Adventitious bud culture medium, rooting culture medium, culture method and tissue culture rapid propagation method for culturing Xanthoceras sorbifolia 'Zhongshi No.4'

Abstract

The invention belongs to the technical field of tissue culture, and particularly relates to an adventitious bud culture medium, a rooting culture medium, a culture method and a tissue culture rapid propagation method for culturing Xanthoceras sorbifolia 'Zhongshi No.4'. The invention provides an adventitious bud culture medium for culturing Xanthoceras sorbifolia 'Zhongshi No.4'. The adventitious bud culture medium comprises an induction culture medium and a proliferation culture medium, under the combined action of 6-BA, NAA and GA3, adventitious bud growth of stem segments with buds of xanthoceras sorbifolia 'Zhongshi 4' is promoted, and the adventitious bud induction rate and the adventitious bud multiplication coefficient are increased. The rooting culture medium provided by the invention promotes rooting of adventitious bud seedlings of Xanthoceras sorbifolia 'Zhongshi No.4' under the action of IBA and IAA with specific concentrations. The invention further provides an adventitious bud culture method of the xanthoceras sorbifolia 'Zhongshi 4', the obtained adventitious buds are high in induction rate and high in bud propagation coefficient, and the invention further provides a tissue culture and rapid propagation method of the xanthoceras sorbifolia 'Zhongshi 4', the rooting rate of tissue culture seedlings is high, and the domestication survival rate is high.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue culture, and in particular relates to an adventitious bud culture medium, a rooting culture medium, a culture method and a tissue culture rapid propagation method for culturing Xanthoceras sorbifolia 'Zhongshi No. 4'. Background Art

[0002] Xanthocerasorbifolium (Xanthocerasorbifolium) is a deciduous shrub or small tree of the Sapindaceae family, widely distributed in northern China. It requires little maintenance and can tolerate extreme conditions, including drought, saline-alkali environments, and temperatures as low as -40°C, making it widely used in soil and water conservation. Xanthocerasorbifolium also has high medicinal and economic value. Its seed kernels contain a high oil content (up to 60%), making it a high-quality raw material for the production of functional medicines, foods, and cosmetics. It has been designated as one of the next generation of alternative biofuel crops.

[0003] In recent years, as people have become increasingly interested in Xanthoceras sorbifolia as an important woody oil plant, selecting and cultivating high-yield or high-quality seed oil germplasm resources is the key to obtaining maximum economic benefits. The oil content and fatty acid composition of oil crop seeds are important biochemical parameters affecting the quality of oil products.

[0004] Xanthoceras sorbifolia 'Zhongshi No. 4' is a new cultivar selected from the Xanthoceras sorbifolia seedling population at the Zhangwu County Germplasm Resource Nursery in Liaoning Province. It is a strong, fast-growing, high-yielding tree with strong stress resistance. The branches of this tree are hairy in its first year, and its leaves are waxy, shiny, and not curled. It has numerous inflorescences, a large proportion of female flowers, over 60% of which are female, and hairy pedicels. The calyx is yellow-green with slight hairs. The capsule is medium-sized, spherical, with three lobes and a medium-thick shell. The seeds are dark brown, weighing approximately 1.5g each. The kernel contains 65.65% oil, 91.67% unsaturated fatty acids, and 3.51% nervonic acid.

[0005] After years of identification and evaluation, the characteristics of Xanthoceras sorbifolia 'Zhongshi No. 4' are stable and consistent. In June 2018, it obtained the Plant Variety Rights Certificate issued by the State Forestry and Grassland Administration.

[0006] There are several varieties of Xanthoceras sorbifolia, and traditional propagation methods primarily rely on seeds or stem cuttings. However, Xanthoceras sorbifolia is a cross-pollinated plant, with high seed heterozygosity and unstable inheritance of desirable traits. Cutting propagation has a low survival rate and cannot provide a sufficient number of propagules year-round to promote the development of improved varieties. Xanthoceras sorbifolia 'Zhongshi No. 4' is primarily propagated by grafting, but this requires a large number of cuttings, which is not easy to achieve. There are no reports in the prior art on tissue culture applied to the specific variety Xanthoceras sorbifolia 'Zhongshi No. 4'. Summary of the Invention

[0007] The present invention provides an adventitious bud culture medium, a culture method and a tissue culture rapid propagation method for culturing Xanthoceras sorbifolia 'Zhongshi No. 4'. The method of the present invention can realize tissue culture of Xanthoceras sorbifolia 'Zhongshi No. 4'.

[0008] In order to solve the above technical problems, the present invention proposes the following technical solutions:

[0009] The present invention provides an adventitious bud culture medium for culturing Xanthoceras sorbifolia 'Zhongshi No. 4', wherein the adventitious bud culture medium comprises an induction culture medium and a proliferation culture medium;

[0010] The induction medium uses MS as a basic medium and also includes 0.5-1.0 mg / L 6-BA, 0.05-0.2 mg / L NAA, 28-30 g / L sucrose and 6-8 g / L agar;

[0011] The proliferation culture medium uses MS as a basic culture medium and further comprises 0.2-0.5 mg / L 6-BA, 0.05-0.1 mg / L NAA, 0.1-0.2 mg / L GA3, 28-30 g / L sucrose and 6-8 g / L agar.

[0012] Preferably, the pH values ​​of the induction medium and the proliferation medium are 5.8-6.0, respectively.

[0013] The present invention provides a method for culturing adventitious buds of Xanthoceras sorbifolia 'Zhongshi No. 4', which uses the induction medium and the proliferation medium in the adventitious bud culture medium described in the above technical solution, comprising the following steps:

[0014] The stem segments with buds of Xanthoceras sorbifolia 'Zhongshi No. 4' were inoculated into induction medium for induction culture to obtain adventitious buds.

[0015] The adventitious buds are inoculated into a proliferation culture medium for proliferation culture to obtain adventitious bud seedlings.

[0016] Preferably, the stem segments with buds are further disinfected before inoculation, and the disinfection includes first soaking in a 5% sodium hypochlorite solution by mass for 5 to 10 minutes, rinsing with sterile distilled water for 2 to 3 times, then soaking in a 75% ethanol by volume for 30 to 40 seconds, and rinsing with sterile water for 3 to 4 times; finally soaking in a 0.1% mercuric chloride solution by mass for 3 to 12 minutes, and rinsing with sterile water for 5 to 6 times.

[0017] Preferably, the temperatures for the induction culture and proliferation culture are 25-27° C., respectively; the time for the induction culture is 2-4 weeks, and the time for the proliferation culture is 4-6 weeks.

[0018] Preferably, the induction culture and proliferation culture are respectively light-cultured; the illumination time in the light-culture is respectively 12 to 16 h / d; and the illumination intensity is respectively 2000 to 3000 lux.

[0019] The present invention provides a rooting medium for culturing adventitious buds of Xanthoceras sorbifolia 'Zhongshi No. 4'. The rooting medium uses 1 / 2MS as a basic medium and further includes 0.5-1.0 mg / L IBA, 0.5-1.0 mg / L IAA, 28-30 g / L sucrose and 6-8 g / L agar, and the contents of IBA and IAA cannot be 0.5 mg / L at the same time.

[0020] The present invention provides a tissue culture rapid propagation method for Xanthoceras sorbifolia 'Zhongshi No. 4', comprising: inoculating adventitious seedlings obtained by culture using the culture method described in the above technical solution into a rooting culture medium for rooting culture to obtain tissue culture seedlings; the rooting culture uses the rooting culture medium described in the above technical solution.

[0021] Preferably, the temperature of the rooting culture is 25-27° C.; the rooting culture is light culture, the light culture time is 12-16 h / d; the light intensity is 2000-3000 lux; and the rooting culture time is 4-6 weeks.

[0022] Preferably, the adventitious buds are cut before inoculation to obtain regenerated buds; the regenerated buds are 3 to 4 cm in length and contain 2 to 4 fully expanded leaves.

[0023] Beneficial effects of the present invention: The present invention provides an adventitious bud culture medium for culturing Xanthoceras sorbifolia 'Zhongshi No. 4', wherein the adventitious bud culture medium includes an induction culture medium and a proliferation culture medium. In the induction culture medium and the proliferation culture medium of the present invention, NAA is 1-naphthaleneacetic acid, and NAA can promote the formation of buds and the growth of adventitious buds. 6-BA is 6-benzylaminopurine, which promotes the growth of adventitious buds of Xanthoceras sorbifolia 'Zhongshi No. 4', and GA3 is gibberellin, which promotes germination. Under the joint action of 6-BA, NAA and GA3, the growth of adventitious buds of the bud-bearing stem segments of Xanthoceras sorbifolia 'Zhongshi No. 4' is promoted, and the induction rate of adventitious buds and the proliferation coefficient of adventitious buds are improved. IBA in the rooting culture medium is indolebutyric acid, and IAA is indoleacetic acid. Under the action of specific concentrations of indolebutyric acid and indoleacetic acid, the rooting of the adventitious bud seedlings of Xanthoceras sorbifolia 'Zhongshi No. 4' is promoted.

[0024] The present invention also provides a method for cultivating adventitious buds of Xanthoceras sorbifolia 'Zhongshi No. 4'. The method comprises inoculating a bud-bearing stem segment of Xanthoceras sorbifolia 'Zhongshi No. 4' into an induction medium for induction culture to obtain adventitious buds; and then inoculating the adventitious buds into a proliferation medium for proliferation culture to obtain adventitious bud seedlings. The resulting adventitious buds have a high induction rate and a high proliferation coefficient.

[0025] The present invention also provides a tissue culture rapid propagation method for Xanthoceras sorbifolia 'Zhongshi No. 4', which uses adventitious buds as explants for rooting culture to obtain tissue culture seedlings. The tissue culture seedlings have a high rooting rate and a high survival rate after acclimation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph showing the effects of different sampling periods on the contamination rate and germination rate of the "Zhongshi No. 4" Xanthoceras sorbifolia variety;

[0027] Figure 2 This is a graph showing the impact of different sampling periods on the contamination rate of "Zhongshi No. 4" Xanthoceras sorbifolia.

[0028] Figure 3 This is a diagram showing the status of the "Zhongshi No. 4" Xanthoceras sorbifolia variety after being cultured on MS medium with different cytokinin concentrations for 4 weeks;

[0029] Figure 4 This is a diagram of the in vitro proliferation of adventitious buds of "Zhongshi No. 4" Xanthoceras sorbifolia in the proliferation medium of comparative example 6 to 14;

[0030] Figure 5 This is a diagram of the proliferation of Xanthoceras sorbifolia buds cultured in the proliferation medium of Example 7 for 4 weeks;

[0031] Figure 6 Figure 2 shows the rooting of "Zhongshi No. 4" Xanthoceras sorbifolia cultured in the rooting medium of Example 9 and Comparative Examples 16-17 for 4 weeks; wherein A shows the rooting of Comparative Example 17; B shows the rooting of Comparative Example 16; C shows the rooting of Example 9; and D shows the rooted seedlings of Example 9. Figure 6 From left to right in the middle are A, B, C, D;

[0032] Figure 7 The regenerated seedlings of "Zhongshi No. 4" Xanthoceras sorbifolia cultured in the greenhouse for 4 weeks in Example 10;

[0033] Figure 8 This is a graph showing the 4-week proliferation culture of the "Zhongshi No. 9" Xanthoceras sorbifolia of Comparative Example 27;

[0034] Figure 9 This is a 4-week rooting culture time chart of the "Zhongshi No. 9" Xanthoceras sorbifolia of Comparative Example 27. DETAILED DESCRIPTION

[0035] The present invention provides an adventitious bud culture medium for culturing Xanthoceras sorbifolia 'Zhongshi No. 4'. The adventitious bud culture medium comprises an induction culture medium and a proliferation culture medium.

[0036] The induction culture medium of the present invention uses MS as a basic culture medium and further comprises 0.5-1.0 mg / L 6-BA, 0.05-0.2 mg / L NAA, 28-30 g / L sucrose and 6-8 g / L agar.

[0037] As an optional embodiment, the concentration of 6-BA in the induction medium provided by the present invention is 0.5-1.0 mg / L; in a specific embodiment of the present invention, the concentration of 6-BA in the induction medium may be 0.5, 0.8 or 1.0 mg / L. The concentration of NAA in the induction medium provided by the present invention is 0.05-0.2 mg / L; in a specific embodiment of the present invention, the concentration of NAA in the induction medium may be 0.05, 0.08, 0.1, 0.15 or 0.2 mg / L. 6-BA and NAA help induce explants to form buds and increase the bud induction rate. The concentration of sucrose in the induction medium provided by the present invention is 28-30 g / L. In a specific embodiment of the present invention, the concentration of sucrose in the induction medium may be 28, 29 or 30 g / L. The concentration of agar in the induction medium provided by the present invention is 6-8 g / L. In a specific embodiment of the present invention, the concentration of agar in the induction medium may be 6, 7 or 8 g / L. In the present invention, the pH value of the induction culture medium may be 5.8-6.0, or 5.9.

[0038] The proliferation culture medium of the present invention uses MS as a basic culture medium and further comprises 0.2-0.5 mg / L 6-BA, 0.05-0.1 mg / L NAA, 0.1-0.2 mg / L GA3, 28-30 g / L sucrose and 6-8 g / L agar.

[0039] As an optional embodiment, the concentration of 6-BA in the proliferation medium provided by the present invention is 0.2-0.5 mg / L, or 0.4-0.5 mg / L; in a specific embodiment of the present invention, the concentration of 6-BA in the proliferation medium is 0.2, 0.3, 0.4 or 0.5 mg / L. The concentration of NAA in the proliferation medium provided by the present invention is 0.05-0.2 mg / L, more preferably 0.1 mg / L; in a specific embodiment of the present invention, the concentration of NAA in the proliferation medium is 0.05, 0.1, 0.16 or 0.2 mg / L. The concentration of GA3 in the proliferation medium provided by the present invention is 0.1-0.2 mg / L, or 0.12-0.18 mg / L; in a specific embodiment of the present invention, the concentration of GA3 in the proliferation medium is 0.1, 0.12, 0.15, 0.18 or 0.2 mg / L. 6-BA, NAA and GA3 jointly promote the proliferation of buds at appropriate concentration ratios, so that the adventitious buds are in good growth condition. The concentration of sucrose in the proliferation culture medium provided by the present invention is 28 to 30 g / L; in a specific embodiment of the present invention, the concentration of sucrose in the proliferation culture medium is 28, 29 or 30 g / L. The concentration of agar in the proliferation culture medium provided by the present invention is 6 to 8 g / L. In a specific embodiment of the present invention, the concentration of agar in the proliferation culture medium can be 6, 7 or 8 g / L. In the present invention, the pH value of the proliferation culture medium can be 5.8 to 6.0, or 5.9.

[0040] The induction medium and proliferation medium provided by the present invention contain MS medium. Using MS medium as the basic medium, the explants have a high regeneration efficiency and vigorous growth. This may be because compared with the other two culture media 1 / 2MS and WPM, MS medium has a higher content of macronutrients, especially nitrogen sources such as KNO3 and NH4NO3. It can be seen that the Xanthoceras sorbifolia "Zhongshi No. 4" prefers to use MS medium containing higher concentrations of inorganic salts and nutrients for in vitro regeneration induction.

[0041] Adding different concentrations of exogenous hormones to the induction medium and proliferation medium of the present invention can effectively stimulate the germination and proliferation of the Xanthoceras sorbifolia "Zhongshi No. 4." The rooting medium formula can effectively promote the rooting of tissue-cultured seedlings of the Xanthoceras sorbifolia "Zhongshi No. 4." After acclimatization, the tissue-cultured seedlings can grow and develop in a normal environment, thereby improving the survival rate of the tissue-cultured seedlings.

[0042] The present invention provides a rooting culture medium for culturing adventitious buds of Xanthoceras sorbifolia 'Zhongshi No. 4', characterized in that the rooting culture medium uses 1 / 2MS as a basic culture medium and further includes 0.5-1.0 mg / L IBA, 0.5-1.0 mg / L IAA, 28-30 g / L sucrose and 6-8 g / L agar, and the IBA and IAA cannot be 0.5 mg / L at the same time.

[0043] As an optional embodiment, the concentration of IBA in the rooting medium provided by the present invention is 0.5-1.0 mg / L, or 0.8-1.0 mg / L, more preferably 1.0 mg / L. The concentration of IAA in the rooting medium provided by the present invention is 0.5-1.0 mg / L, or 0.5-0.7 mg / L, more preferably 0.5 mg / L. IBA and IAA can promote rooting of adventitious buds of Xanthoceras sorbifolia 'Zhongshi No. 4'. The concentration of sucrose in the rooting medium provided by the present invention is 28-30 g / L. In a specific embodiment of the present invention, the concentration of sucrose in the rooting medium may be 28, 29, or 30 g / L. The concentration of agar in the rooting medium provided by the present invention is 6-8 g / L. In a specific embodiment of the present invention, the concentration of agar in the rooting medium may be 6, 7, or 8 g / L. In the present invention, the pH value of the rooting medium may be 5.8-6.0, or 5.9.

[0044] The induction medium, proliferation medium, and rooting medium provided by the present invention all contain sucrose, which can provide the carbon source required for plant growth, promote the formation and proliferation of buds, increase the bud proliferation coefficient and average bud length, and promote bud rooting. The adventitious bud culture medium provided by the present invention has good application effects and can increase the adventitious bud induction rate and adventitious bud proliferation coefficient of bud-bearing stem segments of Xanthoceras sorbifolia 'Zhongshi No. 4'. The present invention does not specifically limit the sources of the components of the induction medium, proliferation medium, and rooting medium; conventional commercially available products can be used.

[0045] The present invention provides a method for culturing adventitious buds of Xanthoceras sorbifolia 'Zhongshi No. 4', using the induction culture medium and proliferation culture medium described in the above technical solution, comprising the following steps:

[0046] The stem segments with buds of Xanthoceras sorbifolia 'Zhongshi No. 4' were inoculated into induction medium for induction culture to obtain adventitious buds.

[0047] The adventitious buds are inoculated into a proliferation culture medium for proliferation culture to obtain adventitious bud seedlings.

[0048] The present invention inoculates the stem segments with buds of Xanthoceras sorbifolia 'Zhongshi No. 4' into an induction culture medium for induction culture to obtain adventitious buds. As an optional embodiment, the preparation method of the stem segments with buds of Xanthoceras sorbifolia 'Zhongshi No. 4' described in the present invention comprises: removing leaves from the branches of Xanthoceras sorbifolia 'Zhongshi No. 4', cleaning and disinfecting them, and then cutting them into stem segments with buds. The branches of Xanthoceras sorbifolia 'Zhongshi No. 4' described in the present invention are well-grown, pest-free, and current-year branches. The present invention does not particularly limit the cleaning method, and conventional methods can be used. The disinfection method of the present invention includes first soaking the Xanthoceras sorbifolia in a 5% sodium hypochlorite solution for 9 to 12 minutes, or 10 minutes; rinsing with sterile distilled water for 2 to 3 times, or 2 times; then soaking in 75% ethanol by volume for 30 to 40 seconds, or 30 seconds; then rinsing with sterile water for 3 to 4 times, and finally soaking in a 0.1% mercuric chloride solution for 3 to 12 minutes, or 5 to 10 minutes, or 7 to 9 minutes, and rinsing with sterile water 5 to 6 times. The disinfection method of the present invention is simple, and the sterilization treatment can effectively reduce the contamination rate during the tissue culture process of Xanthoceras sorbifolia and improve the acquisition rate of sterile seedlings.

[0049] The length of the bud-bearing stem segment of the present invention is 2.0 to 3.0 cm, more preferably 2.5 cm. The bud-bearing stem segment of the present invention includes axillary bud segments and / or terminal bud segments; the axillary bud segments include at least one axillary bud; and the terminal bud segments include at least one terminal bud. Having axillary buds or terminal buds facilitates the formation of adventitious buds.

[0050] As an optional embodiment, the present invention inoculates bud-bearing stem segments of Xanthoceras sorbifolia 'Zhongshi No. 4' into an induction culture medium for induction culture to obtain adventitious buds. The present invention does not particularly limit the inoculation method; conventional methods may be employed. The stem segments of the present invention contain terminal buds or axillary buds. As the induction culture proceeds, the terminal buds or axillary buds germinate to obtain adventitious buds, thereby obtaining stem segments with adventitious buds.

[0051] As an optional embodiment, the temperature of the induction culture is 25-27°C, or 26°C; the time of the induction culture is 2-4 weeks, or 3-4 weeks. After 2 weeks of induction culture with the induction culture medium of the present invention, adventitious buds begin to grow, and the induction culture is then carried out for 4 weeks because after 4 weeks of culture, most of the axillary buds will germinate and grow adventitious buds, which is beneficial to subsequent proliferation culture. As an optional embodiment, the induction culture is light culture. The illumination time of the light culture is 12-16h / d, or 14-16h / d. The intensity of the light culture can be 2000-3000lux, more preferably 3000lux, and the light culture is preferably carried out using cold white light. The time of the induction culture of the present invention is calculated from the time the stem segment is inoculated on the induction culture medium, until the axillary buds or terminal buds germinate and adventitious buds grow, the length of the adventitious buds reaches 3-5cm, the leaves stretch and grow luxuriantly, and it is possible to provide photosynthesis for the next proliferation.

[0052] As an optional embodiment, after obtaining the adventitious buds, the present invention cuts the adventitious buds from the stem segments and transfers them to a proliferation culture medium for adventitious bud proliferation culture to obtain more proliferated buds.

[0053] As an optional embodiment, before the adventitious buds of the present invention are transferred to the proliferation culture medium for proliferation culture, the method further includes: subculturing the stem segments with the adventitious buds. The time of the subculture of the present invention is 4 to 6 weeks, or 4 to 5 weeks, more preferably 4 weeks. The culture medium used for the subculture of the present invention is composed of: MS as the basic culture medium, and also includes 30g / L sucrose, 6g / L agar, 0.2mg / L6-BA and 0.1mg / LNAA. The role of subculture is to promote the proliferation of adventitious buds on the stem segments and obtain a larger number of adventitious buds. Subculture can be performed indefinitely, or no subculture is required.

[0054] As an optional embodiment, the adventitious buds obtained by subculture of the present invention are transferred to a proliferation culture medium for proliferation culture. The temperature of the proliferation culture of the present invention is 25-27°C, or 26°C; the time of the proliferation culture is 4-6 weeks, or 4-5 weeks, and more preferably 4 weeks. The proliferation culture of the present invention is light culture, and the illumination time of the light culture is 12-16h / d, or 14-16h / d; the intensity of the light culture of the present invention can be 2000-3000lux, or 2500-3000lux, and more preferably 3000lux; the light culture of the present invention is preferably cold white light. In the proliferation culture of the present invention, the adventitious buds are proliferated to obtain adventitious seedlings, and the proliferation culture is carried out until the height of the clustered buds is 3-4cm and the buds and leaves grow luxuriantly.

[0055] The present invention provides a tissue culture and rapid propagation method for Xanthoceras sorbifolia 'Zhongshi No. 4', which adopts the adventitious bud culture medium described in the above technical solution, comprising the following steps:

[0056] The adventitious seedlings obtained by the cultivation method of the above technical solution are inoculated into a rooting medium for rooting culture to obtain tissue culture seedlings; the rooting culture adopts the rooting medium of the above technical solution.

[0057] As an optional embodiment, after obtaining the adventitious buds, the present invention cuts the adventitious buds into single buds and transfers them to a rooting medium for rooting culture to obtain rooted seedlings.

[0058] As an optional embodiment, the adventitious buds of the present invention are further divided before inoculation to obtain regenerated buds. The length of the regenerated buds of the present invention is 3 to 4 cm, or 3.5 cm; the regenerated buds of the present invention contain 2 to 4 fully expanded leaves. As an optional embodiment, the temperature of the rooting culture of the present invention is 25 to 27°C, or 26°C; the time of the rooting culture is 2 to 4 weeks, or 3 to 4 weeks, and more preferably 4 weeks. The rooting culture of the present invention is light culture, and the illumination time of the light culture is 12 to 16 h / d, or 14 to 16 h / d; the intensity of the light culture of the present invention can be 2000 to 3000 lux, or 2300 to 2700 lux, and more preferably 2500 lux; the light culture of the present invention is preferably cold white light. The rooting culture of the present invention is carried out until the root length of the rooted seedlings is 5 to 11 cm or the number of roots is 5 to 18.

[0059] As an optional embodiment, the present invention further includes acclimating the rooted seedlings to obtain complete regenerated plants. The acclimation method of the present invention comprises: transferring the culture flask containing the well-rooted Xanthoceras sorbifolia 'Zhongshi No. 4' plants to a normal environment for 3-5 days. The plants are then removed from the culture flask and their roots are gently rinsed with tap water to remove the surrounding culture medium. The plants are then immersed in a 0.2% (w / v) potassium permanganate solution for 1 minute for disinfection before being transplanted into a substrate. The substrate used for transplanting in the present invention comprises sand, peat, and perlite in a 1:1:1 (w / w) ratio. After transplanting, the plants are placed in a greenhouse at 22-25°C for acclimation. As an optional embodiment, the acclimation temperature is 25-27°C, and the acclimation period after transplanting into the substrate is 4-8 weeks, optionally 4-6 weeks, and more preferably 6 weeks. The survival rate of the domesticated Xanthoceras wenguos 'Zhongshi No. 4' plants reaches 76.67%, which is high.

[0060] The technical solution of the present invention can ensure that disease-free propagules containing the same genes are cultivated, and ensure that Xanthoceras sorbifolia 'Zhongshi No. 4' plants with the same genes are obtained during production, thereby avoiding somatic cell clonal variation. Tissue culture propagation plays a vital role in the development of Xanthoceras sorbifolia cultivation. The present invention aims to evaluate the effects of growth regulators on the induction of axillary buds or terminal buds of Xanthoceras sorbifolia, and to provide a reliable solution for the in vitro culture of axillary buds of Xanthoceras sorbifolia, which can be used for the year-round reproduction of genetically uniform and disease-free propagules of this unique species. The tissue technology solution of the present invention is targeted at a specific variety of Xanthoceras sorbifolia 'Zhongshi No. 4', which improves the adventitious bud induction rate, the proliferation coefficient of the adventitious buds, the rooting rate of the adventitious buds, and the acclimation survival rate of Xanthoceras sorbifolia 'Zhongshi No. 4' during tissue culture.

[0061] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0062] In the following examples and comparative examples, BA refers to 6-BA.

[0063] Example 1-1

[0064] 1. Disinfection effect of explants at different disinfection times

[0065] (1) Explant pretreatment and collection: In early April, before the Xanthoceras sorbifolia seedlings germinated in Zhangwu, Liaoning, the "Zhongshi No. 4" Xanthoceras sorbifolia seedlings obtained by grafting and propagation were planted in nutrient pots and covered with a shed to allow them to germinate and grow in the shed to reduce external pollution. In May, after the branches of the Xanthoceras sorbifolia seedlings germinated and elongated, new sprouts with good growth and no pests and diseases were collected. The samples were collected once every month until the branches were completely lignified in October. The leaves and surface impurities of the branches were removed. The branches were cut into small segments about 2.5 cm long with 1-2 axillary buds or terminal buds. A small amount of laundry detergent was added and the dust on the surface of the small segments was cleaned with a soft brush. The stem segments were then placed in a glass bottle and rinsed with running water for 1-2 hours, then rinsed with distilled water 6 times, and placed on a clean bench for later use.

[0066] (2) Explant disinfection: The stem segments were sterilized with a 5% sodium hypochlorite solution for 10 minutes, rinsed twice with sterile distilled water, treated with 75% ethanol by volume for 30 seconds, rinsed with sterile distilled water, and sterilized with a 0.1% mercuric chloride solution for 3, 6, 9, and 12 minutes, and finally rinsed thoroughly with sterile distilled water 5 times to remove residual mercuric chloride. The sterilized stem segments were cut into small stem segments (containing 1 to 2 axillary buds) about 2 to 3 cm in length and inoculated into cylindrical transparent glass bottles containing 30 mL of culture medium (the composition of the culture medium is: MS basal medium, 30 g / L sucrose, 6 g / L agar, 0.5 mg / L 6-BA, and 0.2 mg / L NAA) for induction culture, and the contamination rate was calculated. Contamination rate (%) = number of contaminated adventitious buds / number of inoculated buds × 100%. The results are shown in Table 1-1.

[0067] It can be seen that the best disinfection effect is treatment 3: mercuric chloride (mass concentration 0.1%) disinfection for 9 minutes, with a contamination rate of 54.4%. Although the contamination rate of treatment 4 is lower than that of treatment 3, the germination rate of treatment 3 is 35.6%, which is higher than the 30.3% of treatment 4. Therefore, it can be concluded that although increasing the disinfection time can reduce the contamination rate, too long a treatment time will affect plant germination.

[0068] Table 1-1 Disinfection effect at different disinfection times

[0069]

[0070] Note: For example, if 100 samples were found to be contaminated, 32 germinated, and the remaining 4 samples did not germinate, were not contaminated, or died, then the germination rate would be 32%. Germination rate (%) = number of germinated buds / number of inoculated buds × 100%.

[0071] 2. Explant collection and disinfection

[0072] Treatment 1: (1) Explant pretreatment and collection: same as step 1 (1); (2) Explant disinfection: same as step 1 (2), wherein 0.1% mercuric chloride disinfection is: disinfection with 0.1% mass concentration mercuric chloride solution for 9 minutes;

[0073] Treatment 2: (1) Explant pretreatment and collection: Same as (1) of Treatment 1, with the only difference being that in June, after the branches of the Xanthoceras sorbifolia seedlings sprouted and elongated, new sprouted branches with good growth and no pests and diseases were collected; (2) Explant disinfection: Same as Treatment 1.

[0074] Treatment 3: (1) Explant pretreatment and collection: Same as (1) of Treatment 1, with the only difference being that in July, after the branches of the Xanthoceras sorbifolia seedlings sprouted and elongated, new sprouted branches with good growth and no pests and diseases were collected; (2) Explant disinfection: Same as Treatment 1.

[0075] Different sampling periods have a great impact on explant disinfection and induction, such as Figure 1 As shown, the pollution rate in May was 53.4%, which was higher than that in June and July ( Figure 2 Middle a), the pollution rate is lower in June and July ( Figure 2 b), which were 47.9% and 47.1% respectively. The pollution rates gradually increased in August, September and October, which were 58.73%, 64.73% and 73.81% respectively. Figure 2 The germination rate is higher in June and July, and the germinated tissue culture seedlings grow strong ( Figure 2 f). The germination rate gradually decreases in August, September and October. Most of the contamination rate is caused by fungi carried by the explants. After the explants are disinfected and cultured for a period of time, velvety hyphae will appear on the surface of the explants ( Figure 2 Sometimes, due to improper operation, bacterial contamination will occur, which will generally produce white translucent or milky bacteria around the explants ( Figure 2 Therefore, it is best to collect stem segments for tissue culture explants of Xanthoceras sorbifolia in June or July. Studies on explant collection and disinfection have found that stem segments collected in June and July have a lower contamination rate and better germination. Disinfection with 5% sodium hypochlorite for 10 minutes, 75% ethanol for 30 seconds, and then 0.1% mercuric chloride solution for 9 minutes is most effective.

[0076] Example 1 Optimization of basal culture medium

[0077] 1. Explant Pretreatment and Collection: In early April, before the Xanthoceras sorbifolia seedlings germinated in Zhangwu, Liaoning Province, "Zhongshi No. 4" grafted Xanthoceras sorbifolia seedlings planted in nutrient pots were placed under a canopy to allow them to germinate and grow in the canopy to reduce external contamination. In June, after the branches of the Xanthoceras sorbifolia seedlings germinated and elongated, newly sprouted branches that were well-grown and free of pests and diseases were collected. The branches were cut into small segments approximately 2.5 cm long with 1-2 axillary buds or terminal buds. A small amount of laundry detergent was added and the surface dust of the segments was cleaned with a soft brush. The stem segments were then placed in a glass bottle and rinsed with running water for 1-2 hours, then rinsed six times with distilled water, and placed on a clean bench for later use.

[0078] 2. Explant disinfection: The stem segments were disinfected with 5% sodium hypochlorite solution for 10 minutes, rinsed twice with sterile distilled water, treated with 75% ethanol for 30 seconds, rinsed with sterile distilled water, disinfected with 0.1% mercuric chloride solution for 9 minutes, and finally rinsed thoroughly with sterile distilled water 5 times to remove residual mercuric chloride.

[0079] 3. Cut the sterilized stem into small stem segments about 2-3 cm long, each containing one axillary bud. Inoculate the small stem segment into a cylindrical transparent glass bottle containing 30 mL of induction medium for induction culture.

[0080] The composition of the induction medium is: MS as a basic medium, supplemented with only 30 g / L sucrose, 6 g / L agar, 0.5 mg / L 6-BA and 0.2 mg / L NAA.

[0081] When preparing the induction medium, adjust the pH to 5.8-6.0 with NaOH or HCl solution, pour the pH-adjusted medium solution into a 280 mL cylindrical transparent glass bottle and cover it with a polycarbonate screw cap, then sterilize it under high pressure at 121°C for 20 minutes. The glass bottle is 10 cm high and 7 cm in diameter.

[0082] The induced cultures were placed under 3000 lux of cool white light, with a photoperiod of 14 h / d and a temperature of 25° C. The induction culture lasted for 4 weeks.

[0083] Comparative Example 1

[0084] The same as Example 1, except that the composition of the induction medium is: 1 / 2MS as the basal medium, and only 30 g / L sucrose, 6 g / L agar, 0.5 mg / L 6-BA and 0.2 mg / L NAA are added.

[0085] Comparative Example 2

[0086] The same as Example 1, the only difference is that the composition of the induction medium is: WPM as the basal medium, and only 30 g / L sucrose, 6 g / L agar, 0.5 mg / L 6-BA and 0.2 mg / L NAA are added.

[0087] In Example 1 and Comparative Examples 1-2, two explant stem segments were placed in each culture bottle, 30 bottles were placed in each of Example 1 and Comparative Examples 1-2, and three parallel experiments were set up for each of Example 1 and Comparative Examples 1-2. After 4 weeks of induction culture, the number of adventitious buds induced by each explant was recorded and the induction rate was calculated.

[0088] Table 1 Effects of culture medium type on adventitious bud induction and proliferation

[0089] serial number culture medium Adventitious buds (number) Adventitious bud induction rate (%) 1 MS Example 1 25.33±2.51a 84.34±0.04a 2 1 / 2MS Comparative Example 1 12.67±2.08b 44.50±0.09b 3 WPM Comparative Example 2 9.33±1.52c 31.32±0.06c

[0090] Table 1 shows that the induction rate of adventitious buds was higher when MS was the basal medium. Therefore, MS was used as the basal medium for subsequent culture. Furthermore, adventitious buds induced by MS medium grew better than those induced by 1 / 2MS and WPM. Explants in MS medium generally germinated within 1-2 weeks, while those in 1 / 2MS and WPM medium required 2-4 weeks.

[0091] Example 2 Induction of adventitious buds from isolated stems with axillary buds as explants

[0092] The same as Example 1, the only difference is that the composition of the induction medium is: MS as the basic medium, and only 30 g / L sucrose, 6 g / L agar, 0.5 mg / L 6-BA and 0.05 mg / L NAA are added.

[0093] Example 3

[0094] The same as Example 1, the only difference is that the composition of the induction medium is: MS as the basal medium, and only 30 g / L sucrose, 6 g / L agar, 1.0 mg / L 6-BA and 0.05 mg / L NAA are added.

[0095] Example 4

[0096] The same as Example 1, the only difference is that the composition of the induction medium is: MS as the basal medium, and only 30 g / L sucrose, 6 g / L agar, 0.5 mg / L 6-BA and 0.1 mg / L NAA are added.

[0097] Example 5

[0098] The same as Example 1, except that the composition of the induction medium is: MS as the basal medium, and only 30 g / L sucrose, 6 g / L agar, 1.0 mg / L 6-BA and 0.1 mg / L NAA are added.

[0099] Example 6

[0100] The same as Example 1, the only difference is that the composition of the induction medium is: MS as the basal medium, and only 30 g / L sucrose, 6 g / L agar, 1.0 mg / L 6-BA and 0.2 mg / L NAA are added.

[0101] Example 6-1

[0102] The same as Example 1, except that the composition of the induction medium is: MS as the basal medium, and only 30 g / L sucrose, 6 g / L agar, 0.5 mg / L 6-BA and 0.2 mg / L NAA are added.

[0103] Comparative Example 3

[0104] The same as Example 1, the only difference is that the composition of the induction medium is: MS as the basal medium, and only 30 g / L sucrose, 6 g / L agar, 0.2 mg / L 6-BA and 0.05 mg / L NAA are added.

[0105] Comparative Example 4

[0106] The same as Example 1, except that the composition of the induction medium is: MS as the basal medium, and only 30 g / L sucrose, 6 g / L agar, 0.2 mg / L 6-BA and 0.1 mg / L NAA are added.

[0107] Comparative Example 5

[0108] The same as Example 1, the only difference is that the composition of the induction medium is: MS as the basal medium, and only 30 g / L sucrose, 6 g / L agar, 0.2 mg / L 6-BA and 0.2 mg / L NAA are added.

[0109] After culturing for 4 weeks in Examples 2 to 6, 6-1 and Comparative Examples 3 to 5, the number of axillary buds and the number of buds of each explant were recorded.

[0110] Table 2 Effects of different concentrations of NAA and 6-BA on bud induction of adventitious buds

[0111]

[0112] Note: Different lowercase letters in the same column indicate significant differences (P<0.05) as determined by Duncan's multiple range test. The values ​​presented are the mean ± standard error (SE) of three replicates, each with 30 explants, for a total of three replicates.

[0113] The adventitious bud induction rate (%) in Tables 1 and 2 is obtained by removing contaminated explants and using uncontaminated explants to conduct adventitious bud induction tests with different concentrations of NAA and 6-BA. For example, 150 explants were inoculated, 50 of which were contaminated, and the remaining 100 uncontaminated explants were used for adventitious bud induction. Then, the adventitious bud induction rate = number of germinations / number of inoculated uncontaminated explants × 100%.

[0114] Number of adventitious buds: Inoculate the axillary buds in the induction medium for germination induction. After 4 to 6 weeks of culture, count the number of adventitious buds that have germinated and grown vigorously.

[0115] Method for measuring the length of adventitious buds: Cut off the fully grown adventitious buds along the base of the buds, place them in a transparent sterile culture dish with sterile tweezers, place a measuring ruler under the culture dish, read the value on the measuring ruler to count the adventitious bud lengths, the adventitious bud length of each treatment = the total length of adventitious buds / the number of adventitious buds × 100%. After measurement, the buds can be inoculated into a proliferation culture medium for proliferation culture, or inoculated into a rooting culture medium for rooting culture.

[0116] The effects of different concentrations of 6-BA and NAA combinations were tested (see Table 2). The axillary buds of Xanthoceras sorbifolia cultured on MS medium supplemented with cytokinins began to sprout and grow in about 2 weeks, and then the axillary buds would continue to elongate to form adventitious buds. After about 4 weeks of culture, most of the axillary buds germinated, and the adventitious buds grew to 1-3 cm, and the leaves gradually unfolded. The induction rate of adventitious buds was counted, and the fully grown adventitious buds were cut off and placed in a transparent sterile culture dish. A measuring ruler was placed under the culture dish to measure the length of the adventitious buds. After measurement, the buds could be inoculated into a proliferation culture medium for proliferation culture, or inoculated into a rooting culture medium for rooting culture. Under different hormone conditions, the induction rate (%) and the number of buds were different, which depended on the concentrations of the cytokinins and growth hormones used. When the 6-BA concentration of Example 6-1 was 0.5 mg / L and the NAA concentration was 0.2 mg / L, the bud regeneration effect was best ( Figure 3 In the middle B), the bud induction rate was 84.34±5a and the bud length was 2.87±0.21cm (Table 2). In contrast, the comparative example 5 with a lower concentration of 0.2mg / L 6-BA had a lower germination rate and a longer germination time ( Figure 3 In the medium with more than 0.5 mg / L 6-BA, the response rate and bud regeneration rate dropped sharply. When the 6-BA concentration of Example 6 reached 1 mg / L and was combined with 0.2 mg / L NAA, buds were directly induced at the wound of the explant ( Figure 3 Middle C).

[0117] The adventitious buds induced in Example 1 were subcultured twice, each time for four weeks. The culture medium used for subculture was the same as the proliferation medium used in Comparative Example 8. Subculture effectively induced the formation of more adventitious buds. Subculture was performed to obtain more buds for subsequent experiments. Subculture could be repeated indefinitely or not at all. The resulting adventitious buds were used in the following proliferation culture experiments.

[0118] Example 7

[0119] The adventitious buds obtained from the above subculture were cut off and divided into 1-2 cm small segments containing 2-3 leaves, and inoculated into a proliferation medium for bud proliferation. The proliferation medium was composed of: MS as a basal medium, supplemented with only 30 g / L sucrose, 6 g / L agar, 0.2 mg / L 6-BA, 0.1 mg / L NAA and 0.1 mg / L GA3.

[0120] The culture conditions are as follows: proliferation culture is light culture, the illumination time of light culture is 14 to 16 hours / day, the intensity of light culture is 3000 lux, the light culture is cold white light, and the temperature is 25°C.

[0121] Three replicates were performed, with at least 30 buds in each replicate. After four weeks of culture, the bud proliferation coefficient and average bud length were determined. The elongated stem segments were then used as in vitro explants for further adventitious bud and root induction.

[0122] Example 8

[0123] The same as Example 7, the only difference is that the composition of the proliferation medium is: MS as the basic medium, and only 30g / L sucrose, 6g / L agar, 0.2mg / L 6-BA, 0.1mg / L NAA and 0.2mg / L GA3 are added.

[0124] The results of proliferation and elongation of regenerated shoots after 4 weeks of culture in Examples 7 and 8 are shown in Table 3. The combination of GA3, BA and NAA in Examples 7 and 8 has a certain synergistic effect on stem elongation (see Figure 5 Within four weeks of culture, adventitious bud proliferation and elongation were accompanied by leaf formation. As shown in Table 3, the different combinations of Example 8 produced the highest bud proliferation coefficient (14.67 ± 1.53), with an average bud length of 4.21 ± 0.37. While maintaining constant BA and NAA concentrations, the average bud length of the adventitious buds initially increased and then decreased with increasing GA3 concentration.

[0125] Table 3 Results of proliferation and elongation of regenerated shoots after 4 weeks of culture in Examples 7-8

[0126]

[0127] Shoot proliferation coefficient (%) = number of sprouts after culture / number of inoculated seeds × 100%.

[0128] Average bud length = total bud length / total number of buds.

[0129] Comparative Example 6

[0130] The same as Example 7, the only difference is that the composition of the proliferation medium is: MS as the basic medium, and only 30g / L sucrose, 6g / L agar, 0.1mg / L 6-BA and 0.05mg / L NAA are added.

[0131] Comparative Example 7

[0132] The same as Example 7, except that the composition of the proliferation medium is: MS as the basal medium, supplemented with 30 g / L sucrose, 6 g / L agar, 0.2 mg / L 6-BA and 0.05 mg / L NAA.

[0133] Comparative Example 8

[0134] The same as Example 8, except that the proliferation medium is composed of MS as the basal medium, supplemented with 30 g / L sucrose, 6 g / L agar, 0.2 mg / L 6-BA and 0.1 mg / L NAA.

[0135] Comparative Example 9

[0136] The same as Example 7, except that the proliferation medium is composed of MS as the basal medium, supplemented with 30 g / L sucrose, 6 g / L agar, 0.5 mg / L 6-BA, and 0.05 mg / L NAA.

[0137] Comparative Example 10

[0138] The same as Example 7, except that the composition of the proliferation medium is: MS as the basal medium, supplemented with 30 g / L sucrose, 6 g / L agar, 0.5 mg / L 6-BA and 0.1 mg / L NAA.

[0139] Comparative Example 11

[0140] The same as Example 7, the only difference is that the composition of the proliferation medium is: MS as the basic medium, and only 30 g / L sucrose, 6 g / L agar, 0.5 mg / L 6-BA and 0.2 mg / L NAA are added.

[0141] Comparative Example 12

[0142] The same as Example 7, the only difference is that the composition of the proliferation medium is: MS as the basic medium, and only 30 g / L sucrose, 6 g / L agar, 1.0 mg / L 6-BA and 0.05 mg / L NAA are added.

[0143] Comparative Example 13

[0144] The same as Example 7, except that the composition of the proliferation medium is: MS as the basal medium, supplemented with 30 g / L sucrose, 6 g / L agar, 1.0 mg / L 6-BA and 0.1 mg / L NAA.

[0145] Comparative Example 14

[0146] The same as Example 7, except that the composition of the proliferation medium is: MS as the basal medium, and only 30 g / L sucrose, 6 g / L agar, 1.0 mg / L 6-BA and 0.2 mg / L NAA are added.

[0147] The proliferation of regenerated shoots was observed on MS medium supplemented with different concentrations of 6-BA and NAA, see Comparative Examples 6 to 14, and the results are shown in Table 4. Among them, 0.2 mg / L 6-BA and 0.1 mg / L NAA were most beneficial to the proliferation of multiple shoots, with an average of 14.67 ± 1.53 shoots (average length 2.87 ± 0.15 cm) proliferating per explant within 4 weeks ( Figure 4 The combination of auxin and cytokinin plays an important role in stem regeneration. In different cytokinin tests, low concentrations of 0.1 mg / L 6-BA and 0.05 mg / L NAA were not effective in promoting plant growth ( Figure 4 In the bud induction medium containing 0.5 mg / L 6-BA, the stem nodes and base leaves became thicker and larger, and gradually vitrified (see Figure 4 C). 1.0 mg / L 6-BA medium induced a large amount of callus around the buds, and the buds were full of small buds (see Figure 4 However, increasing the cytokinin concentration significantly reduced shoot length, with average shoot lengths of approximately 0.6 cm for Comparative Examples 12-14. Once the proliferating shoots reached 2-3 cm, a dominant bud formed within the cluster, and robust, lush foliage was obtained, they were cut and cultured for rooting.

[0148] Table 4 Effects of combined concentrations of 6-BA and NAA on proliferation and elongation of regenerated shoots after 4 weeks of culture

[0149]

[0150]

[0151] Note: Different lowercase letters in the same column indicate significant differences (P<0.05) as determined by Duncan's multiple range test. The values ​​presented are the mean ± standard error (SE) of three replicates, with 30 explants per replicate.

[0152] Bud proliferation coefficient = number of buds / number of inoculations;

[0153] Measurement of bud proliferation coefficient: The proliferated regenerated buds generally grow in clusters. Take the clustered buds out of the culture bottle and cut off the buds from the base with scissors. Count the number of growing buds in each cluster, i.e. the number of bud proliferation. The inoculation number, i.e. the number of inoculated buds, is 1.

[0154] Average bud length = total bud length / number of buds;

[0155] Determination of average bud length: Take a group of clustered buds, remove the proliferated clustered buds from the culture bottle, cut the buds from the base with scissors, place the buds in a sterile transparent culture dish, place a measuring ruler under the culture dish, read the sum of the lengths of the buds and the total number of buds, and calculate the average bud length.

[0156] Example 9 Rooting Culture

[0157] The buds elongated to a length of 4-5 cm in Example 7 were cut off for rooting and used as a source of in vitro stem explants for further propagation.

[0158] Slender healthy buds were separated from the bud clusters of Example 7. The slender healthy buds contained 2 to 4 fully expanded leaves and were 3 to 4 cm in length. The slender healthy buds were used as explants for in vitro adventitious root induction.

[0159] Healthy shoots were inoculated into rooting medium for rooting. The culture conditions were: 16 hours / day of illumination, 3000 lux of cool white light, and a temperature of 25°C. Three biological replicates were performed, with at least 30 seedlings collected for each replicate. After four weeks of culture, the root induction frequency (i.e., rooting rate), number of roots per explant, and root length were recorded.

[0160] The composition of the rooting medium is: 1 / 2MS as the basic medium, supplemented with 30 g / L sucrose, 6 g / L agar, 1.0 mg / L IBA and 0.5 mg / L IAA.

[0161] Example 10

[0162] The same as Example 9, the only difference is that the composition of the rooting medium is: 1 / 2MS as the basic medium, and only 30g / L sucrose, 6g / L agar, 0.5mg / L IBA and 1.0mg / L IAA are added.

[0163] Example 11

[0164] The same as Example 9, the only difference is that the composition of the rooting medium is: 1 / 2MS as the basic medium, and only 30g / L sucrose, 6g / L agar, 1.0mg / L 1BA and 1.0mg / L 1AA are added.

[0165] Comparative Example 15

[0166] The same as Example 9, the only difference is that the composition of the rooting medium is: 1 / 2MS as the basic medium, and only 30g / L sucrose, 6g / L agar, 0.5mg / L IBA and 0.5mg / L IAA are added.

[0167] Comparative Example 16

[0168] The same as Example 9, except that the rooting medium was composed of 1 / 2 MS as the basal medium, supplemented with 30 g / L sucrose, 6 g / L agar, 0.5 mg / L IBA, and 2.0 mg / L IAA.

[0169] Comparative Example 17

[0170] The same as Example 9, except that the rooting medium was composed of 1 / 2 MS as the basal medium, supplemented with 30 g / L sucrose, 6 g / L agar, 1.0 mg / L IA and 2.0 mg / L IAA.

[0171] Comparative Example 18

[0172] The same as Example 9, the only difference is that the composition of the rooting medium is: MS as the basic medium, and only 30g / L sucrose and 6g / L agar are added.

[0173] Comparative Example 19

[0174] The same as Example 9, except that the rooting medium was composed of MS as the basal medium, supplemented with 30 g / L sucrose, 6 g / L agar, and 0.50 mg / L IBA.

[0175] Comparative Example 20

[0176] The same as Example 9, the only difference is that the composition of the rooting medium is: MS as the basic medium, and only 30 g / L sucrose, 6 g / L agar and 1.0 mg / L 1 ABA are added.

[0177] Comparative Example 21

[0178] The same as Example 9, the only difference is that the composition of the rooting medium is: MS as the basic medium, and only 30 g / L sucrose, 6 g / L agar and 2.0 mg / L I BA are added.

[0179] Comparative Example 22

[0180] The same as Example 9, the only difference is that the composition of the rooting medium is: 1 / 2MS as the basic medium, and only 30g / L sucrose and 6g / L agar are added.

[0181] Comparative Example 23

[0182] The same as Example 9, except that the rooting medium was composed of 1 / 2 MS as the basal medium, supplemented with 30 g / L sucrose, 6 g / L agar, and 0.5 mg / L IBA.

[0183] Comparative Example 24

[0184] The same as Example 9, except that the rooting medium was composed of 1 / 2 MS as the basal medium, supplemented with 30 g / L sucrose, 6 g / L agar, and 1.0 mg / L IBA.

[0185] Comparative Example 25

[0186] The same as Example 9, except that the rooting medium was composed of 1 / 2 MS as the basal medium, supplemented with 30 g / L sucrose, 6 g / L agar, and 2.0 mg / L IBA.

[0187] The effects of the rooting medium of Examples 9 to 11 and Comparative Examples 15 to 25 on bud rooting are shown in Tables 5 and Figure 6 .

[0188] Table 5 Effect of rooting medium on bud rooting

[0189] basal culture medium deal with Rooting rate Number of roots Root length (cm) MS 0IBA Comparative Example 18 0 0 0 MS 0.5IBA Comparative Example 19 29.67±6.5de 3.33±1.52ef 3.67±0.49g MS 1.0IBA Comparative Example 20 40±10cd 6.67±0.58de 3.53±0.65g MS 2.0IBA Comparative Example 21 22.33±5.0e 6.33±1.53de 4.93±0.709fg 1 / 2MS 0IBA Comparative Example 22 0 0 0 1 / 2MS 0.5IBA Comparative Example 23 42.33±5.03cd 13.67±2.52bc 6.9±0.56cd 1 / 2MS 1.0IBA Comparative Example 24 48.67±8.08bc 16.33±1.53b 10.1±0.35db 1 / 2MS 2.0IBA Comparative Example 25 33.33±8.5cde 12.33±3.21c 5.03±0.30fg 1 / 2MS 0.5IBA+0.5IAA Comparative Example 15 28±10.1de 10.67±1.53cd 7.23±0.30cd 1 / 2MS 0.5IBA+1.0IAA Example 10 64.33±5.13b 15.33±1.53bc 10.9±1.25b 1 / 2MS 0.5IBA+2.0IAA Comparative Example 16 37.33±5.13cde 13.12±3.0c 5.27±0.35efg 1 / 2MS 1.0IBA+0.5IAA Example 9 83.67±3.51a 18.67±3.79a 11.93±0.55a 1 / 2MS 1.0IBA+1.0IAA Example 11 43.28±7.21cd 13.67±3.06bc 7.15±1.61cd 1 / 2MS 1.0IBA+2.0IAA Comparative Example 17 41.34±2.59cd 14.51±2.52bc 7.96±1.4c

[0190] Note: Different lowercase letters in the same column indicate significant differences (P<0.05) as determined by Duncan's multiple range test. The values ​​presented are the mean ± standard error (SE) of three replicates, with 30 explants per replicate.

[0191] Rooting rate (%) = number of rooted buds / number of inoculated seeds × 100%.

[0192] Number of roots: Take a rooted seedling, remove it from the culture medium, remove the surface matrix, and measure the total number of main roots and lateral roots growing from each bud, which is the number of roots.

[0193] Root length: Remove the rooted shoots from the culture medium, remove the surface matrix, measure the length of each root, and calculate the total root length. Root length = total root length / total number of roots.

[0194] Cultivating plants with normal root systems in vitro is the key to establishing regenerated bodies, and auxin is usually used to induce root growth. The effects of exogenous auxin on in vitro rooting of Xanthoceras sorbifolia are shown in Table 5. After 2 weeks, the roots of the buds began to take root. Under the same hormone conditions, the rooting rate and root length of the buds on 1 / 2MS medium were better than those on MS medium. In MS and 1 / 2MS medium, no roots were produced after 4 weeks of culture with 0 mg / L of growth hormone. Under the same hormone conditions, the rooting rate and root length of the buds on 1 / 2MS medium were better than those on MS medium. On 1 / 2MS medium containing 1.0 mg / L IBA, 8 to 12 thick roots were observed to form from the base of the in vitro regenerated buds ( Figure 6 In the test of different concentrations of IBA and IAA, the largest root consisting of 3 to 5 strong main roots and several lateral roots could be formed in 1 / 2MS medium containing IBA (0.5 mg / L) and IAA (1.0 mg / L). Figure 6 In 1 / 2MS medium containing IBA (1.0 mg / L) and IAA (0.5 mg / L), 1 to 3 strong main roots and a large number of lateral roots were formed, and the rooting rate was as high as 83.67±3.51% ( Figure 6 Middle C), the seedlings are growing vigorously ( Figure 6 In the present invention, the combined use of IBA and IAA resulted in a better rooting efficiency than either of them alone. High concentrations of NAA inhibit root formation because NAA exceeding the optimal level stimulates ethylene biosynthesis, which negatively impacts rooting.

[0195] Example 10 Acclimation and Culture

[0196] In order to adapt to the environment, select rooted seedlings with strong bud growth and well-developed root system (main root length 8-12 cm, about 10 fibrous roots) for acclimatization. The rooted seedlings should be 5-7 cm tall and have lush leaves. Specifically:

[0197] The culture bottles containing the well-rooted plants of Example 9 were moved to a normal environment for 3 to 5 days. Subsequently, the plants with well-developed roots were carefully taken out of the culture bottles, and the roots were gently rinsed with tap water to remove the culture medium around the roots, and then immersed in a 0.2% (w / v) potassium permanganate (KMnO4) solution for 1 minute for disinfection. They were then transplanted onto a substrate consisting of sand, peat soil and perlite (the ratio of the three substrates was 1:1:1 (w / w)), and placed in a greenhouse at 22 to 25°C for acclimation. In order to ensure high humidity, the plastic cups were covered with transparent polyethylene bags and sunshade nets, and the seedlings were watered every day to maintain humidity. After two weeks, the bags were gradually opened, and after four weeks, the sunshade nets were removed. The four-week-old regenerated seedlings in the greenhouse were as follows Figure 7 As shown, the plants propagated by this scheme grew well after transplanting. 30 tissue culture seedlings were transplanted in the greenhouse, and the number of explants successfully domesticated was 23 after 6 weeks. The survival rate of the plants after domestication reached 76.67%. In the present invention, the survival rate of Xanthoceras sorbifolia developed by in vitro rooting is higher, which may be due to its complete root system development, many and long lateral roots, which is conducive to better adaptation during the domestication process. Therefore, a healthy and strong root system is crucial for the successful domestication and transplantation of regenerated plants. In addition, an appropriate amount of peat soil can provide essential nutrients such as organic matter for the growth of seedlings, and the enhancement of its soil porosity and water retention capacity is more conducive to the development of seedlings.

[0198] Acclimation survival rate (%) = number of surviving plants / total number of acclimated plants × 100%.

[0199] Comparative Example 27 Zhongshi No. 9 tissue culture

[0200] The tissue culture conditions and culture medium used for Xanthoceras sorbifolia Zhongshi No. 9 are exactly the same as those for Xanthoceras sorbifolia Zhongshi No. 4. The only difference is the variety of Xanthoceras sorbifolia.

[0201] The specific tissue culture of Xanthoceras sorbifolia Zhongshi No. 9 is as follows:

[0202] 1. Explant pretreatment and collection: In early April, before the Xanthoceras sorbifolia seedlings germinate in Zhangwu, Liaoning Province, the grafted Xanthoceras sorbifolia Zhongshi No. 9 seedlings planted in nutrient pots were covered with a canopy to allow them to germinate and grow in the canopy to reduce external contamination. In May, after the branches germinate and elongate, newly sprouted branches with good growth and no pests and diseases were collected. The branches were collected once every month until they were completely lignified in October. Leaves and surface impurities were removed from the branches. The branches were cut into small segments about 2.5 cm long with 1-2 axillary buds or terminal buds. A small amount of laundry detergent was added and the surface dust was cleaned with a soft brush. The stem segments were placed in a wide-mouth bottle and rinsed with running water for 1-2 hours, then rinsed with distilled water 6 times, and placed on a clean bench for later use.

[0203] 2. Explant Disinfection: Stem segments were sterilized with 5% sodium hypochlorite for 10 minutes, then rinsed twice with sterile distilled water. They were then treated with 75% ethanol for 30 seconds, rinsed with sterile distilled water, and sterilized with 0.1% mercuric chloride solution (3, 6, 9, and 12 minutes). Finally, they were thoroughly rinsed five times with sterile distilled water to remove any residual mercuric chloride. Sterilized stem segments were cut into approximately 2-3 cm long segments (containing 1-2 axillary buds) and inoculated into cylindrical transparent glass bottles containing 30 mL of MS basal medium for induction. Contamination rate (%) = number of contaminated adventitious buds / number of inoculated buds (100%). Results are shown in Table 6.

[0204] Table 6 Effects of different disinfection times on the contamination rate and germination rate of Xanthoceras sorbifolia “Zhongshi No. 9”

[0205]

[0206] 3. Bud Proliferation Medium and Growth Conditions: The basal medium of the induction medium was MS, 1 / 2MS, and WPM, respectively. 30 g / L sucrose, 6 g / L agar, 0.5 mg / L BA, and 0.20 mg / L NAA were added to the induction medium. The pH of the induction medium was adjusted to 5.8-6.0 with NaOH or HCl solution. The pH-adjusted medium solution was poured into a 280 mL cylindrical transparent glass bottle (10 cm high, 7 cm diameter), covered with a polycarbonate screw cap, and then autoclaved at 121°C for 20 min. The stem segments disinfected in step 2 were inoculated into the induction medium. All inoculated cultures were placed under cool white light of 2000-3000 lux, a photoperiod of 14-16 h / d, and a temperature of 25-27°C. The results are shown in Table 7.

[0207] Table 7 Effects of exogenous hormones on bud induction of Xanthoceras sorbifolia “Zhongshi No. 9”

[0208]

[0209] Different lowercase letters in the same column indicate significant differences (P < 0.05) as determined by Duncan's multiple range test. Values ​​presented are the mean ± standard error (SE) of three replicates, each with 30 explants. The culture medium was supplemented with 30 g / L sucrose and 6 g / L agar.

[0210] 4. Induction culture: In step 3, adventitious buds were induced from the detached stems with axillary buds. The detached stems were divided into 2-3 cm long segments and inoculated on adventitious bud induction medium 1-3. Two explants were placed in each bottle, with 30 bottles for each treatment, for a total of 9 treatments. After 6 weeks of culture, the number of axillary buds and buds of each explant was recorded. The buds induced from the nodes were then cultured on bud proliferation medium for further bud proliferation. Adventitious bud induction rate (%) = number of induced adventitious buds / number of inoculations × 100%. The results are shown in Table 8.

[0211] The composition of the induction medium 1 is: MS as a basic medium, supplemented with only 30 g / L sucrose, 6 g / L agar, 0.5 mg / L 6-BA and 0.05 mg / L NAA (same as Example 2).

[0212] The composition of induction medium 2 is: MS as the basal medium, supplemented with only 30 g / L sucrose, 6 g / L agar, 0.5 mg / L 6-BA and 0.2 mg / L NAA (same as Example 6-1).

[0213] The composition of induction medium 3 is: MS as a basic medium, supplemented with only 30 g / L sucrose, 6 g / L agar, 1.0 mg / L 6-BA and 0.2 mg / L NAA (same as Example 6).

[0214] Table 8 Effects of NAA and BA concentrations on bud induction

[0215]

[0216] 5. Proliferation culture medium: The adventitious buds obtained by induction culture in step 4 were divided into small segments with a length of 1 to 2 cm, each containing 2 to 3 leaves, and transferred to the proliferation culture medium supplemented with different concentrations of BA and NAA for further proliferation and elongation. The experiment was repeated 3 times, with no less than 30 buds in each repeat. The bud proliferation coefficient and average bud length were determined after 4 to 6 weeks of culture. The elongated buds were then used as the source of in vitro explants for further induction of adventitious bud proliferation and adventitious roots. Bud proliferation coefficient (%) = number of buds after culture / number of inoculations × 100%. The results are shown in Table 9. "Zhongshi No. 9" was not active in hormone treatment. Under the same hormone treatment conditions, the largest number of proliferation was in the treatment with 0.2 mg / L BA and 0.05 mg / L NAA. On average, 5.53 buds proliferated per explant, with an average length of 1.4 cm, and weak growth. Figure 8 .

[0217] Table 9 Effects of combined concentrations of BA and NAA on proliferation and elongation of regenerated shoots after 4 weeks of culture

[0218]

[0219] Note: Each replicate contained 30 explants. The proliferation medium consisted of: MS basal medium supplemented with 30 g / L sucrose, 6 g / L agar, BA (mg / L), and NAA (mg / L).

[0220] 6. Rooting culture: For in vitro adventitious root induction, separate 2 to 4 slender healthy buds (3 to 4 cm high) with fully expanded leaves from the bud cluster obtained in step 5 as explants. Transfer the explants to a glass bottle containing 1 / 2 MS basal medium, to which different concentrations of IBA and IAA are added. The experiment was repeated three times, with at least 30 seedlings collected for each repeat. After 4 to 6 weeks of culture, the root induction frequency, the number of roots per explant, and the root length were recorded. Rooting coefficient (%) = number of roots / number of inoculations × 100%. The results are shown in Table 10.

[0221] Table 10 Effects of IBA and IAA application concentrations on shoot rooting of Zhongshi No. 9

[0222]

[0223] Note: Each replicate contained 30 explants. The rooting medium consisted of 1 / 2 MS medium, 30 g / L sucrose, 6 g / L agar, IBA, and IAA.

[0224] After 2 weeks of culture in treatments 2 to 4, the buds began to root. After 4 weeks of culture in treatment 1, no roots were produced. In 1 / 2MS medium containing 0.5 mg / L IBA and 0.5 mg / L IAA, "Zhongshi No. 9" can form 3 to 5 strong taproots and several lateral roots (see Figure 9 ), but the buds are weak.

[0225] 7. Acclimation: In order to adapt to the environment, the culture bottles containing well-rooted plants obtained in step 6 are moved to a normal environment for 3-5 days for acclimation. Subsequently, the plants with well-developed roots are carefully removed from the culture bottles, and the roots are gently rinsed with tap water to remove the culture medium around the roots, and then immersed in a 0.2% (w / v) potassium permanganate (KMnO4) solution for 1 minute for disinfection. Then transplant them onto a substrate consisting of sand, peat soil and perlite (1:1:1, w / w) and place them in a greenhouse at 22-25°C for acclimation. To ensure high humidity, the plastic cups are covered with transparent polyethylene bags and shade nets, and the seedlings are watered every day to maintain humidity.

[0226] The survival rate of "Zhongshi No. 9" after rooting and acclimation was zero. Analysis suggests that the rooted and propagated seedlings were not robust enough, resulting in weak growth and difficulty adapting to the external environment. Therefore, a healthy and strong root system is crucial for the successful acclimation and transplantation of regenerated plants. In addition, an appropriate amount of peat soil can provide essential nutrients such as organic matter for seedling growth, and its increased porosity and water retention capacity further promote seedling development.

[0227] In summary, the present invention also provides a tissue culture rapid propagation method for Xanthoceras sorbifolia 'Zhongshi No. 4', which uses adventitious buds as explants for rooting culture to obtain tissue culture seedlings. The tissue culture seedlings have a high rooting rate and a high survival rate after acclimation.

[0228] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A culture medium for cultivating adventitious buds of Xanthoceras sorbifolia 'Zhongshi No. 4', characterized in that: The adventitious bud culture medium includes an induction culture medium and a proliferation culture medium; The induction medium uses MS as a basic medium and also includes 0.5-1.0 mg / L 6-BA, 0.05-0.2 mg / L NAA, 28-30 g / L sucrose and 6-8 g / L agar; The proliferation culture medium uses MS as a basic culture medium and further comprises 0.2-0.5 mg / L 6-BA, 0.05-0.1 mg / L NAA, 0.1-0.2 mg / L GA3, 28-30 g / L sucrose and 6-8 g / L agar.

2. The adventitious bud culture medium according to claim 1, characterized in that The pH values ​​of the induction culture medium and the proliferation culture medium are respectively 5.8-6.

0.

3. A method for cultivating adventitious buds of Xanthoceras sorbifolia 'Zhongshi No. 4', characterized in that: Using the induction medium and proliferation medium in the adventitious bud culture medium according to claim 1 or 2, comprising the following steps: The stem segments with buds of Xanthoceras sorbifolia 'Zhongshi No. 4' were inoculated into induction medium for induction culture to obtain adventitious buds. The adventitious buds are inoculated into a proliferation culture medium for proliferation culture to obtain adventitious bud seedlings.

4. The culture method according to claim 3, wherein The stem segments with buds are also disinfected before inoculation, and the disinfection includes first soaking in a sodium hypochlorite solution with a mass concentration of 5% for 5 to 10 minutes, rinsing with sterile distilled water for 2 to 3 times, then soaking in ethanol with a volume concentration of 75% for 30 to 40 seconds, and rinsing with sterile water for 3 to 4 times; and finally soaking in a mercuric chloride solution with a mass concentration of 0.1% for 3 to 12 minutes, and rinsing with sterile water for 5 to 6 times.

5. The culture method according to claim 3, wherein The temperatures for the induction culture and proliferation culture are 25-27° C. respectively; the time for the induction culture is 2-4 weeks, and the time for the proliferation culture is 4-6 weeks.

6. The culture method according to claim 3, wherein The induction culture and proliferation culture are respectively light-cultured; the illumination time in the light-culture is respectively 12 to 16 h / d; and the intensity of the illumination is respectively 2000 to 3000 lux.

7. A culture medium for cultivating adventitious bud rooting of Xanthoceras sorbifolia 'Zhongshi No. 4', characterized in that: The rooting medium uses 1 / 2MS as a basic medium and further comprises 0.5-1.0 mg / L IBA, 0.5-1.0 mg / L IAA, 28-30 g / L sucrose and 6-8 g / L agar, and the IBA and IAA cannot be 0.5 mg / L at the same time.

8. A tissue culture and rapid propagation method for Xanthoceras sorbifolia 'Zhongshi No. 4', characterized in that: include: The adventitious seedlings obtained by the cultivation method according to any one of claims 3 to 6 are inoculated into a rooting medium for rooting culture to obtain tissue culture seedlings; the rooting culture adopts the rooting medium according to claim 7.

9. The tissue culture and rapid propagation method according to claim 8, wherein The rooting culture temperature is 25-27° C.; the rooting culture is light culture, the light culture time is 12-16 h / d; the light intensity is 2000-3000 lux; and the rooting culture time is 4-6 weeks.

10. The tissue culture and rapid propagation method according to claim 7, wherein Before inoculation, the adventitious buds are cut to obtain regenerated buds; the regenerated buds are 3 to 4 cm long and contain 2 to 4 fully expanded leaves.

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