CN116836909A

CN116836909A - Blumea balsamifera growth medium, tissue culture method and application

Info

Publication number: CN116836909A
Application number: CN202311057068.9A
Authority: CN
Inventors: 陈晓鹭; 谌婉赟; 陈振夏; 于福来; 黎行飞; 罗雪婷; 黄梅; 肖永锋; 黎玉兰
Original assignee: Tropical Crops Genetic Resources Institute CATAS
Current assignee: Tropical Crops Genetic Resources Institute CATAS
Priority date: 2023-08-22
Filing date: 2023-08-22
Publication date: 2023-10-03

Abstract

The invention belongs to the technical field of plant cultivation, and particularly relates to a blumea balsamifera growth medium, a tissue culture method and application. The blumea balsamifera growth medium of the invention takes an MS medium as a basic medium and also comprises: 0.2-0.3 mg/LNAA, 0.08-0.16 mg/LDA-6, 10mg/L vitamin C, 20mg/L inositol, 30g/L sucrose and 6g/L agar. The combined application of DA-6 and NAA improves rooting efficiency, root length and plant height of blumea balsamifera. The invention takes the blumea balsamifera germplasm stem segment with axillary bud and length of 6-20 mm as explant to carry out tissue culture seedling, the blumea balsamifera tissue culture seedling has high content of the blumea balsamifera, and can realize the production of fine variety seedling with blumea balsamifera stable inheritance.

Description

Blumea balsamifera growth medium, tissue culture method and application

Technical Field

The invention belongs to the technical field of plant cultivation, and particularly relates to a blumea balsamifera growth medium, a tissue culture method and application.

Background

Blumea balsamifera [ Blumea balsamifera (L.) DC ] is perennial herb of Blumea (Blumea) of Compositae, is a basic source plant of Blumea balsamifera tablet (L-borneol) in Chinese pharmacopoeia, and is prepared by extracting fresh leaves and processing to obtain crystal of Blumea balsamifera tablet (L-borneol). Blumea balsamifera is a native herb plant in tropical and subtropical asia, and has been used as a medicinal plant for over one thousand years, and blumea balsamifera contains various chemical components, wherein volatile oils, especially l-borneol, are the main chemical components of blumea balsamifera. Blumea balsamifera has effects of dispelling pathogenic wind, removing dampness, warming spleen and stomach, relieving diarrhea, promoting blood circulation, removing toxic substance, and various physiological activities such as antiinflammatory, anticancer, antifungal, and antimicrobial.

The L-borneol in blumea balsamifera has higher market value, but the blumea balsamifera lacks improved variety seedlings with stable inheritance at present because individual characters are rich in variation and are easily influenced by environment. The applicant collects a large amount of blumea balsamifera germplasm resources from blumea balsamifera producing areas around the country and stores the blumea balsamifera germplasm resources in a germplasm resource garden, and resource evaluation results show that the content difference of the L-borneol in different germplasm is remarkable, but since each germplasm has only one parent plant, whether the difference is caused by gene difference or influence of environment or plant diseases and insect pests on plants is not known. In addition, the existing blumea balsamifera propagation mode is a mode of mixing sexual reproduction and tillering Miao Moxing propagation of seedlings obtained by cross pollination of the obtained heterozygous seeds, so that the characters of the offspring of the seedlings are separated. The current situations cause barriers to blumea balsamifera germplasm evaluation and fine variety and good seedling breeding.

The tissue culture is a asexual propagation technology, in an artificial environment, tissue culture offspring can eliminate the influence of the character separation and environmental factors of seedlings produced by seed propagation, is an efficient way for rapidly propagating seedlings and retaining the advantages of fine seeds, has great significance for the preservation and evaluation of fine germplasm resources, can overcome the defects of the traditional seedling culture mode by using the tissue culture rapid propagation technology, improves the survival rate and the propagation efficiency, and provides sufficient materials for germplasm preservation, rapid propagation and later regeneration research of the fine seedlings.

Along with the promotion of blumea balsamifera germplasm resource investigation and breeding research work, the current blumea balsamifera tissue culture technology can not meet the current research and production requirements. These deficiencies are mainly manifested in both explants and culture effects.

In the aspect of explants, the existing blumea balsamifera tissue culture technology explants comprise stem segments with axillary buds (Li Lifeng, 2008. Blumea balsamifera tissue culture and GC-MS analysis of main chemical components of volatile oil, university of Guangxi, p.49.), tender stem segments (specifically, stem segments with 1-2 axillary buds or terminal buds 3-4 cm long, see Short5. Blumea balsamifera germplasm resource evaluation and tissue culture fast-growing system research [ D ]. Hainan university, 2023.DOI:10.27073/d.cnki. Ghadu.2021.000970.), leaves (Yan Min, liu Yan, shang Hongmin, 2014, tissue culture of blumea balsamifera, jiangsu agricultural science 42,33-35, chinese patent CN 105309315A) and roots (CN 114375834B). The axillary bud-bearing stem has the advantages of large quantity, availability, large volume, easiness in operation and difficulty in death, and the axillary bud-bearing stem is a mature stem, has developed vascular tissues and medulla, and pathogenic microorganisms easily reach and hide in the vascular bundles, so that detoxification cannot be realized, and particularly the method has high pollution rate and low success rate after in-vitro preservation and tissue culture for a period of time aiming at some important germplasm which grows in the field or in the field and is influenced by plant diseases and insect pests; the second explant is a stem segment with the length of 3-4 cm and containing 1-2 axillary buds or terminal buds, wherein the stem segment also has developed vascular bundles, is not essentially different from the first explant, and also loses the advantages of large quantity and easy availability; the third explant is leaf, which has the problems of developed vascular bundles and brought by the developed vascular bundles as the former two explants, and the culture must be firstly induced to generate callus and then induced to differentiate embryoid to grow into buds finally, and the method inevitably passes through the callus induction stage, which can possibly lead to uncontrollable somatic mutation, and can lead part or all of tissue culture offspring of high-content mother plants to become plants only containing low-concentration L-borneol, and the mutation cannot be intuitively found. The fourth explant is root and there are also vascular bundles and their associated problems. In addition, the stem tip is also an optional explant, and the stem tip culture particularly refers to the separation and the individual culture of a small group of meristematic tissues with a very small range (about 2-3 mm) of the stem tip, and has the advantages of good detoxification effect, high technical requirements on operators, small tissues and easy death. In summary, the explants used in the prior art have problems, either with the presence of vascular bundles leading to high contamination rates or with too small tissue pieces leading to low survival rates. According to the technology disclosed by the invention, according to the problems, the advantages of the two are combined, a tender stem section with the length of 6-20 mm and containing a stem tip is selected as an explant, and the explant avoids all the defects of the explant, so that on one hand, the tissue volume is larger than that of a meristematic region of the stem tip, the operation difficulty is low, the survival rate is high, and on the other hand, developed vascular tissues are not developed yet, pathogenic microorganisms do not arrive yet, the detoxification effect is good, and the pollution rate is low.

In the aspect of culture effect, the prior art also has the defects that: firstly, the prior art does not develop research on tissue culture technology aiming at specific germplasm, and along with the development of blumea balsamifera breeding technology, we find that the old technology can only support the tissue culture requirement of part of germplasm, while other germplasm needs to further research the supporting tissue culture technical support. Secondly, the blumea balsamifera tissue culture method with low rooting coefficient in the prior tissue culture technology, such as the embryoid approach of Chinese patent CN105309315A, uses leaves as explants to induce callus and embryoid, and then induces regeneration buds, wherein the average root number of the blumea balsamifera tissue culture seedlings is 5-7 per seedling. And then inoculating stem sections of the plant seeds into MS and 1/2MS culture media to obtain aseptic seedlings and inducing the aseptic seedlings to proliferate, root and hardening-seedling transplantation, wherein the stem sections are as described in Tang Makino and Yang Shiguan (see, tang Makino, huang Yanfen, kong Li, et al, blumea balsamifera seedling rapid propagation technical research [ J ]. Ind. Guizhou academy of sciences 2015,31 (03): 38-40, yang Shiguan, yang Meichun, blumea balsamifera tissue culture technical research [ J ]. Modern agriculture technology, 2012, (18): 151-153. "; rooting coefficient is generally 4-18 pieces/seedling, and rooting coefficient is low. Thirdly, in the tissue culture proliferation subculture process of blumea balsamifera, plant hormones are used, and the concentration of the hormones often used is high so as to achieve the highest proliferation efficiency, or the regeneration buds are obtained through the way of inducing callus firstly and then inducing embryogenesis of the callus, and uncontrollable somatic mutation is possibly caused, so that the characteristics of the original germplasm are changed. Because blumea balsamifera is a medicinal plant taking L-borneol as a main functional substance, somatic mutation generated in the tissue culture rapid propagation process can lead part or all of tissue culture offspring of a high-content mother plant to become a plant only containing L-borneol with low concentration, and the mutation cannot be intuitively found. However, all blumea balsamifera tissue culture researches before only take the tissue culture offspring as a research target, and the method does not aim at specific germplasm, does not consider the influence on comprehensive factors such as rooting rate, root length, plant height and the like of seedlings, and also does not consider the problem that whether the chemical characteristics of the tissue culture offspring are consistent with those of a parent plant, in particular: the prior tissue culture technology is truly found that the relative content of the L-borneol in the tissue culture offspring volatile oil is 17.2 percent, which is obviously lower than that of a wild strain (46.7 percent) (Li Lifeng, 2008. Tissue culture of blumea balsamifera and GC-MS analysis of main chemical components of the volatile oil. University of Guangxi, abstract and p 24-25), so that the urgent need of developing a new tissue culture technology capable of keeping the chemical characteristics of a mother strain is further enhanced, and the original L-borneol content characteristic of the mother strain which is well continued by the blumea balsamifera tissue culture offspring obtained by the technology disclosed by the invention is overcome the problems of the prior art.

Disclosure of Invention

The invention aims to provide a blumea balsamifera growth medium, which is used for tissue culture of blumea balsamifera and has high rooting coefficient. The invention provides a blumea balsamifera tissue culture medium and a tissue culture method, which can obtain a large number of high-quality blumea balsamifera tissue culture seedlings with maintained genetic stability.

In order to solve the problems, the invention provides the following technical scheme:

the invention provides a blumea balsamifera growth promoting culture medium, which takes an MS culture medium as a basic culture medium and further comprises the following components: 0.2-0.3 mg/LNAA, 0.08-0.16 mg/LDA-6, 10-50 mg/L vitamin C, 20-200 mg/L inositol, 30g/L sucrose and 4-10 g/L agar.

The invention provides the application of the blumea balsamifera growth culture medium in improving one or more of the following 1) to 3),

1) Rooting rate;

2) Plant height;

3) Root length.

The invention provides a blumea balsamifera tissue culture medium, which comprises an adventitious bud induction medium, a proliferation and subculture medium and a growth medium according to the technical scheme;

the adventitious bud induction culture medium takes an MS culture medium as a basic culture medium and further comprises: 0.1-0.5 mg/LNAA, 1.0-3.0 mg/L6-BA, 10-50 mg/L vitamin C, 20-200 mg/L inositol, 30g/L sucrose and 4-10 g/L agar;

The proliferation and secondary culture medium takes MS culture medium as basic culture medium, and further comprises: 0.1-0.5 mg/LNAA, 1.0-3.0 mg/L6-BA, 10-50 mg/L vitamin C, 20-200 mg/L inositol, 30g/L sucrose and 4-10 g/L agar.

The invention provides a blumea balsamifera tissue culture method, which adopts the growth culture medium according to the technical scheme and comprises the following steps:

inoculating blumea balsamifera explants on an adventitious bud induction culture medium to perform adventitious bud induction culture to obtain blumea balsamifera sterile adventitious buds;

inoculating the stem segments with axillary buds of the blumea balsamifera sterile adventitious buds to proliferation and subculture media for subculture proliferation to obtain blumea balsamifera single buds;

inoculating the blumea balsamifera single buds into a growth culture medium for rooting culture to obtain blumea balsamifera tissue culture seedlings;

and hardening off and transplanting the blumea balsamifera tissue culture seedlings.

Preferably, the explant comprises at least one of:

(a) Stem segments with axillary buds;

(b) Tender stem segment with stem tip and length of 6-20 mm.

Preferably, the temperature of the adventitious bud induction culture, the secondary proliferation culture and the rooting culture is 24-26 ℃ respectively.

Preferably, the adventitious bud induction culture, the secondary multiplication culture and the rooting culture are all carried out under the condition of alternate illumination, the illumination time is 11-13 h/d respectively, and the illumination intensity is 1000-1500 lx respectively.

Preferably, the adventitious bud induction culture time is 28-32 d; the time of the secondary proliferation culture is 28-32 d; the rooting culture time is 28-32 d.

Preferably, the proliferation and secondary culture medium takes MS culture medium as basic culture medium, and further comprises: 0.1-0.5 mg/LNAA, 1.0-3.0 mg/L6-BA, 10-50 mg/L vitamin C, 20-200 mg/L inositol, 30g/L sucrose and 4-10 g/L agar;

the adventitious bud induction culture medium takes an MS culture medium as a basic culture medium and further comprises: 0.1-0.5 mg/LNAA, 1.0-3.0 mg/L6-BA, 10-50 mg/L vitamin C, 20-200 mg/L inositol, 30g/L sucrose and 4-10 g/L agar.

The invention provides an application of the blumea balsamifera tissue culture method in blumea balsamifera germplasm breeding for maintaining the genetic stability of the content of the L-borneol.

The invention has the beneficial effects that: the invention provides an blumea balsamifera growth medium, which takes an MS medium as a basic medium and further comprises the following components: 0.2-0.3 mg/LNAA, 0.08-0.16 mg/LDA-6, 10-50 mg/L vitamin C, 20-200 mg/L inositol, 30g/L sucrose and 4-10 g/L agar.

In the growth medium provided by the invention, NAA (1-naphthylacetic acid) is beneficial to rooting of blumea balsamifera tissue culture seedlings and improvement of the plant height of the tissue culture seedlings; DA-6 (diethyl aminoethyl hexanoate) promotes the root growth of blumea balsamifera, improves the plant height of blumea balsamifera regenerated seedlings, and improves the rooting efficiency of the regenerated seedlings; the combined application of DA-6 and NAA synergistically improves the rooting rate of blumea balsamifera germplasm. The results of the examples show that: the rooting rate of blumea balsamifera tissue culture seedlings obtained by using the growth medium is 26.5-30.2 pieces/seedling, and the rooting coefficient is high. The growth medium provided by the invention solves the problem of low rooting coefficient of blumea balsamifera tissue culture seedlings, and has wide application prospect. In addition, the blumea balsamifera tissue culture medium and the tissue culture method provided by the invention can obtain a large number of high-quality blumea balsamifera tissue culture seedlings with maintained genetic stability.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.

FIG. 1 is a graph showing comparison of adventitious bud induction efficiency of 3 blumea balsamifera germplasm in MS medium added with 6-BA of different concentrations;

FIG. 2 shows comparison of adventitious bud rooting efficiency of 3 blumea balsamifera germplasm in MS medium added with NAA of different concentrations;

FIG. 3 is a graph showing comparison of rooting efficiency of adventitious buds of 3 blumea balsamifera germplasm in MS medium added with DA-6 of different concentrations;

FIG. 4 shows the seedling hardening and transplanting process of the blumea balsamifera 3 germplasm offspring tissue culture seedlings;

FIG. 5 is an ion chromatogram of a L-borneol control; in fig. 5, 1 represents a methyl salicylate internal standard, and 2 represents l-borneol;

FIG. 6 shows total ion chromatograms of extracts of 3 germplasm parent plants and offspring leaves of blumea balsamifera; in fig. 6, 1 represents a methyl salicylate internal standard, and 2 represents l-borneol;

FIG. 7 shows comparison of L-borneol content of 3 germplasm parent strains of blumea balsamifera;

FIG. 8 shows comparison of L-borneol content of 3 tissue culture offspring of blumea balsamifera.

Detailed Description

The invention provides an blumea balsamifera growth medium, which takes an MS medium as a basic medium and further comprises the following components: 0.2-0.3 mg/LNAA, 0.08-0.16 mg/LDA-6, 10-50 mg/L vitamin C, 20-200 mg/L inositol, 30g/L sucrose and 4-10 g/L agar.

In the invention, the growth medium takes MS medium as basic medium, and further comprises: 0.2-0.3 mg/LNAA, 0.08-0.16 mg/L DA-6, 10-50 mg/L vitamin C, 20-200 mg/L inositol, 30g/L sucrose and 4-10 g/L agar; preferably, the MS culture medium is used as a basic culture medium, and only contains 0.2-0.3 mg/LNAA, 0.08-0.16 mg/L DA-6, 10-50 mg/L vitamin C, 20-200 mg/L inositol, 30g/L sucrose and 4-10 g/L agar; more preferably, the MS culture medium is used as a basic culture medium, and only contains: 0.3mg/LNAA, 0.16mg/L DA-6, 10mg/L vitamin C, 20mg/L inositol, 30g/L sucrose and 6g/L agar. In the present invention, the concentration of DA-6 in the growth medium is 0.08 to 0.16mg/L, preferably 0.10 to 0.16mg/L, more preferably 0.16mg/L. The NAA concentration in the growth medium is 0.2 to 0.3mg/L, preferably 0.23 to 0.3mg/L, more preferably 0.3mg/L. The concentration of vitamin C in the growth medium is 10-50 mg/L, preferably 10-30 mg/L, more preferably 10mg/L. The concentration of inositol in the growth medium is 20 to 200mg/L, preferably 20 to 120mg/L, more preferably 20 to 60mg/L, and even more preferably 20mg/L. In the present invention, the concentration of agar in the growth medium is 4 to 10g/L, more preferably 5 to 8g/L, still more preferably 6.0g/L. In the present invention, the pH of the growth medium is preferably 6.0 to 6.3, more preferably 6.2.

The NAA in the growth medium provided by the invention is beneficial to rooting of blumea balsamifera tissue culture seedlings and improving the plant height of the tissue culture seedlings, DA-6 promotes the root growth of blumea balsamifera and improves the plant height of blumea balsamifera regenerated seedlings, and the rooting efficiency of the regenerated seedlings is improved. DA-6 and NAA are combined in the growth medium to improve the rooting rate of blumea balsamifera germplasm. Under the combined action of DA-6 and NAA, vitamin C directly participates in the formation of enzyme and protein and fat metabolism, the problem that plant cells cannot synthesize enough vitamin C in the culture process is solved, browning is prevented, inositol is helpful to generate pectin and cell walls, the formation of buds is promoted, the tissue reproduction and differentiation promotion effect is achieved, and sucrose is a necessary carbon source component in a culture medium, meanwhile, the osmotic pressure of the culture medium is maintained, and the bud proliferation of blumea balsamifera is promoted together. According to the invention, the plant growth regulator DA-6 is introduced into the blumea balsamifera tissue culture growth medium for the first time, and the problem of low rooting coefficient of blumea balsamifera tissue culture seedlings in the prior art is solved under the combined action of all components of the growth medium. The growth medium provided by the invention has good application effect, can simultaneously obtain a large number of tissue culture seedlings capable of maintaining genetic stability, and provides high-quality candidate germplasm and technical reference with popularization prospect for breeding blumea balsamifera excellent seedlings by means of asexual propagation.

The sources of NAA, DA-6, inositol, vitamin C, sucrose, agar and MS culture medium are not particularly limited, and conventional commercial products can be adopted.

The growth medium can be used for culturing adventitious buds and original buds of plants (the original buds of the plants comprise terminal buds and lateral buds), the adventitious buds and the original buds of the plants can be cut off, and the adventitious buds and the original buds of the plants can grow and root in the growth medium.

In the present invention, the source of blumea balsamifera is not particularly limited, and conventional blumea balsamifera can be used. In the embodiment of the invention, the blumea balsamifera is preferably 3 germplasm blumea balsamifera with low content of L-borneol, medium content of L-borneol and high content of L-borneol. The content of the low-L-borneol is 2.98-3.62 mg/g, the content of the medium-L-borneol is 4.8-5.42 mg/g, and the content of the high-L-borneol is 8.67-9.57 mg/g.

1) Rooting rate;

2) Plant height;

3) Root length.

The adventitious bud induction culture medium takes MS as a basic culture medium and further comprises: 0.1-0.5 mg/LNAA, 1.0-3.0 mg/L6-BA, 10-50 mg/L vitamin C, 20-200 mg/L inositol, 30g/L sucrose and 4-10 g/L agar;

In the present invention, the adventitious bud induction medium preferably uses MS as a minimal medium, and further comprises: 0.1-0.5 mg/LNAA, 1.0-3.0 mg/L6-BA, 10-50 mg/L vitamin C, 20-200 mg/L inositol, 30g/L sucrose and 4-10 g/L agar; more preferably, the culture medium is MS-based and contains only: 0.1-0.5 mg/LNAA, 1.0-3.0 mg/L6-BA, 10-50 mg/L vitamin C, 20-200 mg/L inositol, 30g/L sucrose and 4-10 g/L agar. In the present invention, the concentration of 6-BA in the adventitious bud induction medium is 1.0 to 3.0mg/L, preferably 1.3 to 2.5mg/L, more preferably 2.0mg/L. The concentration of NAA in the adventitious bud induction culture medium is 0.1-0.5 mg/L, preferably 0.3mg/L. The concentration of vitamin C in the adventitious bud induction culture medium is 10-50 mg/L, preferably 10mg/L. The concentration of inositol in the adventitious bud induction culture medium is 20-200 mg/L, preferably 20mg/L. In the present invention, the concentration of agar in the adventitious bud induction medium is 4 to 10g/L, preferably 6.0 to 6.5g/L, more preferably 6.0g/L. In the present invention, the pH of the adventitious bud induction medium is preferably 6.0 to 6.3, more preferably 6.2.

In the invention, the proliferation and secondary culture medium takes MS culture medium as basic culture medium, and further comprises: 0.1-0.5 mg/LNAA, 1.0-3.0 mg/L6-BA, 10-50 mg/L vitamin C, 20-200 mg/L inositol, 30g/L sucrose and 4-10 g/L agar; more preferably, the MS culture medium is used as a basic culture medium, and only contains: 0.1-0.5 mg/LNAA, 1.0-3.0 mg/L6-BA, 10-50 mg/L vitamin C, 20-200 mg/L inositol, 30g/L sucrose and 4-10 g/L agar.

In the present invention, the concentration of 6-BA in the proliferation and secondary culture medium is 1.0 to 3.0mg/L, preferably 1.3 to 2.0mg/L, more preferably 1.5mg/L. NAA concentration in the proliferation and subculture medium of the present invention is 0.1-0.5 mg/L, preferably 0.3mg/L. The concentration of vitamin C in the proliferation and secondary culture medium is 10-50 mg/L, preferably 10mg/L. The concentration of inositol in the proliferation and secondary culture medium of the invention is 20-200 mg/L, preferably 20mg/L.

In the present invention, the concentration of agar in the proliferation and secondary culture medium is 4 to 10g/L, preferably 6.0 to 6.5g/L, more preferably 6.0g/L.

In the present invention, the pH of the proliferation and secondary culture medium is preferably 6.0 to 6.3, more preferably 6.2.

The proliferation and subculture medium provided by the invention contains 6-BA and NAA, and the combined action of the 6-BA and NAA promotes the bud proliferation of blumea balsamifera. Under the combined action of 6-BA and NAA, vitamin C directly participates in the formation of enzyme and protein and fat metabolism, the problem that plant cells cannot synthesize enough vitamin C in the culture process is solved, browning is prevented, inositol is helpful to generate pectin and cell walls, the formation of buds is promoted, the tissue reproduction and differentiation promotion effect is achieved, and sucrose is a necessary carbon source component in a culture medium, meanwhile, the effect of maintaining osmotic pressure of the culture medium is achieved, and the bud proliferation of blumea balsamifera is promoted together.

According to the invention, the blumea balsamifera explant is inoculated on an adventitious bud induction culture medium to perform adventitious bud induction culture, so that the blumea balsamifera aseptic adventitious bud is obtained. The explant of the present invention preferably comprises at least one of the following:

(a) Stem segments with axillary buds;

(b) Tender stem segment with stem tip and length of 0.6-2 cm.

The length of the stem segment with axillary bud of blumea balsamifera of the invention is preferably 1-3 cm, more preferably 1.5cm. The blumea balsamifera stem section with the axillary buds is preferably obtained by cutting off side leaves of blumea balsamifera branches with the axillary buds. The blumea balsamifera stem segment with axillary bud is selected because the bud is obtained by the growth of the stem Duan Yisheng with axillary bud.

The invention preferably carries out cleaning and disinfection before cutting blumea balsamifera branches with buds into the stem sections with axillary buds of the explants. The cleaning according to the invention preferably comprises washing with a detergent and washing with running water, and the washing time of the detergent according to the invention is preferably 15-25 min, more preferably 20min; the running water washing time is preferably 15 to 25 minutes, more preferably 20 minutes. The detergent of the present invention is not particularly limited, and conventional products may be used. The invention is not particularly limited to water used in the running water flushing, and conventional tap water is adopted.

The sterilization mode of the invention preferably comprises the following steps: after cleaning, the mixture is soaked in sodium hypochlorite solution with the mass concentration of 1% for 14-16 min, and is washed by sterile water for 4-6 times. 2-3 drops of tween 20 are added when the 0.1% sodium hypochlorite solution is soaked, and the tween 20 is used as a surfactant, so that the sterilizing effect of the sodium hypochlorite is enhanced. The soaking time of the invention with 1% sodium hypochlorite solution is preferably 14-16 min, more preferably 15min. After the 1% sodium hypochlorite solution of the present invention is soaked, it is preferably rinsed with sterile water for 4 to 6 times, more preferably 5 times.

The blumea balsamifera stem with the axillary buds is prepared by cleaning and sterilizing the blumea balsamifera stem with the axillary buds, and then the blumea balsamifera stem with the axillary buds is obtained as an explant. The invention preferably inoculates the sterilized blumea balsamifera stem segments with axillary buds on an adventitious bud induction culture medium for adventitious bud induction culture to obtain the blumea balsamifera sterile adventitious buds.

The tender stem section with the stem tip and the length of 0.6-2 cm is prepared as an explant after cleaning and sterilizing the blumea balsamifera shoot. The length of the tender stem segment with the stem tip according to the present invention is preferably 0.6 to 2cm, more preferably 1.0 to 1.5cm, and even more preferably 1.5cm. In the prior art, the stem tip is used as an explant, and although the detoxification effect can be achieved, the tissue used for culturing the stem tip is small, generally smaller than 3mm, the tissue survival rate is low, the technical requirement on an experimenter is high, and the popularization is not easy. The invention uses stem tip and tender stem tissue with the lower range of about 10-15 mm as explant, and the area has no developed vascular bundle, so that the detoxification effect can be achieved, in addition, the tissue volume is large, the technical requirement on experimenters is low during operation, the culture survival rate is high, and the invention has certain advantages. As the explant, the tender stem section with the stem tip can effectively improve the bud number of the unit explant, reduce the pollution rate and the death rate and improve the survival rate. The cleaning and disinfecting modes of the blumea balsamifera shoot with buds are as described above, and are not described herein.

After the sterilized tender stem segment with the stem tip with the length of 0.6-2 cm is obtained, the invention uses the stem segment with the axillary bud or the tender stem segment with the stem tip with the length of 0.6-2 cm as blumea balsamifera explant to be inoculated on an adventitious bud induction culture medium for adventitious bud induction culture, and the blumea balsamifera sterile adventitious bud is obtained.

In the present invention, the temperature of the adventitious bud induction culture is preferably 24 to 26℃and more preferably 25 ℃. The adventitious bud induction culture of the present invention preferably includes a dark culture and an illumination culture stage, and the time of the illumination culture per day is preferably 11 to 13 days, more preferably 12 hours. The illumination intensity of the illumination culture according to the present invention is preferably 1000 to 1500lx, more preferably 1100 to 1400lx, and still more preferably 1300lx. The adventitious bud induction culture time of the present invention is preferably 28 to 32 days, more preferably 30 days. The adventitious bud induction culture of the invention further grows and breeds the blumea balsamifera stem segment with axillary bud or tender stem segment with stem tip to obtain the blumea balsamifera adventitious bud. The composition of the adventitious bud induction medium is the same as that of the blumea balsamifera tissue culture medium described above, and is not summarized here.

After the blumea balsamifera sterile adventitious bud is obtained, the invention preferably cuts the regenerated bud tissue culture seedling obtained by continuously growing the blumea balsamifera sterile adventitious bud into stem segments with axillary buds, and then inoculates the stem segments with axillary buds into proliferation and subculture media for subculture proliferation to obtain blumea balsamifera single buds. The length of the axillary bud-bearing stem segments inoculated in the multiplication and subculture medium of the present invention is preferably 1 to 2cm, more preferably 1.5cm, and each axillary bud-bearing stem segment inoculated in the multiplication and subculture medium of the present invention preferably contains one bud. The adventitious buds are relative to fixed buds, and the fixed buds are buds originally existing in plants and comprise terminal buds and lateral buds (namely axillary buds); adventitious buds are buds which are not originally existed in plants and are generated by dedifferentiation.

In the present invention, the temperature of the secondary proliferation culture is preferably 24 to 26℃and more preferably 25 ℃. The secondary proliferation culture of the invention is preferably performed in dark culture and light culture, and the time of light culture per day is preferably 11-13 hours, more preferably 12 hours. The illumination intensity of the illumination culture according to the present invention is preferably 1000 to 1500lx, more preferably 1100 to 1400lx, and still more preferably 1300lx. The time for the secondary proliferation culture of the present invention is preferably 28 to 32 days, more preferably 30 days. The secondary proliferation culture comprises primary secondary culture. The secondary culture is to replace the culture medium of the secondary proliferation culture. The time of the subculture in the present invention is 14 th to 16 th days, more preferably 15 th day, of the proliferation and subculture medium. The secondary multiplication culture of the invention has the function of promoting the further multiplication of the bud stem section to obtain more regenerated buds and branches, and shearing the regenerated buds and branches to obtain blumea balsamifera single buds. The length of the single bud according to the invention is preferably 2cm. The composition of the proliferation and subculture medium of the present invention is the same as that of the blumea balsamifera tissue culture medium described above, and is not summarized here.

The blumea balsamifera single bud is obtained, and inoculated into a growth culture medium for rooting culture to obtain the blumea balsamifera tissue culture seedling. The inoculation is preferably vertical inoculation with blumea balsamifera single bud base downwards. Preferably, 1 single bud is inoculated in each rooting culture bottle. In the present invention, the rooting culture temperature is preferably 24 to 26 ℃, more preferably 25 ℃. The rooting culture is preferably performed by alternating dark culture and light culture, and the time of light culture per day is preferably 11-13 hours, more preferably 12 hours. The illumination intensity of rooting culture is preferably 1000-1500 lx, more preferably 1100-1300 lx, and even more preferably 1200lx. The rooting culture time is preferably 28-32 d, more preferably 30d. The composition of the growth medium according to the invention is as described above and is not outlined here.

The tissue culture seedling is obtained after rooting culture, the plant height of the tissue culture seedling reaches 4cm, 2-4 true leaves are arranged, the seedling hardening and transplanting culture are carried out after healthy developed root systems are arranged, and the survival rate of the tissue culture seedling is improved. According to the invention, blumea balsamifera tissue culture seedlings are subjected to seedling hardening and transplanting culture.

The seedling hardening is preferably completed in a climatic chamber, and the seedling hardening temperature is preferably Fu Geai sodium fragrance growing temperature. The daytime temperature of the seedling hardening is preferably more than or equal to 20 ℃, more preferably 23 ℃; the nighttime temperature of the seedling hardening is preferably 18 to 20 ℃, more preferably 18 ℃.

The seedling hardening time of the present invention is preferably 3 to 15 days, more preferably 7 days.

In the invention, the transplanting culture is preferably finished in a climatic chamber, and the transplanting culture is preferably carried out by cleaning the roots of the tissue culture seedlings after hardening off, transplanting the tissue culture seedlings to a plug tray for culture, and transplanting the tissue culture seedlings in a large pot for culture while continuing to grow and changing the pot; the matrix in the plug is preferably small-particle turf, and the volume ratio of the matrix in the large basin is preferably turf to sand is 1:1. Before transplanting and culturing, the substrate is preferably sterilized, and the sterilization mode is not particularly limited and conventional methods are adopted.

The source and the specification of the planting pot applied in the transplanting culture are not particularly limited, and conventional products are adopted. According to the invention, 1 blumea balsamifera tissue culture seedling after hardening off is planted in each planting pot.

The blumea balsamifera tissue culture seedling has the advantages that the blumea balsamifera tissue culture seedling has high content of the L-borneol, and can realize the stable inheritance of the blumea balsamifera fine variety seedling.

The application provides an application of the blumea balsamifera tissue culture method in blumea balsamifera germplasm breeding for maintaining the genetic stability of the content of the L-borneol.

The embodiment of the application proves that the content of the parent strain high germplasm L-borneol (9.12+/-0.45) ^a mg/g) is significantly higher than in the middleGermplasm (5.11+ -0.31) ^b mg/g) and low germplasm (3.30.+ -. 0.0.32) ^c mg/g), the content of the tissue culture seedling offspring high germplasm L-borneol (7.28+/-0.34) ^a mg/g) is significantly higher than that of the mesogen (5.71.+ -. 0.38) ^b mg/g) and low germplasm (4.41.+ -. 0.40) ^c mg/g), the leaf of the blumea balsamifera tissue culture seedling offspring obtained by the application has obvious difference with consistent trend on the content of the L-borneol of the blumea balsamifera 3 germplasm mother plant leaf, and the blumea balsamifera tissue culture seedling offspring obtained by the technology still has high content characteristic of the L-borneol and good growth vigor; tissue culture can solve the problem of germplasm degradation of blumea balsamifera in conventional culture.

The inventor conducts early-stage research to collect 235 blumea balsamifera germplasm distributed in a plurality of provincial regions of China, and discovers that the content of the L-borneol is greatly different through agronomic characters and chemical analysis comparison for years, and discovers how long the high content germplasm is in potential difference in research. The application selects germplasm parent strains with different L-borneol contents to develop researches, wherein the L-borneol content is 2.98-3.62 mg/g and is marked as low germplasm (L), the L-borneol content is 4.8-5.42 mg/g and is marked as medium germplasm (M), and the L-borneol content is 8.67-9.57 mg/g and is marked as high germplasm (H). Specifically, the method comprises the steps of firstly preserving in vitro, then expanding propagation and comparing tissue culture characteristic differences, performing chemical characteristic detection on a parent plant and corresponding tissue culture seedlings by utilizing a GC-MS technology after hardening seedlings and transplanting to obtain tissue culture offspring, and investigating whether the high-content chemical characteristics of the parent plant are maintained by the special selected germplasm by taking the low-content germplasm of synchronous culture and field planting as a control. After verifying that the high-content characteristic is continuous, the method is hopeful to carry out demonstration amplification, cultivation and popularization on germplasm and matched tissue culture technology, and achieves the aim of promoting industrial development.

The invention develops tissue culture research aiming at germplasm with different L-borneol contents, improves culture effect, determines that the tissue culture offspring obtained by the technology keeps chemical characteristics of a parent strain through a chemical detection technology, and can determine that the chemical characteristics of the selected parent strain are not accidental manifestations caused by influence of environment, biological stress and the like, but are determined by genetic factors, has certain stability and can be replicated through the technology. The invention establishes a high-efficiency seedling cloning technology system, can obtain a large number of high-quality blumea balsamifera tissue culture seedlings which keep genetic stability, and provides high-quality candidate germplasm and technical reference with popularization prospect for breeding blumea balsamifera excellent seedlings by asexual propagation means.

The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.

The test materials and reagents used in the examples are as follows:

1 reagent

MS culture medium; 6-BA; NAA; diethyl aminoethyl hexanoate (DA-6); inositol; vitamin C; L-Borneol (L-Borneol) standard; the ethyl acetate and methyl salicylate are pure products by conventional chromatography, and the sources are not particularly limited.

2 test materials

The 3 blumea balsamifera germplasm biennial mother plants are stored in the blumea balsamifera germplasm resource nursery of the tropical crop variety research institute of the national academy of agricultural sciences in Tropical crop of the state of the Hainan province in the open air, and are identified as blumea balsamifera (Blumea balsamifera (L.) DC.) of the genus blumea of the family Compositae by a Yu Fu researcher, and unified water and fertilizer management is realized.

Based on earlier studies (XiaoYongfeng, yellow plum, yu Fu, et al, analysis of phenotypic character genetic diversity of blumea balsamifera germplasm resources [ J ]. Fujian agricultural report, 2021,36 (02): 157-167.), it is known that the difference between blumea balsamifera germplasm is large. The invention marks blumea balsamifera L-borneol with the content of 2.98-3.62 mg/g as low germplasm (L), marks L-borneol with the content of 4.8-5.42 mg/g as medium germplasm (M), and marks L-borneol with the content of 8.67-9.57 mg/g as high germplasm (H). The robust 3 germplasm blumea balsamifera shoot branches are cut and used for tissue culture in examples 1-3 and comparative examples 1-2.

Example 1

(1) Sterilization of explants

Cutting the robust 3 germplasm blumea balsamifera branches with buds, cleaning for the first time by using clear water, soaking for 20min by using 2g/L washing powder solution, and washing for 20min by flowing water. Adding 2-3 drops of Tween 20 into 1% sodium hypochlorite solution under an ultra-clean workbench, mixing, soaking for 15min, washing with sterile water for 5 times, cutting off lateral leaves with a sterilized scalpel and forceps, and segmenting the branches into 3-4 cm stem segments with axillary buds to be used as explants for inducing blumea balsamifera tissue culture seedlings.

(2) First generation induction-adventitious bud induction culture

Cutting the obtained 3 blumea balsamifera explants into 1.5cm stem segments with axillary buds, respectively inoculating to a culture medium, starting induction, taking MS as a basic culture medium, adding 2.0 mg/L, NAA 0.3.3 mg/L of 6-BA, 20mg/L of inositol, 10mg/L of vitamin C, 30g/L of sucrose, 6g/L of agar and pH 6.2, and culturing for 30d to obtain the sterile regeneration bud tissue culture seedling. Culture conditions: the temperature is 25 ℃, the photoperiod is carried out for 12 hours, and the illumination intensity is 1000-1500 lx (the culture conditions of the following tissue culture experiments are consistent).

(3) Subculture proliferation

3 germplasm regenerated bud tissue culture seedlings obtained by adventitious bud induction culture are cut into 1.5cm stem segments with axillary buds, each stem segment contains 1 axillary bud, and the stem segments with axillary buds are vertically inoculated into proliferation and subculture media according to the growth direction of the buds for subculture proliferation culture. The regenerated bud tissue culture seedling is required to be strong and grow consistently. Three germplasm (germplasm L, germplasm M and germplasm H) subculture tissue culture seedlings are obtained after subculture multiplication culture.

The proliferation and secondary culture medium takes MS as a basic culture medium, and only contains NAA0.3 mg/L, inositol 20mg/L, vitamin C10mg/L, 6-BA 1.5mg/L, sucrose 30g/L and agar 6g/L, and the pH value of the proliferation and secondary culture medium is 6.2. Culturing for 15d, performing subculture, and replacing the proliferation and subculture medium. The number of buds and the length of buds of the unit explant are counted after 30d of co-culture.

Example 2

The only difference was that in the secondary proliferation culture, the 6-BA in the proliferation and secondary medium was 1.0mg/L as in example 1.

Example 3

The only difference was that in the secondary proliferation culture, 6-BA was 2.0mg/L in proliferation and secondary medium as in example 1.

Comparative example 1

The only difference was that 6-BA was not added to the proliferation and secondary medium in the secondary proliferation culture as in example 1.

Comparative example 2

The only difference was that in the secondary proliferation culture, the 6-BA in the proliferation and secondary medium was 3.0mg/L as in example 1.

The number of buds per unit explant of examples 1 to 3 and comparative examples 1 to 2 was counted as follows: unit number of explant shoots = total shoots/total number of explants, the results are shown in table 1 and figure 1.

TABLE 1 comparison of adventitious bud Induction efficiency of 3 blumea balsamifera germplasm in MS culture medium of different concentrations of 6-BA

Note that: multiple comparisons between the different groups were done using the Duncan's method, with a significant level of p < 0.05, and the end of the same column labeled with different letters indicated significant differences between the different groups of the same germplasm (tables 2-6, infra).

From Table 1 and FIG. 1, it can be seen that the addition of 6-BA at various concentrations had a significant effect on the shoot proliferation of 3 germplasm of blumea balsamifera. First, from the number of unit explant buds, 3 germplasm unit explant buds (L: 4.54.+ -. 0.67) were found at a 6-BA concentration of 1.5mg/L ^a ，M：4.41±0.71 ^a ，H：4.00±0.65 ^a ) All were significantly higher than the other 4 groups, with the highest number of unit explants of 3 germplasm, centered M, lowest H, but the differences between groups were not significant under optimal medium conditions; when the 6-BA concentration was 0mg/L, the number of buds per unit explant at that concentration (L: 0.94.+ -. 0.12) ^e ，M：0.97±0.08 ^d ，H：0.95±0.10 ^e ) At the lowest level, although the original bud point can continue to grow without adding 6-BA, new buds cannot be proliferated, and the proliferation of blumea balsamifera buds is not promoted, so that proper addition of cytokinin 6-BA is necessary for the proliferation of blumea balsamifera buds; when the concentration of 6-BA is 1.0-3.0 mg/L, the blumea balsamifera bud proliferation is promoted to a certain extent;with the increase of the concentration of 6-BA, the bud number of the unit explant shows a trend of increasing and then decreasing, reaches a peak value at the concentration of 1.5mg/L and decreases remarkably at the concentration of 3.0mg/L, which shows that when the concentration of 6-BA is too high, the bud proliferation of blumea balsamifera can be inhibited.

Next, from the bud length point of view, 3 germplasm bud lengths (L: 2.02.+ -. 0.22) when the 6-BA concentration was 1.5mg/L ^a cm，M：2.07±0.12 ^a cm，H：1.88±0.12 ^a cm) were significantly higher than the other 3 groups; secondly, the concentration of 6-BA is 0 and 1.0mg/L, and the bud lengths of the two groups are not obviously different; when the concentration of 6-BA was 2.0mg/L or higher, the bud length of both groups was gradually decreased as the concentration of 6-BA was increased, and when the concentration was 3mg/L, the bud length was minimized (L: 0.49.+ -. 0.10) ^d cm，M：0.41±0.13 ^d cm，H：0.39±0.10 ^e cm). When the bud length is less than 1cm, the bud growth is inhibited by high concentration hormone, and when such buds are not suitable for secondary rooting and are transferred to a new culture medium in a secondary way, the buds are not grown high, do not root and die gradually as the culture time passes, so that the hormone concentration suitable for the proliferation of blumea balsamifera tissue culture seedlings is necessary.

In the comprehensive view, when the concentration of 6-BA is 1.5mg/L, the number of the explant buds and the average bud length of the 3 blumea balsamifera germplasm tissue culture seedling units reach the highest, and the plant appearance is fresh green and healthy under the concentration.

Example 1 three germplasm (germplasm L, germplasm M, germplasm H) were obtained after the subsequent multiplication culture. 11 healthy and orderly subcultured proliferation culture tissue culture seedlings are selected for each germplasm, and 33 germplasm are selected for preparing explants (buds) required by test culture of the next induced rooting. The experiments of examples 4-1 to 4-3 and comparative examples 4-1 to 4-2 were performed by cutting the tissue culture seedlings of the three germplasm into single buds of 2cm length and randomly distributing the single buds.

Example 4-1

Inoculating the single buds into a growth medium for rooting culture. The growth medium takes MS as basic medium, and also contains NAA 0.3mg/L, inositol 20mg/L, vitamin C10mg/L, sucrose 30g/L, agar 6g/L and pH value 6.2 for rooting culture. Each germplasm is inoculated into 20 bottles, each bottle is inoculated with 1 single bud, and rooting rate, root length and plant height are counted after 30 days of culture. Culture conditions: the temperature is 25 ℃, the photoperiod is carried out for 12 hours, and the illumination intensity is 1000-1500 lx.

Example 4-2

The only difference was that NAA concentration in the growth medium was 0.2mg/L as in example 4-1.

Examples 4 to 3

The only difference was that NAA concentration in the growth medium was 0.4mg/L as in example 4-1.

Comparative example 4-1

The only difference was that NAA concentration in the growth medium was 0mg/L as in example 4-1.

Comparative example 4-2

The only difference was that NAA concentration in the growth medium was 0.5mg/L as in example 4-1.

The results of rooting rate, root length and plant height for examples 4-1 to 4-3 and comparative examples 4-1 to 4-2 are shown in Table 2 and FIG. 2.

TABLE 2 comparison of growth effects of 3 blumea balsamifera germplasm in MS medium supplemented with NAA at different concentrations

In plant tissue culture, auxin has the main function of promoting cell division and elongation, and different auxin concentrations have different effects on the growth and development of plants. It can be seen from the combination of Table 2 and FIG. 2 that NAA with different concentrations has a significant effect on rooting conditions of the 3 germplasm tissue culture seedlings of blumea balsamifera. First, from the rooting rate, when NAA concentration was 0.3mg/L, 3 germplasm rooting rates (L: 25.25.+ -. 4.39) ^a Bars/shoots, M: 28.60+ -1.64 ^a Bars/shoots, H: 20.55.+ -. 2.07 ^a Bars/seedlings) are significantly higher than the other four groups, under the optimal medium condition, the rooting rate M of 3 germplasm is highest, L is centered, and H is lowest; when NAA concentration is in the range of 0.2-0.5 mg/L, rooting rate of blumea balsamifera 3 germplasm tissue culture seedlings can be obviously increased, and with increasing NAA concentration, the rooting rate shows a trend of gradually rising and then falling, and reaches at 0.3mg/L The rooting rate is obviously reduced when the concentration is 0.5mg/L to the highest, so that NAA with proper concentration is favorable for rooting of blumea balsamifera tissue culture seedlings, and the rooting rate is inhibited to a certain extent when the concentration is too high; when NAA concentration was 0mg/L, 3 germplasm rooting rates (L: 3.45.+ -. 0.94) ^d Bars/shoots, M: 3.85+ -1.14 ^e Bars/shoots, H: 4.10.+ -. 1.02 ^e Strips/seedlings) are significantly lower than the other four groups, although roots can germinate under the condition of no NAA addition, the quantity of the roots is small, and under the condition of low rooting rate, the plant seedling hardening and transplanting stage growth can be inhibited, and the later stage is difficult to survive, so that the presence of auxin NAA in blumea balsamifera growth medium is necessary.

From the root length aspect, when NAA concentration is 0mg/L, 3 germplasm root lengths of blumea balsamifera (L: 4.86+ -0.23) ^a cm，M：4.65±0.37 ^a cm，H：4.06±0.23 ^a cm) is significantly higher than the other four groups; next, at 0.3mg/L, the root length was highest among the remaining four groups, and it was found that when NAA addition concentration was in the range of 0.2 to 0.5mg/L, the root length was gradually increased and then decreased with increasing NAA concentration, and at 0.5mg/L, the root length was decreased to the lowest, and at 3 germplasm root lengths were (L: 1.25.+ -. 0.21, respectively ^d cm，M：1.18±0.18 ^e cm，H：1.01±0.14 ^d cm)。

From the plant height aspect, when NAA concentration was 0.3mg/L, two of the germplasm plant heights (L: 3.22.+ -. 0.43 ^a cm，H：3.08±0.17 ^a cm) was significantly higher than the other groups, with medium germplasm concentrations of 0mg/L (plant height: 2.95.+ -. 0.25 ^a cm), 0.3mg/L (plant height: 3.02+ -0.17 ^a cm), but overall NAA concentration of 0.3mg/L was most pronounced for plant height promotion; it can be found that when NAA concentration is in the range of 0-0.3 mg/L, plant height can be obviously improved, which indicates that adding NAA with lower concentration is more suitable for 3 blumea balsamifera germplasm in a growth medium; when NAA concentration was 0.5mg/L, 3 germplasm heights (L: 1.41.+ -. 0.19) ^d cm，M：1.38±0.20 ^d cm，H：1.20±0.16 ^e cm), indicating that higher concentrations of NAA have significant inhibitory effects on plant height.

From the comprehensive rooting rate, root length and plant height 3 aspects, when NAA concentration is in the range of 0.2-0.5 mg/L, the rooting rate is highest for 3 blumea balsamifera germplasm rooting, wherein the rooting rate is the highest when NAA is not added, the root length is longer than other groups, but the rooting rate is low under the concentration, when NAA concentration is in the range of 0-0.3 mg/L, the 3 germplasm plant heights seem to have no great difference, the plant height can be increased in the concentration range, but when NAA concentration is 0.3mg/L, the 3 germplasm plant heights are obviously improved, so that when NAA concentration is 0.3mg/L, the rooting rate is the concentration which is most suitable for 3 germplasm growth of blumea balsamifera.

Example 1 after induction culture and subculture of adventitious buds, three tissue culture seedlings of germplasm (germplasm L, germplasm M, germplasm H) were obtained, and the tissue culture seedlings of the subculture were cut into single buds of 2cm for experiments of examples 5-1 to 5-2 and comparative examples 5-1 to 5-2. Examples 5-1 to 5-2 and comparative examples 5-1 to 5-2 are the same batch experiment. The subculture proliferation culture tissue culture seedlings are required to grow consistently and healthily.

Example 5-1

The single buds are vertically inoculated into a growth medium containing DA-6 with the base parts of the buds facing downwards for rooting culture.

The composition of the growth medium was: MS is used as basic culture medium, and contains inositol 20mg/L, vitamin C10mg/L, sucrose 30g/L, agar 6g/L, NAA 0.3mg/L, and DA-6 0.16 mg/L. Culture conditions: the temperature is 25 ℃, the photoperiod is carried out for 12 hours, and the illumination intensity is 1000-1500 lx.

Example 5-2

The only difference was that the DA-6 concentration in the growth medium was 0.08mg/L as in example 5-1.

Comparative example 5-1

The only difference was that the DA-6 concentration in the growth medium was 0mg/L as in example 5-1.

Comparative example 5-2

The only difference was that the DA-6 concentration in the growth medium was 0.32mg/L as in example 5-1.

The rooting rates of examples 5-1 to 5-2 and comparative examples 5-1 to 5-2 were compared, and the results are shown in Table 3 and FIG. 3.

NAA concentrations in the growth media of examples 5-1 to 5-2 and comparative examples 5-1 to 5-2 were 0.3mg/L, and the addition of DA-6 at different concentrations had a certain effect on rooting of various blumea balsamifera qualities, and the results are shown in Table 3 and FIG. 3.

TABLE 3 comparison of blumea balsamifera growth effects in MS Medium supplemented with DA-6 at different concentrations

As can be seen from Table 3 and FIG. 3, the addition of DA-6 at different concentrations can increase plant root rate, root length and plant height. First, from the rooting rate, 3 germplasm rooting rates (L: 29.8.+ -. 2.80) were obtained at a DA-6 concentration of 0.16mg/L ^a Bars/shoots, M: 30.2.+ -. 2.03 ^a Bars/shoots, H: 29.9.+ -. 2.30 ^a Bars/seedlings) were all significantly higher than the other three groups; at 0.08mg/L, the rooting rate of 3 germplasm shows a trend of rising and then falling along with the rising of the DA-6 addition concentration, and the rooting rate is reduced to the lowest at the concentration of 0.32mg/L, and the rooting rate (L: 15.6+/-3.56) ^d Bars/shoots, M: 17.5.+ -. 3.15 ^d Bars/shoots, H: 16.25.+ -. 3.35 ^d Bars/shoots) were significantly lower than the other groups; the rooting rate is remarkably lower when the DA-6 concentration is 0mg/L than when the rooting rate is 0.08mg/L and 0.16mg/L, namely, the rooting rate of 3 germplasm is better when the DA-6 and the NAA are added at a certain concentration than when the NAA is only added.

From the root length aspect, 3 germplasm root lengths (L: 2.41.+ -. 0.20) when the DA-6 concentration is 0.16mg/L ^a cm，M：2.33±0.16 ^a cm，H：2.24±0.20 ^a cm) are all significantly higher than the other three groups; when the DA-6 concentration was 0, 0.8mg/L, there was no significant difference in the length of 3 germplasm roots, which may be due to the lower DA-6 concentration, the rooting effect was not as good as that of the higher concentration; the root length of 3 germplasm was decreased after gradually increasing the concentration of DA-6, and was minimized at a concentration of 0.32mg/L, at which time the root length (L: 1.27.+ -. 0.17) ^c cm，M：1.01±0.10 ^c cm，H：1.26±0.19 ^c cm) is significantly lower than other groups, and the result shows that the root length of blumea balsamifera can be improved by properly adding certain concentration of DA-6.

From the aspect of plant height, when the DA-6 concentration isAt 0.16mg/L, two of the germplasm strains were high (L: 3.46.+ -. 0.27) ^a cm，M：3.17±0.21 ^a cm) was significantly higher than the other groups, with high germplasm at a concentration of 0.08mg/L (plant height: 2.91+ -0.23 ^a cm), 0.16mg/L (plant height: 3.03.+ -. 0.19 ^a cm), but in the sum, the plant height promotion effect is most obvious when the DA-6 concentration is 0.16 mg/L; it was found that when DA-6 was not added, the plant heights of 3 germplasm were significantly lower than that of DA-6 added, so DA-6 was necessary in blumea balsamifera growth medium; when the concentration reached 0.32mg/L, 3 germplasm heights (L: 1.95.+ -. 0.24) ^d cm，M：2.34±0.23 ^d cm，H：2.22±0.18 ^c cm), indicating that the higher concentration of DA-6 has obvious inhibition effect on plant height.

In summary, when the DA-6 concentration is in the range of 0.08-0.16 mg/L, the rooting effect of plants can be obviously improved, but when 0.16mg/L DA-6 is added, the growth effect is optimal, and the rooting rate, root length and plant height of plants at the concentration are obviously increased.

Example 1 regenerated bud tissue culture seedlings obtained through disinfection of explants and adventitious bud induction culture were cut into 1-2 cm axillary bud-carrying stem segments, each stem segment contains 1 axillary bud, and inoculated in proliferation and subculture media for subculture proliferation. Examples 1-1, comparative examples 5-3, comparative examples 5-4, and comparative examples 5-5 were conducted in the same batch.

Comparative examples 5 to 3

The induction and proliferation medium is devoid of vitamin C and inositol. Proliferation and subculture medium composition was: MS is taken as a basic culture medium, and only contains NAA0.3 mg/L,6-BA 1.5mg/L, sucrose 30g/L and agar 6g/L. Other conditions for the secondary proliferation culture were the same as in example 1.

Comparative examples 5 to 4

No inositol was present in the induction and proliferation medium. Proliferation and subculture medium composition was: MS is taken as a basic culture medium, and only contains NAA0.3 mg/L, vitamin C10mg/L,6-BA 1.5mg/L, sucrose 30g/L and agar 6g/L. Other conditions for the secondary proliferation culture were the same as in example 1.

Comparative examples 5 to 5

The induction and proliferation medium is free of vitamins. Proliferation and subculture medium composition was: MS is taken as a basic culture medium, and only contains NAA0.3 mg/L, inositol 20mg/L,6-BA 1.5mg/L, sucrose 30g/L and agar 6g/L. Other conditions for the secondary proliferation culture were the same as in example 1.

Example 1-1

Proliferation and secondary culture medium and secondary proliferation culture conditions of example 1-1 are identical to those of example 1.

The results of the proliferation rate of adventitious buds of the single buds of example 1-1 and comparative examples 5-3 to comparative examples 5-5 in growth media having different concentrations of inositol and vitamin C are shown in Table 4.

TABLE 4 comparison of adventitious bud proliferation efficiency in MS Medium supplemented with inositol and vitamin C

As can be seen from Table 4, the addition of inositol and vitamin C at appropriate concentrations can increase the proliferation efficiency of adventitious buds of plants. First, 3 treatment groups (L: 3.95.+ -. 0.94) in which myo-inositol and vitamin C were added simultaneously to 3 germplasm, as seen from the number of shoots of unit explant ^a ，M：3.68±0.83 ^a ，H：3.80±0.77 ^a ) Are both significantly higher than without both; second, the number of group unit explant shoots with and without myo-inositol and vitamin C added alone is significantly higher than that without both; when inositol and vitamin C were not added simultaneously, 3 germplasm was significantly lower than each group. In the comprehensive view, the addition of appropriate amounts of inositol and vitamin C can promote proliferation of 3 adventitious buds, but the effect is best when both are added at the same time.

Examples 5-3 and comparative examples 5-6 to comparative examples 5-8 are the same batch experiments. The tissue culture seedlings obtained after the subculture of example 1 were cut into single shoots 2cm long, and the obtained single shoots were used for the experiments of examples 5-3 and comparative examples 5-6 to comparative examples 5-8. The subculture proliferation culture tissue culture seedlings are required to grow consistently and healthily.

Comparative examples 5 to 6

The composition of the growth medium was: MS is used as basic culture medium, and only contains 30g/L sucrose, 6g/L agar, 0.3mg/L NAA and 0.16mg/LDA-6. Other conditions for rooting culture were the same as in example 5-1.

Comparative examples 5 to 7

The composition of the growth medium was: MS is used as basic culture medium, and contains vitamin C10mg/L, sucrose 30g/L, agar 6g/L, NAA0.3mg/L, and DA-6 0.16 mg/L. Other conditions for rooting culture were the same as in example 5-1.

Comparative examples 5 to 8

The composition of the growth medium was: MS is used as basic culture medium, and contains inositol 20mg/L, sucrose 30g/L, agar 6g/L, NAA, 0.3mg/L and LDA-6 only 0.16 mg/L. Other conditions for rooting culture were the same as in example 5-1.

Examples 5 to 3

The rooting culture conditions of example 5-3 are exactly the same as those of example 5-1.

TABLE 5 comparison of adventitious bud growth effects in MS Medium supplemented with inositol and vitamin C

As can be seen from table 5, the addition of inositol and vitamin C at appropriate concentrations can significantly enhance the growth effect of adventitious buds of plants. First, from the aspect of rooting rate, when both are added at the same time, 3 germplasm rooting rates (L: 30.05.+ -. 3.41 ^a Bars/shoots, M: 28.95.+ -. 3.10 ^a Bars/shoots, H: 31.00.+ -. 2.88 ^a Bars/shoots) were all significantly higher than the other groups; the rooting rate of the groups added with myo-inositol and vitamin C alone is obviously higher than that of the groups without myo-inositol and vitamin C, which shows that the rooting rate of 3 germplasm can be promoted by adding a proper amount of myo-inositol and vitamin C, but the rooting rate is optimal when the myo-inositol and the vitamin C are added simultaneously.

From the root length aspect, when both are added at the same time, 3 germplasm root lengths (L: 1.17.+ -. 0.09) ^a cm，M：1.21±0.14 ^a cm，H：1.31±0.11 ^a cm) were significantly higher than the other groups; the rooting rate of the groups added with the myo-inositol and the vitamin C alone is obviously higher than that of the groups without the myo-inositol and the vitamin C, but the rooting rate is optimal when the myo-inositol and the vitamin C are added simultaneously.

From the aspect of plant height, when both are added at the same time, only L germplasm plant heights (2.64+ -0.21 ^a cm) is significantly higher than the two groups without addition; of the M, H germplasm, there was no significant difference in the four groups, indicating that the addition of inositol and vitamin C may not have a significant promoting effect on plant height, but also have no negative effect.

Taken together, when a proper amount of inositol and vitamin C are added simultaneously, 3 blumea balsamifera germplasm growth has a remarkable promoting effect, indicating that inositol and vitamin C are necessary in the blumea balsamifera growth medium.

Example 6-1

The explant is a tender stem segment with a stem tip and a length of 1.5cm, and the conditions of sterilization and primary induction culture of the explant are the same as in example 1.

Comparative example 6-1

The explant is a stem tip with the length of 2mm, and the conditions of sterilization and primary induction culture of the explant are the same as in example 1.

Comparative example 6-2

The explant is a stem segment with axillary buds, the length of the stem segment is 1.5cm, and the sterilization and primary induction culture conditions of the explant are the same as those of example 1.

The results of the explant induction efficiency of example 6-1 and comparative examples 6-1 to 6-2 are shown in Table 6.

TABLE 6 comparison of Induction efficiencies of different explants

As can be seen from Table 6Firstly, when 3 germplasm explants are tender stem segments with stem tips and the length is 1.5cm, the pollution rate and the death rate are obviously lower than those of other groups from the aspects of pollution rate and death rate; secondly, from the aspect of survival rate, tender stem segments with stem tips with lengths of 1.5cm are obviously higher than other groups; finally, from the number of the unit explant buds, when the explant is a tender stem segment with a stem tip of 1cm in length, 3 germplasm (L: 5.74.+ -. 2.60) ^a ，M：5.69±2.80 ^a ，H：6.34±2.72 ^a ) All significantly higher than the other groups; the pollution rate of the stem segment with the axillary bud with the length of 1.5cm is highest, and the pollution rate is not very different when the stem tip and the tender stem segment with the length of 1.5cm containing the stem tip are used as explants, which is probably because both vascular bundles are not completely differentiated, a pathogen does not reach the apical meristem along the developed vascular bundles temporarily, the stem segment with the length of 1.5cm forms the developed vascular bundles, the pathogen is suspended in the vascular bundles and grows along with the culture, and the pollution rate of the former two is obviously lower than that of the stem segment with the axillary bud with the length of 1.5 cm. Comprehensively, the 1.5cm bud containing the stem tip is selected as the explant, so that the low pollution rate, the high survival rate, the high induction efficiency and the best culture effect can be achieved.

Example 7 hardening seedlings and transplanting

The culture flask containing the tissue culture seedling, in which each quality of blumea balsamifera obtained in example 5-1 grew healthy roots, was placed in a climatic chamber to exercise the seedlings. Artificial climate box culture conditions: the light intensity is 29000lx when the light is at 23 ℃ in the daytime and 18 ℃ in the night and the light period is 12 hours. The hardening off and transplanting process of the blumea balsamifera 3 germplasm offspring tissue culture seedlings is shown in figure 4. The transplanting culture is completed in a climatic chamber, and the transplanting culture matrix is a mixture of turf and sand; the volume ratio of the matrix is 1:1.

The above-mentioned various germplasm are stored in vitro, then the aseptic seedling is obtained, and 3 germplasm clone offspring groups are obtained by means of tissue culture and propagation, and after hardening seedling and transplanting, the above-mentioned germplasm are planted in flowerpot, and cultured in artificial climatic box, and unified light-water-fertilizer management is implemented.

Example 8 comparison of L-borneol content of 3 germplasm parent strains and offspring tissue culture seedlings of blumea balsamifera

3 germplasm mother strains of blumea balsamifera: picking 3 low (L), medium (M) and high (H) mother plants of blumea balsamifera without plant diseases and insect pests, picking 9 functional leaves at the same position, setting 9 parallel experiments, drying all functional leaves in shade for 7 days, and storing in a refrigerator at-80 ℃ to be tested.

3 germplasm tissue culture seedlings of blumea balsamifera: example 7 after seedling hardening for 338 days, 9 plants with consistent growth vigor and healthy and no plant diseases and insect pests are selected from each germplasm, 1 piece of functional leaf is picked at the same position, and after 7 days of drying in shade, the functional leaf is stored in a refrigerator at the temperature of minus 80 ℃ to be tested.

The method for measuring the content of L-borneol is referred to (Pang Yuxin, yellow plum, yu Fu, etc. the analysis of the difference of the content of main chemical components in blumea balsamifera produced by Qianqiong, J. University of Guangdong medical school, 2014,30 (04): 448-452.) and is finely adjusted. The method for measuring the content of the L-borneol is specifically as follows:

(1) Preparation of test solutions

Taking blumea balsamifera leaf tablets, quickly grinding with liquid nitrogen, precisely weighing 0.2g of powder (sieving with a 20-mesh sieve), adding 2.5mL of ethyl acetate, and weighing the mass m ₁ The method comprises the steps of carrying out a first treatment on the surface of the Ultrasonic extraction is carried out for 30min under the conditions of frequency 40kHz and power 420W. Cooling, weighing mass m ₂ The method comprises the steps of carrying out a first treatment on the surface of the Supplementing the mass to m with ethyl acetate ₁ Shaking, filtering, collecting 1mL of the filtrate, placing in a 10mL volumetric flask, adding 1mL of internal standard solution, fixing volume with ethyl acetate, shaking, and filtering with 0.45 μm microporous membrane.

(2) Preparation of control solution

Accurately weighing appropriate amount of L-borneol reference substance, adding ethyl acetate for dissolution and volume fixing, and preparing reference substance stock solution with mass concentration of 0.998 mg/mL.

(3) Preparation of internal standard solution

Precisely weighing 100mg of methyl salicylate, placing into a 100mL volumetric flask, adding ethyl acetate to fix the volume, and shaking uniformly to prepare an internal standard solution with the mass concentration of 1.016 mg/mL.

(4) Drawing of a Standard Curve

Precisely measuring 0.1, 0.2, 0.5, 0.7, 1 and 2mL of reference substance solution, respectively placing into 10mL volumetric flask, adding 1mL of internal standard solution, fixing volume with ethyl acetate, shaking, filtering with 0.45 μm microporous filter membrane, and taking the mass concentration of reference substance as abscissa (X), wherein the peak areas of reference substance and internal standard methyl salicylateThe ratio is plotted as ordinate (Y) against the standard curve. The linear regression equation y=61.5868x+0.8878, r is obtained ² = 0.9992, the linear range is 0.0101 to 0.202mg/m L.

(5) GC-MS analysis conditions

The chromatographic column is Agilent Cyclosil-B (30 m×0.250mm×0.25 μm) capillary column, mass spectrometry conditions: EI source, solvent delay 6min, ion source temperature 230 ℃, sample inlet temperature 220 ℃, scanning range 45-500 amu. Mass spectrometry conditions: EI source, solvent delay 6min, ion source temperature 230 ℃, sample inlet temperature 220 ℃, scanning range 45-500 amu.

The ion chromatogram of the L-borneol reference substance (figure 5), the total ion superposition chromatogram of the extracts of 3 blumea balsamifera germplasm mother plants and the offspring tissue culture seedling leaves (figure 6), the retention time of 3 germplasm internal standard solutions and the L-borneol peak is consistent with that of the reference substance, and the content comparison of the 3 blumea balsamifera germplasm mother plants and the offspring tissue culture seedling leaves L-borneol is obtained after calculation (figures 7 and 8), and the results show that: the 3 germplasm mother strains and the tissue culture offspring have obvious difference in the content of the L-borneol, and the mother strains have high content of the L-borneol (9.12+/-0.45) ^a mg/g) is significantly higher than M (5.11.+ -. 0.31) ^b mg/g) and L (3.30.+ -. 0.0.32) ^c mg/g), the content of the tissue culture seedling offspring high germplasm L-borneol (7.28+/-0.34) ^a mg/g) is significantly higher than M (5.71.+ -. 0.38) ^b mg/g) and L (4.41.+ -. 0.40) ^c mg/g). The tissue culture scheme provided by the invention can enable the high content of blumea balsamifera high-germplasm L-borneol to be maintained. The raw data corresponding to fig. 7 and 8 are shown in table 7.

TABLE 7 Levoborneol content raw data for 3 germplasm parent strains and tissue culture seedling offspring leaves of blumea balsamifera

Note that: multiple comparisons between different groups were done using Duncan's method with a significant level of p < 0.05, and the end of the same column labeled with different letters indicated significant differences between the different germplasm of the stock or between the different germplasm of the offspring.

In conclusion, the most suitable culture medium for proliferation of 3 blumea balsamifera germplasm is MS+NAA0.3 mg/L+6-BA 1.5 mg/L+vitamin C10 mg/L+inositol 20 mg/L+sucrose 30 g/L+agar 6g/L. The most suitable culture medium for rooting 3 blumea balsamifera germplasm is MS+NAA0.3 mg/L+DA-60.16 mg/L+vitamin C10 mg/L+inositol 20 mg/L+sucrose 30 g/L+agar 6g/L.

According to the invention, on the basis of conventional blumea balsamifera in-vitro preservation and tissue culture technologies, a culture medium is perfected, clone offspring groups are obtained, chemical characteristic detection is carried out on 3 blumea balsamifera germplasm parent plant single plants and clone offspring groups respectively through a GC-MS analysis technology, simultaneously cultured low-content germplasm (L) is used as a control, whether the chemical characteristics of the high content of other 2 germplasm (M and H) parent plants are determined by genetic factors is investigated, and the parent plant single plants which are possibly caused by the increase of the content of L-borneol due to biological stress and the like and cannot be continued through tissue culture are screened. The results show that: the content difference of the L-borneol of each of the 3 germplasm mother strains of blumea balsamifera is obvious and is (L: 3.30+/-0.32) ^c mg/g；M：5.11±0.31 ^b mg/g；H：9.12±0.45 ^a mg/g), the content of the L-borneol in the offspring of the tissue culture seedlings of 3 germplasm after tissue culture cloning (primary culture), proliferation, induced rooting, seedling hardening and transplanting also shows consistent low, medium and high significant differences, which are respectively: 4.41.+ -. 0.40 ^c mg/g)、(M：5.71±0.38 ^b mg/g)、(H：7.28±0.34 ^a mg/g), and the content of the L-borneol of the parent strain are consistent, namely, the content of the L-borneol of 3 germplasm parent strains is continued by the 3 germplasm tissue culture offspring obtained through the scheme.

The result not only can verify that the content difference of the L-borneol of 3 mother strains is determined by genetic factors, but also the genetic characteristics do not cause mutation or other factors in the culture process of the scheme provided by the invention, so that the content difference of the L-borneol of offspring is changed. There is no comparability between the various parent strains and their tissue culture offspring. The reasons are two: firstly, the growth states of the L-borneol and the L-borneol are different, the former is influenced by environmental differences and diseases and insect pests, the synthesis of the L-borneol is possibly promoted or inhibited, and the latter has more uniform growth environment in the incubator and has no diseases and insect pests; secondly, the two strains have different ages, and the existing researches prove that the content difference of the L-borneol in the blumea balsamifera with different ages is obvious and the content of the L-borneol in the blumea balsamifera with different ages is low. It can be seen that the difference between the germplasm can be reflected more realistically only by comparing the difference between plants of different germplasm in the same growing state and of the same age of plant as described in this example. In summary, the tissue culture scheme provided by the invention can enable the property of the blumea balsamifera germplasm with high content of the L-borneol to be maintained.

The invention obtains the blumea balsamifera L-borneol high-content excellent germplasm with popularization prospect, can obtain excellent seedlings through asexual propagation and maintain genetic advantages, establishes a matched efficient tissue culture technology capable of maintaining genetic stability of the germplasm excellent good quality, is expected to popularize the germplasm and the matched tissue culture technology, and has good industrial prospect.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

1. The blumea balsamifera growth medium is characterized in that the growth medium takes an MS medium as a basic medium and further comprises: 0.2-0.3 mg/LNAA, 0.08-0.16 mg/LDA-6, 10-50 mg/L vitamin C, 20-200 mg/L inositol, 30g/L sucrose and 4-10 g/L agar.

2. The use of blumea balsamifera growth medium according to claim 1 for increasing one or more of the following 1) to 3) of blumea balsamifera tissue culture seedlings,

1) Rooting rate;

2) Plant height;

3) Root length.

3. A blumea balsamifera tissue culture medium comprising an adventitious bud induction medium, a proliferation and subculture medium and the growth medium of claim 1;

4. A blumea balsamifera tissue culture method, which is characterized in that the tissue culture method adopts the growth medium as claimed in claim 1 and comprises the following steps:

5. The tissue culture method of claim 4, wherein the explant comprises at least one of:

(a) Stem segments with axillary buds;

(b) Tender stem segment with stem tip and length of 6-20 mm.

6. The tissue culture method according to claim 4, wherein the temperatures of the adventitious bud induction culture, the secondary proliferation culture and the rooting culture are 24 to 26 ℃.

7. The tissue culture method according to claim 4, wherein the adventitious bud induction culture, the secondary proliferation culture and the rooting culture are all carried out under alternate light and dark illumination conditions, the illumination time is 11-13 h/d respectively, and the illumination intensity is 1000-1500 lx respectively.

8. The tissue culture method according to any one of claims 4 to 7, wherein the adventitious bud induction culture time is 28 to 32d; the time of the secondary proliferation culture is 28-32 d; the rooting culture time is 28-32 d.

9. The tissue culture method according to claim 4, wherein the proliferation and subculture medium is a minimal medium of MS medium, further comprising: 0.1-0.5 mg/LNAA, 1.0-3.0 mg/L6-BA, 10-50 mg/L vitamin C, 20-200 mg/L inositol, 30g/L sucrose and 4-10 g/L agar;

10. Use of the blumea balsamifera tissue culture method according to any one of claims 4-9 in blumea balsamifera germplasm breeding for maintaining genetic stability of the content of the l-borneol.