CN116875729A - Method for obtaining aromatic butterfly orchid excellent tissue culture explant - Google Patents
Method for obtaining aromatic butterfly orchid excellent tissue culture explant Download PDFInfo
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Abstract
The application discloses a method for obtaining an excellent tissue culture explant of aromatic butterfly orchid, which comprises the steps of one-time aromatic character screening of a tissue culture explant plant, and three virus detection screening steps of RT-PCR (Reverse transcription-polymerase chain reaction), qRT-PCR (Quantitative Real-time polymerase chain reaction) and small RNA sequencing, wherein the excellent tissue culture explant of the aromatic butterfly orchid obtained through screening can be used for mass production of aromatic butterfly orchid pot plants which are stable in aromatic character and do not carry viruses, the virus transmission source in an aromatic butterfly orchid pot production system is cut off from the aspect of the explant, the problem of aromatic character degradation in the aromatic butterfly orchid pot production process is solved, and important guarantee is provided for mass production of the aromatic butterfly orchid.
Description
Technical Field
The application relates to the field of plant propagation, in particular to a method for obtaining an excellent tissue culture explant of aromatic butterfly orchid.
Background
The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.
The butterfly orchid (Phalaenopsis spp.) is a plant of Phalaenopsis (Orchidaceae) of Orchidaceae, which has rich and gorgeous flower color, unique flower shape, long flowering period, and is a reputation of "Royal" and an important flower species in the past, and has important commercial value. Although there are many kinds of butterfly orchid, there are few kinds of butterfly orchid having aromatic odor, and butterfly orchid aromatic traits are not a stable inherited trait, and they may be degraded during propagation or cross breeding.
The individual plant property of the butterfly orchid plant is strong, tillers are rarely generated, and the butterfly orchid plant can be separated in the cultivation process. The butterfly orchid cultivated in commercial way is propagated by tissue culture, and the long-term asexual propagation causes serious virus accumulation, so that the variety characteristics are seriously degraded, the ornamental value is greatly reduced, and huge economic loss is caused, thereby severely restricting the development of butterfly orchid industry in China. Currently, there are about 50 or more viruses capable of infecting orchids, of which cymbidium mosaic virus (Cymbidium mosaic virus, cymv) and dental ringspot virus (Odontoglossum ringspot virus, ORSV) are two viruses that are more severely and commonly infected in orchids. However, conventional virus detection methods can detect only one or several viruses at a time, and cannot detect all viruses carried by plants at a time. For example, the prior art discloses a method for cultivating and tissue-culturing new strain of detoxified butterfly orchid, which uses RT-PCR to detect cymbidium mosaic virus and odontopathy virus, then selects the plant which does not contain the two viruses to conduct tissue-culturing and propagation to obtain a primary parent bottle, the method can only detect whether the cymbidium mosaic virus and the odontopathy virus are contained, but can not detect whether the plant carries other viruses at the same time, and the single detection result has contingency. Thus, the tissue culture seedlings screened in the prior art may still carry the two viruses and the other various viruses.
The obtained excellent tissue culture explant which is virus-free and has stable aromatic character is the basis for producing excellent seed sources of the aromatic butterfly orchid, and is an important guarantee for keeping stable aromatic character and other ornamental characteristics of potted aromatic butterfly orchid products. However, no related research report aiming at the screening of excellent explants of aromatic butterfly orchid is yet reported.
Disclosure of Invention
The application aims at: aiming at the problems that the variety of the butterfly orchid with aromatic odor is few and inheritance is unstable and aromatic character is seriously influenced by virus in tissue culture breeding at present, the method for obtaining the excellent tissue culture explant of the butterfly orchid is provided, and the excellent tissue culture explant with excellent aromatic character and no virus can be assisted to be rapidly screened out, so that a large number of butterfly orchid plants with excellent aromatic character can be rapidly bred in a tissue culture mode.
The technical scheme of the application is as follows:
a method for obtaining an excellent tissue culture explant of aromatic butterfly orchid mainly comprises the following steps:
step 1: screening for the first time based on the determination of the content of the aromatic main active ingredient of flowers in the full bloom stage of the aromatic butterfly orchid, and selecting aromatic butterfly orchid plants meeting a first condition;
step 2: screening the aromatic butterfly orchid plants selected in the step 1 for the second time based on RT-PCR, and selecting aromatic butterfly orchid plants meeting the second condition;
step 3: thirdly screening the aromatic butterfly orchid plants selected in the step 2 based on qRT-PCR, and selecting aromatic butterfly orchid plants meeting a third condition;
step 4: and (3) screening the aromatic butterfly orchid plants selected in the step (3) for the fourth time based on small RNA sequencing, and selecting the aromatic butterfly orchid plants meeting the fourth condition, wherein each pedicel node position obtained on the plants is the excellent tissue culture explant of the aromatic butterfly orchid.
The fragrance of the aromatic butterfly orchid of different varieties is different, and the main aromatic components are also different. The method is suitable for screening out the excellent tissue culture explants of the aromatic butterfly orchid which have excellent aromatic character and do not carry viruses from the same aromatic butterfly orchid variety. The method can be used for screening the aromatic butterfly orchid of different varieties.
Through the steps, firstly, plants with excellent aromatic character are screened out through the step 1, then whether the plants contain viruses or not is checked, and finally, the plants with excellent aromatic character and no viruses are screened out through two types of elimination, as the obtained plants of the excellent tissue culture explants, the pedicel nodes obtained on the plants are the excellent tissue culture explants, the influence of the viruses on the plant characteristics can be avoided in the tissue culture process of the excellent tissue culture explants, the guarantee is provided for stable expression of the aromatic character, and the probability of cultivating the plants without viruses and with excellent aromatic character is improved. In addition, the cost of the aromatic component measurement is lower than that of the virus detection, and the sample size of the first screening is the largest, so that the aromatic component measurement can save a great deal of cost when being used as the first screening, and plants which do not need to be subjected to the virus detection can be directly eliminated.
Step 2, step 3 and step 4 progressively detect viruses layer by layer through different virus detection methods, wherein the virus detection accuracy and cost of RT-PCR, qRT-PCR and smallRNA are progressively increased layer by layer, the detection range of smallRNA is obviously enlarged, and the number of plant samples to be detected is progressively reduced layer by layer after screening samples are removed layer by layer along with the increase of screening times, so that plants without viruses can be accurately screened out by using lower screening cost, and the cultivation requirements are met while the method is more economical and practical.
The selected excellent explant of the aromatic butterfly orchid is applied to potted production of the aromatic butterfly orchid after being used for tissue culture propagation to obtain tissue culture seedlings, can obtain aromatic butterfly orchid products with stable aromatic character and no virus in batches, and provides important guarantee for large-scale production of the aromatic butterfly orchid.
According to a preferred embodiment, step 1 further comprises the sub-steps of:
step 1.1: determining the main aroma component;
step 1.2: determining the content of aromatic main components of all aromatic butterfly orchid plants in the screening population; preferably, the screening population is greater than 100 strains;
step 1.3: and sequencing all the aromatic butterfly orchid plants in the screening group from high to low based on the content of the aromatic main active ingredient.
According to a preferred embodiment, said step 1.1 further comprises the sub-steps of:
step 1.1.1: detecting the content of all aromatic components in the flowers, and calculating the aroma intensity values of all aromatic components; the aromatic component content detection method is gas chromatography-mass spectrometry (Gas Chromatography-Mass Spectrometer, GC-MS);
step 1.1.2: comparing the fragrance intensity values of all the fragrance components, and selecting the fragrance intensity value with the highest fragrance intensity value as the fragrance main active component.
The aroma intensity value is calculated by the following steps:
according to a preferred embodiment, the first condition is: the content of the aromatic main active ingredients is ranked within the first 30 percent; namely, plants with lower content of aromatic main components are discarded through the first screening, and only plants with higher content of the first 30% are taken as screening groups of the second screening.
The aroma main components which make the greatest contribution to the aroma character can be compared through the calculation of the aroma intensity value, and then the plants with high aroma main components are selected as the candidate plants of tissue culture explant materials, so that the aromatic butterfly orchid plants with excellent aroma character can be cultivated with higher probability.
According to a preferred embodiment, in step 2 and step 3, the detection of cymbidium mosaic virus and odontobamoviruses is performed on aromatic butterfly orchid plants using leaves as material.
According to a preferred embodiment, the fourth screening in step 4 is: and (5) preparing a mixed sample of the bud bracts of each pedicel node of the aromatic butterfly orchid plant, and carrying out small RNA sequencing.
According to a preferred embodiment, the fourth condition is that no virus is carried, specifically that after the 16-28 nt sequences in clean data are assembled and spliced, no virus information is aligned in the Virus RefSeq Nucleotide and Virus RefSeq Protein databases of GenBank.
The virus types which can be detected by the RT-PCR and qRT-PCR are limited, the RT-PCR and qRT-PCR are used for detecting the cymbidium mosaic virus and the odontopathy virus which are widely infected, plants containing the two viruses are removed, the plants without the two viruses are subjected to full virus infection confirmation by adopting a small RNA sequencing method, the fourth screening is carried out to confirm whether the plants contain other viruses except the two viruses, and simultaneously, the small RNA sequencing can also be used for rechecking the detection results of the cymbidium mosaic virus and the odontopathy virus of the plants which pass the RT-PCR and qRT-PCR detection, and the accuracy of the detection results of the viruses is ensured by a plurality of times of confirmation.
In addition, when the small RNA sequencing is performed to detect viruses, the selected material is the stem bract of each stem node of the aromatic butterfly orchid, and the mixed sample is prepared. Because the virus is distributed differently in different parts of the butterfly orchid plant, such as roots, leaves, pedicel and the like, the material used for virus detection also affects the accuracy of the virus detection result. The stem segments are selected as the explants of the tissue culture of the aromatic butterfly orchid, the interior of the explants contains axillary buds which proliferate to form a large number of adventitious buds in the tissue culture process, and then tissue culture seedlings are formed, so that all tissue culture Miao Jun originates from the stem axillary buds, and the stem axillary buds are the most critical parts in the tissue culture explants of the aromatic butterfly orchid. The outside of the peduncle axillary bud is covered with the peduncle bract, the two are closely connected, the virus carrying situation is closest, therefore, the application uses the peduncle bract to detect the virus, can guarantee the accuracy of the virus detection result under the condition that the peduncle axillary bud is not damaged; all the bud slices of the flower stalks of the same plant are sampled to prepare a mixed sample for detection, so that the accidental of a single bud slice detection result can be avoided, and the accuracy of the detection result is further improved; moreover, when the Small RNA detection result shows that the virus is not carried, all pedicel nodes of the plant can be used as excellent explants of the aromatic butterfly orchid.
According to a preferred embodiment, the second condition is that the detection result is negative, in particular that the electrophoresis of RT-PCR detects a virus-free band.
According to a preferred embodiment, the third condition is negative in the detection result, in particular no numerical value for the Cq value.
The cost of single sample detection of RT-PCR and qRT-PCR is about 60-80 yuan, the relative cost of qRT-PCR is slightly higher, the single cost of small RNA sequencing is 1200-1600 yuan, and the cost is 20 times of that of RT-PCR and qRT-PCR, so that the excessive samples are screened by RT-PCR and qRT-PCR before the small RNA sequencing is carried out, and a large amount of detection expenditure can be saved.
According to a preferred embodiment, further comprising step 5: verification of explant screening results. And carrying out offspring breeding by adopting the finally screened aromatic butterfly orchid excellent explants, and carrying out aromatic character stability detection and virus detection on offspring plants to verify the actual use effect of the screened explants.
According to a preferred embodiment, in step 5, the offspring plants are subjected to the determination of the stability of the aroma trait in the following manner: the aromatic main component of the offspring plant is reduced by not more than 15% compared with the explant plant, and the offspring plant is regarded as aromatic character stable.
According to a preferred embodiment, in step 5, the progeny plant virus is detected by: and (3) carrying out virus detection by using a small RNA sequencing method, wherein after 16-28 nt sequences in clean data of all plants are assembled and spliced, virus information is not compared in Virus RefSeq Nucleotide and Virus RefSeq Protein databases of GenBank, and if the virus information is not considered to be provided with viruses, otherwise, the virus information is carried.
Compared with the prior art, the application has the beneficial effects that:
1. a method for obtaining excellent tissue culture explants of aromatic butterfly orchid, through combining aromatic character screening with virus detection screening, can select excellent tissue culture explants with excellent aromatic character and without virus, so as to reduce the probability of remarkable degradation of aromatic character of cultivated plants caused by instability of aromatic character and carried virus, cut off virus transmission sources in an aromatic butterfly orchid potting production system from the aspect of the explants, solve the problem of aromatic character degradation in the aromatic butterfly orchid potting production process, and provide important guarantee for large-scale production of aromatic butterfly orchid;
2. the method for obtaining the excellent tissue culture explant of the aromatic butterfly orchid can repeatedly reduce the contingency of virus detection results and the result difference generated by different methods through multiple virus detection, improve the accuracy of virus detection and avoid the influence of the virus on the aromatic character stability of the aromatic butterfly orchid to the greatest extent; the success rate of cultivating plants with excellent aromatic characters is improved;
3. a method for obtaining excellent tissue culture explants of aromatic butterfly orchid, through setting up the detection sequence, screen layer by layer, reject the unconditional plant with lower cost, carry on the final small RNA sequencing work to the eligible plant after verifying repeatedly, confirm whether it carries the virus finally, and get the excellent tissue culture explant from the eligible plant without virus, get the accurate experimental result with the lowest cost, it is more economical and practical;
4. the method for obtaining the excellent explant of the aromatic butterfly orchid is successfully constructed by combining a gas chromatography-mass spectrometry (GC-MS), a virus detection technology and a small RNA sequencing technology, and fills the technical blank of screening the excellent explant in the tissue culture process of the aromatic butterfly orchid.
Drawings
The arrow in fig. 1 shows the bud of the stem node;
fig. 2 shows a full-bloom stage aromatic butterfly orchid flower for aromatic component detection.
Detailed Description
The following examples facilitate a better understanding of the present application. The test methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples below are all commercially available. The quantitative tests in the following examples were all set up in triplicate and the results averaged.
Example 1 selection of aromatic butterfly orchid 'Xiangnier' Excellent tissue culture explants
The fragrant butterfly orchid and the fragrant naphthalene are used as experimental materials.
A method for obtaining an excellent tissue culture explant of aromatic butterfly orchid,
step 1: selecting aromatic butterfly orchid 'rhizoma Kaempferiae' flowering plants with normal phenotypic character, measuring aromatic component content of flowers in full bloom stage of each plant, and selecting plants with the aromatic main component content of top 30% of all detected plants as candidate plants for secondary screening. As shown in FIG. 2, the line segment in the figure is a scale and the length is 1cm.
Step 2: and (2) detecting the cymbidium mosaic virus and the odontopathy virus by adopting the leaf materials and adopting RT-PCR by adopting the plants screened for the first time in the step (1), and selecting the plants with negative detection results as candidate tissue culture explant plants, wherein the negative detection results refer to electrophoresis detection of virus-free strips by RT-PCR.
Step 3: and (2) detecting the cymbidium mosaic virus and the odontopathy virus by adopting the first-screened plants in the step (1) and adopting the leaf materials and adopting qRT-PCR, and selecting the plants with negative detection results as candidate tissue culture explant plants, wherein the negative detection results are that the Cq value of the qRT-PCR detection results is not numerical.
Step 4: small RNA sequencing
And (3) sampling the stem bracts of the plant by using the candidate tissue culture explant plants obtained in the step (3), wherein the stem bracts are indicated by arrows as shown in fig. 1. The line segment in the figure is a scale, and the length is 1cm. And (3) preparing a mixed sample from a stem bract sample of each stem section of the same plant, and performing small RNA sequencing on the stem bract mixed sample of each plant, wherein sequencing analysis shows that all stem sections of the aromatic butterfly orchid plant without virus can be used as an excellent tissue culture explant of the aromatic butterfly orchid. The bud bract specifically refers to a bract covered outside the axillary bud of the flower stalk. The fact that no virus is carried specifically means that the 16-28 nt sequences in clean data are assembled and spliced and then the virus information is not compared.
Step 5: verification of explant screening results
Culturing the aromatic butterfly orchid explant obtained by the method according to the conventional butterfly orchid tissue culture operation, performing field planting on the obtained tissue culture seedling, randomly selecting 10 plants after the field planting plants are opened, selecting 3 flowers in the full bloom stage from each plant, and determining the aromatic component by adopting GC-MS (gas chromatography-mass spectrometry), thereby identifying the stability of the aromatic character. The aromatic major component is reduced by not more than 15% compared with the explant plant, and is considered as stable in aromatic character.
And simultaneously, virus detection is carried out on 10 selected plants by using a small RNA sequencing method, and after 16-28 nt sequences in clean data of the 10 plants are assembled and spliced, virus information is not compared in Virus RefSeq Nucleotide and Virus RefSeq Protein databases of GenBank, and if the virus information is not compared, viruses are considered to be carried, otherwise, viruses are considered to be carried.
Example 2 selection of Excellent tissue culture explants from aromatic butterfly orchid' cherry tomato
The procedure of example 1 was repeated except that the variety of the aromatic butterfly orchid used in this example was different.
The aromatic butterfly orchid variety selected in this example was 'cherry tomato'.
Example 3 selection of aromatic butterfly orchid' Excellent tissue culture explants
The procedure of example 1 was repeated except that the variety of the aromatic butterfly orchid used in this example was different.
The aromatic butterfly orchid variety selected in this example was 'from the top'.
The aroma stability and virus detection results of examples 1, 2 and 3 are shown in table 1:
TABLE 1 aromatic stability and virus carrying Properties of potted plants obtained by culturing aromatic Phalaenopsis tissue culture explants obtained in examples 1-3
| Project | Example 1 | Example 2 | Example 3 |
| Aromatic butterfly orchid variety used | Butterfly orchid 'Xiangnier' | Butterfly orchid cherry tomato' | Butterfly orchid 'is self-luxury' |
| Aromatic main active ingredient | Myrcene | Eucalyptus oil extract | Hexyl acetate |
| The concentration of the aromatic main component of the explant plant (ng/g) | 31.73 | 297.53 | 87.33 |
| Aromatic main ingredient concentration (ng/g) of offspring plants | 33.21 | 282.65 | 82.90 |
| Whether the aromatic character of the offspring plant is stable | Is that | Is that | Is that |
| Whether the offspring plant carries virus | Whether or not | Whether or not | Whether or not |
As shown in the table above, the aromatic butterfly orchid plants cultivated by the explants screened by the method have stable aromatic character and do not carry virus, and can quickly breed a large number of aromatic butterfly orchid plants with excellent aromatic character.
Comparative example 1
The stem segments of the non-screened aromatic butterfly orchid plants purchased from commercial sources are used as explants, the culture is carried out according to the conventional operation of the tissue culture of the aromatic butterfly orchid, the field planting is carried out on the obtained tissue culture seedlings, and after the flowering of the field planting plants, the stability of the aromatic character and the virus carrying condition of the plants are evaluated.
(1) Study object: after flowering of the field plants, 10 plants were randomly selected as the evaluation population.
(2) Evaluation of aroma stability: 3 flowers in the full bloom stage are selected from each plant, the GC-MS is adopted for aromatic component measurement, the stability of aromatic character is identified, the content of aromatic main active components is reduced by not more than 15 percent compared with the explant plant of the embodiment 3, and the aromatic character is considered to be stable, otherwise, the aromatic character is not stable.
(3) Virus detection: and (3) respectively adopting small RNA sequencing to carry out virus detection on 10 selected plants, assembling and splicing 16-28 nt sequences in clean data, and then, judging that virus information is not compared in Virus RefSeq Nucleotide and Virus RefSeq Protein databases of GenBank, and otherwise, carrying the virus. Plant virus carrying rate (%) =number of plants carrying virus/number of detected plants×100%.
The test results are shown in Table 2:
TABLE 2 fragrance stability and Virus carrying Properties of flowering plants obtained from unscreened aromatic Phalaenopsis 'Ex-Fr' explants
| Project | Case(s) |
| Aromatic main active ingredient | Hexyl acetate |
| Aromatic main ingredient concentration (ng/g) of offspring plants | 53.27 |
| Whether the aromatic character of the offspring plant is stable | Whether or not |
| Viral load of offspring plants | 80％ |
Comparing the data of comparative example 1 with the data of example 3, it can be seen that the explants not screened by the method of the present application have unstable aromatic character and very high virus carrying rate in offspring during tissue culture and breeding.
The above examples merely illustrate specific embodiments of the application, which are described in more detail and are not to be construed as limiting the scope of the application. It should be noted that it is possible for a person skilled in the art to make several variants and modifications without departing from the technical idea of the application, which fall within the scope of protection of the application.
This background section is provided to generally present the context of the present application and the work of the presently named inventors, to the extent it is described in this background section, as well as the description of the present section as not otherwise qualify as prior art at the time of filing, are neither expressly nor impliedly admitted as prior art against the present application.
Claims (10)
1. A method for obtaining an excellent tissue culture explant of aromatic butterfly orchid, comprising the steps of:
step 1: screening for the first time based on the determination of the content of the aromatic main active ingredient of flowers in the full bloom stage of the aromatic butterfly orchid, and selecting aromatic butterfly orchid plants meeting a first condition;
step 2: screening the aromatic butterfly orchid plants selected in the step 1 for the second time based on RT-PCR, and selecting aromatic butterfly orchid plants meeting the second condition;
step 3: thirdly screening the aromatic butterfly orchid plants selected in the step 2 based on qRT-PCR, and selecting aromatic butterfly orchid plants meeting a third condition;
step 4: and (3) screening the aromatic butterfly orchid plants selected in the step (3) for the fourth time based on small RNA sequencing, and selecting the aromatic butterfly orchid plants meeting the fourth condition, wherein each pedicel node position obtained on the plants is the excellent tissue culture explant of the aromatic butterfly orchid.
2. A method for obtaining an excellent tissue culture explant of aromatic butterfly orchid according to claim 1, wherein step 1 further comprises the sub-steps of:
step 1.1: determining the main aroma component;
step 1.2: determining the content of aromatic main components of all aromatic butterfly orchid plants in the screening population;
step 1.3: and sequencing all the aromatic butterfly orchid plants in the screening group from high to low based on the content of the aromatic main active ingredient.
3. A method for obtaining an excellent tissue culture explant of aromatic butterfly orchid according to claim 2, characterized in that said step 1.1 further comprises the sub-steps of:
step 1.1.1: detecting the content of all aromatic components in the flowers, and calculating the aroma intensity values of all aromatic components;
step 1.1.2: comparing the fragrance intensity values of all the fragrance components, and selecting the fragrance intensity value with the highest fragrance intensity value as the fragrance main active component.
4. A method for obtaining an excellent tissue culture explant of aromatic butterfly orchid according to claim 3, characterized in that the aroma intensity value is calculated by:
5. a method for obtaining an excellent tissue culture explant of aromatic butterfly orchid according to claim 1, characterized in that said first condition is: the content of the aromatic main active ingredients is ranked within the first 30 percent.
6. The method for obtaining an excellent tissue culture explant of aromatic butterfly orchid according to claim 1, wherein in the step 2 and the step 3, leaves are used as materials, and the detection of the cymbidium mosaic virus and the odontoram ring spot virus is performed on the aromatic butterfly orchid plants.
7. The method for obtaining an excellent tissue culture explant of aromatic butterfly orchid according to claim 1, wherein the fourth screening in step 4 is: preparing a mixed sample from the bud bracts of the pedicel nodes of the aromatic butterfly orchid plants, and performing small RNA sequencing; the fourth condition is that no virus is carried.
8. The method for obtaining an excellent tissue culture explant of aromatic butterfly orchid according to claim 7, wherein the "no virus carrying" is: after 16-28 nt sequences in clean data are assembled and spliced, virus information is not compared in Virus RefSeq Nucleotide and Virus RefSeq Protein databases of GenBank.
9. The method for obtaining an excellent tissue culture explant of aromatic butterfly orchid according to claim 1, wherein the second condition is negative in detection result.
10. The method for obtaining an excellent tissue culture explant of aromatic butterfly orchid according to claim 1, wherein the third condition is negative in detection result.
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