensity is 3000 lux, and the light duration is 12 to 14 hours.

The hormones used in shoot induction culture of rhododendrons differ from those used for general plants; cytokinins with stronger activity are needed to achieve satisfactory results. Experiments show that the shoot induction effect of using ZT is the most significant; treatments with added KT produce almost no clustered shoots or axillary shoots, only elongation of the original stem tip; while treatment with 6-BA causes intensified browning of the cultured material, and the effect is even inferior to the control. In addition, in the subsequent proliferation culture, rhododendrons' requirement for the type of cytokinin is still best met by adding ZT.

In foreign countries, 2iP is mostly used as an exogenous cytokinin in rhododendron tissue culture, with better results than ZT. However, from an economic perspective, ZT is still recommended.

2. Primary and subculture proliferation culture of rhododendrons

In primary shoot induction culture of rhododendrons, the NAA concentration must not exceed 0.1 mg/L, and increasing the amount of ZT has a clear promoting effect on shoot induction. However, when increasing the ZT concentration, the NAA concentration should not be increased simultaneously; a larger ZT/NAA ratio is better. From the perspective of economical culture, the hormone ratio for primary culture is preferably ZT 5 + NAA 0.01 (mg/L). After about 1 month of culture, the shoot induction rate can reach 100%, and the shoot induction coefficient is 6.5.

In subculture proliferation culture of rhododendrons, the requirement for cytokinin is somewhat lower than in primary culture. Although the proliferation effect still rises with increasing ZT dosage, it is no longer as obvious as in primary culture. In addition, materials of different properties used for proliferation culture also have different proliferation effects, and there are also significant differences among varieties. Among the tested varieties, 'Xiaotaohong' had a higher proliferation coefficient when clustered shoots were used as the proliferation culture material (7.6); while 'Huachunyu' was the opposite, performing better when shoot strips were used (7.5).

In rhododendron tissue culture, the time required from inoculation to shoot induction is significantly longer than for roses and chrysanthemums, so the cycle of subculture proliferation culture is also longer. In the experiment, statistics were conducted using 30 days and 45 days as culture cycles. Under treatments with various hormone level ratios, the total number of proliferated shoots at 45 days of culture was 50% to 80% higher than at 30 days. Therefore, in the rhododendron tissue culture process, cutting and transferring cultures too frequently will instead fail to achieve the purpose of rapid propagation.

3. Rooting culture and transplanting of test-tube plantlets

Although rooting culture of rhododendrons is not very difficult, its requirements for the medium still differ from those of general plants. First, the amount of major inorganic salt elements in the basic medium cannot be halved; second, the amount of sucrose is still 30 g/L, and the amount of auxin NAA added is preferably 1.5 to 2.0 mg/L. The rooting rate at 45 days of culture is about 60%.

Test-tube plantlets used for rooting culture are required to be more than 15 cm tall, have more than 7 true leaves, and be robust in development. Rooting begins to appear 2 weeks after rooting culture, and the highest rooting rate is reached at 6 weeks. In experiments, it was once found that when KT or 6-BA was used to replace ZT during proliferation culture, although the shoot induction and proliferation effects were poorer, the shoot strips developed more thickly and robustly. Therefore, after proliferation culture reaches a certain population size, using KT or 6-BA for subculture treatment can significantly increase the rooting rate and transplanting survival rate. In addition, adding 2 g/L activated charcoal to the rooting medium can significantly enhance the individual development of test-tube plantlets, and after 1 month the rooting rate can reach more than 90%.

Foreign research data show that for some plants that are extremely difficult to root by conventional propagation (cuttings), test-tube plantlets obtained through tissue culture root much more easily. Some can be directly transplanted into rooting media without rooting culture, with a rooting rate reaching 100%, and after 3 to 4 weeks they can be moved into a greenhouse for pot cultivation. This rooting medium is a mixture of peat soil, vermiculite, and perlite, with a pH preferably of 4 to 4.5.

For transplanting rooted test-tube plantlets into soil, the cultivation substrate is a 1:1 mixture of peat + vermiculite. The surface layer should be sieved to facilitate root attachment; the lower layer should be laid with coarse particles to facilitate water seepage through the substrate. During transplanting, use tweezers to remove the medium attached to the roots (without damaging the root system). Use the tip of the tweezers to poke a small hole in the substrate, place the roots in, cover with soil, press gently to firm, and water thoroughly. The key to the survival of transplanted test-tube plantlets is ensuring certain temperature and humidity conditions. The transplanting environment differs greatly from the conditions during test-tube culture, often causing discomfort or death of transplanted seedlings.

Although transplanting rooted test-tube plantlets can be carried out throughout the year, in the absence of a rooting room, it is still safer and more convenient to carry it out in spring and autumn.

4. Application of tissue culture technology in breeding new rhododendron varieties

In the early stage of the experiment, given the difficulty of sterilizing rhododendron explants, the inoculable population available for testing was too small to meet the needs of normal experimentation. In order to screen out suitable medium formulas and corresponding culture conditions as quickly as possible, the author combined hybrid breeding, adopted the method of test-tube germination of hybrid seeds, and then used the epicotyls of test-tube seedlings as explants for research, achieving twice the result with half the effort. The author found that hybrid rhododendron seedlings propagated by conventional sowing require 3 to 5 years to flower, while hybrid seedlings cultured from epicotyls can flower in the second year after transplanting, greatly shortening the juvenile phase of hybrid seedlings, which is of great significance for accelerating the breeding process of flowering and fruiting plants.

Generally speaking, sterilization treatment of seeds is much easier than that of other organs or tissues. The sterilization treatment of rhododendron hybrid seeds is: soak the surface in 70% alcohol for 1 minute, rinse with sterile water, then transfer to 0.1% mercuric chloride solution and soak for 10 minutes, rinse with sterile water 3 times, each time for more than 1 minute. After sterilization, cut open the fruit to take seeds and inoculate for culture; germination begins to appear two weeks after inoculation. After seedlings emerge, epicotyls are taken for rapid shoot induction propagation, and the propagation coefficient is relatively high. After plantlets form, stem tips or stem nodes can be taken for further culture, and the culture conditions and methods are the same as before. In China, tissue culture involving new variety breeding is not common, especially in ornamental horticultural plants. From the perspective of development, if tissue culture technology is to maintain strong vitality, it should not be separated from new variety breeding. Therefore, organically combining tissue culture technology with the breeding of new rhododendron varieties is very beneficial for improving the construction level of green space plant landscapes and promoting the industrialized development of rhododendrons.