Search

CN-121970683-A - Efficient tissue culture and rapid propagation seedling method for shinyleaf yellowhorn

CN121970683ACN 121970683 ACN121970683 ACN 121970683ACN-121970683-A

Abstract

The invention relates to the technical field of seedling raising, and discloses a high-efficiency tissue culture and rapid seedling raising method for shinyleaf yellowhorn, which comprises the following steps: the method comprises the steps of selecting and preprocessing an explant, selecting the current healthy branch of shinyleaf yellowhorn, shearing a stem section with axillary buds as the explant, cleaning and trimming the stem section, disinfecting the surface of the preprocessed explant, and establishing an operation standard which takes thorough sterilization and tissue activity protection into consideration by standardizing the selection standard of the explant and executing a step surface disinfection program when the high-efficiency tissue culture and rapid propagation seedling culture of the shinyleaf yellowhorn is carried out, so that the microbial contamination rate of the explant caused by incomplete sterilization can be reduced, and the tissue activity damage caused by excessive sterilization is reduced, thereby ensuring that a sufficient and healthy sterile initial material is obtained, solving the problems of high initial culture contamination rate and induction start failure and laying a reliable foundation for the establishment of the whole tissue culture and rapid propagation system.

Inventors

  • Li Aoga
  • WANG SIRAN
  • QING JUN
  • LI HUI
  • JI RENHUA
  • BAI YUE
  • WANG YAPING

Assignees

  • 内蒙古农业大学

Dates

Publication Date
20260505
Application Date
20260226

Claims (10)

  1. 1. The high-efficiency tissue culture and rapid propagation seedling method for shinyleaf yellowhorn is characterized by comprising the following steps of: Step one, selecting and preprocessing an explant, selecting the current healthy branches of shinyleaf yellowhorn, cutting off stem segments with axillary buds as the explant, and cleaning and trimming the explant; Step two, sterilizing the surface of the pretreated explant; preparing a primary culture medium, namely preparing a solid culture medium serving as primary induction; inoculating and primary culturing, namely inoculating the sterilized explant to a primary culture medium, and performing primary induction culture in a controllable environment; Step five, proliferation culture, namely transferring cluster buds or adventitious buds obtained by primary culture into a proliferation culture medium for proliferation; Rooting culture, namely transferring the propagated unrooted seedlings into a rooting culture medium to induce root system formation; Hardening off and transplanting, hardening off the tissue culture seedlings with complete roots, and transplanting the tissue culture seedlings into a seedling culture matrix; In the third step, the primary culture medium is prepared from the following components of MS basic culture medium, 6-benzyladenine 0.5-2.0mg/L, naphthylacetic acid 0.1-0.5mg/L, sucrose 25-35g/L, agar 5-7g/L and pH value regulated to 5.7-5.9; In the fifth step, the proliferation culture medium is prepared from the following components of MS basic culture medium, 6-benzyladenine 1.0-3.0mg/L, naphthylacetic acid 0.1-0.5mg/L, sucrose 25-35g/L, agar 5-7g/L, and pH value adjusted to 5.7-5.9; In the step six, the rooting culture medium is prepared from the following components of 1/2MS basic culture medium, 0.5-1.5mg/L indolebutyric acid, 15-25g/L sucrose, 5-7g/L agar and regulating the pH to 5.7-5.9.
  2. 2. The method for efficient tissue culture and rapid propagation of shinyleaf yellowhorn according to claim 1, wherein in the first step, the current annual robust branches of shinyleaf yellowhorn are semi-lignified branches with the diameter of 0.3-0.6cm, the length of the sheared stem segments is 1.0-2.0cm, 1-2 axillary buds are reserved in each stem segment, and the shinyleaf yellowhorn is washed for 30-60 minutes by flowing water after being trimmed.
  3. 3. The method for high-efficiency tissue culture and rapid propagation of shinyleaf yellowhorn according to claim 1, wherein in the second step, the disinfection treatment comprises the steps of placing the pretreated explant in an ultra-clean workbench, firstly soaking the explant in 70-75% ethanol solution for 30-60 seconds for preliminary disinfection, then soaking and disinfecting the explant in 0.1-0.2% mercuric chloride solution or 2-4% sodium hypochlorite solution for 8-15 minutes, and finally flushing the explant with sterile distilled water for 3-5 times.
  4. 4. The method for high-efficiency tissue culture and rapid propagation of shinyleaf yellowhorn according to claim 1, wherein in the third step, the preparation method of the primary culture medium comprises the steps of dissolving MS basic culture medium powder in distilled water, adding the plant growth regulator solution of 6-benzyl adenine and naphthylacetic acid, adding sucrose and agar, stirring and mixing uniformly, regulating pH to 5.7-5.9, subpackaging into a culture container, and sterilizing at 121 ℃ for 15-20 minutes.
  5. 5. The method for efficient tissue culture and rapid propagation of shinyleaf yellowhorn according to claim 1, wherein in the fourth step, the primary culture condition comprises setting the culture temperature to be 24-26 ℃, the illumination intensity to be 2000-3000lux, the light period to be 14-16 hours of illumination/8-10 hours of darkness, and the culture time to be 20-35 days until the explant induces cluster buds or adventitious buds.
  6. 6. The method for high-efficiency tissue culture and rapid propagation of shinyleaf yellowhorn according to claim 1, wherein in the fifth step, the proliferation culture condition comprises the steps of setting the culture temperature to be 24-26 ℃, setting the illumination intensity to be 2000-3000lux, setting the light period to be 14-16 hours of illumination/8-10 hours of darkness, setting the culture period to be 25-35 days, setting the proliferation coefficient of each generation to be 3-5 times, cutting cluster buds into bud segments in the proliferation process, and transferring the bud segments into a fresh proliferation culture medium for subculture.
  7. 7. The method for efficient tissue culture and rapid propagation of shinyleaf yellowhorn according to claim 1, wherein in the step six, rooting culture conditions comprise a culture temperature of 24-26 ℃, an illumination intensity of 2000-3000lux, a light period of 14-16 hours of illumination/8-10 hours of darkness, a culture time of 15-25 days until root systems with the length of 2-3cm are induced at the root parts of the shinyleaf yellowhorn, and 0.5-1.0g/L of active carbon is added into the rooting culture medium.
  8. 8. The method for high-efficiency tissue culture and rapid propagation of shinyleaf yellowhorn according to claim 1, wherein in the seventh step, the seedling hardening and transplanting comprises the steps of moving a culture container to a natural illumination environment for hardening the seedling for 3-5 days when the rooting seedling grows to 3-5cm high, taking out the tissue culture seedling, cleaning a root culture medium, transplanting the tissue culture seedling into a seedling culture medium mixed by vermiculite, perlite and turf according to a volume ratio of 1:1:1, keeping the humidity at 80-90% in an initial stage, keeping the temperature at 20-25 ℃, and carrying out normal management after 7-10 days of seedling reviving.
  9. 9. The method for efficient tissue culture and rapid propagation of shinyleaf yellowhorn according to claim 1, wherein organic additives are added into the primary culture medium, the proliferation culture medium and the rooting culture medium, wherein the organic additives are 50-100ml/L of coconut juice or 50-100g/L of banana puree, and the organic additives are added aseptically when the culture medium is cooled to 50-60 ℃ after sterilization.
  10. 10. The method for high-efficiency tissue culture and rapid propagation of shinyleaf yellowhorn according to claim 1, wherein the culture environments in the fourth step, the fifth step and the sixth step are controlled to have relative humidity of 60-70%, the culture container is covered by a breathable sealing film, the whole tissue culture process is operated under a sterile condition, and the explant inoculation density is 3-5 stem segments per bottle.

Description

Efficient tissue culture and rapid propagation seedling method for shinyleaf yellowhorn Technical Field The invention relates to the technical field of seedling raising, in particular to a high-efficiency tissue culture and rapid seedling raising method for shinyleaf yellowhorn. Background The shinyleaf yellowhorn is a fallen leaf shrub or small arbor of shinyleaf yellowhorn of sapinduaceae, the shinyleaf yellowhorn is an excellent ornamental tree species, stems or branches and leaves of the shinyleaf yellowhorn can be used as medicines, the shinyleaf yellowhorn has the effects of dispelling wind and removing dampness, reducing swelling and relieving pain, the root system of the adult shinyleaf yellowhorn is developed, the shinyleaf yellowhorn is deep in root taking and wide in distribution, the shinyleaf yellowhorn is an industrial raw material of wind prevention and sand fixation, small-river-area treatment and pioneering tree species, the kernel oil content is high, the fresh leaves of the shinyleaf yellowhorn can also be used for preparing tea, and the seed coats and the pericarps can be used for preparing active carbon. At present, in the actual operation of tissue culture and rapid propagation of shinyleaf yellowhorn, because the explant carries endophytes and has a complex surface structure, the concentration and time combination of a conventional disinfectant are difficult to achieve balance between thorough sterilization and tissue protection activity when the disinfection treatment is carried out, when the disinfection is incomplete or the explant is excessively damaged, the primary pollution rate is high or the induction start fails, sufficient sterile materials cannot be obtained, meanwhile, in the proliferation culture stage, the hormone proportion of the conventional culture medium is relatively fixed with the culture mode, the growth difference of different genotypes or clones in the culture process can not be dynamically adapted, the physiological abnormality of partial excellent plant proliferation coefficient, cluster bud growth weakness or vitrification appears, and an effective regulation means is lacked when the growth abnormality appears, in the rooting and transplanting stage, the problems of root development failure, root hair scarcity or long slow seedling transplanting period and large survival fluctuation rate often exist in the conventional rooting induction and seedling hardening method due to the fact that the tissue culture seedlings are in high-wet, constant temperature and heterotrophic environment exist for a long time, and the overall tissue culture efficiency and the economic benefit are further influenced. Therefore, a high-efficiency tissue culture and rapid propagation seedling method for shinyleaf yellowhorn is provided to solve the problems. Disclosure of Invention Aiming at the defects of the prior art, the invention provides a high-efficiency tissue culture and rapid propagation seedling method for shinyleaf yellowhorn, which solves the problem that the concentration and time combination of the conventional disinfectant proposed in the background art are difficult to achieve balance between thorough sterilization and tissue activity protection. In order to achieve the purpose, the invention provides the following technical scheme for realizing the high-efficiency tissue culture and rapid propagation seedling method for shinyleaf yellowhorn, which comprises the following steps: Step one, selecting and preprocessing an explant, selecting the current healthy branches of shinyleaf yellowhorn, cutting off stem segments with axillary buds as the explant, and cleaning and trimming the explant; Sterilizing the surface of the pretreated explant to remove microbial contamination; preparing a primary culture medium, namely preparing a solid culture medium serving as primary induction; inoculating and primary culturing, namely inoculating the sterilized explant to a primary culture medium, and performing primary induction culture in a controllable environment; Step five, proliferation culture, namely transferring cluster buds or adventitious buds obtained by primary culture into a proliferation culture medium for proliferation; Rooting culture, namely transferring the propagated unrooted seedlings into a rooting culture medium to induce root system formation; Hardening off and transplanting, hardening off the tissue culture seedlings with complete roots, and transplanting the tissue culture seedlings into a seedling culture matrix; In the third step, the primary culture medium is prepared from the following components of MS basic culture medium, 6-benzyladenine 0.5-2.0mg/L, naphthylacetic acid 0.1-0.5mg/L, sucrose 25-35g/L, agar 5-7g/L and pH value regulated to 5.7-5.9; In the fifth step, the proliferation culture medium is prepared from the following components of MS basic culture medium, 6-benzyladenine 1.0-3.0mg/L, naphthylacetic acid 0.1-0.5mg/L, sucrose 25-35g/L, agar 5-7