Rapid propagation method for obtaining clumpy buds and rooting seedlings by taking four-season bamboo stalk buds as explants

Abstract

The invention discloses a rapid propagation method for obtaining cluster buds and rooting seedlings by taking four-season bamboo stalk buds as explants, and belongs to the technical field of bamboo plant tissue culture. According to the method, the blank that four-season bamboo shoots and rooting seedlings are efficiently obtained through plant tissue culture is filled, and the culture medium suitable for inducing the four-season bamboo shoots to proliferate and root is screened out by using the four-season bamboo shoots as the explants and combining different growth regulator combinations. The method is simple and easy to implement, overcomes the defects of low propagation coefficient, seasonal limitation, waste of manpower and material resources and the like of a traditional propagation mode of the bambusa everyday, can efficiently induce to obtain a large number of cluster buds and rooting plants, greatly shortens the propagation cycle of the bambusa everyday, and lays a foundation for high-yield afforestation and garden application of the bambusa everyday.

Description

Technical Field

[0001] The invention relates to the technical field of bamboo plant tissue culture, in particular to a rapid propagation method for obtaining clustered buds and rooted seedlings by using four-season bamboo stalk buds as explants. Background Art

[0002] Bamboo has significant economic, ecological and social benefits, and is one of the most promising and profitable renewable forestry resources. With the continuous improvement of people's aesthetic appreciation and cultural confidence, bamboo has become more and more ornamental and aesthetically pleasing due to its special beauty, natural characteristics and cultural heritage. The market demand for garden bamboo, especially the scarce precious ornamental bamboo, is increasing day by day, and its germplasm resource breeding and large-scale development have become one of the important directions and hotspots in the current bamboo research field. Bamboo itself has special reproductive biological characteristics, such as long flowering cycle, irregular flowering, no fruiting after flowering, and death after flowering. Therefore, bamboo rarely reproduces sexually, and traditional asexual reproduction methods such as mother bamboo transplantation, whip propagation, basket propagation and cutting propagation are generally adopted. However, the traditional propagation method consumes a lot of bamboo, has high labor intensity, low propagation coefficient, and the obtained seedlings have large differences in individual size and state. Plant tissue culture technology provides feasibility for the large-scale rapid propagation of bamboo, which can be applied to factory seedling cultivation, germplasm preservation, mutant screening, and improved variety breeding. In recent years, the research on bamboo tissue culture technology has made great progress. At present, we have successfully obtained the tissue culture and rapid propagation technology systems of several important economic bamboo species, such as Malayan sweet dragon bamboo, latam bamboo, and Xishuangbanna sweet dragon bamboo.

[0003] Oligostachyum lubricum belongs to the genus Oligostachyum of the subfamily Bambusoideae of the family Gramineae. It is an excellent economic and ornamental bamboo with important economic benefits and garden application value. Oligostachyum lubricum is named for its long shoot period (May-October). The shoots are edible and are rare summer and autumn bamboo shoots in subtropical areas. The underground stems of Oligostachyum lubricum are of a compound axis mixed type, with a stalk height of 4 to 6m, a ground diameter of 1 to 3.5cm, and 3 branches of bamboo branches. It is luxuriant and can be used for landscaping and shelterbelt construction. At present, the breeding of Oligostachyum lubricum generally adopts the traditional propagation method of mother bamboo transplantation. Based on this, the present invention specifically proposes to establish a tissue culture rapid propagation technology system using the stalk buds of Oligostachyum lubricum as explants, laying a foundation for its high-yield afforestation and garden application. Summary of the invention

[0004] The purpose of the present invention is to provide a rapid propagation method for obtaining clustered buds and rooted seedlings using four-season bamboo stalk buds as explants, so as to solve the problems existing in the above-mentioned prior art. The method provided by the present invention is simple and easy to implement, and overcomes the shortcomings of the traditional propagation method of four-season bamboo, such as low propagation coefficient, seasonal restrictions, waste of manpower and material resources, etc. It can efficiently induce a large number of clustered buds and rooted plants, greatly shorten the propagation cycle of four-season bamboo, and lay the foundation for its high-yield afforestation and gardening applications.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a rapid propagation method for obtaining clustered buds and rooted seedlings by using four-season bamboo stalk buds as explants, comprising the following steps:

[0007] (1) Using 1-2 year old bamboo shoots with stalk buds as explants, and sterilizing them;

[0008] (2) inoculating the sterilized explants on a basic culture medium, wherein the basic culture medium is MS + 3 mg / L 6-BA + 30 g / L sucrose + 2.5 g / L phytagel;

[0009] (3) After two weeks of culture, the explants were transferred to a proliferation medium for culture, wherein the proliferation medium was MS + 3-4 mg / L 6-BA + 0.8-1.2 mg / L KT + 0.3-0.5 mg / L TDZ + 30 g / L sucrose + 2.5 g / L phytagel;

[0010] (4) Cultivating on a proliferation medium for four to eight weeks until clusters of buds form;

[0011] (5) The proliferated buds are inoculated onto a rooting medium and cultured until roots are formed. The rooting medium is MS+3 mg / L 6-BA+3 mg / L NAA+1.5 mg / L IBA+30 g / L sucrose+2.5 g / L plant gel.

[0012] Preferably, the proliferation medium is MS+3.5 mg / L 6-BA+0.8 mg / L KT+0.4 mg / L TDZ+30 g / L sucrose+2.5 g / L phytagel.

[0013] Optionally, the branch segments with stem buds are cut into 1.5 cm to 2 cm branch segments with buds, and then sterilized.

[0014] Optionally, the sterilization operation is to treat with alcohol, rinse with sterile water for 3 to 5 times, then treat with 2% sodium hypochlorite solution, and rinse with sterile water for 3 to 5 times.

[0015] Optionally, the alcohol treatment time is 60 seconds, the 2% sodium hypochlorite solution treatment time is 8 minutes, and the sterile water flushing time is 2 minutes each time.

[0016] Optionally, the culture conditions are a temperature of 25±1°C, a light intensity of 1200-1500lx, and a light duration of 14h / d.

[0017] The invention also provides application of the rapid propagation method in preparing four-season bamboo buds and rooted seedlings.

[0018] The present invention discloses the following technical effects:

[0019] The present invention shortens the reproduction period of four-season bamboo, effectively improves tissue culture efficiency, and provides a feasible solution for large-scale rapid propagation of four-season bamboo by establishing and continuously optimizing the tissue culture technology system of four-season bamboo.

[0020] The present invention fills the gap of efficiently obtaining four-season bamboo buds and rooted seedlings through plant tissue culture. By using four-season bamboo stalk buds as explants and combining different growth regulator combinations, a culture medium suitable for inducing four-season bamboo bud proliferation and rooting is screened. The method is simple and easy to implement, overcomes the shortcomings of the traditional four-season bamboo propagation method, such as low propagation coefficient, seasonal restrictions, waste of manpower and material resources, etc., can efficiently induce a large number of buds and rooted plants, greatly shorten the propagation cycle of four-season bamboo, and lay a foundation for its high-yield afforestation and gardening applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 This is a picture of four seasons bamboo branches with buds;

[0023] Figure 2 This is a picture of the growth of four-season bamboo on the induction medium after two weeks;

[0024] Figure 3 This is a picture of the growth of four-season bamboo on the proliferation medium after two weeks;

[0025] Figure 4 This is a picture of the buds of four-season bamboo after four weeks on the proliferation medium;

[0026] Figure 5 This is a picture of the buds of four-season bamboo after six weeks on the proliferation medium;

[0027] Figure 6 This is a picture of the buds of four-season bamboo after eight weeks on the proliferation medium;

[0028] Figure 7 This is a picture of the rooting of four-season bamboo after four weeks on the rooting medium;

[0029] Figure 8 This is a comparison chart of long-leaved bitter bamboo branches with buds;

[0030] Fig. 9 This is a comparison chart of the growth of long-leaved bitter bamboo on the induction medium after two weeks;

[0031] Fig.10 This is a comparison chart of the growth of long-leaved bitter bamboo on the proliferation medium after two weeks;

[0032] Fig.11 This is a comparison chart of the growth of long-leaved bitter bamboo after four weeks on the proliferation medium;

[0033] Fig.12 This is a comparison chart of the growth of long-leaved bitter bamboo after six weeks on the proliferation medium. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] Example 1 Rapid propagation method for obtaining bud clusters and rooted seedlings using four-season bamboo stalk buds as explants

[0040] A rapid propagation method for obtaining clustered buds and rooted seedlings using four-season bamboo stalk buds as explants comprises the following steps:

[0041] (1) Selection of explants: Figure 1 The four-season bamboo explant material selected in this embodiment is shown. Cut the semi-lignified branches of four-season bamboo that are 1 to 2 years old, healthy and disease-free on a sunny day. Put the branches with unsprouted full stalk buds neatly into a fresh-keeping bag and seal it. Cut off the excess leaves, remove the sheath ring under the stalk bud, cut the branches into 1.5cm to 2cm stem segments with buds, shake and clean them with detergent for 5 minutes, and rinse them with running water for 2 hours.

[0042] (2) Establishment of sterile system: Place the cleaned stem segments with buds in a sterile glass bottle, soak them in 75% alcohol for 60 seconds, pour out the alcohol, shake and rinse them with sterile water for 3 to 5 times, 2 minutes each time, and then treat the stem segments with 2% sodium hypochlorite solution for 8 minutes. During this period, shake the culture bottle continuously to fully sterilize the explants. Pour out the sodium hypochlorite solution, shake and rinse them with sterile water for 3 to 5 times, 2 minutes each time, wash off the residual disinfectant, and use sterile filter paper to absorb the surface moisture. After inoculation, the explants treated by the above disinfection methods have the highest bud survival rate and the lowest contamination rate.

[0043] Table 1 Effects of different disinfection methods on the survival rate and contamination rate of four-season bamboo explants

[0044] deal with 75% alcohol(s) 2% NaClO solution (min) Contamination rate (%) mortality rate(%) Survival rate (%) 1 45 8 80.0 10.0 10.0 2 45 12 55.4 14.3 30.3 3 45 16 64.0 10.9 25.1 4 60 8 48.0 15.2 36.8 5 60 12 55.5 17.2 27.3 6 60 16 50.1 17.8 32.1

[0045] As shown in Table 1, considering the contamination rate, mortality rate and survival rate, treatment group 4 used 75% alcohol for 60 seconds, rinsed with sterile water 3-5 times, and then treated with 2% NaClO solution for 8 minutes, and rinsed with sterile water 3-5 times, which was the best solution for disinfecting four-season bamboo stalk buds. After inoculation, the four-season bamboo stalk buds treated by the above disinfection methods had the lowest contamination rate of 48.0%, and the highest bud induction survival rate of 36.8%. The survival rate of explants treated with 75% alcohol for 45 seconds and 2% sodium hypochlorite for 12 minutes in treatment group 2 was also high, but the contamination rate of alcohol treatment for 60 seconds was obviously lower than that of 45 seconds, and the effect of alcohol on the contamination rate was greater than that of sodium hypochlorite solution. For sodium hypochlorite solution, the survival rate was the highest and the contamination rate was the lowest after 8 minutes of treatment.

[0046] (3) Cluster bud induction: The sterilized four-season bamboo shoots were inoculated on a basic culture medium composed of MS + 3 mg / L 6-BA. The growth was observed after two weeks of culture. Figure 2 shown.

[0047] (4) Cluster bud proliferation: The four-season bamboo stalk buds after induction culture were transferred to the proliferation medium until a large number of cluster buds and rooted seedlings were formed. Among the 9 formulas, based on the proliferation coefficient and cluster bud growth, the optimal proliferation medium was treatment group 4: MS + 3.5mg / L6-BA + 0.8mg / L KT + 0.4mg / L TDZ. Analysis of the data in Table 2 shows that with the increase of 6-BA concentration, the proliferation coefficient first increased and then decreased. When the concentration was 3mg / L, the proliferation coefficient was low; when the concentration was 3.5mg / L, the proliferation coefficient was the highest and the proliferation effect was the best; when the concentration was 4mg / L, the proliferation coefficient was low, the bud cluster growth was poor, and the leaves turned yellow. When the KT concentration was 1.2mg / L, the proliferation coefficient was low; when the TDZ concentration was 0.5mg / L, the proliferation coefficient was low. The results showed that when the 6-BA concentration was 3.5mg / L, the KT concentration was 0.8mg / L, and the TDZ concentration was 0.4mg / L, the new bud clusters were the largest, the bud clusters were strong, and the proliferation effect was the best. Figure 3 This is the situation after two weeks of proliferation, no proliferation buds are produced; Figure 4 This is the situation after four weeks of proliferation, with new proliferation buds appearing at the base of the culm; Figure 5 This is the situation after six weeks of proliferation. Proliferation buds continue to sprout at the base of the culm, and there are signs of budding at the bottom of the bud cluster. Figure 6 This is the situation after eight weeks of proliferation, with new proliferation buds continuously sprouting from the bottom of the bud cluster.

[0048] Table 2 Effects of different plant growth regulator ratios on the proliferation of four-season bamboo shoots

[0049]

[0050] Note: a, b, c, d, e, f, and g indicate significant differences at the α=0.05 level.

[0051] (5) Rooting of buds: The proliferated buds were inoculated into a rooting medium for rooting culture. The rooting medium composition was: MS + 3 mg / L 6-BA + 3 mg / L NAA + 1.5 mg / L IBA. Figure 7 This is the rooting condition after four weeks of cultivation. Several small roots have emerged.

[0052] All the above culture media were added with 30 g / L sucrose, 2.5 g / L phytagel, pH=5.8-6.0, and sterilized at 121°C for 20 min. The culture conditions were culture temperature 25±1°C, light intensity 1200-1500 lx, and light duration 14 h / d.

[0053] Example 2 Control experiment of plant tissue culture with long-leaved bitter bamboo stalk bud explants

[0054] A control experiment of plant tissue culture using long-leaf bitter bamboo stalk bud explants comprises the following steps:

[0055] (1) Selection of explants: Figure 8 The explant material of the long-leaf bitter bamboo selected in this embodiment is shown. Cut 1-2 year-old, healthy, disease-free semi-lignified branches of the long-leaf bitter bamboo on a sunny day. Put the branches with unsprouted full stalk buds neatly into a fresh-keeping bag and seal it. The processing method is the same as in Example 1. Cut off the excess leaves, remove the sheath ring under the stalk bud, cut the branches into 1.5cm-2cm stem segments with buds, shake and clean with detergent for 5 minutes, and rinse with running water for 2 hours.

[0056] (2) Establishment of sterile system: Place the cleaned stem segments with buds in a sterile glass bottle, soak them in 75% alcohol for 45 seconds, pour out the alcohol, shake and rinse them with sterile water for 3 to 5 times, 2 minutes each time, and then treat the stem segments with 2% sodium hypochlorite solution for 10 minutes. During this period, shake the culture bottle continuously to fully sterilize the explants. Pour out the sodium hypochlorite solution, shake and rinse them with sterile water for 3 to 5 times, 2 minutes each time, wash off the residual disinfectant, and use sterile filter paper to absorb the surface moisture. After inoculation of the explants treated by the above disinfection methods, the survival rate of long-leaved bitter bamboo buds was the highest and the contamination rate was low.

[0057] Table 3 Effects of different disinfection methods on the survival rate and contamination rate of long-leaved bitter bamboo explants

[0058] Treatment Group 75% alcohol(s) 2% NaClO solution (min) Contamination rate (%) mortality rate(%) Survival rate (%) 1 45 8 40.0 3.3 56.7 2 45 10 18.3 5.8 75.9 3 45 12 16.3 11.4 72.3 4 60 8 22.7 10.2 67.1 5 60 10 14.8 15.6 69.6 6 60 12 12.7 24.1 63.2

[0059] As shown in Table 3, considering the contamination rate, mortality rate and survival rate, treatment 2 uses 75% alcohol for 45 seconds, rinses with sterile water 3-5 times, and then treats with 2% NaClO solution for 10 minutes, and rinses with sterile water 3-5 times, which is the best solution for disinfecting the long-leaf bitter bamboo stalk buds. After inoculation, the long-leaf bitter bamboo stalk buds treated by the above disinfection methods have a low contamination rate of 18.3%, and the bud induction survival rate is the highest, which is 75.9%, which is different from the best solution for disinfecting the four-season bamboo stalk buds. The survival rate of the explants treated with 75% alcohol for 45 seconds and 2% sodium hypochlorite for 12 minutes in treatment 3 is also high, but considering that the mortality rate of the explants treated with sodium hypochlorite for 12 minutes is obviously higher than that of 10 minutes, for sodium hypochlorite solution, the effect of 10 minutes treatment is the best.

[0060] (3) Cluster bud induction: The sterilized long-leaved bitter bamboo stalk buds were inoculated on the same basic culture medium as the four-season bamboo and cultured for two weeks. Fig. 9 shown.

[0061] (4) Cluster bud proliferation: After induced culture, the buds of the long-leaf bitter bamboo stalks were transferred to the same 9 proliferation culture media as the four-season bamboo. The base of the long-leaf bitter bamboo stalks turned black, no proliferation buds were produced, the leaves turned yellow and dried, and gradually died. Fig.10 This is the situation of long-leaved bitter bamboo after two weeks of proliferation under the same plant growth regulator ratio as treatment group 4 in Table 2. No new proliferation buds were produced. Fig.11 This is the situation of long-leaf bitter bamboo after four weeks of proliferation under the same plant growth regulator ratio as treatment group 4 in Table 2. New buds sprouted at the bud points on the stalks of long-leaf bitter bamboo, but no proliferation buds were produced. Fig.12 The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows: The results are as follows:

[0062] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A rapid propagation method for obtaining clustered buds and rooted seedlings using four-season bamboo stalk buds as explants, characterized in that: The following steps are involved: (1) Using 1-2 year old bamboo shoots with stalk buds as explants, and sterilizing them; (2) inoculating the sterilized explants on a basic culture medium, wherein the basic culture medium is MS + 3 mg / L 6-BA + 30 g / L sucrose + 2.5 g / L phytagel; (3) After two weeks of culture, the explants were transferred to a proliferation medium containing MS + 3-4 mg / L 6-BA + 0.8-1.2 mg / L KT + 0.3-0.5 mg / L TDZ + 30 g / L sucrose + 2.5 g / L phytagel; (4) Cultivating on a proliferation medium for four to eight weeks until clusters of buds form; (5) The proliferated buds are inoculated onto a rooting medium and cultured until roots are formed. The rooting medium is MS+3 mg / L 6-BA+3 mg / L NAA+1.5 mg / L IBA+30 g / L sucrose+2.5 g / L plant gel.

2. The rapid propagation method as claimed in claim 1, characterized in that The proliferation culture medium is MS+3.5 mg / L 6-BA+0.8 mg / L KT+0.4 mg / L TDZ+30 g / L sucrose+2.5 g / L plant gel.

3. The rapid propagation method as claimed in claim 1, characterized in that The branch segments with stalk buds are cut into 1.5 cm to 2 cm branch segments with buds, and then sterilized.

4. The rapid propagation method as claimed in claim 1, characterized in that The sterilization process is as follows: after treating with alcohol, rinsing with sterile water for 3 to 5 times, treating with 2% sodium hypochlorite solution, and rinsing with sterile water for 3 to 5 times.

5. The rapid propagation method as claimed in claim 4, characterized in that The alcohol treatment time is 60 seconds, the 2% sodium hypochlorite solution treatment time is 8 minutes, and the sterile water washing time is 2 minutes each time.

6. The rapid propagation method as claimed in claim 1, characterized in that The culture conditions are as follows: temperature 25±1°C, light intensity 1200-1500lx, and light duration 14h / d.

7. Use of the rapid propagation method as described in any one of claims 1 to 6 in preparing four-season bamboo clump buds and rooted seedlings.

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