Method for inducing dendrocalamus malus polyploidy by taking callus as material

Abstract

The invention relates to a dendrocalamus brandisii polyploidy induction method taking calluses as materials, which comprises the following steps: 1) using shoot tips of propagation buds cultured by dendrocalamus brandisii embryos as explants, and inducing the calluses; (2) in a liquid suspension environment, colchicine is used for treating the dendrocalamus brandisii calluses; and 3) subculturing the treated calluses and domesticating regenerated seedlings. The method for performing polyploid induction on the dendrocalamus brandisii in the liquid suspension environment by using the callus as the material is explored, the optimal result is obtained by treating the embryogenic callus of the dendrocalamus brandisii for 48 hours by using 100 mgL <-1 > colchicine, and a new thought and method can be provided for bamboo breeding problems.

Description

Technical Field

[0001] The invention belongs to the technical field of plant polyploid induction, and specifically designs a polyploid induction method of Malayan sweet bamboo using callus tissue as material. Background Art

[0002] As one of the world's important forest resources, bamboo has important ecological, economic, and cultural values ​​in many countries around the world. China is one of the countries with the richest bamboo resources in the world. In 2023, the total output value of the bamboo industry was about 500 billion yuan, and the bamboo industry has become one of China's important forest pillar industries. Bamboo is mainly propagated asexually, so there is little genetic variation within the bamboo population and bamboo resources are scarce. And due to its biological characteristics such as long nutritional cycle and unstable flowering period, it is difficult and time-consuming to carry out traditional breeding in bamboo, so there are few reports on artificial breeding of bamboo.

[0003] After undergoing chromosome doubling, plants usually show advantages such as strong plants and strong stress resistance. Therefore, ploidy breeding technology is often used to improve fruit quality, enhance plant resistance to lodging and enlarge leaves. Artificial induction of polyploidy has become an important way to improve the quality of original varieties. As one of the mature technologies of plant artificial breeding, polyploidy has been widely used in various plants. Commonly used polyploidy breeding methods include physical mutagenesis and chemical mutagenesis. Due to factors such as the low mutagenesis rate of physical mutagenesis, current research mostly uses chemical mutagenesis for polyploidy induction, among which colchicine is more widely used. However, in bamboo plants, the research is still at the stage of inducing seeds and anthers. Due to factors such as the long flowering cycle of bamboo plants, the difficulty in predicting the flowering period, and the low fruiting rate, this method is time-consuming and laborious, and the appearance of chimeras leads to problems such as low induction rate and unstable traits.

[0004] Dendrocalamus asper is a large clumping bamboo species in the genus Dendrocalamus of the subfamily Bambusoideae of the Poaceae family. Its shoots are delicious and nutritious, and it is a delicious and healthy green vegetable. Its stems are tall and straight, and are often used as raw materials for construction, handicrafts and paper in Southeast Asian countries. The whole bamboo has a beautiful shape and is used for landscaping, with extremely high economic and ornamental value. However, due to the biological characteristics of bamboo plants, there are few reports on the breeding of Dendrocalamus asper, and its reproduction method is mostly asexual, resulting in a shortage of its improved variety resources. This invention is based on the mature in vitro regeneration system of Dendrocalamus asper in the laboratory, takes Dendrocalamus asper as the research object, uses bud-induced embryonic callus as the material, uses colchicine for chemical mutagenesis, and doubles its chromosome number. Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide a method for inducing polyploidy of Malayan sweet bamboo using callus as material, which is specifically achieved through the following technical solutions:

[0006] Obtaining embryonic callus of Malayan sweet bamboo includes the following steps:

[0007] 1) Obtaining tissue culture seedlings of Malayan sweet dragon bamboo: Peel off the shell of Malayan sweet dragon bamboo seeds and place them in a beaker, soak them in running water for 24 hours, use 0.5% sodium hypochlorite solution to vacuum filter for 20 minutes, rinse with sterile water, peel off the embryo under a stereo microscope, inoculate it into MS culture medium, culture for 28 days, and move it to bud proliferation culture medium (containing 1mg·L -1 6-Benzylaminopurine (6-BA), 1 mg·L -1 Kinetin (KT), 0.25 mg·L -1 The culture was continued in a wide-mouth bottle containing naphthaleneacetic acid (NAA), 30 g / L sucrose and 0.36% agar by weight of the medium, pH 5.7, and the culture conditions were a light intensity of 45 μmol·m -2 ·s -1 , photoperiod: 16 h light / 8 h dark, temperature 25 ± 2 °C, 0.4-0.6 cm sterile stem segments with buds were used as explants for subsequent experiments;

[0008] 2) Callus induction and subculture of D. truncatum: Plants with good growth status were selected from D. truncatum tissue culture seedlings, and stem segments with buds were cut as explants and inoculated into callus induction medium (containing 3 mg·L -1 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.5 mg·L -1 Naphthaleneacetic acid (NAA), 30 g / L sucrose and 0.36% agar by weight of the medium MS solid medium, pH 5.7), callus was initially formed after 3 weeks of induction. Milky white, dense granular callus was selected and inoculated into proliferation medium (containing 3 mg·L -1 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.5 mg·L -1 The culture was carried out on MS solid medium containing 6-benzylaminopurine (6-BA), 30 g / L sucrose and 0.36% agar by weight of the medium, pH 5.7. After 30 days of proliferation culture, callus tissue in good condition was selected for subsequent experiments.

[0009] The application of polyploid induction of Malayan sweet dragon bamboo using callus tissue as material in a liquid suspension environment includes the following steps:

[0010] 1) Treating the callus of Malayan sweet dragon bamboo with colchicine in a liquid suspension environment: Using a liquid medium containing colchicine to suspend the callus of Malayan sweet dragon bamboo. The liquid medium uses MS medium as the basic medium, supplemented with 1.0 mg·L -1 6-Benzylpurine (6-BA), 1.0 mg·L -1 Kinetin (KT), 0.25 mg·L -1 Naphthaleneacetic acid (NAA) and 30 g / L sucrose, pH 5.7;

[0011] 2) Subculture of treated callus and acclimatization of regenerated seedlings: The treated callus was transferred to differentiation medium (containing 2.0 mg·L -1 6-BA, 1.0mg·L -1 KT, 0.25mg·L -1 NAA, 30 g / L sucrose and 0.36% agar by weight of the medium MS solid medium, pH 5.7) for differentiation culture. After the callus tissue differentiated and grew into robust regenerated seedlings, the regenerated seedlings were transferred to the rooting medium (containing 3.0 mg·L -1 Indole-3-acetic acid (IAA), 30 g / L sucrose and 0.36% agar by weight of the medium MS solid medium, pH 5.7) were used to induce rooting. After a strong root system was grown, the regenerated seedlings were taken out and acclimatized in a mixed matrix (perlite, peat soil, vermiculite, prepared in a ratio of 1:1:1) and covered with a plastic bag to prevent water loss. After 7 days of cultivation, a small gap was cut in the plastic bag, and the gap was continuously enlarged in the next 7 days until the plastic bag was removed;

[0012] 3) DNA content determination and polyploid phenotype analysis of Malayan sweet dragon bamboo regeneration seedlings: The leaves of Malayan sweet dragon bamboo regeneration seedlings differentiated by colchicine treatment were collected, and the DNA content was determined by flow cytometry, and the samples that met the peak of polyploid Malayan sweet dragon bamboo were marked. Morphological observations were compared between the successfully domesticated Malayan sweet dragon bamboo polyploid regeneration seedlings and ordinary regeneration seedlings, such as plant height, leaf length and leaf width, and measurements and statistics were performed to obtain accurate data.

[0013] Furthermore, in step 1), the embryonic callus induced from the bud-bearing stem segments of Dracaena serrata is used as the test material to carry out the polyploid induction experiment.

[0014] Furthermore, in step 1), the callus tissue was induced to polyploidy by liquid suspension method, and the colchicine concentration was set to 100 mg·L -1 , the processing time is 48 hours.

[0015] Furthermore, the specific steps of step 1) inducing callus tissue of Dracaena serrata are as follows:

[0016] 1-1) Select callus tissues of Phyllostachys truncatulae with good growth condition and place them in a sterilized conical flask.

[0017] 1-2) Add 50 ml of 100 mg·L -1 Colchicine, 1mg·L -1 6-Benzylaminopurine (6-BA), 1 mg·L -1 Kinetin (KT), 0.25 mg·L -1 MS liquid culture medium containing naphthaleneacetic acid (NAA) and 30 g / L sucrose, wherein the pH value of the culture medium is 5.7, is sealed in a conical flask and treated in a constant temperature shaker at 25°C for 48 hours.

[0018] 1-3) After the callus was treated, it was rinsed with sterile water for 3 times, and the excess water was removed with filter paper and then inoculated into the differentiation medium of Malayan sweet bamboo (containing 2.0 mg·L -1 6-BA, 1.0mg·L -1 KT, 0.25mg·L -1 Differentiation culture was carried out on MS solid medium containing NAA, 30 g / L sucrose and 0.36% agar by weight of the medium, pH 5.7).

[0019] 1-4) in differentiation medium (containing 2.0 mg·L -1 6-BA, 1.0mg·L -1 KT, 0.25mg·L -1 The adventitious bud induction rate and callus mortality rate were calculated after culturing on MS solid medium containing NAA, 30 g / L sucrose and 0.36% agar by weight of the medium, pH 5.7 for 30 days.

[0020] Furthermore, flow cytometry was used to screen out the Malayan sweet bamboo plants whose DNA content was double that of the control group.

[0021] The present invention uses a concentration of 100 mg·L -1 The callus tissue of Malayan sweet dragon bamboo was treated with colchicine for 48 hours to induce polyploidy, thereby obtaining a higher polyploidy induction rate, and the regenerated seedlings of Malayan sweet dragon bamboo with doubling chromosomes were identified by flow cytometry.

[0022] The present invention proves that the embryonic callus of Malayan sweet dragon bamboo is treated with 100mg·L -1The colchicine liquid medium was dark cultured and oscillated at 1200rpm to treat the callus for 48 hours. Under controllable browning and vitrification conditions, this scheme can most effectively produce chromosome-doubling regenerated plants, and obtain the largest number of chromosome-doubling plants at a low mosaic rate. At the same time, the colchicine concentration is also close to the semi-lethal concentration of the callus. Compared with wild-type plants, the guard cell size, leaf thickness and leaf size of the twelve-ploid Malayan sweet dragon bamboo have increased, suggesting that it may be superior to the wild type in terms of energy utilization and stress resistance. In the future, the stress resistance adaptability and promotion potential of polyploid sweet dragon bamboo can be explored by combining polyploid cultivation, physiological and biochemical analysis and molecular biotechnology. The present application provides a polyploid breeding technology based on a bamboo in vitro indirect regeneration system, which provides a new solution for the efficient cultivation of new polyploid bamboo germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Results of colchicine treatment of callus tissue in liquid suspension method; ((A) Browning rate of callus tissue at different colchicine concentrations and different treatment times; (B) Differentiation rate of callus tissue at different colchicine concentrations and different treatment times. In the figure, different lowercase letters indicate significant differences between different materials and concentrations at the p<0.05 level);

[0024] Figure 2 The results of flow cytometric analysis of Malayan sweet dragon bamboo and the morphological observation of different polyploids ((A), (D): hexaploid in the control group; (B), (E): twelve ploid in the experimental group; (C), (F): eighteen ploid in the experimental group);

[0025] Figure 3 The morphological characteristics of the leaves of Malayan sweet dragon bamboo with different ploidy ((A): comparison of leaf length; (B): comparison of leaf width; (C): comparison of length-to-width ratio; in the figure, different lowercase letters indicate significant differences between different materials and concentrations at the p<0.05 level);

[0026] Figure 4 Observation of guard cells of different ploidy in Phyllostachys truncatula (A: hexaploid in the control group; B: dodecaploid in the experimental group; C: octaploid in the experimental group; D: deformed guard cells; E: deformity rate of guard cells of different ploidy). DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with specific embodiments to facilitate a better understanding of the present technical solution.

[0028] Example

[0029] 1. Use the bud tips of the proliferating buds of Malayan sweet dragon bamboo embryo culture as explants to induce callus tissue according to the following steps:

[0030] 1) Obtaining tissue culture seedlings of Malayan sweet dragon bamboo: Peel off the shell of Malayan sweet dragon bamboo seeds and place them in a beaker, soak them in running water for 24 hours, use 0.5% sodium hypochlorite solution to vacuum filter for 20 minutes, rinse with sterile water, peel off the embryo under a stereo microscope, inoculate it into MS medium, culture for 28 days, and move it to bud proliferation medium (containing 1mg·L -1 6-Benzylaminopurine (6-BA), 1 mg·L -1 Kinetin (KT), 0.25 mg·L -1 The culture was continued in a wide-mouth bottle containing naphthaleneacetic acid (NAA), 30 g / L sucrose and 0.36% agar by weight of the medium, pH 5.7, and the culture conditions were a light intensity of 45 μmol·m -2 ·s -1 , photoperiod: 16 h light / 8 h dark, temperature 25±2℃, sterile stem segments with buds of about 0.5 cm were used as explants for subsequent experiments;

[0031] 2) Callus induction and subculture of D. truncatum: Plants with good growth status were selected from D. truncatum tissue culture seedlings, and stem segments with buds were cut as explants and inoculated into callus induction medium (containing 3 mg·L -1 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.5 mg·L -1 Naphthaleneacetic acid (NAA), 30 g / L sucrose and 0.36% agar by weight of the medium MS solid medium, pH 5.7), callus was initially formed after 3 weeks of induction. Milky white, dense granular callus was selected and inoculated into proliferation medium (containing 3 mg·L -1 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.5 mg·L -1 The culture was carried out on MS solid medium containing 6-benzylaminopurine (6-BA), 30 g / L sucrose and 0.36% agar by weight of the medium, pH 5.7. After 30 days of proliferation culture, callus tissue in good condition was selected for subsequent experiments.

[0032] 2. Using callus as material, polyploid induction of Malayan sweet dragon bamboo was carried out in a liquid suspension environment according to the following steps:

[0033] 1) Use colchicine to induce polyploidy in callus tissue: add 100 mg L -1 The callus of Malayan sweet bamboo was treated with liquid culture medium of colchicine for 48 hours. The liquid culture medium was Murashige and Skoog medium (MS) as the basic culture medium, supplemented with 1.0 mg·L -16-Benzylpurine (6-BA), 1.0 mg·L -1 Kinetin (KT), 0.25 mg·L -1 After the above treatment, the callus tissue was transferred to a medium containing 2.0 mg·L -1 6-BA, 1.0mg·L -1 KT, 0.25mg·L -1 Differentiation culture was carried out on MS solid medium containing NAA, 30 g / L sucrose and 0.36% agar by weight of the medium. After 30 days, the callus browning rate and differentiation rate were counted (browning rate = number of browning callus / total number of callus × 100%; differentiation rate = number of green callus / total number of callus × 100%).

[0034] 2) Subculture of callus tissue of D. truncatum and acclimatization of regenerated seedlings: The induced adventitious buds were cultured in adventitious bud induction medium for 30 days and then transferred to the subculture medium for regenerated seedlings of D. truncatum for subculture. -1 The plants were cultured in a 1 / 2MS solid medium containing indole-3-acetic acid (IAA), 30 g / L sucrose and 0.36% agar by weight of the medium for 30 days. After the plants grew strong roots, the regenerated seedlings were taken out, the medium on the roots was carefully cleaned and removed, and the plants were planted in a matrix mixed with peat and perlite, and covered with a plastic bag to prevent water loss. After 7 days of culture, a small gap was cut in the plastic bag, and the gap was continuously enlarged in the next 7 days until the plastic bag was removed.

[0035] Control Example 1: The callus tissue was subjected to polyploid induction treatment using a liquid culture medium without colchicine, and the other steps were the same as those in the embodiment.

[0036] Experimental Example 1: Polyploidy induction of callus tissue using different concentrations of colchicine

[0037] Under liquid culture conditions, the changes in colchicine concentration and treatment time had a significant effect on the growth state of callus tissue of Malayan sweet bamboo. With the increase of colchicine concentration, the callus browning rate showed an upward trend, and the differentiation rate also decreased. Under the same treatment time, with the increase of colchicine concentration in liquid culture medium, the differentiation rate of callus tissue showed a certain degree of decline ( Figure 1 , A), while the browning rate gradually increased ( Figure 1 , B). Under the condition of fixed treatment concentration, the browning rate showed an increasing trend with the extension of treatment time. Therefore, under the premise of not affecting the subsequent differentiation of callus into seedlings as much as possible, referring to factors such as chromosome doubling rate, the browning rate was 100 mg·L -1 The best colchicine concentration was used to treat callus for 48 hours. The results are shown in Table 1.

[0038] Table 1 Chromosome doubling rate of regenerated seedlings

[0039]

[0040] Test Example 2: Determination of DNA content in regenerated seedlings of Malayan sweet bamboo

[0041] Take the heart leaves of the regenerated seedlings of the embodiment as materials, use The samples were processed by PI Absolute P ploidy analysis kit and analyzed on cytoFLEX flow cytometer. The specific operation is as follows: take the leaf about 1 cm from the base of the core leaf and place it in a culture dish, add 3-4 drops of nuclear extract (total 500μl), use a sharp blade to quickly cut for 60 seconds, then add the remaining nuclear lysis solution to lyse and incubate for 60 seconds. After lysis, the sample was filtered with a 50μm filter membrane, and 2ml of nuclear staining solution was added. It was incubated in the dark for 30-60 minutes before analysis on the machine.

[0042] The results showed that the relative fluorescence intensity peak of some plants in the experimental example was located at 320, indicating that the DNA content in the cell nucleus doubled after colchicine treatment ( Figure 2 , B), i.e., chromosome-doubling plants (twelve ploid). In addition, the relative fluorescence intensity peaks of two experimental plants appeared near 500, indicating that the DNA in their cells increased by about two times compared with the control group ( Figure 2 , C), i.e., a plant that has doubled many times (octaploid).

[0043] In addition, compared with the control, the 18-ploid Malayan sweet bamboo was observed to be significantly deformed, with the plant being short overall, fewer tillers, shorter internodes, significantly smaller leaves, thicker and more fragile, with curled edges. The plant subsequently died due to poor growth. This may be the result of chromosome synapsis disorder ( Figure 2 , F).

[0044] Experimental Example 3: Comparison of leaf parameters of Phyllostachys truncatula with chromosome doubling

[0045] The leaves of the regenerated seedlings were collected for cytological comparison. 2As the material, 502 glue was used to stick the lower epidermis of the leaves on a glass slide. After the glue solidified, 50% sodium hypochlorite was used to bleach it for 2 hours. After the sodium hypochlorite was rinsed with running water, the mesophyll and other excess tissues were brushed off under a gentle water flow with a toothbrush to obtain the lower epidermis of the leaves. After air drying, the stomata of the leaves were observed and compared under a microscope. The middle part of the heart leaf of Malayan sweet dragon bamboo was taken, and sliced ​​by hand with a sharp blade. The thin and uniform sample was placed on a glass slide, distilled water was dripped, and a cover glass was covered to make a temporary slide, which was observed under a microscope. The wild type and chromosome-doubled Malayan sweet dragon bamboo that were domesticated and transplanted to the greenhouse for 90 days were selected as materials. The leaf length and width of the second to sixth mature leaves on the branches with a diameter of about 5 mm that grew at the same time this year were measured, and the leaf length-width ratio was counted and calculated.

[0046] Microscopic observation of the stomata on the lower epidermis of leaves of dodecaploid and octaploid Malayan sweet bamboo showed that compared with the hexaploid control group, dodecaploid and octaploid plants showed a typical phenomenon of enlarged guard cells ( Figure 4 , A~C). The length and width of guard cells were quantitatively analyzed using ImageJ software. The statistical results are shown in Table 2. The guard cell length of the dodecaploid plant increased by 40.10% and the width increased by 19.32%. In addition, guard cell deformities were observed in the stomata of the lower epidermis of the leaves of the 18-ploid Malayan sweet dragon bamboo ( Figure 4 , D), where the deformity rate of guard cells also increases synchronously with the increase of ploidy ( Figure 4 , E).

[0047] The cross-sections of the leaves of the dodecaploid and 18-ploid Malayan sweet bamboo were observed under a microscope, and the leaf thickness, upper epidermal thickness and lower epidermal thickness were measured and counted. The results are shown in Table 3. Compared with the hexaploid control group, the total leaf thickness and the thickness of the upper and lower epidermal cells of the dodecaploid and 18-ploid Malayan sweet bamboo showed a significant increase. Specifically, the leaf thickness of the dodecaploid plants increased by 47.12%, the upper epidermal thickness increased by 60.40%, and the lower epidermal thickness increased by 11.54%; the leaf thickness of the 18-ploid plants increased by 103.72%, the upper epidermal thickness increased by 144.14%, and the lower epidermal thickness increased by 109.46%. The above data results show that chromosome doubling can lead to a synchronous increase in the overall thickness of the leaves of Malayan sweet bamboo and the thickness of the upper and lower epidermal cells, and this increase effect is positively correlated with the ploidy level.

[0048] Table 2 Analysis of pore parameters

[0049]

[0050] Table 3 Leaf tissue parameter analysis

[0051]

[0052] Through comparative analysis with the hexaploid Malayan sweet bamboo, it was found that the leaves of the dodecaploid and 18ploid Malayan sweet bamboo had certain differences in morphological characteristics compared with the hexaploid plants ( Figure 3 , A~C). Specifically, the leaves of dodecaploid plants showed a significant increase trend, while the leaves of octaploid plants were relatively small. However, in terms of leaf length-width ratio, both dodecaploid and octaploid plants were higher than the hexaploid control group. The above results indicate that chromosome doubling has a significant effect on the size and morphology of the leaves of D. serrata, but the effect varies with the ploidy level.

Claims

1. A method for inducing polyploidy of Malayan sweet bamboo using callus as material, characterized in that The following steps are involved: 1) Obtaining tissue culture seedlings of Malayan sweet dragon bamboo: Peel off the shell of Malayan sweet dragon bamboo seeds and place them in a beaker, soak them in running water for 24 hours, use 0.5% sodium hypochlorite solution for vacuum filtration for 20 minutes, rinse them with sterile water, peel off the embryos under a stereo microscope, inoculate them into MS culture medium, culture them for 28 days, and move them to bud proliferation culture medium for further culture after the seedlings grow to 3-7 cm. The culture conditions are light intensity of 45 μmol·m -2 ·s -1 , photoperiod: 16 h light / 8 h dark, temperature 25 ± 2 °C, 0.4-0.6 cm sterile stem segments with buds were used as explants for subsequent experiments; 2) Induction and subculture of callus of D. truncatum: Select plants with good growth status from D. truncatum tissue culture seedlings, cut stem segments with buds as explants and inoculate them into callus induction medium. After 3 weeks of induction, callus is initially formed; select milky white, dense granular callus and inoculate it into proliferation medium for culture. After 30 days of proliferation culture, select embryonic callus with good status for subsequent experiments; 3) Using colchicine to treat the callus of Malayan sweet dragon bamboo in a liquid suspension environment: Using a liquid medium containing colchicine to suspend and induce the callus of Malayan sweet dragon bamboo; the liquid medium uses MS medium as the basic medium and adds 1.0 mg·L -1 6-Benzylpurine 6-BA, 1.0 mg·L -1 Kinetin KT, 0.25mg·L -1 Naphthaleneacetic acid NAA and 30 g / L sucrose, pH 5.7; 4) Subculture of treated callus and acclimatization of regenerated seedlings: the treated callus is transferred to a differentiation medium for differentiation culture; after the callus differentiates and grows to form robust regenerated seedlings, the regenerated seedlings are transferred to a rooting medium to induce rooting, and after a robust root system is grown, the regenerated seedlings are taken out, acclimatized in a mixed matrix, and the plants are covered with a plastic bag to prevent water loss. After 7 days of culture, a small gap is cut in the plastic bag, and the gap is continuously enlarged within the next 7 days until the plastic bag is removed to obtain a polyploid Malayan sweet dragon bamboo polyploid plant.

2. A method for inducing polyploidy of Malayan sweet bamboo using callus as material as claimed in claim 1, characterized in that In step 1), the bud proliferation medium contains 1 mg·L -1 6-Benzylaminopurine 6-BA, 1 mg·L -1 Kinetin KT, 0.25mg·L -1 MS solid medium containing naphthylacetic acid NAA, 30 g / L sucrose and 0.36% agar by weight of the medium, with a pH value of 5.

7.

3. The polyploid induction method of Malayan sweet bamboo using callus as material as claimed in claim 1, characterized in that In step 2), the induction medium contains 3 mg·L -1 2,4-Dichlorophenoxyacetic acid 2,4-D, 0.5mg·L -1 MS solid medium containing naphthylacetic acid NAA, 30 g / L sucrose and 0.36% agar by weight of the medium, with a pH value of 5.

7.

4. The polyploid induction method of a Malayan sweet bamboo using callus as material as claimed in claim 1, characterized in that In step 2), the proliferation medium contains 3 mg·L -1 2,4-Dichlorophenoxyacetic acid 2,4-D, 0.5mg·L -1 MS solid medium containing 6-benzylaminopurine 6-BA, 30 g / L sucrose and 0.36% agar by weight of the medium, with a pH value of 5.

7.

5. The polyploid induction method of Malayan sweet bamboo using callus as material as claimed in claim 1, characterized in that In step 3), the callus tissue was induced to polyploidy using the liquid suspension method, and the colchicine concentration was set to 100 mg·L -1 , the processing time is 48 hours.

6. The polyploid induction method of Bamboo Sphaerocarpa using callus as material as claimed in claim 1, characterized in that In step 3), the specific steps of inducing callus are: 6-1) Select callus tissues of Phyllostachys truncatulae with good growth condition and place them into sterilized conical flasks; 6-2) Add 50 ml of 100 mg·L -1 Colchicine, 1mg·L -1 6-Benzylaminopurine 6-BA, 1 mg·L -1 Kinetin KT, 0.25mg·L -1 MS liquid culture medium containing NAA and 30 g / L sucrose, wherein the pH value of the culture medium is 5.7, and the conical flask is sealed and treated in a constant temperature shaker at 25°C for 48 hours; 6-3) After the callus treatment, it was rinsed with sterile water for 3 times, and the excess water was removed with filter paper, and then inoculated on the differentiation medium of Malayan sweet dragon bamboo for differentiation culture; 6-4) After 30 days of culture on differentiation medium, the adventitious bud induction rate and callus mortality were counted.

7. A method for inducing polyploidy of Phyllostachys truncatula using callus as material as claimed in claim 1 or 6, characterized in that The differentiation medium contains 2.0 mg·L -1 6-BA, 1.0mg·L -1 KT, 0.25mg·L -1 MS solid medium containing NAA, 30 g / L sucrose and 0.36% agar by weight of the medium, with a pH value of 5.

7.

8. The polyploid induction method of Bamboo Sphaerocarpa using callus as material as claimed in claim 1, characterized in that In step 4), the rooting medium contains 3.0 mg·L -1 MS solid medium containing indole-3-acetic acid IAA, 30 g / L sucrose and 0.36% agar by weight of the medium, with a pH value of 5.

7.

9. The polyploid induction method of Bamboo Sphaerocarpa using callus as material as claimed in claim 1, characterized in that In step 4), the mixed matrix is ​​perlite, peat soil and vermiculite, which are prepared in a ratio of 1:1:1.

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