Detailed Explanation of Shoot-Tip Tissue Culture Technology for Chinese Cymbidium
The new shoot growth period of Chinese cymbidium coincides with the hot, humid, rainy season in the south, when miscellaneous fungi proliferate rampantly, and the soil and media are especially severely contaminated with fungi. The potting medium used for inoculation sampling should carry as few miscellaneous fungi as possible, receive no organic fertilizer, and be placed in a light-transmitting, rain-sheltered location; when the new shoots first emerge, let the young shoots be exposed above the surface. Take new shoots 6-13 cm long, cut them high from the base of the plant, use a scraper to remove roots, debris and 2-3 outer sheath leaves; after thorough washing, cut the material again to 2-3 cm in length, and sterilize in a 10% hypochlorous acid solution for 10 minutes. If contamination is severe, cross-sterilize with 0.I1% mercuric chloride and saturated bleaching powder supernatant. After sterilization, rinse several times with sterile water, then place on sterile filter paper to blot dry the residual water, and then under a dissecting microscope perform aseptic dissection to excise the shoot tip and axillary buds. If the purpose is virus elimination, the excised shoot tip should be below O.2 mm; otherwise, a shoot tip of 2 mm or more with 2 leaf primordia may be excised, which favors survival.
(1) Induction of Protocorm-Like Bodies
Isolated tissues from various parts of orchids can all induce the formation of protocorm-like bodies, which then form plants through differentiation culture. Once protocorm-like bodies are formed, they can be continuously divided and subcultured for proliferation, that is, a clonal propagation line is established. Orchid protocorm-like bodies have strong vitality and stable genetic traits, and are extremely advantageous for rapid propagation and germplasm resource conservation.
After inoculation, the explants are placed under dark conditions at 23-25 degrees for culture; after 1-2 months, 1 to several milky-white protocorm-like bodies can differentiate, appearing under the dissecting microscope as round spherical protrusions similar in shape to mulberries; they later turn green, and upon further culture readily become rhizomes (or form dendritic clustered shapes). The factors affecting the success of orchid protocorm-like body induction include:
1. Influence of variety: Due to differences among varieties in genetic type, endogenous hormones and polyphenol content and other factors, the difficulty of inducing initiation also differs.
2. Influence of culture medium: Each variety has a different degree of adaptability to different culture media. Cymbidium goeringii varieties do well with White + BA; + NAA5 + CM8.5% and B11 + BA + NAA2. "Xiahui", "Qiusu" and others do well with MS + BA0.5 + NAA1 plus activated charcoal 0.5% medium. Preliminary observations show that Cymbidium goeringii varieties adapt to media with low inorganic salt concentration and low ammoniacal nitrogen content but high auxin content. Varieties such as "Xiahui" and "Qiusu" adapt to media with higher inorganic salt concentration and lower auxin content.
3. Influence of new shoot length and material type: The initiation rate and success rate of shoot tips are both higher than those of lateral buds; induction with 9-13 cm new shoots gives the highest success rate.
4. Influence of browning death after initiation: After inoculation of Chinese cymbidium shoot tips, those that survive and swell into mulberry-shaped protocorm-like bodies are considered initiated, and the success rate is fairly good, but after initiation they readily undergo browning and death, which is one of the reasons for failure in Chinese cymbidium tissue culture. According to data from the Biotechnology Research Institute of the Sichuan Academy of Agricultural Sciences, browning deaths account for nearly 3/4. Because Chinese cymbidium shoot tips contain relatively large amounts of polyphenol oxidase, they readily brown and die during shoot-tip culture. Using larger explants for inoculation, lowering the culture temperature, dark culture, minimizing wound surface area, and adding browning inhibitors to the medium (commonly used antioxidants such as rutin, citric acid, etc.), or using activated charcoal in combination, can reduce browning death.
(2) Orchidaceae plants mostly differentiate to form plants through the protocorm-like body pathway. Tropical orchid protocorm-like bodies are mostly spherical, while those of Chinese cymbidium are clustered (rhizomatous). The protocorm-like body formation stage is a favorable stage for mass propagation of orchids and is an important link in rapid propagation and increasing the multiplication rate. After shoot tips and lateral buds are inoculated for 3-6 months, when rhizomes have formed they can be divided for subculture; the proliferation medium is preferably a liquid medium with W and B11 as the basic medium, supplemented with NAA1-2. Place on a low-speed rotating shaker with light for culture (1-2 rotations/min), replace the medium once every 15 days; after 3-4 consecutive subcultures, transfer again to a solid medium of the same composition supplemented with activated charcoal (0.3%) and citric acid (500 mg/l), subculturing once a month. Liquid culture and solid culture are carried out alternately. During proliferation culture, the division of protocorm-like bodies should not be too small; the cultured population should not be too few; the culture liquid should not be too much; and the subculture time should not be too long, otherwise the protocorm-like bodies grow poorly or even die. Protocorm-like bodies can be continuously re-divided and proliferated, thus establishing a clonal line. The number and duration of subcultures for Chinese cymbidium protocorm-like bodies remain to be explored, but from protocorm-like bodies that have been subcultured for 3-5 years, they still maintain normal proliferation and differentiation ability.