CN110192528B

CN110192528B - Method for inducing adventitious buds of cyclocarya paliurus and subculture multiplication culture

Info

Publication number: CN110192528B
Application number: CN201910591809.9A
Authority: CN
Inventors: 洑香香; 成圆; 尚旭岚; 张洋; 方升佐; 杨万霞
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2019-07-01
Filing date: 2019-07-01
Publication date: 2020-10-16
Anticipated expiration: 2039-07-01
Also published as: CN110192528A

Abstract

The invention discloses an induction and subculture multiplication method for an adventitious bud of cyclocarya paliurus, and belongs to the technical field of plant tissue culture. Selecting young twigs of cyclocarya paliurus as explants, and sterilizing the explants before inducing and value-added culturing the explants; the sterilization and disinfection treatment adopts alcohol treatment for 25-30s and sodium hypochlorite treatment for 3-4 min; the formula of the cyclocarya paliurus adventitious bud induction culture medium is as follows: WPM +6-BA + sucrose + agar powder, and adjusting pH to 5.75-5.85; the formula of the cyclocarya paliurus adventitious bud multiplication culture medium is as follows: WPM + IBA + sucrose + agar powder. According to the invention, by optimizing materials, sterilizing and disinfecting treatment and culture formulas, a large amount of adventitious bud materials with strong growth vigor, extended leaves and high growth rate can be obtained in a short time, a foundation is laid for establishing a high-efficiency cyclocarya paliurus in-vitro rapid propagation system, and meanwhile, the method has important significance for the protection, development and utilization of cyclocarya paliurus germplasm resources.

Description

Method for inducing adventitious buds of cyclocarya paliurus and subculture multiplication culture

Technical Field

The invention belongs to the technical field of plant tissue culture. In particular to a method for inducing adventitious buds of cyclocarya paliurus and carrying out subculture multiplication.

Background

Cyclocarya paliurus (Batal) Iljinskaja is a Cyclocarya plant of Juglandaceae (Jugladaceae) and widely distributed in 420-5200 m (Chinese plant lineage, 1979) of southern sea level in Yangtze river. The active substances contained in the bark and leaves of the Chinese medicinal composition have the functions of clearing away heat and toxic materials, relieving pain and diminishing inflammation (Chinese journal of Chinese medicine resources, 1994). Recent research shows that the special secondary metabolites of the compound have good pharmacological effects of reducing blood fat (WU ZF et al, 2017), reducing blood fat (Jiang et al, 2015; Ma et al, 2015), reducing blood sugar (fulvine and the like, 2017; Wufang and the like, 2014), enhancing immunity (Rankine and the like, 2016), resisting oxidation (Wang et al, 2013; Tang et al, 2016), resisting tumors (Liu Xin and the like, 2007) and the like. In addition, it can also inhibit cardiovascular diseases and diabetes complications (Xiexingyong and Xiehua, 2007).

Cyclocarya paliurus is a multifunctional economic tree which integrates medicinal, health-care, material and ornamental values, and has wide application prospect. Due to the fact that natural resources are few, the cyclocarya paliurus is scattered in a deep mountain old forest and some natural protection areas mostly at odds and ends, and the fructification rate and the plump rate of the cyclocarya paliurus in a natural state are low ( fragrance, etc., 2010), the current cyclocarya paliurus propagation mode still takes sexual propagation as a main mode, but the natural germination rate of the cyclocarya paliurus in propagation in survival is low (Xiansinghua and Xianshuan, 2008), the seed demand is large (Yanwanxia and Fangzhuo, 2008), the dormancy relieving operation is complicated (Shanghai, etc., 2011), the seeding and seedling raising germination rate is low (Yuyongfu, etc., 2017) and the like, and the natural resources are main factors for limiting the scale planting of. In order to better protect and develop and utilize the existing wild cyclocarya paliurus resources, the current hot topic is to replace the wild resources by artificial cultivation on the basis of selection of the superior trees, and the asexual propagation is not limited by time and space, can be produced annually, has high multiplication coefficient, can keep the excellent characters of the variety, and is considered to be a better method for realizing the large-scale propagation of the cyclocarya paliurus. Cyclocarya paliurus has no report on grafting, few researches on cuttage and unsatisfactory effects (Li Xian Min et al, 2014; Wang Xiaoning, 2012). The tissue culture has made a periodic breakthrough in the aspect of tissue culture, and a large number of adventitious bud induction and subculture multiplication formulas and culture formulas for callus induction and subculture multiplication (Zhang Jinfeng, etc., 2012; Raney's bracelet, etc., 2014; Zhang wenquan, etc., 2018) exist at present, the adventitious bud induction rate can reach more than 90%, the multiplication coefficient can reach as high as 7.33, and the callus induction rate and the differentiation rate can respectively reach more than 85% and 66.3%. However, the existing formula generally has the problems of poor repeatability, poor reported adventitious bud and callus growth state, low callus adventitious bud differentiation rate and the like.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to overcome the defects of the prior art and provides a cyclocarya paliurus adventitious bud induction and subculture multiplication culture method, which can obtain a large number of healthy and disease-free cyclocarya paliurus leaves growing strongly in a short time, provides an effective way for preserving cyclocarya paliurus germplasm resources and lays a foundation for the subsequent industrialized extraction of secondary metabolites such as cyclocarya paliurus triterpenes, flavones and polysaccharides.

The technical scheme is as follows: in order to solve the problems, the technical scheme adopted by the invention is as follows:

a cyclocarya paliurus adventitious bud induction and subculture multiplication culture method comprises the steps of selecting young and tender branches of cyclocarya paliurus as explants, and performing sterilization and disinfection treatment on the explants before performing induction and multiplication culture on the explants: the sterilization and disinfection treatment adopts alcohol treatment for 25-30s and sodium hypochlorite treatment for 3-4 min; the formula of the cyclocarya paliurus adventitious bud induction culture medium is as follows: WPM +6-BA + sucrose + agar powder, and adjusting pH to 5.75-5.85, wherein the concentration of 6-BA is 4.0-6.0 mg.L^-1The concentration of sucrose is 25000-^-1The concentration of the agar powder is 6000-6500 mg.L^-1(ii) a The formula of the cyclocarya paliurus adventitious bud multiplication culture medium is as follows: WPM + IBA + sucrose + agar powder, and adjusting pH to 5.75-5.85, wherein IBA concentration is 1.5-2.0 mg.L^-1The concentration of sucrose is 25000-^-1The concentration of the agar powder is 6000-6500 mg.L^-1. The method specifically comprises the following steps:

(1) selection of explants: sampling for 4-9 months, selecting a 3-4cm long tender branch which is disease-free and strong in growth and has 1-2 axillary bud points and grows in the current year from the three-year-old stumped seedlings of cyclocarya paliurus, and pretreating an explant; the specific method for explant selection comprises the following steps: cutting 3-4cm long semi-lignified stem with buds from 1 and 2 nodes of the top end, and reserving 1-2 axillary bud points; the axillary buds are required to be enlarged but the bud scales are not opened. The environment condition of a greenhouse is preferably adopted, because the greenhouse environment is easy to control, plant diseases and insect pests are easy to manage, and the pollution rate of explants can be reduced from the source; the stem section material selected by the invention has good state, is not easy to pollute and has good formula reaction effect.

(2) And (3) sterilizing and disinfecting explants: putting the cyclocarya paliurus explant on an ultra-clean workbench for sterilization and disinfection, wherein the sterilization and disinfection treatment adopts alcohol and sodium hypochlorite, the alcohol treatment lasts for 25-30s, and the sodium hypochlorite treatment lasts for 3-4 min;

(3) adventitious bud induction culture: inoculating the sterilized explant into an adventitious bud induction culture medium, and culturing for 20-25 d;

(4) and (3) adventitious bud multiplication culture: after the explant is subjected to induced culture for 20-25d, cutting healthy adventitious buds with the length of 1.4-2.1cm and vigorous growth, transferring the adventitious buds to a subculture multiplication medium for subculture multiplication for 30d, sealing the culture medium with a sealing film, and then putting the culture medium into a culture room for culture.

According to the method for inducing adventitious buds of cyclocarya paliurus and subculture proliferation, the explant is pretreated after being selected: adding 1-2 drops of detergent into running water for cleaning, adding 2-3 drops of 84 disinfectant into the detergent, soaking the detergent for 1-2min, washing the detergent for 1-2 h by the running water, and transferring the detergent to a clean bench for sterilization and disinfection.

According to the method for inducing adventitious buds of cyclocarya paliurus and subculture multiplication, the concentration of alcohol used for sterilizing explants is 75%, the sterilizing time is 25s, and the explant is washed by sterile water for 3 times; the concentration of sodium hypochlorite is 10%, the disinfection time is 3.5min, and the solution is washed by sterile water for 5 times.

The method for inducing and subculturing the cyclocarya paliurus adventitious bud has the concentration of 6-BA in the cyclocarya paliurus adventitious bud induction culture medium of 6.0 mg.L^-1And adjusting the pH to 5.80-5.82.

The method for inducing and subculturing the cyclocarya paliurus adventitious bud, wherein the concentration of IBA in the cyclocarya paliurus adventitious bud multiplication culture medium is 1.5 mg.L^-1And adjusting the pH to 5.80-5.82.

The method for inducing and subculturing the cyclocarya paliurus adventitious bud comprises the step of adding 4.5-5.0 mg.L of cyclocarya paliurus adventitious bud proliferation culture medium^-1The rare earth lanthanum is beneficial to strengthening the seedlings.

The method for inducing the adventitious bud of the cyclocarya paliurus and carrying out subculture multiplication comprises the following steps: the temperature is 21 +/-2 ℃, the illumination time is 16h/d, and the illumination intensity is about 50 mu mol.m^-2·s^-1The relative humidity is 50% -65%, the illumination and light intensity of the environment simulate the illumination condition of cyclocarya paliurus under natural conditions, the environment temperature of 21 +/-2 ℃ is suitable for the growth of tissue culture materials of cyclocarya paliurus, and meanwhile, the tissue culture materials can be inhibited from growingBacteria in the breeding room breed.

The method for inducing adventitious buds of cyclocarya paliurus and subculture proliferation comprises the steps of spreading the sterilized and disinfected cyclocarya paliurus explants on sterilized and disinfected filter paper, airing the filter paper, and then inoculating the filter paper. The explant body surface after disinfection has moisture, arranges on disinfected filter paper and blots up and dries in the air, can detach the surperficial moisture of stem segment, prevents that the bacterium in the air and the operation brought from adsorbing on the stem segment surface, influences experimental success rate.

The method for inducing the adventitious bud of the cyclocarya paliurus and carrying out subculture multiplication culture comprises the following steps: removing the part of the morphological lower end of the stem segment, which is oxidized by the disinfectant, by using a sterilized scalpel, so as to avoid that the disinfectant is not completely washed; the cut is an inclined plane, so that the contact area between the cut surface and the culture medium can be increased, and the nutrient absorption is facilitated. When the trimmed stem segments are horizontally and vertically connected into a culture medium which is sterilized at high temperature, 1/5-1/4 of the depth of the inserted culture medium accounting for the length of the stem segments is inserted, 1 stem segment is connected into each bottle of culture medium, the insertion depth of the stem segments is not too deep, the air permeability is poor if the stem segments are too deep, and the stem segments are easy to die; if the stem is too shallow, the nutrient components in the culture are not easily absorbed.

Has the advantages that: compared with the prior art, the invention has the advantages that:

(1) the method optimizes the operation method of the cyclocarya paliurus tissue culture in each stage, effectively reduces the pollution rate, improves the proliferation rate, and obtains a large amount of healthy and vigorously growing adventitious buds. The treatment combination of 75% alcohol and 10% sodium hypochlorite can kill microorganisms attached to the surface of the cyclocarya paliurus explant, and the material pollution rate and the death rate are reduced.

(2) The adventitious bud induction formula adopted by the invention ensures that the stem segment has the juvenile state characteristic in the growth and germination period, the growth is vigorous, the bacteria is less, the adventitious bud induction rate is higher, and the tissue is not easy to necrotize.

(3) The adventitious bud multiplication formula adopted by the invention better meets the requirement of rapid multiplication of the adventitious buds of cyclocarya paliurus, the seedling height can reach 5.3cm, the vitrification rate is 0, the multiplication coefficient of cluster buds is 2.4, the cyclocarya paliurus grows robustly, and the cyclocarya paliurus can grow at high speed.

(4) By adopting the disinfection combination, the minimal medium and the hormone combination, the adventitious buds with high multiplication coefficient and good growth state can be obtained, and the number of the adventitious buds obtained by the formula can be stably repeated.

(5) The method obtains a large amount of healthy and disease-free cyclocarya paliurus leaves in a short period, has special theoretical and practical significance for realizing industrialized extraction of secondary metabolites, and provides an effective way for saving wild germplasm resources of cyclocarya paliurus.

(6) The method for inducing and proliferating the adventitious buds of cyclocarya paliurus simplifies the operation steps, reduces the vitrification rate, improves the adventitious bud induction rate, can obtain a large amount of strong, disease-free and vigorous materials in a short period, can realize large-scale production, and can effectively solve the contradiction between the requirement of industrial extraction of secondary metabolites of cyclocarya paliurus on raw materials and the shortage of wild germplasm resources.

Drawings

FIG. 1 is a diagram showing the result of screening a minimal medium, wherein FIG. 1A is a diagram showing the state of axillary buds at 3D of inoculation, FIG. 1B is a diagram showing the state of axillary buds at 7D of inoculation, FIG. 1C is a diagram showing the state of axillary buds at 15D of inoculation, and FIG. 1D is a diagram showing the axillary buds induced by an MS medium after 35D of inoculation; FIG. 1E shows axillary buds induced by WPM medium after 35d inoculation, FIG. 1F shows axillary buds induced by B5 medium after 35d inoculation, FIG. 1G shows axillary buds induced by DKW medium after 35d inoculation, and FIG. 1H shows axillary buds induced by 1/2MS medium after 35d inoculation;

FIG. 2 shows the effect of different concentrations of 6-BA on the induction of adventitious buds of cyclocarya paliurus (cultured for 15 d): wherein FIG. 2A is 2.0 mg.L^-1Adventitious bud induced under 6-BA, FIG. 2B is 4.0 mg. L^-1Adventitious bud induced under 6-BA, FIG. 2C is 6.0 mg. L^-1Adventitious bud induced under 6-BA, FIG. 2D is 8.0 mg. L^-1Adventitious bud induced under 6-BA, FIG. 2E is 10.0 mg. L^-1Adventitious bud induced under 6-BA, FIG. 2F-12.0 mg. L^-1Adventitious buds induced under 6-BA;

FIG. 3 is a graph showing the effect of culturing the proliferation of adventitious buds of cyclocarya paliurus with a 6-BA + GA3 hormone combination, in which FIGS. 3A and 3B: stem segment, leaf vitrification, partial elongation growth, fig. 3C: adventitious buds inoculated in WPM minimal medium have thin and weak stem segments and inhibited basal callus, and the growth is slow, the stem segments are thin and weak, leaves are narrow and shriveled, 3F: long and narrow leaf, partly detached and dead, fig. 3G and fig. 3H: the adventitious bud is small, only leaves are grown, the growth is slow, and abnormal seedlings are grown;

FIG. 4 shows the effect of culturing the adventitious bud growth of cyclocarya paliurus by IBA, FIGS. 4A and 4B show the effect of culturing for inducing adventitious buds for 25D, FIGS. 4C, 4D, 4E and 4F show the effect of culturing for adventitious buds for 30D, and FIGS. 4G, 4H, 4I and 4J show the adventitious buds for rooting.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.

Example 1

Two disinfectants of alcohol and sodium hypochlorite are selected to study the disinfection treatment of cyclocarya paliurus, wherein the disinfection time of 75% alcohol is as follows: 25s and 30 s. 10% sodium hypochlorite treatment time: 3min, 3.5min and 4 min. The experimental design is detailed in table 1. The stem segments are trimmed and washed, then placed on a superclean bench, further disinfected by ethanol and sodium hypochlorite, and washed by sterile water for 4-5 times after disinfection. Cutting off the part oxidized by the disinfectant on the sterilized stem segment with buds on the sterilized filter paper, placing the stem segment in a culture medium, observing the pollution condition of the stem segment after inoculation after 10 days, observing the pollution characteristics of the explant, further judging the cause of pollution, and counting the pollution rate and the death rate, wherein the results are shown in table 2.

As can be seen from Table 2, the difference between different combinations of treatments was significant in the case of contamination and death of explants under different treatment conditions. The contamination and mortality of treatment 2 was lowest at 4-7 months of sampling time, and the contamination of treatment 2 was lowest at only 13% at 8-9 months of sampling time, but the mortality was slightly higher than that of treatment 1. Therefore, considering the comprehensive consideration of the contamination rate, death rate and sampling time of explants, the treatment 2 is considered to be the optimal disinfection treatment scheme of cyclocarya paliurus stem segments with buds.

Therefore, the optimal disinfection combination of cyclocarya paliurus stem with buds is selected to be disinfected by 75% alcohol for 25s + 10% sodium hypochlorite for 3.5min, wherein the pollution rate of the explants is 11% -13%, and the death rate is 2% -6%.

TABLE 1 Effect of Disinfection combinations on shoot Germination

Disinfection combination	Alcohol treatment time(s)	Sodium hypochlorite treatment time (min)
			1	25	3
2	25	3.5
			3	25	4
4	30	3
			5	30	3.5
6	30	4

TABLE 2 Effect of Disinfection combination and sampling time on shoot Germination

Example 2

In order to study the influence of the basic culture medium on the induction and proliferation growth of the adventitious bud of cyclocarya paliurus, 5 basic culture media are set in the experiment: MS, WPM, B5, DKW, 1/2MS (except for macroelements and CaCl)₂1/2 of total amount, all others are total amount) the experiment is carried out by adopting a single-factor random block experiment, putting the disinfected cyclocarya paliurus stem segments on disinfected filter paper (11 × 11cm) to absorb water, removing the oxidized parts of the head and the tail ends of the stem segments and the leaf stalks of leaf axils by using disinfected scissors, inoculating the trimmed stem segments into a culture medium, and adding the same hormone combination into the basic culture medium respectively, wherein the pruning method comprises the steps of removing the part oxidized by the disinfectant at the lower end of the stem segments morphologically by using a disinfected scalpel, increasing the contact area between a tangent plane and the culture medium by using a cut as a slope, when the trimmed stem segments are horizontally and vertically inoculated into the high-temperature disinfected culture medium, the insertion depth of the stem segments is about 1/5-1/4 of the length of the stem segments, 1 stem segment is inoculated into each bottle of the culture medium, 20 bottles are inoculated for one time, and the repeated 3 times, in the process, the environmental conditions in a culture room are controlled, and the contaminated stem segments are cleaned in time, and the adventitious bud induction rate, the germination induction rate and the growth condition are counted after 30d culture.

TABLE 3 Effect of basal Medium on shoot Germination

Kind of culture Medium	Callus rate (%)	Germination Rate (%)	Growth state
				MS	16.8±1.8ab	85.6±2.2a	Good axillary bud growth, light green leaf, leaf extension
WPM	8.3±2.5b	93.0±2.8ab	Strong axillary bud growth and light green leaf
				B5	9.8±4.9b	88.7±3.5ab	Strong axillary bud growth and dark green leaf
DKW	23.3±3.3a	82.0±1.0b	Good growth of axillary buds and light green leaves
				1/2MS	13.5±3.8ab	63.4±1.9c	Axillary buds grow normally, the stems are thin and weak, and the leaves are yellow

Note: the. + -. front is the mean value followed by the standard error, and the same lower case letters in the figure indicate that there is no significant difference at a level of p < 0.05 between treatments under the same index, as follows.

As can be seen from Table 3, the axillary buds of different culture media in the invention have different growth states, and the callus rate and the germination rate have obvious difference. From the callus induction rate, the cyclocarya paliurus stem segments have callus on the base parts in 5 basic culture media, the callus quantity is small, the callus induction rate of the DKW culture medium is the highest and reaches 23.3%, and the callus induction rate of the WPM culture medium is the lowest and is 8.3%. From the germination rate of the axillary buds, the germination rate of the axillary buds of the rest culture media is higher and reaches 82-93 percent except that 1/2MS culture media are only 63.4 percent, and the highest germination rate is WPM culture media. From the growth state, the axillary buds start to sprout after the stem segments of the cyclocarya paliurus are inoculated for 3D (figure 1A), the axillary buds grow after 7D (figure 1B), the leaves stretch after 15D (figure 1C), and the growth state is generally good in 4 kinds of basic culture media except 1/2MS, wherein the axillary buds grow robustly in WPM (figure 1E) and B5 (figure 1F) culture media, the leaves are light green or dark green, the leaves grow well in MS (figure 1D) and DKW (figure 1G), the leaves stretch, and the axillary buds can grow normally, and in 1/2MS culture media, the stem segments are thin and weak, the leaves are small and yellow, and the growth state is general (figure 1H).

The callus induction rate, the germination rate and the growth state are comprehensively considered, and the WPM basic culture medium is considered to be most suitable for being used as a basic culture medium for inducing and proliferating the adventitious buds of cyclocarya paliurus.

Example 3

In order to induce the stem segments of cyclocarya paliurus to differentiate into primary adventitious buds, the experiment adds plant growth regulator 6-BA in a WPM basic culture medium, mainly researches the influence of the concentration of 6-BA on the induction of the adventitious buds of cyclocarya paliurus, and the concentration treatment combination is shown in the following table 4. The explant selects stem segments which are sterilized, disinfected and trimmed in the previous stage, is vertically inserted into an adventitious bud induction culture medium, the insertion depth is about 1/4 of the length of the stem segments, each bottle of culture medium is inserted into 1 stem segment, 20 bottles of culture medium are inoculated once and repeated for 3 times, and in the process, the environmental conditions in a culture room are controlled, and polluted materials and culture medium are cleaned in time. After culturing for 20-25 days, counting the induction condition of adventitious buds, and calculating the induction rate of cluster buds, the number of new buds and the seedling height. (Cluster shoot induction (%). the number of explants which produced cluster shoots/the number of explants which were not contaminated after inoculation. times.100%)

TABLE 4 influence of hormone combinations on the Induction of primary adventitious buds of cyclocarya paliurus

As can be seen from Table 4 above, cluster buds are induced on all the treated culture media, the adventitious bud induction rate is over 85%, but the induction rate, the number, the seedling height and the growth condition of the cluster buds induced by different treatments are obviously different. From the induction rate, the induction rate of the multiple shoots of the stem section tended to increase and then decrease with the increase of the concentration of 6-BA, and was 6.0 mg.L^-1The highest content is 97.7 percent and is 2.0-12.0 mg.L^-1Within the concentration range, the inductivity of the primary cluster buds is between 88.6 percent and 97.7 percent, and no obvious difference exists between the primary cluster buds (p is more than 0.05); from the number of new shoots, 6.0 mg.L^-1And 12.0 mg. L^-1At the concentration of 6-BA, the number of new buds is obviously higher than other concentrations, namely 5.3 buds and 4.5 buds respectively; from the aspect of seedling height, 4.0mg L of the seed crystal^-1At the concentration, the height of the seedlings reaches 1.9cm, which is remarkably higher than that of other treatments, but part of the buds show a shrivelled state of leaves, 6.0 mg.L^-1、8.0mg·L^-1And 10.0 mg. L^-1The seedling height difference is small under the concentration of 6-BA, and is 1.4-1.5 cm; from the growth state, the concentration is 2.0 mg.L^-1At 6-BA concentration, the bud was small, light green, and the leaf color was yellowish (FIG. 2A), and 4.0 mg.L^-1At 6-BA concentration, the bud is light green, the leaf color is light green (FIG. 2B), 6.0 mg. L^-16-BA, pale green bud, extended leaf, vigorous growth (FIG. 2C), 8.0 mg. L^-1And 10.0 mg. L^-1The buds were light green and the leaves were light green at 6-BA concentration, and they grew normally (FIGS. 2D and 2E). Comprehensively considering the number of cluster buds induced by stem segments, the height of seedlings, the growth condition and the like, the formula of the adventitious bud induction culture medium is selected to be WPM +6.0 mg.L^-16-BA, the stem section under the formula induces the largest number of adventitious buds, the growth is vigorous, and the leaves are extended and can grow normally. Further, the concentration of 6-BA was 12.0 mg.L^-1When the number of the cluster buds is higher than 6.0 mg.L, the induction rate of the cluster buds, the number of new buds and the height of seedlings are slightly lower than^-1The color of the leaves is normal, the leaves are extended but slight vitrification occurs (figure 2F), the subculture time can be considered to be shortened, the vitrification phenomenon can be reduced, and the formula can be considered.

Example 4

Proliferation and subculture: after a period of culture, selecting healthy and strong adventitious buds with normal color, separating the buds by using a scalpel, transferring the buds into a subculture multiplication medium (the formula of the subculture multiplication medium is shown in table 5), mainly researching the influence of the concentrations of growth regulators 6-BA, IBA and GA3 on the proliferation of the adventitious buds (taking WPM as a basic medium), inoculating 1 material into each bottle, sealing a sealing film, placing the bottles into a culture chamber for culture, inoculating 20 bottles once every 35d of subculture, repeating for 3 times, and counting the seedling height, the vitrification rate, the multiplication coefficient and the growth state. TABLE 5 Effect of subculture multiplication formula on multiplication of adventitious bud of cyclocarya paliurus

As can be seen from Table 5, the growth regulating substances GA3, IBA and 6-BA all have a certain promoting effect on the proliferation culture of adventitious buds, wherein, in the hormone combination of 6-BA and GA3, the growth vigor of the adventitious buds is more common or worse, the stem segments are weak and easy to generate callus inhibition, and abnormal seedlings (figure 3A-3H) can appear, the proliferation coefficient of the cluster buds is increased along with the increase of the concentration of 6-BA, and is 2.0 mg.L^-16-BA and 0.1 mg. L^-1The combination of GA3 gave a maximum growth factor of 3.2, but the vitrification ratio increased with increasing hormone concentration. In the formulation with only IBA hormone, the seedling height and the multiplication coefficient generally show the tendency of increasing firstly and then decreasing with the increase of the IBA concentration, and are 1.5 mg.L^-1The highest growth rate is achieved, and the growth condition is better. Overall, WPM +1.5 mg-L^-1Compared with other proliferation culture formulas, the IBA formula has obvious advantages that the height of adventitious buds can reach 5.3cm on average and is obviously higher than that of other formulas, no vitrification phenomenon occurs, the proliferation coefficient of cluster buds is also obviously higher than that of other formulas and can reach about 2.4cm, the adventitious buds obtained in the formula grow vigorously and can grow highly (figures 4C, 4D, 4E and 4F), part of adventitious buds grow vigorously, and adventitious buds with thicker stem segments can be used for further rooting culture (figures 4I, 4J and 4K). The experiment shows that the optimal successive transfer multiplication formula of the cyclocarya paliurus adventitious bud is WPM +1.5 mg.L^-1IBA. In addition, 4.5-5.0 mg.L is added into the formula^-1The rare earth lanthanum may be advantageousAnd (5) strengthening seedlings.

The culture mediums of examples 1, 2, 3 and 4 were added at 3000-35000 mg.L^-1Sucrose and 6000-6500 mg.L^-1The pH value of the agar powder is adjusted to be within the range of 5.80-5.82, the culture condition is that the illumination time of a culture room is 16h/d, and the illumination intensity is about 50 mu mol.m^-2·s^-1The relative humidity is 50-65%, and the temperature is 21 +/-2 ℃.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and the protection scope of the present invention should be regarded as follows.

Claims

1. A cyclocarya paliurus adventitious bud induction and subculture multiplication culture method is characterized in that young and tender branches of cyclocarya paliurus are selected as explants, the sampling time of the explants is 4-9 months, and the explants are sterilized and disinfected before induction and multiplication culture of the explants; the sterilization and disinfection treatment adopts alcohol treatment for 25-30s and sodium hypochlorite treatment for 3-4 min; the formula of the cyclocarya paliurus adventitious bud induction culture medium is as follows: WPM +6-BA + sucrose + agar powder, and adjusting pH to 5.75-5.85, wherein the concentration of 6-BA is 4.0-6.0 mg.L^-1The concentration of sucrose is 25000-^-1The concentration of the agar powder is 6000-6500 mg.L^-1(ii) a The formula of the cyclocarya paliurus adventitious bud multiplication culture medium is as follows: WPM + IBA + sucrose + agar powder, and adjusting pH to 5.75-5.85, wherein IBA concentration is 1.5-2.0 mg.L^-1The concentration of sucrose is 25000-^-1The concentration of the agar powder is 6000-6500 mg.L^-1(ii) a The environmental conditions of the culture chamber are as follows: the temperature is 21 +/-2 ℃, the illumination time is 16h/d, and the illumination intensity is about 50 mu mol.m^-2•s^-1The relative humidity is 50% -65%; adding the concentration of 4.5-5.0 mg.L into the formula of the cyclocarya paliurus adventitious bud multiplication culture medium^-1The rare earth lanthanum of (1).

2. The method for inducing adventitious buds and carrying out subculture proliferation according to claim 1, which comprises the following steps:

(1) selecting a 3-4cm long tender branch which is disease-free and strong in growth and has 1-2 axillary bud points, from a three-year-old stumped seedling of cyclocarya paliurus, and pretreating the explant;

(2) placing the explant on a superclean workbench for sterilization and disinfection, and treating with alcohol for 25-30s and sodium hypochlorite for 3-4 min;

(3) inoculating the sterilized and disinfected explant into an adventitious bud induction culture medium, and culturing for 20-25 d;

(4) and after the explant is subjected to induced culture for 20-25d, cutting healthy adventitious buds with vigorous growth by 1.4-2.1cm, transferring the adventitious buds to an adventitious bud multiplication culture medium for subculture multiplication culture for 30 d.

3. The method for inducing adventitious buds and carrying out subculture proliferation of cyclocarya paliurus according to claim 1 or 2, wherein explant selection is followed by pretreatment: adding 1-2 drops of detergent into running water for cleaning, adding 2-3 drops of 84 disinfectant into the cleaning solution, soaking for 1-2min, washing for 1-2 h with the running water, and transferring the cleaning solution to a clean bench for sterilization and disinfection.

4. The method for inducing adventitious buds and carrying out subculture proliferation on cyclocarya paliurus according to claim 1 or 2, wherein the alcohol concentration for explant disinfection is 75%, the disinfection time is 25s, and the explant is washed with sterile water for 3 times; the concentration of sodium hypochlorite is 10%, the disinfection time is 3.5min, and the sterile water is washed for 5 times.

5. The method for inducing adventitious bud and subculture proliferation of cyclocarya paliurus according to claim 1 or 2, wherein the concentration of 6-BA in the formula of the culture medium for inducing adventitious bud of cyclocarya paliurus is 6.0 mg.L^-1And adjusting the pH value of the adventitious bud induction culture medium to 5.80-5.82.

6. The method for inducing and subculturing an adventitious bud of cyclocarya paliurus according to claim 1 or 2, wherein the concentration of IBA in the formula of the propagation medium for adventitious bud of cyclocarya paliurus is 1.5 mg.L^-1And is variably adjustedThe pH value of the bud multiplication culture medium is 5.80-5.82.

7. The method for inducing adventitious buds and carrying out subculture proliferation of cyclocarya paliurus according to claim 1 or 2, wherein the sterilized cyclocarya paliurus explants are spread on sterilized filter paper, dried in the air and then inoculated.

8. The method for inducing adventitious buds and carrying out subculture proliferation on cyclocarya paliurus according to claim 1 or 2, wherein the pruning method of the cyclocarya paliurus explants is as follows: removing the part of the morphological lower end of the stem segment oxidized by the disinfectant by using a sterilized scalpel; the contact area between the section and the culture medium can be increased by the cut being an inclined plane; and when the trimmed stem segments are horizontally and vertically inoculated into a culture medium which is sterilized at high temperature, 1/5-1/4 of the length of the stem segments is inserted into the culture medium.