A rapid propagation method for obtaining clumps and rooted seedlings by using bamboo shoots of four seasons bamboo as explants

Abstract

This invention discloses a rapid propagation method for obtaining shoot clusters and rooted seedlings of Bambusa textilis using stem shoots as explants, belonging to the field of bamboo plant tissue culture technology. This invention fills the gap in the efficient acquisition of Bambusa textilis shoot clusters and rooted seedlings through plant tissue culture. By utilizing Bambusa textilis stem shoots as explants and combining different growth regulators, suitable culture media for inducing shoot cluster proliferation and rooting in Bambusa textilis are screened. This method is simple and easy to implement, overcoming the shortcomings of traditional Bambusa textilis propagation methods, such as low propagation coefficient, seasonal limitations, and waste of manpower and resources. It can efficiently induce the acquisition of a large number of shoot clusters and rooted plants, greatly shortening the propagation cycle of Bambusa textilis and laying the foundation for its high-yield afforestation and landscaping applications.

Description

Technical Field

[0001] This invention relates to the field of bamboo plant tissue culture technology, and in particular to a rapid propagation method for obtaining bud clusters and rooted seedlings using bamboo culm buds as explants. Background Technology

[0002] Bamboo possesses significant economic, ecological, and social benefits, making it one of the most promising and profitable renewable forestry resources. With the increasing aesthetic appreciation and cultural confidence of the people, the ornamental and aesthetic value of bamboo, due to its unique beauty, natural characteristics, and cultural connotations, is becoming increasingly prominent. The market demand for garden bamboo, especially rare and precious ornamental bamboo, is growing rapidly, making its germplasm resource propagation and large-scale development one of the important directions and hot topics in bamboo research. Bamboo itself has unique reproductive biological characteristics, such as a long flowering cycle, irregular flowering, no fruit set after flowering, and death after flowering. Therefore, bamboo rarely reproduces sexually, and traditional asexual propagation methods such as transplanting mother bamboo, whip propagation, tuft propagation, and cutting propagation are commonly used. However, traditional propagation methods consume a lot of resources, are labor-intensive, have a low propagation coefficient, and produce seedlings with significant differences in individual size and condition. Plant tissue culture technology provides a feasibility for large-scale rapid propagation of bamboo, and can be applied to various aspects such as factory seedling production, germplasm preservation, mutant screening, and improved variety selection. In recent years, significant progress has been made in bamboo tissue culture technology research. Currently, rapid propagation technology systems for several important economic bamboo species have been successfully obtained, such as Malayan sweet bamboo, bamboo fern, and Xishuangbanna sweet bamboo.

[0003] *Oligostachyum lubricum*, belonging to the subfamily Bambusoideae of the family Gramineae, is a valuable dual-purpose bamboo for both economic and ornamental purposes, possessing significant economic benefits and horticultural applications. Named for its long shoot-producing season (May-October), the shoots are edible and are a rare summer-autumn bamboo shoot variety in subtropical regions. The rhizomes of *Oligostachyum lubricum* are multi-axial mixed-crop type, with culms 4-6m tall and 1-3.5cm in diameter at ground level. The bamboo has three branches, producing abundant branches and leaves, making it suitable for landscaping and shelterbelt construction. Currently, the traditional propagation method of *Oligostachyum lubricum* is commonly used, involving transplanting mother bamboo. Therefore, this invention proposes a tissue culture rapid propagation technology system using *Oligostachyum lubricum* culm buds as explants, laying the foundation for its high-yield afforestation and horticultural applications. Summary of the Invention

[0004] The purpose of this invention is to provide a rapid propagation method for obtaining bud clusters and rooted seedlings using bamboo culm buds as explants, in order to solve the problems existing in the prior art. The method provided by this invention is simple and easy to implement, overcoming the shortcomings of traditional bamboo propagation methods such as low propagation coefficient, seasonal limitation, and waste of manpower and resources. It can efficiently induce a large number of bud clusters and rooted plants, greatly shortening the propagation cycle of bamboo and laying the foundation for its high-yield afforestation and landscaping applications.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a rapid propagation method for obtaining clustered buds and rooted seedlings using bamboo stalk buds as explants, comprising the following steps:

[0007] (1) Use 1-2 year old bamboo shoots with culm buds as explants and sterilize them;

[0008] (2) The sterilized explants were inoculated on a basic culture medium, which was MS + 3 mg / L 6-BA + 30 g / L sucrose + 2.5 g / L plant gel;

[0009] (3) After two weeks of culture, the explants were transferred to a proliferation medium for culture, which 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 plant gel;

[0010] (4) Culture on proliferation medium for four to eight weeks until shoot clusters form;

[0011] (5) The proliferated shoots 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 plant gel.

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

[0014] Optionally, the sterilization process involves treating with alcohol, rinsing with sterile water 3-5 times, then treating with 2% sodium hypochlorite solution and rinsing with sterile water 3-5 times.

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

[0016] Optionally, the cultivation conditions are a temperature of 25±1℃, a light intensity of 1200~1500lx, and a light duration of 14h / d.

[0017] The present invention also provides the application of the rapid propagation method described above in the preparation of four-season bamboo clumps and rooted seedlings.

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

[0019] This invention establishes and continuously optimizes a tissue culture technology system for propagating bamboo from buds, thereby shortening the propagation cycle of bamboo and effectively improving tissue culture efficiency, providing a feasible solution for the large-scale rapid propagation of bamboo.

[0020] This invention fills the gap in the efficient acquisition of *Phyllostachys edulis* buds and rooted seedlings through plant tissue culture. By using *Phyllostachys edulis* culm buds as explants and combining different growth regulators, a suitable culture medium for inducing the proliferation and rooting of *Phyllostachys edulis* buds was screened. This method is simple and easy to implement, overcoming the shortcomings of traditional *Phyllostachys edulis* propagation methods, such as low propagation coefficient, seasonal limitations, and waste of manpower and resources. It can efficiently induce the acquisition of a large number of buds and rooted plants, greatly shortening the propagation cycle of *Phyllostachys edulis* and laying the foundation for its high-yield afforestation and landscaping applications. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 A diagram of a bamboo shoot with buds in all four seasons;

[0023] Figure 2 This image shows the growth of Bambusa textilis on an induction medium after two weeks.

[0024] Figure 3 This image shows the growth of Bambusa textilis on a growth medium after two weeks.

[0025] Figure 4 A diagram of the shoot clusters of Bambusa textilis after four weeks on a growth medium;

[0026] Figure 5 Image of shoot clusters of Bambusa textilis after six weeks on a growth medium;

[0027] Figure 6 Image of shoot clusters of Bambusa textilis after eight weeks on a growth medium;

[0028] Figure 7 This image shows the rooting status of Bamboo stalks after four weeks on the rooting medium.

[0029] Figure 8 Comparison diagram of a branch segment with buds from the long-leaved bitter bamboo;

[0030] Figure 9 A control diagram showing the growth of long-leaved bitter bamboo on induction medium after two weeks;

[0031] Figure 10 A comparison diagram showing the growth of long-leaved bitter bamboo on the proliferation medium after two weeks;

[0032] Figure 11 A comparison diagram showing the growth of long-leaved bitter bamboo on the proliferation medium after four weeks;

[0033] Figure 12 This is a comparison chart showing the growth of long-leaved bitter bamboo on a proliferation medium after six weeks. Detailed Implementation

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

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0039] Example 1: A rapid propagation method for obtaining clustered shoots and rooted seedlings using bamboo culm shoots as explants.

[0040] A rapid propagation method for obtaining shoot clusters and rooted seedlings using bamboo stalk buds as explants includes the following steps:

[0041] (1) Selection of explants: Figure 1 The explant material of *Phyllostachys edulis* selected in this embodiment is shown. On a sunny day, semi-lignified branches of 1-2 year old, healthy, and disease-free *Phyllostachys edulis* were cut. The branches with plump, un-emerged buds were neatly placed in a plastic bag and sealed. Excess leaves were removed, and the sheaths below the buds were removed. The branches were cut into 1.5-2 cm stem segments with buds, cleaned with detergent and shaken for 5 minutes, then rinsed under running water for 2 hours.

[0042] (2) Establishment of a sterile system: Cleaned stem segments with buds were placed in sterile glass bottles and soaked in 75% alcohol for 60 seconds. After pouring out the alcohol, the segments were rinsed with sterile water, shaking and swirling 3-5 times for 2 minutes each time. Then, the stem segments were treated with 2% sodium hypochlorite solution for 8 minutes, continuously shaking the culture bottle to ensure thorough sterilization of the explants. The sodium hypochlorite solution was then poured out, and the segments were rinsed with sterile water, shaking and swirling 3-5 times for 2 minutes each time to remove residual disinfectant. The surface moisture was then blotted dry with sterile filter paper. Explants treated with the above sterilization method showed the highest bud survival rate and the lowest contamination rate after inoculation.

[0043] Table 1. Effects of different disinfection methods on the survival rate and contamination rate of Bambusa textilis explants.

[0044] deal with 75% alcohol(s) 2% NaClO solution (min) Pollution 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 overall contamination rate, mortality rate, and survival rate, treatment group 4, using 75% alcohol for 60 seconds, rinsing with sterile water 3-5 times, followed by treatment with 2% NaClO solution for 8 minutes and rinsing with sterile water 3-5 times, represents the optimal disinfection scheme for *Phyllostachys edulis* buds. After inoculation with *Phyllostachys edulis* buds treated with this method, the contamination rate was the lowest (48.0%), and the bud induction survival rate was the highest (36.8%). In treatment group 2, the explant survival rate was also relatively high after treatment with 75% alcohol for 45 seconds and 2% sodium hypochlorite for 12 minutes. However, the contamination rate was significantly lower with 60 seconds of alcohol treatment compared to 45 seconds, and alcohol had a greater impact on the contamination rate than sodium hypochlorite solution. For sodium hypochlorite solution, treatment for 8 minutes resulted in the highest survival rate and the lowest contamination rate.

[0046] (3) Bud induction: Sterilized bamboo stalk buds were inoculated onto a basic culture medium consisting of MS + 3 mg / L 6-BA. After two weeks of culture, their growth was observed as follows. Figure 2 As shown.

[0047] (4) Bud proliferation: The induced bamboo shoots were transferred to the proliferation medium until a large number of bud clusters and rooted seedlings were formed. Among the nine formulations, based on the proliferation coefficient and bud cluster growth, the optimal proliferation medium was treatment group 4: MS + 3.5 mg / L 6-BA + 0.8 mg / L KT + 0.4 mg / L TDZ. Analyzing the data in Table 2, as the concentration of 6-BA increased, the proliferation coefficient first increased and then decreased. The proliferation coefficient was low at a concentration of 3 mg / L; the proliferation coefficient was highest and the proliferation effect was best at a concentration of 3.5 mg / L; the proliferation coefficient was low at a concentration of 4 mg / L, the bud cluster growth was poor, and the leaves turned yellow. The proliferation coefficient was low at a KT concentration of 1.2 mg / L and low at a TDZ concentration of 0.5 mg / L. The results showed that when the concentration of 6-BA was 3.5 mg / L, the concentration of KT was 0.8 mg / L, and the concentration of TDZ was 0.4 mg / L, the number of new bud clusters was the largest, the bud clusters were robust, and the proliferation effect was the best. Figure 3 This is the situation two weeks after proliferation; no proliferating buds have been produced. Figure 4 This is the situation four weeks after propagation, with new propagation buds emerging at the base of the stem; Figure 5 This is the situation after six weeks of propagation. Propagation buds continue to sprout from the base of the stem, and there are signs of sprouting at the bottom of the bud cluster. Figure 6 This is the situation after eight weeks of propagation, with new propagating buds continuously sprouting from the bottom of the bud cluster.

[0048] Table 2 Effects of different plant growth regulator ratios on the proliferation of bamboo shoot segments in *Phyllostachys edulis*

[0049]

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

[0051] (5) Rooting of shoot clusters: The proliferated shoot clusters were inoculated into a rooting medium for rooting culture. The composition of the rooting medium was: MS + 3 mg / L 6-BA + 3 mg / L NAA + 1.5 mg / L IBA. Figure 7 To observe the rooting process after four weeks, several small roots have emerged.

[0052] All culture media were supplemented with 30 g / L sucrose and 2.5 g / L plant gel, with a pH of 5.8-6.0, and autoclaved at 121℃ for 20 min. The culture conditions were 25±1℃, light intensity of 1200-1500 lx, and a photoperiod of 14 h / d.

[0053] Example 2: Control experiment of plant tissue culture using explants of *Phyllostachys edulis* culm buds.

[0054] A control experiment using explants of *Phyllostachys edulis* culm buds for plant tissue culture includes the following steps:

[0055] (1) Selection of explants: Figure 8 The explant material of *Phyllostachys longipes* selected in this embodiment is shown. On a sunny day, 1-2 year old, healthy, and disease-free semi-lignified branches of *Phyllostachys longipes* were cut. The branches with plump, un-emerged buds were neatly placed in a plastic bag and sealed. The treatment method was the same as in Example 1. Excess leaves were cut off, the sheath ring under the bud was removed, and the branches were cut into 1.5-2 cm stem segments with buds. The segments were cleaned by shaking with detergent for 5 minutes and then rinsed under running water for 2 hours.

[0056] (2) Establishment of a sterile system: Cleaned stem segments with buds were placed in sterile glass bottles and soaked in 75% alcohol for 45 seconds. After pouring out the alcohol, the segments were rinsed with sterile water, shaking and swirling 3-5 times for 2 minutes each time. Then, the stem segments were treated with 2% sodium hypochlorite solution for 10 minutes, continuously shaking the culture bottle to ensure thorough sterilization of the explants. The sodium hypochlorite solution was then poured out, and the segments were rinsed with sterile water, shaking and swirling 3-5 times for 2 minutes each time to remove residual disinfectant. The surface moisture was then absorbed using sterile filter paper. After inoculation with explants treated using the above sterilization method, the survival rate of *Phyllostachys edulis* 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 explants from *Phyllostachys edulis*.

[0058] Processing group 75% alcohol(s) 2% NaClO solution (min) Pollution 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 overall contamination rate, mortality rate, and survival rate, Treatment 2, which involves using 75% alcohol for 45 seconds, rinsing with sterile water 3-5 times, followed by treatment with 2% NaClO solution for 10 minutes and rinsing with sterile water 3-5 times, is the optimal disinfection scheme for *Phyllostachys edulis* culm buds. After inoculation with *Phyllostachys edulis* culm buds treated with this method, the contamination rate was low (18.3%), and the bud induction survival rate was the highest (75.9%), which differs from the optimal disinfection scheme for *Phyllostachys edulis* culm buds. Treatment 3, which involved 75% alcohol for 45 seconds and 2% sodium hypochlorite for 12 minutes, also showed a high survival rate for explants. However, considering that the mortality rate of explants treated with sodium hypochlorite for 12 minutes was significantly higher than that treated for 10 minutes, the 10-minute treatment with sodium hypochlorite solution was the most effective.

[0060] (3) Budding induction: Sterilized long-leaved bitter bamboo culm buds were inoculated onto the same basic culture medium as that of four-season bamboo and cultured for two weeks. Their growth was as follows: Figure 9 As shown.

[0061] (4) Bud proliferation: The induced buds of long-leaf bitter bamboo were transferred to 9 different proliferation media, the same as those of four-season bamboo. The base of the long-leaf bitter bamboo culms turned black and no buds were produced. The leaves turned yellow and dried up, and gradually died. Figure 10 This represents the situation of *Phyllostachys longifolia* after two weeks of propagation under the same plant growth regulator ratio as treatment group 4 in Table 2, with no new propagating buds produced. Figure 11 The results show the situation of *Phyllostachys longifolia* after four weeks of propagation under the same plant growth regulator ratio as treatment group 4 in Table 2. New buds sprouted from the bud points on the culms of *Phyllostachys longifolia*, but no proliferating buds were produced. Figure 12 The results show the situation of *Phyllostachys longifolia* after six weeks of propagation under the same plant growth regulator ratio as treatment group 4 in Table 2. No new buds were produced at the bud points on the culms, the leaves withered and turned yellow, growth deteriorated, and the plant gradually died. These results demonstrate that the unique formula suitable for the propagation of *Phyllostachys longifolia* buds is not suitable for the propagation of buds in other bamboo species such as *Phyllostachys longifolia*.

[0062] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A rapid propagation method for obtaining clumps and rooted seedlings from seasonal bamboo culm buds, characterized by, The method comprises the following steps: (1) taking 1-2 year old branches of Oligostachyum lubricum with culm buds as explants and sterilizing the explants; (2) inoculating the sterilized explants on a basic medium, wherein the basic medium is MS+3 mg / L 6-BA+30 g / L sucrose+2.5 g / L phytagel; (3) after two weeks of culture, transferring the explants to a proliferation medium, wherein the proliferation medium is 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) culturing on the proliferation medium for four to eight weeks to form a cluster of buds; (5) inoculating the proliferated cluster of buds on a rooting medium to root, wherein 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 phytagel.

2. The method of claim 1, wherein, 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.

3. The method of claim 1, wherein, The branch with culm buds is cut into 1.5-2 cm long branches with buds and then sterilized.

4. The method of claim 1, wherein, The sterilization operation is as follows: after alcohol treatment, washing with sterile water for 3-5 times, then treating with 2% sodium hypochlorite solution, and washing with sterile water for 3-5 times.

5. The method of claim 4, wherein, The alcohol treatment time is 60 s, the 2% sodium hypochlorite solution treatment time is 8 min, and the sterile water washing time is 2 min each time.

6. The method of claim 1, wherein, The culture condition is 25±1℃, light intensity of 1200-1500 lx, and light duration of 14 h / d.

7. Application of the rapid propagation method according to any one of claims 1-6 in the preparation of a cluster of Oligostachyum lubricum buds and rooted seedlings.

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