Tissue culture rapid propagation method of clematis' Qianwei '

Abstract

The invention discloses a tissue culture and rapid propagation method of clematis' Qianhui ', which comprises the following steps: inoculating a disinfected explant into an induction culture medium, inoculating the explant into an adventitious bud proliferation culture medium after multiple shoots grow, inoculating the multiple shoots subjected to proliferation culture into a rooting culture medium, and domesticating and transplanting when the root length is 2-4cm. Tissue culture and rapid propagation are performed on the clematis variety 'Qianhui' by adopting a method of inducing stem tips to generate cluster buds for seedling, a culture medium formula and an operation method for tissue culture are simple, the repeatability is high, and the method has the beneficial effects of high propagation coefficient, low pollution rate, easiness in rooting, strong plant growth vigor and the like.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a tissue culture and rapid propagation method of Clematis 'Qianwei'. Background Art

[0002] Clematis, a vine plant of the genus Clematis in the family Ranunculaceae, is known as the "Queen of Vines" for its large and colorful flowers, rich and varied flower shapes, and long flowering period. This unique plant resource has been widely used in the landscape layout of vertical greening and home gardening, greatly enriching the diversity and aesthetic value of green landscapes. Globally, Clematis has rich germplasm resources and numerous cultivated varieties, and my country has successfully introduced thousands of species, which has not only greatly expanded the domestic Clematis germplasm resource bank, but also promoted the vigorous development of home gardening.

[0003] However, the reproduction of Clematis faces a series of challenges. Its seeds have a high abortion rate and a low germination rate, resulting in low seed reproduction efficiency, which has become a major obstacle to the widespread promotion of the variety. At present, although the reproduction of Clematis mainly relies on traditional methods such as cuttings and layering, these methods also have limitations such as low reproduction coefficients and long reproduction cycles, which are difficult to meet the needs of large-scale expansion and factory production.

[0004] In order to solve these breeding problems, tissue culture rapid propagation technology came into being. This technology can rapidly increase the amount of plant materials in a short period of time through plant tissue culture methods while maintaining the excellent genetic characteristics of the mother plant. In addition, tissue culture rapid propagation technology also provides the possibility of establishing a genetic transformation system, laying a solid foundation for subsequent transgenic technology research.

[0005] As a representative variety of southern European clematis, Clematis 'Qianwei' occupies an important position in my country's home gardening market with its bright color and beautiful flower shape, and is deeply loved by consumers. However, the existing propagation methods limit its rapid promotion and large-scale production. Therefore, exploring and establishing a set of efficient and stable tissue culture and rapid propagation system for Clematis 'Qianwei' is not only of great significance for the realization of factory production of this variety, but also helps to promote the establishment of its genetic transformation system, providing strong support for future genetic improvement and new variety breeding.

[0006] The present invention aims to provide a tissue culture rapid propagation method for the Clematis 'Qianwei' variety, so as to solve the problems of low efficiency and long cycle in the existing propagation technology, meet the large market demand for this variety, and lay a solid foundation for its genetic improvement and transgenic research. Summary of the invention

[0007] The purpose of the present invention is to provide a tissue culture rapid propagation method for Clematis 'Qianwei', which can improve the tissue culture rapid propagation efficiency of Clematis 'Qianwei'.

[0008] To achieve the above object, the present invention provides a tissue culture rapid propagation method of Clematis 'Qianwei', comprising the following steps:

[0009] Step S1, explant treatment and disinfection: taking an explant of Clematis odorata, disinfecting it and setting it aside for later use; wherein the variety of Clematis odorata is Clematis odorata 'Qianwei';

[0010] Step S2, primary culture: inoculating the explants in an induction medium, culturing for more than 10 days, and growing clustered shoots; the induction medium comprises MS+6-BA 1.0 mg / L+NAA 0.05 mg / L+3% sucrose+0.7% agar, with a pH of 5.6-5.8;

[0011] Step S3, proliferation culture: inoculating the Clematis with clustered buds into an adventitious bud proliferation medium and culturing for more than 40 days; the adventitious bud proliferation medium comprises MS+6-BA 1.0 mg / L+TDZ 0.5 mg / L+3% sucrose+0.7% agar, with a pH of 5.6-5.8;

[0012] Step S4, rooting and seedling cultivation: the clustered buds after proliferation culture are cut into stem segments of 2 to 3 cm, inoculated into rooting medium, dark cultured for 7 to 10 days and then light cultured, and when the root length is 2 to 4 cm, acclimatization and transplanting can be carried out; the composition of the rooting medium is 1 / 2WPM+NAA 0.05mg / L+IBA 0.2mg / L+3% sucrose+0.7% agar, and the pH is 5.6 to 5.8.

[0013] Wherein, in step S1, the explant is a young and tender top stem segment with buds on the new branches of Clematis that year, the leaves are removed, and the stem segment is cut into 2-3 cm.

[0014] Among them, in step S1, the disinfection method is: soak in 1% carbendazim solution for 5-7 minutes, rinse with sterile water for 3 to 5 times, wipe dry the water, place on the clean bench, soak in 75% alcohol solution for 20 seconds, rinse with sterile water for 3 to 4 times, soak in 0.1% mercuric chloride for 6 to 8 minutes, and rinse with sterile water for 4 to 5 times.

[0015] Wherein, in step S2, firstly cut off the 0.5 cm portions at both ends of the stem segment, and then inoculate them into the induction medium.

[0016] Wherein, in step S3, the temperature of proliferation culture is 23-27°C, and the light intensity is 1000-2000 Lux.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention adopts a method of inducing stem tips to generate clustered buds and seedlings to carry out tissue culture rapid propagation of the Clematis variety 'Qianwei'. The medium formula and operation method of tissue culture are simple, highly repeatable, and have the beneficial effects of high propagation coefficient, low contamination rate, easy rooting, strong plant growth, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a photo of the primary culture of Clematis 'Qianwei' explants;

[0020] Figure 2 Actual picture of Clematis 'Qianwei' explant proliferation culture;

[0021] Figure 3 Actual photo of the rooting and seedling cultivation of Clematis 'Qianwei' explants. DETAILED DESCRIPTION

[0022] The specific implementation modes of the present invention are described in detail below in conjunction with the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific implementation modes.

[0023] In this embodiment, Clematis 'Qianwei' comes from the Institute of Flowers, Guangxi Academy of Agricultural Sciences. Basic culture medium MS medium and WPM medium: The basic culture medium used for primary and proliferation culture is MS medium solid powder, and the basic culture medium used for rooting culture is WPM medium solid powder.

[0024] Example 1 Rapid propagation of Clematis 'Qianwei' by tissue culture

[0025] (1) Explant treatment and disinfection

[0026] Select the young and tender top stem segments with buds on the new branches of the year of the strong and pest-free Clematis 'Qianwei', remove the leaves, cut the stem segments into 2 cm, gently brush the dirt on the surface of the stem segments with a toothbrush, then rinse with running water for 80 minutes, soak in 1% carbendazim solution for 5-7 minutes, rinse with sterile water 4 times, wipe dry, place on a clean bench, soak in 75% alcohol for 20 seconds, rinse with sterile water 4 times, then soak in 0.1% mercuric chloride for 7 minutes, rinse with sterile water 4 times, and dry the surface moisture of the explants on sterile filter paper for use;

[0027] (2) Primary Culture

[0028] Take the sterile stem segments with buds after the above treatment, cut off the parts of the bud segments in contact with mercuric chloride at both ends, that is, 0.5 cm of the stem segments above and below, and leave 1 cm of the stem segments to be inoculated into the induction medium. After 10 days of induction in the induction medium, the stem segments with buds begin to produce clustered buds. After 40 days of culture, the clustered buds grow robustly. Figure 1 As shown; the composition of the induction medium is MS+6-BA1.0mg / L+NAA 0.05mg / L+3% (mass concentration) sucrose+0.7% (mass concentration) agar, pH is 5.8;

[0029] (3) Proliferation culture

[0030] The Clematis with clustered buds was inoculated into adventitious bud proliferation medium and cultured for more than 40 days for adventitious bud proliferation. Figure 2 As shown; the composition of the adventitious bud proliferation medium is MS+6-BA 1.0mg / L+TDZ 0.5mg / L+3% (mass concentration) sucrose+0.7% (mass concentration) agar, pH is 5.8; the culture temperature is 23-27°C, and the light intensity is 1000-2000Lux;

[0031] (4) Rooting and seedling cultivation

[0032] After proliferation culture, the clustered buds were cut into 2 cm stem segments, inoculated into rooting medium, and cultured in the dark for 8 days before light culture. Figure 3 As shown, acclimatization and transplanting were carried out when the root length was 3 cm; the composition of the rooting medium was 1 / 2WPM+NAA 0.05 mg / L+IBA 0.2 mg / L+3% (mass concentration) sucrose+0.7% (mass concentration) agar, and the pH was 5.8.

[0033] Example 2 Experiment on the effect of different disinfection treatments on survival rate, contamination rate and browning rate

[0034] Select the young and tender top stem segments with buds from the new branches of the year of the robust and pest-free Clematis 'Qianwei', remove the leaves, and cut the stem segments into 2 cm pieces as test materials.

[0035] The experiment was divided into two groups of single factor experiments. For treatment groups 1 to 3, the test materials were soaked in 75% ethanol for 30s, 20s and 10s respectively, then rinsed with sterile water 3 to 4 times, then soaked in 0.1% mercuric chloride for 5min, and rinsed with sterile water 4 to 5 times; for treatment groups 4 to 6, the test materials were soaked in 75% ethanol for 20s, then rinsed with sterile water 3 to 4 times, then soaked in 0.1% mercuric chloride for 6min, 8min, 10min, and rinsed with sterile water 4 to 5 times. After the treatment, the surface moisture of the explants was dried on sterile filter paper, the parts of the bud segments that were in contact with mercuric chloride at both ends were cut off, and the upper and lower 0.5cm stem segments were left. The 1cm long stem segment was inoculated in the induction medium with MS as the basic medium and pH 5.6-5.8 and cultured according to the normal process. The statistical results are shown in Table 1.

[0036] Table 1 Effects of different disinfection treatments on survival rate, contamination rate and browning rate

[0037]

[0038]

[0039] As shown in Table 1, among the different treatments with 75% ethanol, treatment 2 had the highest survival rate of 58.3% and the lowest browning rate of 25%. Although the contamination rate was not the lowest, considering the disinfection effect comprehensively, immersion in 75% ethanol for 20 seconds was the most suitable. In the 0.1% HgCl treatment, the survival rates of treatments 4 and 5 were both high, reaching more than 50%. The browning rate of treatment 4 was the lowest, at 22.3%, but the contamination rate was high. Considering the disinfection effect comprehensively, the 0.1% HgCl treatment for 6 to 8 minutes had a higher survival rate and the best disinfection effect.

[0040] Example 2 Experiment on the induction effect of different culture media on axillary buds

[0041] The experiment used three commonly used culture media, namely MS, 1 / 2MS and B5, as the basic culture media. Each culture medium was supplemented with 0.5mg / L 6-BA and 0.05mg / L NAA. The young stems of Clematis 'Qianwei' that had been disinfected were inoculated in three different culture media. The stem segments were about 1cm long and were vertically inserted into the culture media. Two were inoculated in each bottle, 15 bottles were treated in each case, and each treatment group was repeated 3 times. After 30 days, the induction rate of axillary buds of explants, the average seedling height and germination rate were calculated. The experimental results are shown in Table 2.

[0042] Table 2 Results of axillary bud induction on different culture media

[0043]

[0044] As shown in Table 2, the results of axillary bud induction in different culture media were significantly different. The germination rates of MS, 1 / 2MS and B5 were 74.21%, 56.8% and 46.75% respectively, and the differences were significant. The average height of the seedlings was 2.56cm, 1.25cm and 0.86cm respectively. The differences between MS and 1 / 2MS and B5 were significant, while the differences between 1 / 2MS and B5 were not significant. The germination of stem segments in different culture media was observed, and it was found that the axillary buds of the stem segments in B5 culture medium were short and strong, but no leaves sprouted; the axillary buds of 1 / 2MS culture medium were short and thin, and the leaves were yellow-green, showing poor development; the axillary buds of the stem segments in MS culture medium were well developed, which was the best culture medium for inducing axillary bud development.

[0045] Example 3 Effect of 6-BA+NAA on axillary bud induction of Clematis 'Qianwei'

[0046] According to the method of Example 1, tissue culture rapid propagation of Clematis 'Qianwei' was carried out, and 6-BA and NAA in the induction medium of (2) primary culture were set to 9 groups of different concentrations. The test results are shown in Table 3.

[0047] Table 3 Effects of different concentrations of hormones on the induction of axillary buds of Clematis

[0048]

[0049] As shown in Table 3, the axillary bud germination rate of treatments 1, 2, and 4 was high. As the concentration of NAA increased, the axillary bud germination rate of Clematis decreased. It can be seen that the optimal concentration of NAA was 0.05 mg / L. As the concentration of 6-BA increased, the axillary bud germination rate generally showed a trend of gradual decrease. Based on the results of the comprehensive germination rate, the optimal concentration of 6-BA was 1.0 mg / L. Therefore, 6-BA 1.0 mg / L + NAA 0.05 mg / L is the best hormone combination for axillary bud induction of Clematis 'Qianwei'.

[0050] Example 4 Effect of 6-BA+TDZ on the proliferation of axillary buds of Clematis 'Qianwei'

[0051] According to the method of Example 1, tissue culture rapid propagation of Clematis 'Qianwei' was carried out, and 6-BA and TDZ in the adventitious bud proliferation medium of (3) proliferation culture were set to 9 groups of different concentrations. The test results are shown in Table 4.

[0052] Table 4 Effects of different hormone ratios on axillary bud proliferation

[0053]

[0054]

[0055] As shown in Table 4, all nine different hormone treatments can promote the proliferation of buds, but the proliferation effects are different when the concentrations of 6-BA and TDZ are different. Among them, when 6-BA is 1.0 mg / L and TDZ is 0.5 mg / L, the proliferation coefficient of Clematis is the highest, there are many clustered buds, and the stem segments are thick and the growth rate is fast. The proliferation effect is significantly better than other treatments, which is the best proliferation hormone ratio. Therefore, considering the comprehensive proliferation coefficient and seedling growth, MS+6-BA 1.0 mg / L+TDZ 0.5 mg / L is the best culture scheme for Clematis bud proliferation.

[0056] Example 5 Effect of NAA+IBA on Rooting Culture of Clematis 'Qianwei'

[0057] According to the method of Example 1, tissue culture and rapid propagation of Clematis 'Qianwei' were carried out. The concentrations of NAA and IBA in the rooting medium for (4) rooting and seedling cultivation were set to three different groups. The test results are shown in Table 5.

[0058] Table 5 Effects of different hormone concentrations on rooting culture

[0059] deal with NAA(mg / L) IBA(mg / L) Rooting rate (%) Plant growth status 1 0.05 0.1 42.8b The plant is average, with yellow-green leaves and short roots. 2 0.05 0.2 65.7a The plant is strong, with dark green leaves and thick and long roots. 3 0.05 0.3 32.6b The plant is strong, the leaves turn purple-red, and the roots are slender

[0060] It can be seen from Table 5 that when the NAA concentration is 0.05 mg / L, the rooting rates of plants with different IBA concentrations are significantly different, among which Treatment 2 has the highest rooting rate, reaching 65.7%, and the growth condition is excellent, the plants are strong, the leaves are dark green, and the roots are thick and long. Therefore, WPM+NAA 0.05 mg / L+IBA 0.2 mg / L is the best culture formula for inducing rooting.

[0061] Comparative Example 6

[0062] (1) Explant treatment and disinfection

[0063] Select the young and tender top stem segments with buds from the new branches of the year of Clematis 'Qianwei' that are healthy and free of diseases and insect pests, remove the leaves, cut the stem segments into 2 cm, rinse with running water for 45 minutes, soak in 75% alcohol for 10 seconds, rinse with sterile water for 3 times, soak in 1% sodium hypochlorite solution for 4 minutes, rinse with sterile water for 5 times, and dry the surface moisture of the explants on sterile filter paper for use;

[0064] (2) Primary Culture

[0065] Take the sterile stem segments with buds after the above treatment, cut off the parts of the bud segments in contact with sodium chlorate at both ends, i.e., 0.5 cm of the stem segments above and below, and leave 1 cm of the stem segments to be inoculated in the induction medium. After 10 days of induction in the induction medium, the stem segments with buds begin to produce clustered buds. After 40 days of culture, the clustered buds grow robustly. Figure 1As shown; the composition of the induction medium is MS+6-BA1.0mg / L+NAA 0.05mg / L+3% (mass concentration) sucrose+0.7% (mass concentration) agar, pH is 5.8;

[0066] (3) Proliferation culture

[0067] The Clematis with clustered buds was inoculated into adventitious bud proliferation medium and cultured for more than 40 days for adventitious bud proliferation. Figure 2 As shown; the composition of the adventitious bud proliferation medium is MS+6-BA 1.0mg / L+TDZ 0.5mg / L+3% (mass concentration) sucrose+0.7% (mass concentration) agar, pH is 5.8; the culture temperature is 23-27°C, and the light intensity is 1000-2000Lux;

[0068] (4) Rooting and seedling cultivation

[0069] After proliferation culture, the clustered buds were cut into 2 cm stem segments, inoculated into rooting medium, and cultured in the dark for 8 days before light culture. Figure 3 As shown, acclimatization and transplanting were carried out when the root length was 3 cm; the composition of the rooting medium was 1 / 2WPM+NAA 0.05 mg / L+IBA 0.2 mg / L+3% sucrose+0.7% agar, and the pH was 5.8.

[0070] Comparative Example 7

[0071] (1) Explant treatment and disinfection

[0072] Select the young and tender top stem segments with buds from the new branches of the year of Clematis 'Qianwei' that are healthy and pest-free, remove the leaves, cut the stem segments into 2 cm, rinse with running water for 45 minutes, soak in 75% alcohol for 20 seconds, rinse with sterile water for 3 times, soak in 1% sodium hypochlorite solution for 1.5 minutes, rinse with sterile water for 5 times, and dry the surface moisture of the explants on sterile filter paper for use;

[0073] (2) Primary Culture

[0074] Take the sterile stem segments with buds after the above treatment, cut off the parts of the bud segments in contact with sodium chlorate at both ends, i.e., 0.5 cm of the stem segments above and below, and leave 1 cm of the stem segments to be inoculated in the induction medium. After 10 days of induction in the induction medium, the stem segments with buds begin to produce clustered buds. After 40 days of culture, the clustered buds grow robustly. Figure 1 As shown; the composition of the induction medium is MS+6-BA1.0mg / L+NAA 0.05mg / L+3% (mass concentration) sucrose+0.7% (mass concentration) agar, pH is 5.8;

[0075] (3) Proliferation culture

[0076] The Clematis with clustered buds was inoculated into adventitious bud proliferation medium and cultured for more than 40 days for adventitious bud proliferation. Figure 2 As shown; the composition of the adventitious bud proliferation medium is MS+6-BA 1.0mg / L+TDZ 0.5mg / L+3% (mass concentration) sucrose+0.7% (mass concentration) agar, pH is 5.8; the culture temperature is 23-27°C, and the light intensity is 1000-2000Lux;

[0077] (4) Rooting and seedling cultivation

[0078] After proliferation culture, the clustered buds were cut into 2 cm stem segments, inoculated into rooting medium, and cultured in the dark for 8 days before light culture. Figure 3 As shown, acclimatization and transplanting were carried out when the root length was 3 cm; the composition of the rooting medium was 1 / 2WPM+NAA 0.05 mg / L+IBA 0.2 mg / L+3% sucrose+0.7% agar, and the pH was 5.8.

[0079] The tissue culture test results of Comparative Example 1, Comparative Example 6 and Comparative Example 7 are shown in Table 6

[0080] Reproduction coefficient Contamination rate Browning rate Example 2.6 26.7％ 22.3％ Comparative Example 6 - 98％ - Comparative Example 7 - 96％ -

[0081] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A tissue culture rapid propagation method for Clematis 'Qianwei', characterized in that: The following steps are involved: Step S1, explant treatment and disinfection: taking explants of Clematis odoratissimus, disinfecting and setting aside for later use; the variety of Clematis odoratissimus is Clematis odoratissimus 'Qianwei'; Step S2, primary culture: inoculating the explants in an induction medium, culturing for more than 10 days, and growing clustered shoots; the induction medium comprises MS+6-BA 1.0 mg / L+NAA 0.05 mg / L+3% sucrose+0.7% agar, with a pH of 5.6-5.8; Step S3, proliferation culture: inoculating the Clematis with clustered buds into an adventitious bud proliferation medium and culturing for more than 40 days; the adventitious bud proliferation medium comprises MS+6-BA 1.0 mg / L+TDZ 0.5 mg / L+3% sucrose+0.7% agar, with a pH of 5.6-5.8; Step S4, rooting and seedling cultivation: the clustered buds after proliferation culture are cut into stem segments of 2 to 3 cm, inoculated into rooting medium, dark cultured for 7 to 10 days and then light cultured, and when the root length is 2 to 4 cm, acclimatization and transplanting can be carried out; the composition of the rooting medium is 1 / 2WPM+NAA 0.05mg / L+IBA 0.2mg / L+3% sucrose+0.7% agar, and the pH is 5.6 to 5.

8.

2. The tissue culture rapid propagation method according to claim 1, characterized in that: In step S1, the explant is a young and tender top stem segment with buds on the new branches of Clematis that year, the leaves are removed, and the stem segment is cut into 2-3 cm.

3. The tissue culture rapid propagation method according to claim 1, characterized in that: In step S1, the disinfection method is: soak in 1% carbendazim solution for 5 to 7 minutes, rinse with sterile water for 3 to 5 times, wipe dry, place on a clean bench, soak in 75% alcohol solution for 20 seconds, rinse with sterile water for 3 to 4 times, soak in 0.1% mercuric chloride for 6 to 8 minutes, and rinse with sterile water for 4 to 5 times.

4. The tissue culture rapid propagation method according to claim 2, characterized in that: In step S2, first cut off 0.5 cm of each end of the stem segment, and then inoculate it into the induction medium.

5. The tissue culture rapid propagation method according to claim 1, characterized in that: In step S3, the temperature of the proliferation culture is 23-27°C, and the light intensity is 1000-2000 Lux.

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