CN115669540B - Cultivation method of orchid seedlings free of endophyte pollution - Google Patents
Cultivation method of orchid seedlings free of endophyte pollution Download PDFInfo
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Abstract
The invention provides a cultivation method of orchid seedlings without endophytic pollution, which is characterized in that the stem tip of orchid test-tube seedlings with the size of 0.5mm is cut and cultivated, intermediate propagules are induced by the stem tip cultivation, and then the intermediate propagules are proliferated and differentiated to obtain orchid seedlings without endophytic pollution. The method thoroughly removes endophytes polluted in the orchid by cutting the stem tip with proper size for culture, can lead the tissue culture and rapid propagation of the orchid to be carried out in proper culture conditions and culture mediums, reduces the influence of antibiotics and the like on the tissue culture and rapid propagation of the orchid, and further improves the quality and benefit of 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 endophytic pollution.
Background
Orchid (Cymbidium) is a plant of the genus orchid of the family orchid and is a world-wide flower of great ornamental, economic, cultural and ecological value. About 68 orchids have been found in the world, 49 of which are found in our country, accounting for 72.1% of the world. Orchid has long cultivation history and rich cultural connotation in China, is called as 'monarch in flower', is also deeply favored by people in all countries of the world, and can be used as high-grade cut flowers and high-grade potted flowers. With the improvement of the living standard and cultural quality of people, orchids start to enter common families, are favored by more and more consumers, and have wide market prospect.
Endophyte pollution refers to pollution caused by the fact that endophytes exist in plants and a general surface disinfection method is difficult to thoroughly remove and enters a culture process along with an explant material. Endophyte contamination often occurs in the industrial production of ornamental seedlings, generally after 5d inoculation or in subculture, forms "filaments" or "halos" in the medium at an early stage, is not easily perceived by the naked eye, is easily found when examined under backlit conditions, and after multiple subcultures, bacterial load accumulates before it emerges in the medium (Kritzi 9ger et al, 1997; zhou Junhui et al, 2003; shang Xueyan et al, 2014;Srivastava et al et al, 2021). Heretofore, endophyte contamination has been found in tissue culture rapid propagation of 47 (genus) plants, and about 51 endophytes have been isolated therefrom and identified. Research results show that endophyte pollution has adverse effects on each link of 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 is of great significance for further improving the factory production efficiency and benefit of 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 bacterial content of explants, changing the components or pH value of a culture medium, adding antibiotics into the culture medium, culturing degerming seedlings and the like (Zhou Quan men and the like 2012; zhou Junhui and the like 2003; wen Luhua and Wu Ruina, 2020). In the method, the culture of the degerming seedlings is a fundamental method for controlling endophyte pollution, chen Chuangong and the like (2019) take stem tip meristem of the cluster buds of Gannan navel oranges as explants, the aseptic seedling of fructus aurantii is taken as stock, and the method of combining heat treatment with stem tip culture degerming is adopted, so that the degerming seedlings are successfully cultured, the degerming rate can reach 93.2% (Chen Chuangong, gu Xiao, yingjia, and the like).
As 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. How to degerming orchid is not reported at present. The existing endophyte pollution prevention and control technology is mainly used for reducing the influence of endophytes on culture materials, so that endophyte pollution is difficult to thoroughly eliminate, and some prevention and control measures can also have adverse effects on proliferation and differentiation of orchid intermediate propagules and test tube seedling production. Therefore, the technology for removing the endophytes of the orchid is developed, the problem of endophyte pollution is thoroughly solved, and the method has important significance for improving the competitiveness of orchid seedling industry and turning over the walking stick in seed industry.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings in the prior art and provide a cultivation method of 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 pollution comprises the following steps:
s1, stem tip culture: taking strong orchid test-tube plantlets as materials, cutting stem tips with the size of 0.5mm under aseptic condition, and culturing on an induction culture medium to obtain intermediate propagules;
s2, proliferation and differentiation of intermediate propagules: and (3) 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, so as to obtain orchid seedlings.
The invention cuts the stem tip of the orchid with proper size for culture, thereby thoroughly removing specific polluted endophytes in the orchid, enabling the tissue culture and rapid propagation of the orchid to be carried out in a proper culture condition and culture medium, and reducing the influence of antibiotics and the like on the tissue culture and rapid propagation of the orchid.
Preferably, the method further comprises the step of verifying whether the orchid seedlings obtained in the step S2 are polluted by endophytes. The verification method can be used for separating and culturing endophytes in the process of orchid seedling tissue, or can be used for PCR detection by using detection primers of endophytes by taking total DNA of orchid seedlings as a template.
Further preferably, the verification is that the DNA of orchid seedlings is extracted, the endophyte detection primer is used for carrying out PCR amplification reaction, and the amplified product is detected through electrophoresis, so as to judge whether the endophyte pollution exists or not. Specifically, the CTAB method is adopted to extract the total DNA of the test tube plantlet, the specific primer design is carried out according to the 16S rDNA sequence of the endophyte, the specific primer is utilized to carry out PCR amplification on the total DNA of the test tube plantlet, and the amplified product is subjected to 1.0% agarose gel electrophoresis to detect the degerming effect.
The invention mainly aims at removing endophytes of specific orchid by cutting stem tips with proper sizes for culture; the three steps of induction, proliferation and differentiation of the intermediate propagules have no influence on the removal of endophytes, but have influence on whether degerming seedlings can be produced.
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 juice+30.0 g/L white granulated sugar+7.0-8.0 g/L carrageenan+0.2-1.0 g/L AC, and the pH is 5.6-6.0; the culture is carried out for 2-3 months in the dark at 24-26 ℃.
Preferably, the stem tip is cultured into a strong orchid test tube seedling with the selected plant height of about 8cm, a sterile normal temperature surgical knife and a dissecting needle are used for removing the leaf blade in a sterile culture dish under a dissecting mirror on an ultra-clean workbench to strip the sheath blade and the leaf blade, and the stem tip with the size of 0.5mm is cut for induction culture.
Preferably, the culture medium for the proliferation 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+7.0 g/L carrageenan+0.3-0.5 g/L AC, and the pH is 5.6-6.0; the proliferation culture is carried out in darkness at 24-26 ℃ or in light for 11-13 hours every day, the light intensity is 600-1000 lux, and the culture is carried out for 40-60 days for 1 time.
Further preferably, the proliferation culture is performed by cutting the intermediate propagules in step S1 to a size of 0.5 cm. Times.0.5 cm in a large scale protocorm or less within 1cm, inoculating the cut protocorm to a proliferation medium, and inoculating 8 to 15 pieces (pieces) 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+7.0 g/L carrageenan+0.00-0.05 g/L AC, and the pH is 5.6-6.0; the differentiation culture is carried out at 24-26 ℃ under the illumination for 11-13 h every day with the illumination intensity of 1000-2000 lux for 40-60 d.
Further preferably, the differentiation culture is that the root-like stems with the length of 0.8-1.0 cm without topping or the protocorms cut into the size of 0.5cm multiplied by 0.5cm are inoculated into a differentiation culture medium, 8-12 (blocks) are inoculated into each bottle, and the culture is carried out at 25 ℃ for 11-13 hours in daily illumination with the illumination intensity of 1000-2000 lux for 40-60 days; inoculating the seedling with the plant height of 3.0-5.0 cm into rooting and seedling strengthening culture medium, and culturing for 40-60 d under the same condition.
Preferably, the rooting and seedling-strengthening culture medium formula is MS+0.1-0.2 mg/L6-BA+0.5-1.0 mg/L NAA+30.0g/L white granulated sugar+7.0 g/L carrageenan+0.3-0.5 g/L AC, and the pH is 5.6-6.0.
Preferably, the orchid is 'small kalimeris'.
Preferably, the endophyte is an endophyte XF-NSJ.
Preferably, the specific primer for detecting the endophytic bacteria XF-NSJ comprises an upstream primer and a downstream primer, and the sequences of the upstream primer and the downstream primer are respectively: 5'-GGAAAGATTTTTTGGTTGGGG-3', downstream primer: 5'-CCTTTGAGTTTTAGCCTTGCG-3'.
Further preferably, the PCR amplification reaction system for detecting endogenous bacteria XF-NSJ is as follows: mix 12.0. Mu.L, 1.0. Mu.L of upstream primer, 1.0. Mu.L of downstream primer, 10.0. Mu.L of ddH2O, 1.0. Mu.L of template DNA, 25.0. Mu.L in total; amplification procedure: 5mi9 at 90 ℃;94℃30s,54.4℃30s,72℃2 mm 9 30s,34 cycles; 10mi9 at 72 ℃;4 ℃ is infinity.
Compared with the prior art, the invention has the following beneficial effects:
the invention can thoroughly remove the polluted endophytes in orchid by cutting the stem tip of orchid with proper size for culture, and can lead the tissue culture and rapid propagation of orchid to be carried out in proper culture conditions and culture medium, thus obtaining orchid seedlings without endophyte pollution, thereby eliminating the influence of endophyte pollution on the tissue culture and rapid propagation of orchid, improving the efficiency and benefit of tissue culture and rapid propagation, avoiding the influence of antibiotics and the like on the tissue culture and rapid propagation of orchid, and further improving the quality and benefit of seedlings.
Drawings
FIG. 1 is the effect of shoot tip size on the induction of intermediate propagules in 'small allium'. Wherein A is the stem tip with different sizes and the root-like stems formed by the induction of the stem tip, and B is the root-like stem induction rate of the stem tip with different sizes.
FIG. 2 shows the effect of shoot tip size on proliferation, differentiation and rooting of the root stem of 'Dragon's own. Wherein A is the proliferation root-like stem number of the stem tips with different sizes, B is the differentiation seedling number of the stem tips with different sizes, C is the test tube seedling number produced by the stem tips with different sizes, and D is the proliferation root-like stem and differentiation seedling of the stem tips with different sizes.
FIG. 3 shows the test tube seedling degerming effect detection of 'small phoenix' cultured by stem tips with different sizes. Wherein M: DL2000Marker,1: culturing test tube plantlets with 0.5mm stem tip, 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 seedling of 'small phoenix' contaminated by endophyte, 6: negative blank control.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The invention is further illustrated by taking an orchid variety of 'small phoenix' as an example, wherein the 'small phoenix' is a new orchid variety obtained by hybridization of 'large phoenix' hybrid orchid serving as a female parent and 'penguin Bai Mo' black orchid serving as a male parent, and the strain is similar to 'penguin Bai Mo', and is easy to be quickly propagated by tissue culture and divided propagation. The single branches are evenly colored with 9 flowers, the flower diameter is 7.4cm, the flowers are orange, and the fragrance is provided. Flowers are started in the beginning of the greenhouse cultivation in the last ten days of 1 month, and the flowering period is 35-45 d.
Bacteria XF-NSJ are endophytes polluted by orchid which are self-separated by the inventor, are one of the genus Brevibacterium (Curtibacterium), and the 16S rDNA sequence is as follows:
AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGATGAAGCCCAGCTTGCTGGGTGGATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCCTGACTCTGGGATAAGCGTTGGAAACGACGTCTAATACTGGATATGACCGCCGATCGCATGGTCTGGTGGTGGAAAGATTTTTTGGTTGGGGATGGACTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCAACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTTAGTAGGGAAGAAGCGTAAGTGACGGTACCTGCAGAAAAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCTGTGAAATCCCGAGGCTCAACCTCGGGCTTGCAGTGGGTACGGGCAGACTAGAGTGCGGTAGGGGAGATTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGATCTCTGGGCCGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGTTGGGCGCTAGATGTAGGGACCTTTCCACGGTTTCTGTGTCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAACGGCCAGAGATGGTTGCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCTATGTTGCCAGCGCGTTATGGCGGGGACTCATAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGCTGCGATACCGTAAGGTGGAGCGAATCCCAAAAAGCCGGTCTCAGTTCGGATTGAGGTCTGCAACTCGACCTCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCTTGTGGAAGGAGCCGTCGAAGGTGGGATCGGTGATTAGGACTAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGATCACCT。
example 1 cultivation method of orchid seedlings free of Endoconcha XF-NSJ contamination
S1, stem tip culture: taking a strong test-tube seedling of the 'small kalimeris' as a material, cutting off a stem tip under aseptic condition, and culturing on an induction culture medium to obtain a root-shaped stem (intermediate propagule); the method comprises the following steps:
s11, test tube seedling selection and stem tip separation: selecting a strong test-tube plantlet of 'small kalimeris' with a plant height of about 8cm, removing the sheath leaves and the leaves from a sterile culture dish under a dissecting mirror on an ultra-clean workbench by using a sterile normal-temperature surgical knife and a dissecting needle, and cutting off stem tips of 0.5, 1.0, 2.0 and 3.0mm respectively (upper diagram of FIG. 1A);
s12, root-like stem induction: the stem tip is quickly inoculated into an induction culture medium MS+2.0mg/L6-BA+0.5mg/LNAA+7.0 g/L carrageenan+30.0 g/L white granulated sugar+0.5 g/L AC+10.0% (w/w) coconut juice, pH is 5.8, and the stem tip is cultured for 90d in the dark at about 25 ℃ and the induced rhizomes are shown in the lower graph of FIG. 1A. Statistical root-like stem inductances found that the root-like stems of different sizes were different, the larger the stem tip was, the higher the root-like stem inductances were (fig. 1B).
S2, proliferation and differentiation of the rhizomes to produce test tube plantlets: performing proliferation culture on the rhizomes, and performing differentiation culture when 10 rhizomes with the length of about 1.0cm are propagated from each stem tip to obtain test tube plantlets; the method comprises the following steps:
s21, root and stem proliferation: cutting the rhizomes into 0.8-1.0 cm, inoculating the rhizomes into a proliferation culture medium MS+1.5mg/L6-BA+0.2mg/L NAA+30.0g/L white granulated sugar+7.0 g/L carrageenan+0.5 g/L AC, carrying out proliferation culture under the conditions that the pH is 5.8, the temperature is 2421 ℃ and the illumination intensity is 800-1000 lux, carrying out 45D subculture for 1 time, and carrying out subculture to form the rhizomes, wherein the rhizomes are shown in a figure 2D;
s22, differentiating the root-shaped stems to produce test tube plantlets: inoculating a root stem with the length of 0.8-1.0 cm without topping to a differentiation culture medium MS+1.5mg/L6-BA+0.2mg/L NAA+30.0g/L white sugar+7.0 g/L carrageenan+0.02 g/L AC, wherein 8-10 root stems are inoculated to each bottle, and culturing for 45 days at 2421 ℃ under the conditions of illumination intensity of 1000-1500 lux for about 45 days under the condition of illumination intensity of 11-13 h per day, wherein differentiated seedlings are shown in a figure 2D; seedling of about 3.0-5.0 cm is inoculated to rooting and seedling strengthening culture medium MS+0.1 mg/L6-BA+0.5 mg/L NAA+7.0g/L carrageenan+30.0 g/L white granulated sugar+0.5 g/L AC, pH is 5.8, and cultured for about 45D under the same condition, and the produced test-tube seedling is shown in figure 2D.
In addition, the number of the proliferation root-like stems of the stem tips with different sizes and the number of the test-tube seedlings produced by the stem tips with different sizes are shown in fig. 2A-C, and it can be seen that the proliferation root-like stems of the stem tips with different sizes, the number of the differentiation seedlings and the test-tube seedlings produced by the stem tips with different sizes are different, the larger the stem tips are, the more the proliferation root-like stems, the number of the differentiation seedlings and the test-tube seedlings produced are, but the larger the stem tips are, which means that the risk of endophyte pollution is larger.
S3, identifying the effect of removing the polluted endophytes: extracting total DNA of test-tube plantlets of 'small kalimeris' by adopting a CTAB method, designing a specific primer according to a 16S rDNA sequence of polluted endophyte XF-NSJ of 'small kalimeris', carrying out PCR amplification of DNA by using the specific primer, detecting a degerming effect of an amplification product by 1.0% agarose gel electrophoresis, and obtaining pollution-free endophyte test-tube plantlets; the method comprises the following steps:
s31, extracting DNA of test tube plantlets: about 200.0mg of single test tube plantlet is taken, ground into powder by liquid nitrogen, and then total DNA is extracted by a CTAB method.
S32, designing and synthesizing a primer: the specific Primer is designed by adopting Primer 5.0 software according to the 16S rDNA sequence of the polluted endophytic bacteria XF-NSJ, and the specifically designed Primer is as follows:
an upstream primer: 5'-GGAAAGATTTTTTGGTTGGGG-3' the number of the individual pieces of the plastic,
a downstream primer: 5'-CCTTTGAGTTTTAGCCTTGCG-3';
the primers were synthesized by the Cangzhou Praeparata and used for PCR amplification.
S33, PCR amplification: the DNA of test tube seedling cultured by the stem tip of 'small Fenglan' is used as a template, and a 16S rDNA specific primer of polluted endophytic bacteria XF-NSJ is adopted for PCR amplification. Amplification reaction system (25.0 μl): 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. Amplification procedure: 5mi9 at 90 ℃;94℃30s,54.4℃30s,72℃2 mm 9 30s,34 cycles; 10mi9 at 72 ℃;4 ℃ is infinity.
S34, electrophoresis detection: taking 10.0 mu L of PCR reaction products, electrophoresis on 1.0% agarose gel (150V, 30mi 9), taking DL2000 DNA Marker as a molecular weight control, observing the result in a UVP gel imaging system and photographing, judging that the degerming is failed if a specific band of polluted endophyte is amplified, and judging that the degerming is successful if no specific band exists.
As shown in FIG. 3 and Table 1, the effect of removing the contaminated endophyte XF-NSJ of the test tube plantlet produced by culturing the stem tip of the test tube plantlet of 0.5mm 'Xiaofang' is 100.0%, so that the contaminated endophyte in orchid can be thoroughly removed by selecting and cutting the stem tip of orchid with the size of 0.5mm for culturing.
TABLE 1 success rate of degerming test tube plantlets of 'small-phoenix' cultured with different sizes of stem tips
Example 2 cultivation method of orchid seedlings free of Endoconcha XF-NSJ contamination
Substantially the same as in example 1, except that each medium was formulated differently. Specifically, the induction culture medium is MS+1.0mg/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 juice, and the pH is 5.6.
The proliferation culture medium is MS+0.5 mg/L6-BA+0.5 mg/L NAA+30.0g/L white granulated sugar+7.0 g/L carrageenan+0.3 g/L AC, and the pH is 5.6.
The differentiation medium is MS+1.0mg/L6-BA+0.1mg/L NAA+30.0g/L white sugar+7.0g/L carrageenan+0.00 g/L AC, and the pH is 5.6.
Rooting and seedling strengthening culture medium MS+0.2 mg/L6-BA+1.0 mg/L NAA+7.0g/L carrageenan+30.0 g/L white granulated sugar+0.3 g/L AC, and pH is 5.6.
Example 3 cultivation method of orchid seedlings free of Endoconcha XF-NSJ contamination
Substantially the same as in example 1, except that each medium was formulated differently. Specifically, the induction medium is MS+1.5 mg/L6-BA+0.3 mg/L NAA+7.5g/L carrageenan+30.0 g/L white granulated sugar+1.0 g/L AC+20.0% (w/w) coconut juice, and the pH is 6.0.
The proliferation medium is MS+2.0mg/L6-BA+0.35 mg/L NAA+30.0g/L white granulated sugar+7.0 g/L carrageenan+0.4 g/L AC, and the pH is 6.0.
The differentiation medium is MS+2.0mg/L6-BA+0.15mg/L NAA+30.0g/L white sugar+7.0g/L carrageenan+0.05 g/L AC, and the pH is 6.0.
Rooting and seedling strengthening culture medium MS+0.15 mg/L6-BA+0.75 mg/L NAA+7.0g/L carrageenan+30.0 g/L white granulated sugar+0.4 g/L AC, and pH is 6.0.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (5)
1. The cultivation method of orchid seedlings is characterized by comprising the following steps:
s1, taking strong orchid test-tube plantlets as materials, cutting stem tips with the size of 0.5mm under the sterile condition, and culturing on an induction culture medium to obtain intermediate propagules;
s2, performing proliferation culture on the intermediate propagules, and performing differentiation culture when more than 10 intermediate propagules are propagated from each stem tip to obtain orchid seedlings;
extracting orchid seedling DNA, carrying out PCR amplification reaction by using endophyte detection primers, and carrying out electrophoresis detection on an amplification product, wherein the seedling without an amplification strip is the orchid seedling with successful degerming;
the endophyte detection primer comprises an upstream primer and a downstream primer, and the sequences of the upstream primer and the downstream primer are respectively: 5'-GGAAAGATTTTTTGGTTGGGG-3', downstream primer: 5'-CCTTTGAGTTTTAGCCTTGCG-3';
the orchid is 'small phoenix blue'.
2. The cultivation method according to claim 1, wherein the induction 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 juice+30.0 g/L white granulated sugar+7.0-8.0 g/L carrageenan+0.2-1.0 g/L AC, and the pH is 5.6-6.0; the culture is carried out for 2-3 months in the dark at 24-26 ℃.
3. The cultivation method according to claim 1, wherein the culture medium for the proliferation 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+7.0 g/L carrageenan+0.3-0.5 g/L AC, and the pH is 5.6-6.0; the proliferation culture is carried out in darkness at 24-26 ℃ or under the condition of illumination of 11-13 h per day, the illumination intensity is 600-1000 lux, and the culture is carried out for 40-60 d subcultures for 1 time.
4. The cultivation method according to claim 1, wherein 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+7.0 g/L carrageenan+0.00-0.05 g/L AC, and the pH is 5.6-6.0; the differentiation culture is carried out at 24-26 ℃ under the illumination of 11-13 h per day under the illumination intensity of 1000-2000 lux for 40-60 d.
5. The method according to claim 1, wherein the PCR amplification reaction system for detecting endophytes is as follows: 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, 25.0. Mu.L in total; amplification procedure: 90 ℃ for 5 min;94℃for 30s,54.4℃for 30s,72℃for 2 min for 30s,34 cycles; 72 ℃ for 10min;4 ℃ is infinity.
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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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