Tissue Culture technique of Stem Tip of Orchid

The growing period of orchid sprouts in China coincides with the rainy season of high temperature and humidity in the south, and the multiplication of miscellaneous bacteria is rampant, especially in soil and medium. The orchid basin medium for inoculation and sampling should carry as few miscellaneous bacteria as possible, do not apply organic fertilizer, put it in a place of light and shelter from the rain, and let the buds bare when the new buds are emerging. Take the new buds of 6~13cm, cut high from the base of the plant, remove the roots, stolen goods and wrapped leaves with a scraper, wash the material thoroughly, cut the 2~3cm length, and disinfect it in 10% hypochloric acid solution for 10 minutes. If the carrier is serious, cross-disinfect with 0.1% liter of mercury and the supernatant of saturated bleach. After sterilization, rinse with sterile water several times, then put it on sterilized filter paper to absorb dry water, and then operate aseptically under anatomical microscope to obtain stem tips and axillary buds. For the purpose of removing virus, the stem tip should be below O.2mm, otherwise the stem above 2mm can be stripped with 2 leaf primordia, which is beneficial to survival.

(1) Induction of protocorm

The in vitro tissue part of each part of orchid can be induced to form protocorm, and then differentiated and cultured to form plants. Once the protocorm is formed, it can be continuously divided, cultivated and multiplied, that is, the asexual reproduction system is established. The protocorm of orchid has strong vitality and stable genetic characters, which is very beneficial to rapid propagation and germplasm conservation.

After inoculation, the explants were cultured in the dark of 23-25 degrees. After 1-2 months, one to several milky protocorms could be distinguished. The protuberances similar to mulberry fruit were observed under anatomical microscope, and then turned green. After culture, it is easy to show a rhizome (or a tufted branch-like shape). The factors affecting the success of orchid protocorm induction are as follows:

1. The influence of varieties: the difficulty of inducing initiation is also different in different varieties due to the differences in genetic types, endogenous hormones and polyphenols content.

2. The influence of culture medium: the adaptation degree of each variety to different culture medium is different. White+BA;+NAA5+CM8.5% and B11+BA+NAA2 are the best varieties of Cymbidium. "Xia Hui", "Qiusu" and so on are better on the medium of 0.5% of MS+BA0.5+ NAA1 + active carbon. Preliminary observation showed that Chunlan varieties were suitable for the medium with low inorganic salt concentration and ammonia nitrogen content and high auxin content. Varieties such as "Xiahui" and "Qiusu" are suitable for the medium with higher concentration of inorganic salt and lower content of auxin.

3. The influence of new bud degree and material type: both the initiation rate and success rate of stem tip were higher than those of bud induction at 9cm and 13cm, and the success rate was the highest.

4. The effect of browning death after induction: the stem tip of Guolan variety survived and expanded into mulberry-shaped protocorm because the success rate was good, but it was easy to browning and died after start-up, which was one of the reasons for the failure of Guolan tissue culture. According to the data of Biotechnology Research Institute of Sichuan Academy of Agricultural Sciences, browning accounts for almost 3% of deaths. Because the bud tip of Chinese orchid has more polyphenol oxidase, it is easy to brown and die after stem item culture, such as using larger explants, reducing the culture temperature, dark culture, reducing the wound surface as much as possible, and adding browning inhibitors (commonly used antioxidants, such as rutin, citric acid, etc.) in the culture medium, or combined with the application of activated carbon, it has the effect of reducing browning death.

(2) Orchidaceae plants often differentiate into plants through the protocorm pathway. Most of the tropical orchid protocorms are orchid-shaped, while the protocorms of Guolan are tufted (rhizomes). The stage of protocorm formation is a favorable stage for mass propagation of orchids and an important link for rapid propagation and improvement of proliferation rate. After 3-6 months of inoculation of stem tip and lateral bud, the rhizome could be divided and subcultured. W and B11 were used as the basic medium for proliferation, and liquid medium with NAA1~2 was suitable. The culture medium was changed every 15 days and subcultured for 3 or 4 times, then transferred to the solid medium with 0.3% activated carbon and citric acid (500mg/1), which was subcultured once a month. Liquid culture and solid culture are carried out alternately. In multiplication culture, the division of protocorm should not be too small, the culture population should not be too small, the culture medium should not be too much, and the subculture time should not be too long, otherwise the protocorm should not grow badly or even die. Protocorms can be continuously subdivided and proliferated, thus establishing clones. The succession times and time of Guolan protocorm still need to be explored, but from the protocorm which has been subcultured for 3-5 years, it still maintains the normal ability of proliferation and proportion.