CN115362940A - Rapid seedling culture method for pitcher plant tissue culture and application thereof - Google Patents

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a method for quickly culturing seedlings of pitcher plant by tissue culture and application thereof, and the method comprises the following steps: cleaning explants, (2) disinfecting explants, (3) inducing cluster buds, (4) proliferating cluster buds and (5) rooting cluster buds. The invention provides a method for quickly culturing the seedlings of pitcher plant by tissue culture, which has the advantages of low induced browning rate, high subculture multiplication coefficient, short multiplication period, capability of getting out of the pitcher during rooting and the like.

Description

Rapid seedling raising method for nepenthes tissue culture and application thereof

Technical Field

The invention relates to the technical field of plant tissue culture, in particular to a method for quickly culturing seedlings of pitcher plant by tissue culture and application thereof.

Background

Nepenthes (Nepenthes misabis), also known as pigling cages, catchfly worms, doll bottles, money and treasures, bao and the like are perennial herbaceous plants of the Nepenthes, china is mainly distributed in the west and south of Guangdong and Hainan, and abroad is mainly distributed in various countries such as Cambodia, burma, laos, thailand, vietnam, north Australia and great oceanics. Common nepenthes generally grow in swamp, roadside, mountain waist, mountain top and other shrubs with the elevation of 50-400 m, on grasslands or under forests.

The written description of nepenthes began with the ethine de flareal, the dominator in madagaska, france, 1658, and Joseph Banks, 1789, introduced it for the first time to the royal garden of plants, uk. The coquettish tendrils, beautiful leaf cages and peculiar predation mode arouse people's interest, it is often used in potted plant or hanging the basin and admire, very elegant and unique, also have the function of eliminating indoor winged insects, air-purifying, it is one of the main ornamental insect-eating plants in our country at present. The nepenthes are recognized by people at the earliest in China because of medicinal value, and famous medical books such as Chinese materia medica, guangdong traditional Chinese medicine, guangxi materia medica selection edition, national Chinese herbal medicine compilation and the like have more detailed records on the nature, taste and meridian tropism of the nepenthes. The nepenthes are used as a medicine by whole plants, have the effects of moistening lung and relieving cough, clearing heat and promoting diuresis and removing dampness, and relieving fever and swelling, are mainly used for treating lung dryness and hemoptysis, common cold and cough, whooping cough, jaundice, dysentery, lithangiuria, gastric and duodenal ulcer, hypertension, diabetes, sore and carbuncle and other diseases, and are shown to contain effective components such as flavonoid glycoside, phenols, amino acid, polysaccharides, anthraquinone glycoside and the like through modern medical research.

Due to the ornamental value and the medicinal value, the market demand of the pitcher plant is increased day by day, the damage of the wild resource to the ecological environment is reduced rapidly, and the artificial cultivation is the inevitable development of the pitcher plant. However, the nepenthes are male and female, the seed setting rate is low, the seed germination rate is extremely low, the germination period is long, and seedlings grow slowly after germination, so that the nepenthes seed propagation does not become a main mode of nepenthes propagation. The common nepenthes are bred in a cutting way, but the common nepenthes are bred in a cutting way, and seedlings are easy to obtain due to the rapid growth of the common nepenthes, so that the common nepenthes are the most important breeding way at present, but the defects of low breeding rate, long breeding period and the like still exist.

With the development of modern biotechnology, the research of pitcher plant tissue culture technology is gradually developed, and China has reports on the culture of relevant pitcher plant tissues in the 90 s of the last century, but the pitcher plant tissue culture technology still has the defects of serious initial induced browning, low successive multiplication coefficient, long multiplication period, almost no cage generation during rooting and the like (figure 1).

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the pitcher plant tissue culture method which has the advantages of low induced browning rate, high subculture multiplication coefficient, short multiplication period, capability of getting out of a cage during rooting and the like.

In order to realize the purpose, the invention adopts the technical scheme that: a method for tissue culture and rapid seedling culture of pitcher plant comprises the following steps:

(1) Cleaning explants: selecting tender stems of pitcher plant, cutting off leaves, placing in saturated soap water solution for rinsing for 8-10 min, then cleaning soap liquid on the surface with tap water, then dripping 2-3 drops of Tween-80, shaking for 10-15 min, and finally showering with tap water for 60-80 min;

(2) Explant disinfection: cleaning the explant, transferring the explant to a super clean workbench, disinfecting the explant by using 75% alcohol for 20-30 s, rinsing the explant by using sterile water for 2-3 times, disinfecting the explant by using a saturated bleaching powder solution for 20-25 min, rinsing the explant by using the sterile water for 3-4 times, disinfecting the explant by using a 0.1% mercuric chloride solution for 3-4 min, rinsing the explant by using the sterile water for 3-4 times, disinfecting the explant by using a 0.05% mercuric chloride solution for 8-9 min, and rinsing the explant by using the sterile water for 6-7 times;

(3) Inducing cluster buds: removing surface moisture of a sterilized explant, cutting wounds at two ends, cutting the sterilized explant into 1-2 cm long bud small segments, inoculating the bud small segments into 1/3MS + NAA 0.05-0.1 mg/L + CPPU 0.4-0.5 mg/L + KT 2.0-3.0 mg/L + silver nitrate 1.5-2.0 mu g/L + activated carbon 1.0g/L cluster bud induction culture medium, after inoculation, performing dark culture at the low temperature of 5 +/-2 ℃ for 6 days, transferring the culture medium to the environment at the temperature of 25 +/-2 ℃, continuing dark culture for 10 days, and performing alternate light-dark culture for 30-40 days under the illumination intensity of 2000-3000 LX and the sunlight intensity of 12 h;

(4) And (3) cluster bud multiplication: inducing the obtained cluster buds to cut into single buds, inoculating the single buds into a cluster bud proliferation culture medium of improved WPM, 0.1-0.15 mg/L NAA, 1.0-1.5 mg/L TDZ, 0.1-0.2 mg/L BR, 20-30 mg/L CH, 1.0-2.0 g/L beef extract and 0.5g/L active carbon, and culturing for 50-60 days under the environment of 25 +/-2 ℃ and the illumination intensity of 2000-3000 LX and the light-dark alternation of 12 hours of light;

(5) Rooting of cluster buds: the obtained cluster buds are multiplied and cut into single buds, the single buds are inoculated into a rooting culture medium of improved WPM, 0.5-0.6 mg/L NAA, 0.2-0.3 mg/L IBA, 3.0-4.0 g/L beef extract, 60-80 mg/L CH and 0.3g/L active carbon, and the single buds are cultured for 50-60 days under the condition of 25 +/-2 ℃ and light intensity of 3000-4000 LX and light-dark alternation of 12 hours of sunlight;

further, on the basis of the original WPM culture medium, the improved WPM is characterized in that monopotassium phosphate is changed to 40mg/L, ammonium nitrate is changed to 210mg/L, magnesium sulfate is changed to 40mg/L, potassium sulfate is changed to 340mg/L, calcium nitrate is changed to 70mg/L, thiamine hydrochloride is changed to 1.0mg/L, glycine is changed to 2.5mg/L, and the rest components are unchanged.

The beneficial technical effects of the invention are as follows:

1. according to the invention, when the condition of preliminary induction browning of pitcher plant is solved, 75% alcohol, saturated bleaching powder solution and high-concentration mercury bichloride are adopted for short-term disinfection combined with low-concentration mercury bichloride for long-term disinfection during explant disinfection, so that the disinfection pollution rate and the disinfection death rate can be effectively reduced, the damage of a disinfectant to the explant can be reduced, the browning rate of the explant in the culture process is reduced, and meanwhile, silver nitrate with a certain concentration and high-concentration active carbon are adopted for preliminary induction culture, so that the preliminary induction browning rate can hardly occur in the behavior period after inoculation is completed, and the complicated procedure of continuously rotating bottles in a short period is reduced. The browning of the nepenthes during primary induction is a very common problem, which is the key to whether the explants can induce the cluster buds, and the prior art can only solve the problem that the browning does not influence the induction of the cluster buds by a mode of continuously rotating the bottles for a period of 5-7 days.

2. According to the invention, NAA, KT and CPPU with extremely high activity are adopted for induction during cluster bud induction culture, the induction rate can reach 90%, the formula is the best induction culture medium formula of the induction rate of the pitcher plant at present, and the reason is that on one hand, the proper matching of the species and concentration of each plant growth regulator is realized, and meanwhile, the low browning rate can also be one of the reasons for improving the induction rate.

3. According to the invention, the effective collocation of NAA, TDZ and BR is adopted in the enrichment medium, so that the redifferentiation capability of the cluster buds is greatly improved, a large amount of cluster buds can grow in a short period, meanwhile, the enrichment medium adopts the improved WPM medium as a basic medium, and two proper organic nutritional ingredients, namely CH and beef extract, are added according to the autotrophic and heterotypic characteristics of the common nepenthes, so that the rapid growth of the cluster buds is effectively promoted, the proliferation period is greatly shortened, and the proliferation coefficient is improved. The prior nepenthes tissue culture technology has the advantages that the proliferation coefficient is mostly 2-3 during proliferation culture, and the proliferation period is generally 60-90 days, so that the production efficiency of the nepenthes during tissue culture production is reduced, and the production cost is increased.

4. In the tissue culture process of the pitcher plant, the pitcher plant can hardly be formed, but the pitcher plant without the cage generally needs a period of at least one year to be formed during the domestication process, so that the domestication period of the pitcher plant is overlong. In the invention, during rooting culture, improved WPM is adopted, CH and beef extract with higher concentration are added, and NAA and IBA with proper concentration are matched, so that nepenthes can be bound into a cage with a certain probability in the tissue culture process, the domestication period of the nepenthes is finally shortened, and the commodity cost of the nepenthes is reduced.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a graph of the results of seedlings cultured by prior art methods, with no "cages" present for tissue culture seedlings;

FIG. 2 is a graph showing the result of rooting culture of the multiple shoots in example 1, in which "cages" have been developed.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Test example 1

1. Multiplication culture of cluster buds

The application of 0.1mg/L NAA as the only auxin, and the matching of CPPU, KT, 6-BA, TDZ, ZT, BR, GA3 and 2-iP with different concentrations finds that the nepenthes induced bud is extremely sensitive to CPPU, TDZ and BR, three factors are selected for carrying out orthogonal test, and the specific test design is as follows:

TABLE 1 Experimental design

TABLE 2 results of the experiment

Z2, Z3 and Z8 are respectively selected to carry out repeated tests for many times, and the result shows that only Z3 and Z8 have high repeatability, but Z2 has poor repeatability, but Z8 has serious vitrification phenomenon, and the seedling after transferring is easy to cause stiff seedling.

2. Organic matter additive test for cluster buds

Because the Z3 and Z8 seedlings with high multiplication coefficients are weak and have vitrification phenomena in different degrees, the addition tests of CH, YE (yeast extract), beef extract, LH (hydrolyzed milk protein), coconut milk, coconut juice, banana and potato with different concentrations are carried out on the basis of the two formulas, and the CH, YE, beef extract and LH are found to have certain effects of strengthening seedlings and reducing vitrification rates.

Orthogonal experiments were performed on the basis of the above, and the specific experimental design is shown in table 3:

TABLE 3 Experimental design

The results of the Z3 addition organics test are shown in table 4:

TABLE 4 results of the experiment

From the above table, it can be seen that CH with an appropriate concentration can significantly reduce the occurrence of vitrification and also can strengthen the seedling to a certain extent, and YE with a low concentration can also reduce vitrification and seedling to a certain extent, but a certain number of leaves can be blackened no matter how much the concentration is, so that a part of the seedling can not continue to grow, thereby reducing the multiplication coefficient, and the beef extract with an appropriate concentration has a significant seedling strengthening effect, while LH is added to have a certain seedling strengthening effect, but greatly reduce the multiplication coefficient. And CH and beef extract are added in a matching manner, so that the seedlings can be strengthened, and the multiplication coefficient can be improved to a great extent.

The results of the Z8 addition organics test are shown in table 5:

TABLE 5 results of the experiment

Treatment ofThe result	Coefficient of multiplication	Others are	Processed \ result	Coefficient of proliferation	Others
						Y1	3.9	Weak seedling with much vitrification	Y6	3.0	The seedlings were weak, and there was little vitrification and little leaf blackening.
Y2	3.2	The seedlings are strong, have more vitrification, and have certain seedling leaves which turn black.	Y7	2.7	The seedlings were slightly robust and had a small amount of vitrification.
						Y3	2.5	Weak seedling, more vitrification and blackening seedling leaves.	Y8	1.9	The seedlings are strong, a small amount of vitrification occurs, but more leaves become black.
Y4	3.7	The seedlings are strong and have a small amount of vitrification,	Y9	3.1	the seedlings are strong, a small amount of vitrification occurs, and more seedling leaves become black.
						Y5	4.0	The seedling is strong, has a small amount of vitrification, and has a small amount of seedling leaf blackening

It can be seen from table 5 above that no matter what kind and concentration of organic matter is matched, the occurrence of vitrification can not be completely avoided, and the proliferation coefficient is also significantly lower than that of the plant growth regulator combination of Z3 and the organic matter addition combination of Y5.

3. Rooting culture of cluster buds

In the proliferation culture research, CH and beef extract are found to enable the proliferated seedlings to be stronger, so that the vitrification phenomenon is avoided, and the 'cage' can be promoted to a certain extent.

During rooting culture, a combination of no organic matter and CH and beef extract with certain concentration is adopted for testing, and the specific test design is as follows.

TABLE 6 Experimental design

Serial number/organic matter	CH（mg/L）	Beef extract (g/L)	Serial number/organic matter	CH（mg/L）	Beef extract (g/L)
						S1	0	0	S9	50	0
S2	0	1.0	S10	50	1.0
						S3	0	2.0	S11	50	2.0
S4	0	4.0	S12	50	4.0
						S5	20	0	S13	100	0
S6	20	1.0	S14	100	1.0
						S7	20	2.0	S15	100	2.0
S8	20	4.0	S16	100	4.0

TABLE 7 test results

Numbering	Rooting rate	Root state	60 days knot rate	Numbering	Rooting rate	Root state	Rate of formation of cage in 60 days
								S1	100%	Is extremely thin and weak	1.4%	S9	100%	Is relatively thin and weak	4.9%
S2	100%	Thin and weak	1.6%	S10	100%	Is relatively thick and strong	6.7%
								S3	100%	Is relatively thin and weak	2.3%	S11	100%	Rough and strong	7.7%
S4	100%	Is relatively thick and strong	3.6%	S12	100%	Is very thick and strong	15.3%
								S5	100%	Is extremely thin and weak	2.8%	S13	100%	Is relatively thick and strong	5.4%
S6	100%	Thin and weak	2.3%	S14	100%	Is relatively thick and strong	7.9%
								S7	100%	Is relatively thin and weak	3.9%	S15	100%	Rough and strong	10.8%
S8	100%	Is relatively thick and strong	5.8%	S16	100%	Is extremely thick and strong	16.7%

As can be seen from the above Table 7, during rooting culture, the root system is made stronger by adding organic matters with the concentrations and types of S12 and S16, and the caging rate is the highest in 60 days.

Example 1

A method for quickly culturing the seedlings of pitcher plant by tissue culture comprises the following steps:

1. cleaning explants: selecting tender stem segments of nepenthes, cutting off leaves, washing in saturated soap water solution for 8min, cleaning soap solution on the surface with tap water, dripping 2 drops of Tween-80, shaking for 10min, and finally showering with tap water for 60min;

2. explant disinfection: cleaning explant, transferring to a super clean workbench, sterilizing with 75% alcohol for 20s, rinsing with sterile water for 3 times, sterilizing with saturated bleaching powder solution for 20min, rinsing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride solution for 3min, rinsing with sterile water for 3 times, sterilizing with 0.05% mercuric chloride solution for 9min, and rinsing with sterile water for 7 times;

3. inducing cluster buds: absorbing surface moisture of a sterilized explant by using sterile paper, cutting wounds at two ends, cutting the sterilized explant into small sections with buds of 1cm in length, inoculating the small sections with buds to a 1/3MS + NAA 0.05mg/L + CPPU 0.4mg/L + KT 2.0mg/L + silver nitrate 1.5 mu g/L + activated carbon 1.0g/L cluster bud induction culture medium, carrying out dark culture at the low temperature of 5 ℃ for 6 days after inoculation, transferring the culture medium to the environment with the temperature of 25 ℃, continuing dark culture for 10 days, and then carrying out LX alternate culture for 40 days with the light intensity of 2000LX and the light intensity of 12h in sunlight;

4. and (3) cluster bud multiplication: inducing the obtained cluster buds to cut into single buds, inoculating the single buds into a cluster bud proliferation culture medium of improved WPM + NAA 0.1mg/L + TDZ 1.0mg/L + BR 0.1mg/L + CH 20mg/L + beef extract 1.0g/L + active carbon 0.5g/L, and culturing for 60 days under the light-dark alternation of illumination intensity of 2000LX and daily illumination of 12h at the temperature of 25 ℃;

5. rooting of cluster buds: cutting the obtained cluster buds into single buds, inoculating the single buds into a rooting culture medium of modified WPM + NAA 0.5mg/L + IBA 0.2mg/L + + beef extract 3.0g/L + CH 60mg/L + activated carbon 0.3g/L, and culturing for 60 days under the conditions of light intensity of 3000LX and light and dark alternation of daily illumination for 12h at the temperature of 25 ℃ (figure 2);

6. domesticating rooted seedlings: cleaning the rhizomes of the pitcher plant, planting the rhizomes of the pitcher plant in a basal culture medium, domesticating the rhizomes of the pitcher plant in an environment with a shading rate of 75 percent, keeping the air humidity of 100 percent in the early stage of domestication, then gradually reducing the air humidity, and keeping the humidity of the water moss of 70 percent in the whole domestication period.

Example 2

A method for quickly culturing the seedlings of pitcher plant by tissue culture comprises the following steps:

1. cleaning explants: selecting tender stem segments of nepenthes, cutting off leaves, washing in saturated soap water solution for 10min, cleaning soap solution on the surface with tap water, dripping 3 drops of Tween-80, shaking for 15min, and finally showering with tap water for 80min;

2. and (3) disinfection of explants: cleaning the explant, transferring the explant to a super clean workbench, disinfecting the explant by using 75% alcohol for 30s, rinsing the explant by using sterile water for 3 times, disinfecting a saturated bleaching powder solution for 25min, rinsing the explant by using sterile water for 3 times, disinfecting a 0.1% mercuric chloride solution for 4min, rinsing the explant by using sterile water for 4 times, disinfecting a 0.05% mercuric chloride solution for 8min, and rinsing the explant by using sterile water for 7 times;

3. inducing cluster buds: absorbing surface moisture of a sterilized explant by using sterile paper, cutting wounds at two ends, cutting the sterilized explant into 2 cm-long small sections with buds, inoculating the small sections with buds to a 1/3MS + NAA 0.1mg/L + CPPU 0.5mg/L + KT 3.0mg/L + silver nitrate 2.0 mu g/L + activated carbon 1.0g/L primary cluster bud induction culture medium, carrying out dark culture at a low temperature of 3 ℃ for 6 days after inoculation, transferring the culture medium to an environment with a temperature of 23 ℃, continuing dark culture for 10 days, and then carrying out light-dark alternate culture for 40 days with a light intensity of 3000LX and a light intensity of 12h in sunlight;

4. and (3) cluster bud multiplication: inducing the obtained cluster buds to cut into single buds, inoculating the single buds into a cluster bud proliferation culture medium of improved WPM + NAA 0.15mg/L + TDZ 1.5mg/L + BR 0.2mg/L + CH 30mg/L + beef extract 2.0g/L + active carbon 0.5g/L, and culturing for 60 days under the condition of 23 ℃ illumination intensity of 3000LX and 12h illumination light and dark alternation;

5. rooting of cluster buds: cutting the obtained cluster buds into single buds, inoculating the single buds into a rooting culture medium of improved WPM + NAA 0.6mg/L + IBA 0.3mg/L + beef extract 4.0g/L + CH 80mg/L + active carbon 0.3g/L, and culturing for 60 days under the condition of 23 ℃ and light intensity of 4000LX and light-dark alternation of 12h of sunlight;

6. domestication of rooted seedlings: cleaning the rhizogenesis seedlings of the nepenthes, cleaning a base culture medium, planting the rhizogenesis seedlings in the water moss, domesticating the rhizogenesis seedlings in an environment with a shading rate of 75%, keeping the air humidity of 100% in the early stage of domestication, then gradually reducing the air humidity, and keeping the water moss humidity of 70% in the whole domestication period.

Comparative example 1

The domestication is carried out according to the best technology in the research on the fast propagation technology of the tissue culture of pitcher plant (Guangxi agricultural science [ J ],2007, 38 (2): 131 to 132) by the methods of Xieqing and Xianxie, etc., and the domestication is carried out according to the domestication technology in the embodiment 1 of the invention.

Comparative example 2

The domestication was carried out according to the best technique in the research on the fast propagation technique of pitcher plant (tropical forestry [ J ],2003,31 (2): 50 to 52) and according to the domestication technique in example 1 of the present invention.

Comparative example 3

The culture was performed according to the best technique in nepenthes tissue culture (agricultural science [ J ],2017, 5.

Comparative example 4

The method is implemented according to example 1 in the patent application No. CN1442044A, and domestication is carried out according to the domestication technology in example 1 of the invention.

Comparative example 5

The method is implemented according to example 1 in the patent of a method for quickly breeding pitcher plant in vitro (application number: CN 105519445A), and domestication is carried out according to the domestication technology in the example 1 of the invention.

Comparative statistics of the methods of examples 1 and 2, comparative examples 1-5, from disinfection contamination rate, disinfection mortality rate, browning rate during induction without flask rotation, 60-day proliferation factor, 30-day rooting rate, 60-day caging rate and acclimatization 120-day caging rate are given in the following table:

TABLE 8 comparative results

From table 8 above, it can be seen that the disinfection contamination rate of the methods disclosed in examples 2 and 3 is only higher than that of comparative example 5, although the disinfection death rate is higher than that of comparative example 3, the contamination rate of comparative example 3 is very high, and the browning rate of non-spinner bottles during induction, the proliferation coefficient of 60 days, the caging rate of 60 days and the caging rate of domesticated 120 days in the methods of examples 1 and 2 are all higher than those of the prior art comparative examples.

Finally, it should be noted that: although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and it is intended to cover in the claims the invention as defined in the appended claims.

Claims (7)

1. The method for tissue culture and rapid seedling raising of pitcher plant is characterized by comprising the following steps:

(1) Cleaning explants: selecting tender stems of pitcher plant, cutting off leaves, washing in saturated soap water solution for 8-10 min, washing with tap water, dripping 2-3 drops of Tween-80, shaking for 10-15 min, and finally showering with tap water for 60-80 min;

(2) Explant disinfection: cleaning the explant, transferring the explant to an ultra-clean workbench, and sequentially sterilizing the explant by using 75% alcohol, saturated bleaching powder solution, 0.1% mercuric chloride solution and 0.05% mercuric chloride solution;

(3) Inducing cluster buds: removing surface moisture of a sterilized explant, cutting wounds at two ends, cutting the sterilized explant into 1-2 cm long bud segments, inoculating the bud segments into a cluster bud induction culture medium, carrying out dark culture at the low temperature of 5 +/-2 ℃ for 6 days, transferring the culture medium to the environment at the temperature of 25 +/-2 ℃, continuing the dark culture for 10 days, and then carrying out dark culture for 30-40 days under the light-dark alternation of the illumination intensity of 2000-3000 LX and the sunlight of 12 h;

(4) And (3) cluster bud multiplication: inducing the obtained cluster buds to cut into single buds, inoculating the single buds into a cluster bud multiplication culture medium, and culturing for 50-60 days under the conditions of illumination intensity of 2000-3000 LX and 12h of sunlight in dark and light alternation at the temperature of 25 +/-2 ℃;

(5) Rooting cluster buds: the obtained cluster buds are cut into single buds, inoculated in a rooting culture medium and cultured for 50 to 60 days under the condition of illumination intensity of 3000 to 4000LX and illumination of 12 hours in sunlight and darkness alternately at the temperature of 25 +/-2 ℃.

2. The method according to claim 1, wherein in the step (2), the disinfection is carried out by: sterilizing with 75% alcohol for 20-30 s, rinsing with sterile water for 2-3 times, sterilizing with saturated bleaching powder solution for 20-25 min, rinsing with sterile water for 3-4 times, sterilizing with 0.1% mercuric chloride solution for 3-4 min, rinsing with sterile water for 3-4 times, sterilizing with 0.05% mercuric chloride solution for 8-9 min, and rinsing with sterile water for 6-7 times.

3. The method according to claim 1, wherein in step (3), the formula of the cluster bud induction medium is: 1/3MS + NAA 0.05-0.1 mg/L + CPPU 0.4-0.5 mg/L + KT 2.0-3.0 mg/L + silver nitrate 1.5-2.0 mug/L + activated carbon 1.0g/L.

4. The method according to claim 1, wherein in step (4), the formula of the culture medium for proliferating the multiple shoots is: improved WPM, 0.1-0.15 mg/L NAA, 1.0-1.5 mg/L TDZ, 0.1-0.2 mg/L BR, 20-30 mg/L CH, 1.0-2.0 g/L beef extract and 0.5g/L active carbon.

5. The method of claim 1, wherein in step (5), the rooting medium has the following formula: the improved WPM, NAA 0.5-0.6 mg/L, IBA 0.2-0.3 mg/L, beef extract 3.0-4.0 g/L, CH 60-80 mg/L and active carbon 0.3g/L.

6. The method according to claim 4 or 5, wherein the modified WPM is prepared by modifying potassium dihydrogen phosphate to 40mg/L, ammonium nitrate to 210mg/L, magnesium sulfate to 40mg/L, potassium sulfate to 340mg/L, calcium nitrate to 70mg/L, thiamine hydrochloride to 1.0mg/L, glycine to 2.5mg/L, and the balance of the ingredients to be unchanged, based on the original WPM medium.

7. Use of the method according to any one of claims 1 to 6 in the breeding of nepenthes.

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