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CN-121970684-A - Leaf tissue culture and rapid propagation method for shinyleaf yellowhorn

CN121970684ACN 121970684 ACN121970684 ACN 121970684ACN-121970684-A

Abstract

The invention provides a leaf tissue culture and rapid propagation method of shinyleaf yellowhorn, which comprises the steps of leaf explant preparation, induced differentiation culture, proliferation culture, rooting culture, seedling hardening and transplanting. In the invention, shinyleaf yellowhorn leaf is used as an explant, adventitious buds are directly induced, the callus induction stage is not needed, the rapid propagation efficiency is improved, and the seedling speed is high. And can be used for gene transformation by agrobacterium mediation or other methods based on the invention.

Inventors

  • WU DAN
  • ZHAO YONGJUN
  • LU LU
  • LIU KUN
  • HAN QINGJUN
  • LI NINGNING
  • HAN YI
  • YANG HAIPING
  • ZHUANG ZHENJIE

Assignees

  • 山东省林草种质资源中心(山东省药乡林场)

Dates

Publication Date
20260505
Application Date
20260328

Claims (5)

  1. 1. A method for tissue culture and rapid propagation of shinyleaf yellowhorn leaves is characterized by comprising the following steps: s1, preparing a leaf explant: selecting healthy leaves of shinyleaf yellowhorn without plant diseases and insect pests, cutting the complex leaves into single leaves, brushing the surfaces of the single leaves by using a brush, placing the single leaves on an ultra-clean workbench, treating the single leaves for 30s by using an ethanol water solution with the volume fraction of 75%, then washing the single leaves by using sterile water for 2 times, treating the single leaves by using a sodium dichloroisocyanate water solution with the mass fraction of 0.5% for 5min, and then washing the single leaves by using sterile water for 5 times to obtain a sterilized explant; s2, induced differentiation culture: Placing the sterilized explant obtained in the step S1 on an inoculation tray, drawing out wounds at positions where the leaf backs of the explant are perpendicular to main veins by using a blade, inoculating 1-3 to each bottle of the sterilized explant in an induced differentiation culture medium with the leaf backs facing downwards, and carrying out induced differentiation culture for 25-28 days, wherein adventitious buds are generated at leaf stems, leaf veins and wounds to obtain the induced differentiated explant; The induced differentiation culture medium is prepared by adding the following raw materials with the final concentration into an improved DKW culture medium, wherein the raw materials comprise 0.5mg/L of 6 benzylaminoadenine, 1.0 mg/L of indolebutyric acid and 0.05mg/L of indoleacetic acid; The improved DKW culture medium is prepared by adding raw materials with the following final concentration into distilled water and adjusting the pH value to 5.8-6.0, wherein the DKW culture medium comprises 3.81g/L of dry powder, 5mL/L of calcium salt solution, 1g/L of ammonium nitrate, 30g/L of sucrose and 6.5g/L of agar; S3, proliferation culture: transferring the induced and differentiated explant obtained in the step S2 with the leaf back surface upwards to a proliferation culture medium, inoculating 2 adventitious buds per bottle for proliferation culture to obtain clustered adventitious buds, or cutting off leaves from adventitious buds with the height of more than 1.5cm in the adventitious buds obtained in the step S2 along stems, cutting into stem sections with the length of 1.5cm, transferring to the proliferation culture medium, inoculating 2 adventitious buds per bottle for proliferation culture to obtain clustered adventitious buds; The proliferation culture medium is prepared by adding the following raw materials with the final concentration of 0.75mg/L of 6 benzylaminoadenine and 0.75mg/L of indolebutyric acid into a DKW culture medium; The DKW culture medium is prepared by adding raw materials with the following final concentration into distilled water and adjusting the pH value to 5.8-6.0, wherein the DKW culture medium comprises 3.81g/L of dry powder, 5mL/L of calcium salt solution, 30g/L of sucrose and 6.5g/L of agar; S4, rooting culture: Cutting off the leaves along the stems of the adventitious buds with the height of more than 1.5cm of the leaves which are obtained in the step S2 and are induced to differentiate, cutting into stem sections with the length of 1.5cm, transferring the stem sections into rooting culture medium, inoculating 2 of the stem sections into each bottle, and carrying out rooting culture to obtain shinyleaf yellowhorn rooting seedlings; Or cutting off leaves from adventitious buds with the height of more than 1.5cm along stems, cutting into stem sections with the length of 1.5cm, transferring into rooting culture medium, inoculating 2 adventitious buds per bottle, and rooting culture to obtain shinyleaf yellowhorn rooting bottle seedlings; The rooting culture medium is prepared by adding raw materials with the following final concentration into a DKW basic culture medium, wherein the raw materials comprise 0.5mg/L of indolebutyric acid, 0.2mg/L of naphthylacetic acid and 30 g/L of sucrose; the DKW basic culture medium is prepared by adding raw materials with the following final concentration into distilled water and adjusting the pH value to 5.8-6.0, wherein the DKW basic culture medium comprises 3.81g/L of dry powder, 5mL/L of calcium salt solution and 6.5g/L of agar; S5, hardening seedlings and transplanting: And (3) transferring the shinyleaf yellowhorn rooting bottle seedlings obtained in the step (S4) to a greenhouse for hardening seedlings with the bottles, adapting to 3d under natural conditions at the temperature of 25-28 ℃ and the humidity of 80%, uncovering to adapt to 3d, cleaning a culture medium on a root system, transplanting the culture medium into a matrix in a plug tray, growing new leaves, and continuously culturing the culture medium in the greenhouse for 30d under the conditions of the temperature of 25-35 ℃ and the humidity of 35-80%, and transplanting the culture medium into a field.
  2. 2. The method for quickly propagating shinyleaf yellowhorn leaf tissue according to claim 1, wherein the induced differentiation culture time in S2 is 25-28 d, the culture conditions are that dark culture is performed for 7d, the dark culture conditions are that 25 ℃ and 14h are alternately performed, 18 ℃ and 10h are alternately performed, then illumination-dark alternate culture is performed, the illumination condition is that the illumination time is 14h, the illumination intensity is 3000lx, the temperature is 25 ℃, the dark condition is that the dark time is 10h, and the temperature is 18 ℃.
  3. 3. The method for quickly propagating shinyleaf yellowhorn leaf tissue according to claim 1, wherein the proliferation culture time in S3 is 21-25 d, the illumination-darkness alternate culture is carried out under the condition that the illumination time is 14h, the illumination intensity is 3000lx and the temperature is 25 ℃, and the darkness condition is that the darkness time is 10h and the temperature is 18 ℃.
  4. 4. The method for quickly propagating shinyleaf yellowhorn leaf tissue according to claim 1, wherein the rooting culture time in S4 is 25-28 d, the culturing condition is that dark culture is performed for 7d, the dark culture condition is that 25 ℃ and 14h are alternately performed, and 18 ℃ and 10h are alternately performed, then illumination-dark culture is performed, the illumination condition is that the illumination time is 14h, the illumination intensity is 3000lx, the temperature is 25 ℃, the dark condition is that the dark time is 10h, and the temperature is 18 ℃.
  5. 5. The method for rapid propagation of shinyleaf yellowhorn leaf tissue culture according to claim 1, wherein the matrix in S5 is a mixture of turf, vermiculite and perlite in a mass ratio of 1:1:1.

Description

Leaf tissue culture and rapid propagation method for shinyleaf yellowhorn Technical Field The invention belongs to the technical field of tissue culture, and particularly relates to a method for quickly propagating shinyleaf yellowhorn by leaf tissue culture. Background The xanthoceras sorbifolia (Xanthoceras sorbifolium Bunge) is deciduous shrubs or small trees of xanthoceras genus of Sapindaceae family. The wild resource distribution area is mainly loess plateau of warm zone and warm zone climate area, and the wild resource diversity of the shinyleaf yellowhorn is higher. In order to ensure consistent varieties, the shinyleaf yellowhorn is mainly subjected to propagation production by means of grafting, cutting and the like, and the technical means are limited by shortage of parent materials, so that the operation is complicated, the propagation efficiency of the shinyleaf yellowhorn is low, and the social demand problems of shortage of shinyleaf yellowhorn seedlings, rapid variety propagation and the like can not be solved. Therefore, establishing a shinyleaf yellowhorn efficient propagation technology system is a core problem to be solved in order to rapidly improve the yield of shinyleaf yellowhorn seedlings. Disclosure of Invention The invention aims to solve the technical problems of the prior art and provides a method for tissue culture and rapid propagation of shinyleaf yellowhorn leaves, in the method, the shinyleaf yellowhorn leaf can be directly subdivided into adventitious buds, one step is saved compared with the leaf differentiated into callus, and the callus differentiated into the adventitious buds, so that the rapid propagation efficiency is improved. And can be used for gene transformation by agrobacterium mediation or other methods based on the invention. In order to solve the technical problems, the invention adopts the technical scheme that the method for tissue culture and rapid propagation of the shinyleaf yellowhorn leaves comprises the following steps: s1, preparing a leaf explant: Selecting young leaves (healthy leaves without plant diseases and insect pests) sprouting in shiny-leaved yellowhorn fields in 4 months, cutting the compound leaves into single leaves, brushing the surfaces of the single leaves with a brush, placing the single leaves on an ultra-clean workbench, treating the single leaves with an ethanol water solution with the volume fraction of 75 percent for 30s, washing the single leaves with sterile water for 2 times, treating the single leaves with a sodium dichloroisocyanate water solution with the mass fraction of 0.5 percent for 5min, and washing the single leaves with sterile water for 5 times to obtain the sterilized explants; s2, induced differentiation culture: placing the sterilized explant obtained in the step S1 on an inoculation tray, drawing a wound at a position where the back surface of the explant is vertical to a main vein by using a blade, inoculating 1-3 to 1-28 of the sterilized explant into an induced differentiation culture medium with the back surface facing downwards, and carrying out induced differentiation culture for 25-28 d, wherein a large number of adventitious buds are generated at petioles, veins and wound positions, and the length of part of the adventitious buds can reach more than 1.5cm, so that the induced differentiated explant is obtained; the induced differentiation culture medium is prepared by adding the following raw materials with the final concentration into an improved DKW culture medium, wherein the raw materials comprise 0.5mg/L of 6 benzylaminoadenine, 1.0 mg/L of indolebutyric acid and 0.05mg/L of indoleacetic acid (IAA); The improved DKW culture medium is prepared by adding raw materials with the following final concentration into distilled water and adjusting the pH value to 5.8-6.0, wherein the DKW culture medium comprises 3.81g/L of dry powder, 5mL/L of calcium salt solution, 1g/L of ammonium nitrate, 30g/L of sucrose and 6.5g/L of agar; S3, proliferation culture: transferring the induced and differentiated explant obtained in the step S2 with the leaf back surface upwards to a proliferation culture medium, inoculating 2 adventitious buds per bottle for proliferation culture to obtain clustered adventitious buds, or cutting off leaves from adventitious buds with the height of more than 1.5cm in the adventitious buds obtained in the step S2 along stems, cutting into stem sections with the length of 1.5cm, transferring to the proliferation culture medium, inoculating 2 adventitious buds per bottle for proliferation culture to obtain clustered adventitious buds; The proliferation culture medium is prepared by adding the following raw materials with the final concentration of 0.75mg/L of 6 benzylaminoadenine and 0.75mg/L of indolebutyric acid into a DKW culture medium; The DKW culture medium is prepared by adding raw materials with the following final concentration into distilled water and adjusting the pH value to 5.