CN115669540A - Cultivation method of orchid seedlings without endophyte pollution - Google Patents
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Abstract
The invention provides a method for cultivating orchid seedlings without endophyte pollution, which comprises the steps of cutting and cultivating the stem tips of test-tube plantlets of orchid with the size of 0.5mm, inducing intermediate propagules through stem tip cultivation, and then proliferating and differentiating the intermediate propagules to obtain the orchid seedlings without endophyte pollution. The method thoroughly removes the polluted endophytes in the orchid by cutting the stem tips with proper sizes for culture, can carry out the tissue culture and the rapid propagation of the orchid in proper culture conditions and culture media, reduces the influence of antibiotics and the like on the tissue culture and the rapid propagation of the orchid, and further improves the quality and the benefit of the orchid seedlings.
Description
Technical Field
The invention relates to the technical field of plant seedling production. More particularly, it relates to a cultivation method of orchid seedlings without endophyte pollution.
Background
Orchid (Cymbidium) is a kind of orchid of the orchid family, and is a kind of famous flowers in the world with important ornamental, economic, cultural and ecological values. About 68 cymbidium plants are found in the world, and 49 plants are found in China and account for 72.1 percent of the world. The orchid has a long cultivation history and rich cultural connotation in China, is called as 'monarch in flower', is well popular with people all over the world, can be used as high-grade cut flower and also can be used as high-grade potted flower. With the improvement of living standard and cultural quality of people, orchids begin to enter common families, are popular with more and more consumers and have wide market prospect.
Endophyte contamination refers to contamination caused by the fact that endophytes exist in plants, generally, surface disinfection methods are difficult to thoroughly remove, and enter a culture process along with explant materials. Endophyte contamination often occurs in the industrialized production of ornamental plant seedlings, generally occurs after 5 days of inoculation or in subculture, forms 'filaments' or 'halos' in a culture medium at the initial stage, is not easy to be detected by naked eyes, is easy to be found by examination under a backlight condition, and is shown in the culture medium after bacterial load is accumulated after a plurality of subcultures (Kritzi 9ger et al, 1997; zhoujunhui et al, 2003; thoguassian et al, 2014 Srivastava et al, 2021). To date, endophyte contamination has been found in tissue culture rapid propagation of 47 (genus) plants, from which about 51 endophytes were isolated and identified. The existing research results show that the endophyte pollution has adverse effects on each link of the plant tissue culture and rapid propagation, and the tissue culture and rapid propagation efficiency and benefit are reduced. Therefore, the research on the prevention and control technology of endophyte pollution has important significance for further improving the factory production efficiency and benefit of the plant seedlings and the competitiveness of the seedling industry in China.
The prevention and control measures of endophyte pollution in plant tissue culture and rapid propagation mainly comprise the steps of reducing the content of the explant, changing the components or pH value of a culture medium, adding antibiotics into the culture medium, culturing and removing bacterins and the like (Zhou-quan et al, 2012; zhou-Jun et al, 2003; wenlua and Wu-Rina, 2020). In the method, the culture of the sterile seedlings is a fundamental method for controlling the pollution of endophytes, the germchit red and the like (2019) take stem tip meristem of caespitous buds of Gannan navel orange as an explant, and sterile seedling of fructus aurantii as a rootstock, and the sterile seedlings are successfully cultured by adopting a heat treatment combined with stem tip culture and sterilization method, wherein the sterilization rate can reach 93.2 percent (the tissue culture sterilization and micro-bud grafting rapid propagation technology [ J ] for the production of the germchit red, ancient bin, yijiaxuan, and the like, gannan navel orange sterile seedlings [ J ] northern horticulture, 2019 (18): 26-31.).
Because endophyte pollution often occurs in the industrial production of orchid seedlings, the tissue culture rapid propagation benefit and efficiency are obviously reduced when the endophyte pollution is serious. There is no report on the elimination of orchid bacteria. And the existing technology for preventing and controlling endophyte pollution mainly aims to reduce the influence of endophytes on culture materials, is difficult to thoroughly eliminate the endophyte pollution, and some prevention and control measures can also have adverse effects on proliferation and differentiation of orchid intermediate propagules and production of test-tube plantlets. Therefore, the development of the technique for removing the endophytes of the orchid can thoroughly solve the problem of the pollution of the endophytes, and has important significance for improving the competitiveness of orchid seedling industry and turning over stick in planting industry.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for cultivating orchid seedlings without endophyte pollution.
The above object of the present invention is achieved by the following technical solutions:
a cultivation method of orchid seedlings without endophyte contamination comprises the following steps:
s1, stem tip culture: taking a strong orchid test-tube plantlet as a material, aseptically cutting a stem tip with the size of 0.5mm, and culturing on an induction culture medium to obtain an intermediate propagule;
s2, intermediate propagule proliferation and differentiation: and carrying out proliferation culture on the intermediate propagules, and carrying out differentiation culture when more than 10 intermediate propagules are propagated from each stem tip to obtain orchid seedlings.
The method thoroughly removes specific pollution endophytes in the orchid by cutting the stem tip of the orchid with proper size for culture, can ensure that the tissue culture and rapid propagation of the orchid are carried out in proper culture conditions and culture medium, and reduces the influence of antibiotics and the like on the tissue culture and rapid propagation of the orchid.
Preferably, the method further comprises verifying the orchid seedlings obtained in the step S2 for the existence of endophyte contamination. The verification method can be used for separating and culturing endophytes in orchid seedling tissues, and can also be used for carrying out PCR detection by using the detection primers of the endophytes by using the total DNA of orchid seedlings as a template.
Preferably, the verification comprises extracting orchid seedling DNA, performing PCR amplification reaction by using an endophyte detection primer, and detecting an amplification product by electrophoresis so as to judge whether the orchid seedling DNA is polluted by the endophyte. Specifically, total DNA of the test-tube plantlet is extracted by a CTAB method, a specific primer is designed according to an endophyte 16S rDNA sequence, the specific primer is utilized to carry out PCR amplification on the total DNA of the test-tube plantlet, and an amplification product is subjected to 1.0% agarose gel electrophoresis to detect the bacteria removal effect.
The method for removing the endophytes of the specific orchid mainly comprises the steps of cutting stem tips with proper sizes and culturing to remove the endophytes; the three steps of induction, proliferation and differentiation of the intermediate propagules have no influence on the removal of endophytes, but have influence on the production of the sterile seedlings.
Preferably, the induction culture medium in the step S1 is MS + 1.0-2.0 mg/L6-BA + 0.1-0.5 mg/L NAA + 5.0-20.0% (w/w) coconut milk +30.0g/L white granulated sugar + 7.0-8.0 g/L carrageenan + 0.2-1.0 g/L AC, and the pH value is 5.6-6.0; the culture is carried out for 2 to 3 months at a temperature of between 24 and 26 ℃ in the dark.
Preferably, the stem tip culture is to select a strong orchid test-tube seedling with a plant height of about 8cm, remove leaves in a sterile culture dish under an anatomical lens on an ultra-clean workbench, strip sheath leaves and leaves with a sterile normal-temperature scalpel and an dissecting needle, and cut a stem tip with a size of 0.5mm for induction culture.
Preferably, the culture medium for enrichment culture in the step S2 is MS + 0.5-2.0 mg/L6-BA + 0.2-0.5 mg/L NAA +30.0g/L white granulated sugar +7.0g/L carrageenan + 0.3-0.5 g/L AC, and the pH is 5.6-6.0; the proliferation culture is carried out in the dark at the temperature of 24-26 ℃ or under the illumination of 11-13 h every day and under the illumination intensity of 600-1000 lux, and the subculture is carried out for 1 time at 40-60 d.
More preferably, the proliferation culture is performed by controlling the intermediate propagules in step S1 within 1cm or cutting large protocorms into 0.5cm × 0.5cm, inoculating into proliferation culture medium, and inoculating 8-15 pieces (blocks) per bottle.
Preferably, the culture medium for the differentiation culture in the step S2 is MS + 1.0-2.0 mg/L6-BA + 0.1-0.2 mg/L NAA +30.0g/L white granulated sugar +7.0g/L carrageenan + 0.00-0.05 g/L AC, and the pH value is 5.6-6.0; the differential culture is carried out for 40-60 days at 24-26 ℃ under the illumination intensity of 1000-2000 lux for 11-13 h per day.
More preferably, the differentiation culture is to inoculate the rhizome with the length of 0.8 to 1.0cm without top removal or the protocorm-like body cut into 0.5cm multiplied by 0.5cm into a differentiation culture medium, 8 to 12 (blocks) are inoculated into each bottle, and the differentiation culture is carried out for 40 to 60 days at the temperature of about 25 ℃, the illumination time of each day is 11 to 13 hours and the illumination intensity is 1000 to 2000 lux; inoculating the seedling with the plant height of 3.0-5.0 cm into a rooting and strong seedling culture medium, and culturing for 40-60 days under the same condition.
Preferably, the formula of the rooting and seedling strengthening culture medium is MS + 0.1-0.2 mg/L6-BA + 0.5-1.0 mg/L NAA +30.0g/L white granulated sugar +7.0g/L carrageenan + 0.3-0.5 g/L AC, and the pH value is 5.6-6.0.
Preferably, the orchid is 'Xiaofenglan'.
Preferably, the endophyte is an endophyte XF-NSJ.
Preferably, the specific primers for detecting the endophytic bacterium XF-NSJ comprise an upstream primer and a downstream primer, and the sequences of the upstream primer and the downstream primer are respectively: 5 'GGAAAGATTTTTTGGTTGGGGGG-3', a downstream primer: 5 'CCTTTGAGTTTAGCCTTGCG-doped 3'.
Further preferably, the PCR amplification reaction system for detecting the endogenous bacterium XF-NSJ is as follows: 12.0 mu L of Mix, 1.0 mu L of upstream primer, 1.0 mu L of downstream primer, 10.0 mu L of ddH2O and 1.0 mu L of template DNA, and the total amount is 25.0 mu L; and (3) amplification procedure: 5mi9 at 90 ℃; 30s at 94 ℃, 30s at 54.4 ℃, 2mi9 30s at 72 ℃ and 34 cycles; 10mi9 at 72 ℃; infinity at 4 ℃.
Compared with the prior art, the invention has the following beneficial effects:
according to the method, the stem tips of the orchid with proper size are cut for culture, so that the polluted endophyte in the orchid can be thoroughly removed, the tissue culture and rapid propagation of the orchid can be carried out under proper culture conditions and culture medium, and the orchid seedlings without endophyte pollution are obtained, so that the influence of the endophyte pollution on the tissue culture and rapid propagation of the orchid is eliminated, the tissue culture and rapid propagation efficiency and benefit are improved, meanwhile, the influence of antibiotics and the like on the tissue culture and rapid propagation of the orchid is avoided, and the seedling quality and benefit are further improved.
Drawings
FIG. 1 is a graph showing the effect of stem tip size on the induction of the 'Xiaofenglan' intermediate propagules. Wherein A is the stem tips with different sizes and the rhizome formed by the stem tips through induction, and B is the induction rate of the rhizome of the stem tips with different sizes.
FIG. 2 is the effect of stem tip size on the proliferation, differentiation and rooting and seedling strengthening of 'Xiaofenglan' rhizome. Wherein A is the number of the proliferated rhizome of the stem tip with different sizes, B is the number of the differentiated seedling of the stem tip with different sizes, C is the number of the test tube seedling produced by the stem tip with different sizes, and D is the number of the proliferated rhizome and the differentiated seedling of the stem tip with different sizes.
FIG. 3 is the germ-shedding effect detection of test-tube plantlets of 'small phoenix-like orchid' cultured by stem tips of different sizes. Wherein, M: DL2000Marker,1:0.5mm stem tip culture test-tube plantlet, 2:1.0mm stem tip culture test-tube plantlet, 3:2.0mm stem tip culture test-tube plantlet, 4:3.0mm stem tip culture test-tube plantlet, 5: positive control: tissue culture seedlings of 'Xiaofenglan' contaminated by endophytes, 6: negative blank control.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
The present invention is further illustrated in the following examples by taking the 'small phoenix orchid' as an example, wherein the 'small phoenix orchid' is a new orchid variety bred by hybridization by taking the 'big phoenix' hybrid orchid as a female parent and taking the 'penguin white black' black orchid as a male parent, and the plant shape is similar to 'penguin white black', and the rapid propagation and the separate propagation by tissue culture are easy. The average diameter of each branch is 7.4cm, and the flower is orange and fragrant. Beginning to flower in 1 last ten days of month in greenhouse cultivation, and the flowering period is 35-45 d.
The bacterium XF-NSJ is an orchid pollution endophyte which is separated by the inventor, is one of the brevibacterium (Curtobacterium), and has the 16S rDNA sequence as follows:
AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGATGAAGCCCAGCTTGCTGGGTGGATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCCTGACTCTGGGATAAGCGTTGGAAACGACGTCTAATACTGGATATGACCGCCGATCGCATGGTCTGGTGGTGGAAAGATTTTTTGGTTGGGGATGGACTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCAACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTTAGTAGGGAAGAAGCGTAAGTGACGGTACCTGCAGAAAAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCTGTGAAATCCCGAGGCTCAACCTCGGGCTTGCAGTGGGTACGGGCAGACTAGAGTGCGGTAGGGGAGATTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGATCTCTGGGCCGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGCGCTAGATGTAGGGACCTTTCCACGGTTTCTGTGTCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAACGGCCAGAGATGGTTGCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGTTGCCAGCGCGTTATGGCGGGGACTCATAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGCTGCGATACCGTAAGGTGGAGCGAATCCCAAAAAGCCGGTCTCAGTTCGGATTGAGGTCTGCAACTCGACCTCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCTTGTGGAAGGAGCCGTCGAAGGTGGGATCGGTGATTAGGACTAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGATCACCT。
example 1 cultivation method of orchid seedlings without endophytic bacterium XF-NSJ contamination
S1, stem tip culture: taking a strong test-tube seedling of 'Xiaofenglan' as a material, cutting a stem tip under aseptic condition, and culturing on an induction culture medium to obtain a rhizome (a middle propagule); the method specifically comprises the following steps:
s11, test-tube plantlet selection and stem tip separation: selecting a test-tube seedling of 'Xiaofenglan' with the plant height of about 8cm and strong growth, removing leaves and sheaths in a sterile culture dish under an anatomical lens on a super clean workbench by using a sterile normal-temperature scalpel and an dissecting needle, and cutting 0.5mm, 1.0mm, 2.0mm and 3.0mm stem tips respectively (the upper picture of a picture 1A);
s12, rhizome induction: quickly inoculating the stem tip into an induction culture medium MS +2.0 mg/L6-BA +0.5mg/LNAA +7.0g/L carrageenan +30.0g/L white granulated sugar +0.5g/L AC +10.0% (w/w) coconut juice, culturing for 90 days at the temperature of about 25 ℃ in the dark at the pH of 5.8, and inducing the rhizome as shown in the lower graph of FIG. 1A. Statistics of the rhizoid induction rate shows that the rhizoid induction rates of the stem tips with different sizes are different, and the higher the stem tip is, the higher the rhizoid induction rate is (fig. 1B).
S2, propagating and differentiating the rhizome to produce test-tube plantlets: carrying out enrichment culture on the rhizome, and carrying out differentiation culture when about 10 rhizomes with the length of 1.0cm are propagated from each stem tip to obtain a test-tube plantlet; the method specifically comprises the following steps:
s21, propagating rhizome: cutting the rhizome into 0.8-1.0 cm, inoculating the rhizome into a multiplication culture medium MS +1.5mg/L6-BA +0.2mg/L NAA +30.0g/L white granulated sugar +7.0g/L carrageenan +0.5g/L AC, pH 5.8, inoculating 8-10 pieces of the rhizome into each bottle, carrying out multiplication culture at 2421 ℃ under the condition that the illumination intensity is 800-1000 lux, carrying out subculture for 1 time for 45D, and showing the rhizome formed by subculture as a figure 2D;
s22, differentiation production of test-tube plantlets by using rhizomes: inoculating a rhizome with the length of 0.8-1.0 cm without top removal to a differentiation culture medium MS +1.5mg/L6-BA +0.2mg/L NAA +30.0g/L white granulated sugar +7.0g/L carrageenan +0.02g/L AC, the pH is 5.8, 8-10 pieces of the rhizome are inoculated to each bottle, and the rhizome is cultured for about 45 days at 2421 ℃, the illumination time is 11-13 h per day and the illumination intensity is 1000-1500 lux, and the seedling formed by differentiation is shown in a picture 2D; inoculating the seedling of about 3.0-5.0 cm to a rooting and strong seedling culture medium MS +0.1 mg/L6-BA +0.5mg/L NAA +7.0g/L carrageenan +30.0g/L white granulated sugar +0.5g/L AC, culturing for about 45 days under the same condition, and producing the test-tube seedling as shown in the lower part of a figure 2D.
In addition, the number of the propagated rhizome of the stem tip with different sizes, the number of the differentiated seedlings of the stem tip with different sizes and the number of the test tube seedlings produced by the stem tip with different sizes are shown in fig. 2A-C, and it can be seen that the number of the propagated rhizome, the number of the differentiated seedlings and the produced test tube seedlings of the stem tip with different sizes are all different, and the larger the stem tip is, the more the number of the propagated rhizome, the number of the differentiated seedlings and the produced test tube seedlings are, but the larger the stem tip is, the larger the contamination risk of endophytes is meant to be.
S3, identifying the effect of the pollution-removing endophyte: extracting total DNA of a test-tube plantlet of 'small cymbidium goeringii' by adopting a CTAB method, designing a specific primer according to a 16S rDNA sequence of XF-NSJ of the 'small cymbidium goeringii' polluted endophytic bacteria, carrying out PCR amplification on the DNA by using the specific primer, and detecting the bacteria removal effect of an amplification product through 1.0% agarose gel electrophoresis to obtain a pollution-free endophytic bacteria test-tube plantlet; the method comprises the following specific steps:
s31, DNA extraction of test-tube plantlets: about 200.0mg of each test-tube plantlet was taken, ground into powder with liquid nitrogen, and then total DNA was extracted by CTAB method.
S32, primer design and synthesis: designing specific primers by using Primer 5.0 software according to a 16S rDNA sequence of the contamination endophyte XF-NSJ, wherein the specifically designed primers are as follows:
an upstream primer: 5 'GGAAAGATTTTTTGGTTGGGGG-3',
a downstream primer: 5-;
the primers are synthesized by Guangzhou Ongke organism and used for PCR amplification.
S33, PCR amplification: the DNA of a test-tube plantlet cultured by a stem tip of the small cymbidium is taken as a template, and a 16S rDNA specific primer of the polluted endophytic bacterium XF-NSJ is adopted for PCR amplification. Amplification reaction system (25.0. Mu.L): mix 12.0. Mu.L, upstream primer 1.0. Mu.L, downstream primer 1.0. Mu.L, ddH 2 O10.0. Mu.L, template DNA 1.0. Mu.L. And (3) amplification procedure: 5mi9 at 90 ℃; 30s at 94 ℃, 30s at 54.4 ℃, 2mi9 30s at 72 ℃ and 34 cycles; 10mi9 at 72 ℃; infinity at 4 ℃.
S34, electrophoresis detection: 10.0 mu L of PCR reaction product is electrophoresed on 1.0% agarose gel (150V, 30mi 9), DL2000 DNA Marker is taken as molecular weight control, the result is observed and photographed in a UVP gel imaging system, if a specific band of the polluted endophyte is amplified, the bacteria removal failure is judged, and if no specific band exists, the bacteria removal success is shown.
As shown in FIG. 3 and Table 1, the effect of removing the contaminating endophyte XF-NSJ from the test-tube plantlet cultured and produced by using the stem tip of the test-tube plantlet of 0.5mm small cymbidium is 100.0%, so that the stem tip of the orchid with the size of 0.5mm is selected to be cut and cultured, and the contaminating endophyte in the orchid can be completely removed.
TABLE 1 success rate of germ removal of test-tube plantlet of 'small phoenix-like orchid' cultured by stem tips of different sizes
Example 2 cultivation method of orchid seedlings without endophytic bacteria XF-NSJ pollution
The same as example 1 except that the media formulations were different. Specifically, the induction culture medium is MS +1.0 mg/L6-BA +0.1mg/L NAA +8.0g/L carrageenan +30.0g/L white granulated sugar +0.2g/L AC +5.0% (w/w) coconut milk, and the pH value is 5.6.
The enrichment medium is MS +0.5 mg/L6-BA +0.5mg/L NAA +30.0g/L white granulated sugar +7.0g/L carrageenan +0.3g/L AC, and the pH is 5.6.
The differentiation medium is MS +1.0 mg/L6-BA +0.1mg/L NAA +30.0g/L white granulated sugar +7.0g/L carrageenan +0.00g/L AC, pH 5.6.
Rooting and seedling strengthening culture medium MS +0.2 mg/L6-BA +1.0mg/L NAA +7.0g/L carrageenan +30.0g/L white granulated sugar +0.3g/L AC, pH 5.6.
Example 3 cultivation method of orchid seedlings without endophytic bacterium XF-NSJ contamination
The same as example 1 except that the media formulations were different. Specifically, the induction culture medium is MS +1.5mg/L6-BA +0.3mg/L NAA +7.5g/L carrageenan +30.0g/L white granulated sugar +1.0g/L AC +20.0% (w/w) coconut milk, and the pH value is 6.0.
The enrichment medium is MS +2.0 mg/L6-BA +0.35mg/L NAA +30.0g/L white granulated sugar +7.0g/L carrageenan +0.4g/L AC, and the pH is 6.0.
The differentiation medium is MS +2.0 mg/L6-BA +0.15mg/L NAA +30.0g/L white granulated sugar +7.0g/L carrageenan +0.05g/L AC, and the pH is 6.0.
Rooting and seedling strengthening culture medium MS +0.15 mg/L6-BA +0.75mg/L NAA +7.0g/L carrageenan +30.0g/L white granulated sugar +0.4g/L AC, pH 6.0.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A cultivation method of orchid seedlings without endophyte pollution is characterized by comprising the following steps:
s1, taking a strong orchid test-tube plantlet as a material, aseptically cutting a stem tip with the size of 0.5mm, and culturing on an induction culture medium to obtain an intermediate propagule;
s2, carrying out enrichment culture on the intermediate propagules, and carrying out differentiation culture when more than 10 intermediate propagules are propagated from each stem tip to obtain orchid seedlings.
2. The cultivation method according to claim 1, further comprising verifying the presence or absence of endophyte contamination of the orchid seedlings obtained in step S2.
3. The cultivation method according to claim 2, wherein the verification is performed by extracting orchid seedling DNA, performing PCR amplification reaction with an endophyte detection primer, and detecting the amplification product by electrophoresis to determine whether there is endophyte contamination.
4. The cultivation method according to claim 1, wherein the inducing medium of step S1 is MS + 1.0-2.0 mg/L6-BA + 0.1-0.5 mg/L NAA + 5.0-20.0% (w/w) coconut milk +30.0g/L white granulated sugar + 7.0-8.0 g/L carrageenan + 0.2-1.0 g/L AC, pH 5.6-6.0; the culture is carried out for 2 to 3 months at a temperature of between 24 and 26 ℃ in the dark.
5. The cultivation method according to claim 1, wherein the culture medium for enrichment culture in step S2 is MS + 0.5-2.0 mg/L6-BA + 0.2-0.5 mg/L NAA +30.0g/L white granulated sugar +7.0g/L carrageenan + 0.3-0.5 g/L AC, pH 5.6-6.0; the proliferation culture is carried out in the dark at the temperature of 24-26 ℃ or under the illumination of 11-13 h every day and under the illumination intensity of 600-1000 lux, and the subculture is carried out for 1 time at 40-60 d.
6. The cultivation method according to claim 1, wherein the culture medium for the differentiation culture in step S2 is MS + 1.0-2.0 mg/L6-BA + 0.1-0.2 mg/L NAA +30.0g/L white sugar +7.0g/L carrageenan + 0.00-0.05 g/L AC, pH 5.6-6.0; the differential culture is carried out for 40-60 days at 24-26 ℃ under the illumination intensity of 1000-2000 lux for 11-13 h per day.
7. The cultivation method as claimed in claim 1, wherein the orchid is a 'Xiaofenglan'.
8. The cultivation method as claimed in claim 1, wherein the endophyte is an endophyte XF-NSJ.
9. The cultivation method according to claim 8, wherein the specific primers for detecting and verifying the endogenous bacterial XF-NSJ comprise an upstream primer and a downstream primer, and the sequences of the upstream primer and the downstream primer are respectively: 5 'GGAAAGATTTTTTGGTTGGGG-3', a downstream primer: 5 'CCTTTGAGTTTAGCCTTGCG-doped 3'.
10. The culture method according to claim 9, wherein the PCR amplification reaction system for detecting and verifying the endophytic bacterium XF-NSJ is as follows: 12.0 mu L of Mix, 1.0 mu L of upstream primer, 1.0 mu L of downstream primer, 10.0 mu L of ddH2O and 1.0 mu L of template DNA, and the total amount is 25.0 mu L; and (3) amplification procedure: 5min at 90 ℃; 30s at 94 ℃, 30s at 54.4 ℃, 2min at 72 ℃, 30s, and 34 cycles; 10min at 72 ℃; infinity at 4 ℃.
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| CN105165612A (en) * | 2015-09-25 | 2015-12-23 | 临沂大学 | Cleisostoma scolopendrifolium tissue culture and rapid propagation method |
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| CN106258997A (en) * | 2016-11-01 | 2017-01-04 | 玉林师范学院 | Without away from calanthe high quality seedling method for quickly breeding |
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