CN116686715A - Butterfly orchid tissue culture seedling cultivation method without carrying cymMV and ORSV - Google Patents
Butterfly orchid tissue culture seedling cultivation method without carrying cymMV and ORSV Download PDFInfo
- Publication number
- CN116686715A CN116686715A CN202310917822.5A CN202310917822A CN116686715A CN 116686715 A CN116686715 A CN 116686715A CN 202310917822 A CN202310917822 A CN 202310917822A CN 116686715 A CN116686715 A CN 116686715A
- Authority
- CN
- China
- Prior art keywords
- tissue culture
- orsv
- butterfly orchid
- cymmv
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000723826 Odontoglossum ringspot virus Species 0.000 title claims abstract description 79
- 238000012364 cultivation method Methods 0.000 title claims description 7
- 244000079298 Epidendrum obrienianum Species 0.000 title 1
- 238000001514 detection method Methods 0.000 claims abstract description 85
- 241000700605 Viruses Species 0.000 claims abstract description 60
- 230000006698 induction Effects 0.000 claims abstract description 38
- 238000000034 method Methods 0.000 claims abstract description 34
- 241000710019 Cymbidium mosaic virus Species 0.000 claims abstract description 33
- 239000000463 material Substances 0.000 claims abstract description 20
- 238000012216 screening Methods 0.000 claims abstract description 16
- 238000004519 manufacturing process Methods 0.000 claims abstract description 6
- 240000002292 Psychopsis papilio Species 0.000 claims abstract 15
- 241000196324 Embryophyta Species 0.000 claims description 48
- 238000003757 reverse transcription PCR Methods 0.000 claims description 33
- 239000001963 growth medium Substances 0.000 claims description 25
- 238000011529 RT qPCR Methods 0.000 claims description 21
- 229920001817 Agar Polymers 0.000 claims description 12
- 244000061456 Solanum tuberosum Species 0.000 claims description 12
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 12
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 12
- 229930006000 Sucrose Natural products 0.000 claims description 12
- 239000008272 agar Substances 0.000 claims description 12
- 239000005720 sucrose Substances 0.000 claims description 12
- 235000020415 coconut juice Nutrition 0.000 claims description 9
- 238000001962 electrophoresis Methods 0.000 claims description 8
- 230000001939 inductive effect Effects 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- 238000005286 illumination Methods 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 238000004382 potting Methods 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 abstract description 5
- 244000127818 Phalaenopsis amabilis Species 0.000 description 45
- 241000218922 Magnoliophyta Species 0.000 description 6
- 241000208293 Capsicum Species 0.000 description 5
- 235000002566 Capsicum Nutrition 0.000 description 5
- 241001505935 Phalaenopsis Species 0.000 description 5
- 239000001390 capsicum minimum Substances 0.000 description 5
- 229910052594 sapphire Inorganic materials 0.000 description 5
- 239000010980 sapphire Substances 0.000 description 5
- 238000001784 detoxification Methods 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 3
- 241000233855 Orchidaceae Species 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000004304 visual acuity Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The application discloses a method for cultivating butterfly orchid tissue culture seedlings without carrying cymMV and ORSV, which comprises the following steps: screening of tissue culture explants, induction of axillary buds of pedicel, induction and screening of adventitious buds of stem tip, induction of cluster buds, culture of tissue culture seedlings and detection of the tissue culture seedlings. The application combines the tissue culture propagation technology and the sensitive and reliable virus detection technology, establishes a reliable method for cultivating the butterfly orchid tissue culture seedlings without carrying the cymMV (Cymbidium mosaic virus, cymMV) and the dental annular spot virus (Odontoglossum ringspot virus, ORSV), blocks the propagation of the cymMV and the ORSV in the downstream production link of the butterfly orchid from the seed source, improves the quality of the butterfly orchid products, saves cultivation time, manpower and material resources and has high cultivation efficiency.
Description
Technical Field
The application relates to the field of plant propagation, in particular to a method for cultivating butterfly orchid tissue culture seedlings without carrying cymv and ORSV.
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.
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 commercially is propagated by tissue culture, and the long-term asexual propagation causes serious virus accumulation, so that the variety characteristic is seriously degraded and the quality is reduced. Cymbidium mosaic virus (Cymbidium mosaic virus, cymv) and odontopathy virus (Odontoglossum ringspot virus, ORSV) are two viruses that are serious and common in butterfly orchid and are the two most critical viruses affecting the development of the butterfly orchid industry. Most of the butterfly orchid tissue culture seedlings are infected with cymv and ORSV singly or in combination, so that the butterfly orchid potted plants produced later carry the cymv and the ORSV. After the butterfly orchid plant is infected by cymv and ORSV, yellow ring spots and streak spots can appear on the leaf respectively, and the flowers fade, so that the ornamental value is greatly reduced, and huge economic loss is caused, thereby severely restricting the development of the butterfly orchid industry in China.
A butterfly orchid detoxification and rapid propagation technology in the prior art discloses: through the steps of primary culture, stem tip elongation culture, detoxification culture, seedling strengthening, rooting culture and the like, a set of method and a rapid propagation way for carrying out secondary phalaenopsis detoxification by combining a physical method with a chemical reagent are obtained, and the method can obtain the detoxified seedling, but the chemical reagent is needed to have adverse effect on the growth of the detoxified seedling, and can possibly cause plant variation to influence the ornamental character of the phalaenopsis; in addition, the detoxification rate of the prior art is only 90%, and the situation that the tissue culture seedlings obtained after final cultivation still carry the CymMV and the ORSV exists in the method, so that when the tissue culture seedlings are put into use, virus detection is still required to be carried out on all the tissue culture seedlings in each batch, the cost is very huge, and the time, the materials and the manpower spent in the cultivation process of the tissue culture seedlings still carrying the CymMV and the ORSV after detection are wasted.
The method for constructing the butterfly orchid tissue culture seedling without carrying the cymMV and the ORSV has important significance for the quick and healthy development of the butterfly orchid industry in China. At present, no report of combining a tissue culture propagation technology and a sensitive and reliable virus detection technology exists in China, and a culture method of the butterfly orchid tissue culture seedlings without carrying cymMV and ORSV is established from the source screening of explants.
Disclosure of Invention
The application aims at: aiming at the problem that the current butterfly orchid tissue culture seedlings carry serious cymv and ORSV, the butterfly orchid tissue culture seedling cultivation method without carrying cymv and ORSV is provided, the planting materials without carrying cymv and ORSV are screened layer by layer in each step for further cultivation, and finally, the cultivated tissue culture seedlings do not carry cymv and ORSV.
The technical scheme of the application is as follows:
the butterfly orchid tissue culture seedling cultivation method without carrying cymMV and ORSV comprises the following steps:
step 1: screening of tissue culture explants
Step 1.1: virus detection was performed based on RT-PCR (Reverse transcription-polymerase chain reaction): selecting butterfly orchid plants with normal phenotype, taking leaves as materials, and respectively adopting RT-PCR to detect CymMV and ORSV;
step 1.2: virus detection was performed based on qRT-PCR (Quantitative Real-time polymerase chain reaction): and (3) taking the stem sections which grow well for plants with negative RT-PCR detection results in the step (1.1), respectively adopting qRT-PCR to detect CymMV and ORSV for the stem bracts of the stem sections, wherein the stem sections corresponding to the stem bracts with the Cq value of more than or equal to 35 can be used as tissue culture explants. The bud bracts specifically refer to bracts covered on the outer sides of axillary buds of each bud node. The RT-PCR detection result is negative, and is that: electrophoresis by RT-PCR detects virus-free bands.
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. All butterfly orchids tissue culture Miao Jun originates from the peduncles axillary buds inside the peduncles, which are the most critical sites in the tissue culture explants. The outside of the axillary bud of the peduncles is covered with the peduncles, the growing positions of the peduncles and the peduncles are closely connected, the virus carrying condition is closest, but the tissue sample quantity of the peduncles is very limited. According to the application, firstly, leaves are used as materials, RT-PCR is used for detecting CymMV and ORSV, plants with negative detection results are obtained through preliminary screening, then qRT-PCR is carried out for taking out the stem bracts from the stem sections used as candidate explants to detect CymMV and ORSV, and under the condition of not damaging the axillary buds of the stems, the stem bract materials are fully utilized, the value of the limited stem bract materials is exerted, and the accuracy of the detection results is ensured.
Most plants with higher virus content can be removed after primary screening by RT-PCR, and for plants without virus bands displayed by RT-PCR, the plants are not necessarily completely plants without CymMV and ORSV due to limitation of visual acuity and position reasons of sampling in the detection process; and detecting plants without virus bands in RT-PCR (reverse transcription-polymerase chain reaction) by qRT-PCR, and respectively sampling the stem bracts of each stem node used as a candidate explant as detection materials, so that errors caused by sampling position difference can be eliminated, the content of viruses can be quantitatively displayed, detection errors can be avoided from the samples, the detection precision is improved from a detection method, the accuracy of the detection result of the viruses is integrally improved, and the tissue culture explants with extremely low or no virus content are screened in an assisted manner. The screening of the explants is used as the first step of the tissue culture seedling cultivation process, the subsequent cultivation result is obviously influenced, and the improvement of the screening accuracy of the explants has an important influence on obtaining a good cultivation result.
Step 2: induction of axillary buds of pedicel
Sterilizing the tissue culture explant selected in the step 1, inoculating the tissue culture explant to a pedicel axillary bud induction culture medium, performing dark culture for 2 weeks, and then performing pedicel axillary bud induction in a dim light environment; the peduncle axillary bud induction culture medium is 1/2MS+2.0-3.0 mg/L6-BA+0.2 mg/L NAA+30g/L potato+50 ml/L coconut water+25 g/L sucrose+7.0 g/L agar, and the pH value is 5.70.
Step 3: induction and screening of adventitious buds of stem tip
Step 3.1: peeling stem tips of the strong axillary buds obtained by induction in the step 2, inoculating the stem tips on an adventitious bud induction culture medium, performing dark culture for 2-3 weeks, and then transferring the culture medium into a weak light environment for culture; the adventitious bud induction culture medium is 1/2MS+1.5-3.0 mg/L TDZ+0.2mg/L NAA+30g/L potato+50 ml/L coconut water+25 g/L sucrose+7.0 g/L agar, and the pH is 5.70.
Step 3.2: and (3) detecting all adventitious buds obtained through induction by adopting a double detection method, namely performing CymMV and ORSV detection, and selecting adventitious buds with double negative detection results for induction of cluster buds. The "dual detection method" is to detect cymv and ORSV using both RT-PCR and qRT-PCR. The detection result is double negative, which is that: the electrophoresis detection of RT-PCR has no virus band and the Cq value of qRT-PCR detection result has no numerical value.
RT-PCR judges whether viruses are carried or not by detecting whether virus bands exist or not through electrophoresis, and belongs to semi-quantitative detection; in the case where the virus content is too low to observe the fluorescence carried by naked eyes, it cannot be said that the virus is not contained. The qRT-PCR can quantitatively detect the virus content, the quantized data can assist in confirming whether plants without virus bands in the RT-PCR carry viruses, and based on the virus quantification, tissue culture intermediate propagation materials or tissue culture plants meeting the continuous culture requirement can be screened out in an assisted mode. Meanwhile, by adopting two virus detection methods to carry out virus detection, the result of the virus detection can be interactively verified, the contingency of the virus detection result is reduced, the reliability of the detection result is higher, and the success rate of cultivating the tissue culture seedlings without carrying the CymMV and the ORSV is improved.
After the operations based on step 1, step 2 and step 3, tissue culture intermediate propagation material without carrying cymv and ORSV can be screened out. The probability that the cultivated tissue culture seedlings do not carry CymMV and ORSV can be further improved by breeding the tissue culture intermediate propagation material.
Step 4: induction of clumped buds
Inoculating the adventitious buds obtained by screening in the step 3 on a clustered bud induction culture medium, and inducing clustered buds in a low-light environment; the cluster bud induction culture medium is as follows: 1/2MS+3.0-4.0 mg/L6-BA+0.2 mg/L NAA+30g/L potato+50 ml/L coconut water+25 g/L sucrose+7.0 g/L agar, pH 5.70.
Step 5: culture of tissue culture seedlings
Dividing the cluster buds induced in the step 4 into single buds, and inoculating the single buds on a tissue culture seedling culture medium to culture the tissue culture seedlings; the tissue culture seedling culture medium is as follows: flower treasure +1/2MS +0.1-0.2 mg/L NAA +30g/L potato +1-2 g/L active carbon +25g/L sucrose +7.0g/L agar, pH is 5.70.
Step 6: detection of tissue culture seedlings
And (3) performing spot check on the tissue culture seedlings obtained in the step (5) according to the proportion of 10%, taking the leaves as materials, and adopting a double detection method to detect the CymMV and the ORSV, wherein the detection results of the spot check on the tissue culture seedlings are double negative, so that the batch of tissue culture seedlings can be used as the tissue culture seedlings without the CymMV and the ORSV for subsequent pot production.
Through the steps, in the process of selecting the explants in the step 1, RT-PCR and qRT-PCR are sequentially adopted to detect the CymMV and the ORSV, the explants with high virus content can be removed from the source, and the explants with extremely low virus content and even no virus are selected for further culture, so that the success rate of culturing the tissue culture seedlings without carrying the CymMV and the ORSV is improved. In the step 3, the adventitious buds cultured by the stem tip are detected by adopting a double detection method, and the adventitious buds which do not carry the cymv and the ORSV are selected for the next induction. And virus detection is carried out again in the cultivation process, a small amount of adventitious buds possibly carrying the CymMV and the ORSV are removed, and the probability that the cultivated tissue culture seedlings do not carry the CymMV and the ORSV is further improved. The culture process of the tissue culture seedlings is interfered for a plurality of times by means of virus detection, the culture of the tissue culture intermediate propagation material which does not meet the continuous culture requirement is stopped, only the tissue culture intermediate propagation material which meets the requirement is continuously cultured, and the waste of manpower, material resources and time is avoided and the culture efficiency is improved while the fact that the finally cultured tissue culture seedlings do not carry the CymMV and the ORSV is ensured.
According to a preferred embodiment, the weaknessThe light environment is: the temperature is 20-25 ℃, and the illumination intensity is 5-10 mu mol.m -2 ·s -1 The light irradiation time per day was 12 hours, and the relative humidity of air was 40%.
According to a preferred embodiment, further comprising step 7: virus detection of plants at each growth stage: and (3) detecting cymv and ORSV of the butterfly orchid tissue culture seedling obtained by the method, the plant of 2 months of field planting of the tissue culture seedling and the flowering plant respectively.
According to a preferred embodiment, the detection method of cymv and ORSV in step 7 is: 30 plants are selected from the butterfly orchid tissue culture seedling, the tissue culture seedling field planting 2 months old plant and the flowering plant obtained by the method, cymv and ORSV detection is carried out on the leaves of the butterfly orchid tissue culture seedling, the double detection method is adopted in the step 3, the virus carrying rate of the plant is counted, and the virus carrying rate (%) = number of plants carrying virus/number of detected plants multiplied by 100%.
According to a preferred embodiment, the virus-carrying plants of step 7 are: in the detection of cymv and ORSV, the electrophoretic detection of RT-PCR has viral bands or qRT-PCR has numerical values for the Cq value of the detection result.
Compared with the prior art, the application has the beneficial effects that:
1. the butterfly orchid tissue culture seedling cultivation method without carrying the cymMV and the ORSV can obtain butterfly orchid tissue culture seedlings without carrying the cymMV and the ORSV in batches, provide reliable high-quality seed sources for the production of downstream butterfly orchid potted products, block the propagation of the cymMV and the ORSV in the downstream butterfly orchid production links from the seed sources, and improve the quality of butterfly orchid products;
2. the method for cultivating the butterfly orchid tissue culture seedlings without carrying the cymMV and the ORSV can be used for cultivating the butterfly orchid tissue culture seedlings without carrying the cymMV and the ORSV in batches by means of an accurate virus detection method and reliable steps only by means of a conventional operation method, has low operation requirements and high breeding efficiency, saves manpower, material resources and time, and provides a new idea for cultivating the butterfly orchid high-quality tissue culture seedlings.
Drawings
The arrow in FIG. 1 shows the bud bract covered outside the axillary bud of the pedicel node of the present application.
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.
The preparation method of the culture medium in each of the following examples is a conventional method, namely: mixing the components according to the content of the components according to the formula of the culture medium, regulating the pH to 5.70, and performing conventional disinfection and sterilization operation on the culture medium.
The features and capabilities of the present application are described in further detail below in connection with examples.
Example 1 cultivation method of Phalaenopsis tissue culture seedlings without CymMV and ORSV
Step 1: screening of tissue culture explants
Step 1.1: virus detection based on RT-PCR: selecting butterfly orchid plants with normal phenotype, taking leaves as materials, and respectively adopting RT-PCR to detect CymMV and ORSV;
step 1.2: virus detection based on qRT-PCR: and (2) taking each stem segment which grows robustly from plants with negative RT-PCR detection results (electrophoresis detection of virus-free bands of RT-PCR) in the step (1.1), respectively adopting qRT-PCR to detect CymMV and ORSV on the stem bracts of each stem segment, and using the stem segment corresponding to the stem bracts with Cq value more than or equal to 35 as a tissue culture explant. The bud bracts specifically refer to bracts covered outside axillary buds of each bud node, as shown by arrows in fig. 1. The line segments in the figure are scale bars and have a length of 5mm.
Step 2: induction of axillary buds of pedicel
Sterilizing the pedicel node selected in the step 1 as a tissue culture explant, inoculating on a pedicel axillary bud induction culture medium, performing dark culture for 2 weeks, and then performing illumination intensity of 5 mu mol.m at 20 DEG C -2 ·s -1 The induction of the axillary buds of the pedicel is carried out in a low-light environment with the air relative humidity of 40% under the condition that the illumination time of each day is 12 hours. The culture medium for inducing the axillary buds of the pedicel is1/2MS+2.0 mg/L6-BA+0.2 mg/L NAA+30g/L potato+50 ml/L coconut water+25 g/L sucrose+7.0 g/L agar, pH 5.70.
Step 3: induction and screening of adventitious buds of stem tip
And (3) peeling stem tips of the strong axillary buds obtained by induction in the step (2), inoculating the stem tips on an adventitious bud induction culture medium, performing dark culture for 2-3 weeks, and then transferring the culture medium into the weak light environment in the step (2) for culture. And (3) performing CymMV and ORSV detection on the induced adventitious buds by adopting a double detection method consisting of RT-PCR and qRT-PCR, wherein the detection result is double negative (namely, the electrophoresis detection of RT-PCR is free of virus stripes and the qRT-PCR detection result has no number of Cq values), and the adventitious buds can be used for inducing cluster buds. The adventitious bud induction culture medium is 1/2MS+1.5mg/L TDZ+0.2mg/L NAA+30g/L potato+50 ml/L coconut water+25 g/L sucrose+7.0 g/L agar, and the pH is 5.70.
Step 4: induction of clumped buds
Inoculating the adventitious buds obtained by screening in the step 3 onto a cluster bud induction medium of 1/2MS+3.0mg/L6-BA+0.2 mg/LNAA+30g/L potato+50 ml/L coconut water+25 g/L sucrose+7.0 g/L agar, wherein the pH of the medium is 5.70, and performing cluster bud induction in the low light environment described in the step 2.
Step 5: culture of tissue culture seedlings
Dividing the cluster buds induced in the step 4 into single buds, inoculating the single buds to a culture medium of Huabao+1/2MS+0.1 mg/LNAA+30g/L potato+1 g/L active carbon+25 g/L sucrose+7.0 g/L agar, and culturing tissue culture seedlings, wherein the pH of the culture medium is 5.70.
Step 6: detection of tissue culture seedlings
And (3) performing spot check on the tissue culture seedlings obtained in the step (5) according to the proportion of 10%, and respectively performing CymMV and ORSV detection by adopting a double detection method, wherein the detection results are double negative as described in the step (3), so that the batch of tissue culture seedlings can be used as the tissue culture seedlings without the CymMV and ORSV for subsequent potting production.
Step 7: virus detection of plants at various growth stages
30 plants are selected from the butterfly orchid tissue culture seedling, the tissue culture seedling field planting 2 month old plant and the flowering plant obtained by the method, cymv and ORSV detection is carried out on the leaves of the butterfly orchid tissue culture seedling, the virus carrying rate of the plant is counted, the virus carrying rate (%) = virus carrying plant number/detection plant number multiplied by 100%, wherein the virus carrying plant refers to: in the detection of cymv and ORSV, the electrophoretic detection of RT-PCR has viral bands or qRT-PCR has numerical values for the Cq value of the detection result.
Example 2 and example 3 are methods for cultivating butterfly orchid tissue culture seedlings without carrying cymv and ORSV
Examples 2 and 3 are further modifications of example 1; the same contents are not described in detail.
Example 2 and example 3 differ from example 1 in that table 1 shows:
table 1 example 2 and example 3 differ from example 1
The virus carrying rates of the butterfly orchid tissue culture seedlings, the 2 month old plants for the tissue culture seedlings and the flowering plants in the example 1, the example 2 and the example 3 are shown in table 2:
TABLE 2 viral load of plants at each stage of growth of butterfly orchid obtained in examples 1 to 3 (%)
| Project | Example 1 | Example 2 | Example 3 |
| Tissue culture seedling | 0 | 0 | 0 |
| Tissue culture seedling field planting 2 month old plant | 0 | 0 | 0 |
| Plants already flowering | 0 | 0 | 0 |
According to table 2, the tissue culture seedlings cultivated by the method of the application do not detect cymv and ORSV at each stage after cultivation, and the method of the application can keep that the tissue culture seedlings cultivated by the method of the application do not carry cymv and ORSV at each growth stage because all the tissue culture seedlings do not carry cymv and ORSV in the continuous growth process and do not mutually infect in the cultivation process.
Comparative example 1
Comparative example 1 is a case where two varieties of the tissue culture seedlings of butterfly orchid 'large capsicum' and 'sapphire' purchased from commercial sources and the field planting plants thereof carry cymv and ORSV.
(1) Study object: the butterfly orchid 'big capsicum' and 'sapphire' tissue culture seedlings purchased in commercial channels are planted in a fixed mode, 2 months old plants and flowering plants are planted in the tissue culture seedlings, and 30 plants are selected from each stage of each variety.
(2) The virus detection method comprises the following steps: the 'double detection method' in the step 3 of the application is adopted to detect the CymMV and the ORSV of the leaf, namely: and simultaneously, detecting by adopting RT-PCR and qRT-PCR.
(3) Data statistics: the plant virus carrying ratio (%) =the number of plants carrying virus/the number of detected plants×100% was counted for each growth stage of two varieties, wherein the plants carrying virus means: in the detection of CymMV and ORSV, the electrophoresis detection of RT-PCR has virus strips or qRT-PCR detection results Cq has numerical values.
(4) The virus-carrying conditions of the butterfly orchid 'large capsicum' and 'sapphire' tissue culture seedlings purchased commercially, and the 2 month old plants and flowering plants of the tissue culture seedlings were set as shown in table 3:
TABLE 3 plant viral load Rate for each growth stage of butterfly orchid big Capsicum and sapphire
| Project | Butterfly orchid big capsicum' | Butterfly orchid 'sapphire' |
| Tissue culture seedling | 100％ | 60％ |
| Tissue culture seedling field planting 2 month old plant | 100％ | 63.33％ |
| Plants already flowering | 100％ | 63.33％ |
As can be seen from table 3, the existing tissue culture seedling plants have a higher virus-carrying ratio, and even in a better case, the plant virus-carrying ratio reaches 60%, and the probability of carrying cymv and ORSV increases further due to the mutual infection during cultivation.
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.
Claims (10)
1. The butterfly orchid tissue culture seedling cultivation method without carrying cymMV and ORSV is characterized by comprising the following steps:
step 1: screening of tissue culture explants
Step 1.1: virus detection based on RT-PCR: selecting butterfly orchid plants with normal phenotype, taking leaves as materials, and respectively adopting RT-PCR to detect CymMV and ORSV;
step 1.2: virus detection based on qRT-PCR: for plants with negative RT-PCR detection results in the step 1.1, taking the stem sections which grow robustly, respectively adopting qRT-PCR to detect CymMV and ORSV for the stem bracts of the stem sections, and selecting the stem sections corresponding to the stem bracts with the Cq value of the detection results of more than or equal to 35 as tissue culture explants;
step 2: induction of axillary buds of pedicel
Sterilizing the tissue culture explant selected in the step 1, inoculating the tissue culture explant to a pedicel axillary bud induction culture medium, performing dark culture for 2 weeks, and then performing pedicel axillary bud induction in a dim light environment;
step 3: induction and screening of adventitious buds of stem tip
Step 3.1: peeling stem tips of the strong axillary buds obtained by induction in the step 2, inoculating the stem tips on an adventitious bud induction culture medium, performing dark culture for 2-3 weeks, and then transferring the culture medium into a weak light environment for culture;
step 3.2: detecting the induced adventitious buds by adopting a double detection method, namely performing CymMV and ORSV detection, and selecting adventitious buds with double negative detection results for inducing cluster buds;
step 4: induction of clumped buds
Inoculating the adventitious buds obtained by screening in the step 3 on a clustered bud induction culture medium, and inducing clustered buds in a low-light environment;
step 5: culture of tissue culture seedlings
Dividing the cluster buds induced in the step 4 into single buds, and inoculating the single buds on a tissue culture seedling culture medium to culture the tissue culture seedlings;
step 6: detection of tissue culture seedlings
And (3) performing spot check on the tissue culture seedlings obtained in the step (5), and detecting the CymMV and the ORSV by adopting a double detection method, wherein the detection results of the spot check on the tissue culture seedlings are double negative, so that the batch of tissue culture seedlings can be used as the tissue culture seedlings without the CymMV and the ORSV for subsequent potting production.
2. The method for cultivating butterfly orchid tissue culture seedlings without carrying cymv and ORSV according to claim 1, wherein said "double detection method" is to detect by using RT-PCR and qRT-PCR simultaneously.
3. The method for cultivating butterfly orchid tissue culture seedlings without carrying cymv and ORSV according to claim 1, wherein in said step 1, said "RT-PCR detection result is negative" is: electrophoresis by RT-PCR detects virus-free bands.
4. The method for cultivating butterfly orchid tissue culture seedlings without carrying cymv and ORSV according to claim 1, wherein in said step 3.2, said "detection result is double negative" is: the electrophoresis detection of RT-PCR has no virus band and the Cq value of qRT-PCR detection result has no numerical value.
5. The method for cultivating butterfly orchid tissue culture seedlings without cymv and ORSV according to claim 1, wherein the proportion of the spot check in said step 6 is 10%.
6. The method for cultivating butterfly orchid tissue culture seedlings without carrying cymv and ORSV according to claim 1, wherein in step 2, the pedicel axillary bud induction medium is: 1/2MS+2.0-3.0 mg/L6-BA+0.2 mg/L NAA+30g/L potato+50 ml/L coconut water+25 g/L sucrose+7.0 g/L agar, pH 5.70.
7. The method for cultivating butterfly orchid tissue culture seedlings without carrying cymv and ORSV according to claim 1, wherein said low light environment is: the temperature is 20-25 ℃, and the illumination intensity is 5-10 mu mol.m -2 ·s -1 The light irradiation time per day was 12 hours, and the relative humidity of air was 40%.
8. The method for cultivating butterfly orchid tissue culture seedlings without carrying cymv and ORSV according to claim 1, wherein said adventitious bud induction medium is: 1/2MS+1.5-3.0 mg/L TDZ+0.2mg/L NAA+30g/L potato+50 ml/L coconut water+25 g/L sucrose+7.0 g/L agar, pH 5.70.
9. The method for cultivating butterfly orchid tissue culture seedlings without carrying cymv and ORSV according to claim 1, wherein said cluster bud induction medium is: 1/2MS+3.0-4.0 mg/L6-BA+0.2 mg/L NAA+30g/L potato+50 ml/L coconut water+25 g/L sucrose+7.0 g/L agar, pH 5.70.
10. The method for cultivating butterfly orchid tissue culture seedlings without carrying cymv and ORSV according to claim 1, wherein said tissue culture seedling culture medium is: flower treasure +1/2MS +0.1-0.2 mg/L NAA +30g/L potato +1-2 g/L active carbon +25g/L sucrose +7.0g/L agar, pH is 5.70.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310917822.5A CN116686715A (en) | 2023-07-25 | 2023-07-25 | Butterfly orchid tissue culture seedling cultivation method without carrying cymMV and ORSV |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310917822.5A CN116686715A (en) | 2023-07-25 | 2023-07-25 | Butterfly orchid tissue culture seedling cultivation method without carrying cymMV and ORSV |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116686715A true CN116686715A (en) | 2023-09-05 |
Family
ID=87827906
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310917822.5A Pending CN116686715A (en) | 2023-07-25 | 2023-07-25 | Butterfly orchid tissue culture seedling cultivation method without carrying cymMV and ORSV |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116686715A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070220633A1 (en) * | 2006-03-15 | 2007-09-20 | National Taiwan University. | Viral vector for inducing gene inactivation of plants and application thereof |
| CN102676695A (en) * | 2012-04-16 | 2012-09-19 | 杭州师范大学 | RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit capable of detecting CymMV (Cymbidium Mosaic Virus) and ORSV (Odontoglossum Ringspot Virus) simultaneously and method thereof |
| CN104604687A (en) * | 2015-01-29 | 2015-05-13 | 赤峰市农牧科学研究院 | Method for inducing clustered shoots to rapidly propagate butterfly orchid by utilizing pedicle stem segments after bud cutting |
| CN114946654A (en) * | 2022-05-13 | 2022-08-30 | 佛山昕锴生物科技有限公司 | Phalaenopsis stem tip growth point culture virus removal technology |
| CN115852052A (en) * | 2022-11-14 | 2023-03-28 | 云南省农业科学院花卉研究所 | Real-time fluorescent quantitative PCR primer probe combination for detecting CymRSV and method thereof |
-
2023
- 2023-07-25 CN CN202310917822.5A patent/CN116686715A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070220633A1 (en) * | 2006-03-15 | 2007-09-20 | National Taiwan University. | Viral vector for inducing gene inactivation of plants and application thereof |
| CN102676695A (en) * | 2012-04-16 | 2012-09-19 | 杭州师范大学 | RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection kit capable of detecting CymMV (Cymbidium Mosaic Virus) and ORSV (Odontoglossum Ringspot Virus) simultaneously and method thereof |
| CN104604687A (en) * | 2015-01-29 | 2015-05-13 | 赤峰市农牧科学研究院 | Method for inducing clustered shoots to rapidly propagate butterfly orchid by utilizing pedicle stem segments after bud cutting |
| CN114946654A (en) * | 2022-05-13 | 2022-08-30 | 佛山昕锴生物科技有限公司 | Phalaenopsis stem tip growth point culture virus removal technology |
| CN115852052A (en) * | 2022-11-14 | 2023-03-28 | 云南省农业科学院花卉研究所 | Real-time fluorescent quantitative PCR primer probe combination for detecting CymRSV and method thereof |
Non-Patent Citations (3)
| Title |
|---|
| 刘沛然;王四清;: "建兰花叶病毒及齿兰环斑病毒的检测方法(综述)", 热带作物学报, no. 01, 15 February 2009 (2009-02-15), pages 99 - 103 * |
| 张妙彬;潘丽晶;肖杨;杨玉环;: "双重RT-PCR同时检测兰花两种主要病毒的研究", 广东农业科学, no. 05, 10 May 2010 (2010-05-10), pages 166 - 167 * |
| 王仁睿等: ""蝴蝶兰组织培养体系的建立及其关键技术研究"", 《中国农学通报》, vol. 26, no. 10, 20 May 2010 (2010-05-20), pages 197 - 201 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bajaj et al. | Interspecific hybridization of Brassica napus and B. juncea through ovary, ovule and embryo culture | |
| CN100553445C (en) | New lilium longiflorum production of hybrid seeds parent's tissue-culturing rapid propagation and cross-breeding method | |
| US11432487B2 (en) | Systems, methods and kits for micropropagation of Cannabis | |
| CN115537346B (en) | Mucillus mucilaginosus for promoting growth and differentiation of sansevieria trifasciata and application thereof | |
| CN104429953A (en) | Stem tip detoxification method for sweet potato virus seedling | |
| CN101715730B (en) | Tissue culture rapid propagation method for sugarcane new Taisugar No.10 | |
| CN110506634B (en) | Iris chemical mutagenesis dose screening method | |
| CN105660404A (en) | Method for PEG-resisting alfalfa callus dedifferentiation | |
| CN110741901B (en) | Cultivation method of heavy valve hippeastrum rutilum | |
| CN107155874A (en) | A kind of utilization Anther Culture obtains the method and its culture medium of Dihaploid Potato plant | |
| CN116686715A (en) | Butterfly orchid tissue culture seedling cultivation method without carrying cymMV and ORSV | |
| CN110036908A (en) | A method of accelerating corn inbred lines | |
| CN114027180B (en) | Cultivation method and application of shinyleaf yellowhorn polyploid | |
| CN108308017B (en) | Cultivation method of virus-resistant interspecific introgression line of sweet potatoes | |
| CN114424749B (en) | In-vitro rapid propagation method for liriope spicata | |
| CN111727879B (en) | Culture medium and method for oil peony propagation | |
| Kengkarj et al. | Assessment of somaclonal variation in Chrysan-themum (Dendranthema grandiflora Kitam.) using RAPD and morphological analysis | |
| CN103651078A (en) | Method for screening transgenic plant seeds | |
| CN111480572B (en) | Method for screening rice tissue culture material | |
| CN111937742A (en) | Method for creating intergeneric distant hybrid of hibiscus and chamomile | |
| CN116616179B (en) | Tissue culture rapid propagation method of excellent clone of Gardenia jasminoides ellis with high content of geniposide | |
| Saruwatari et al. | Interspecific lily hybrids with the ability to flower precociously and to produce multiple flower stalks from Lilium formosanum | |
| KR102309624B1 (en) | Lily tissue somatic cell meristem Culture quick proliferation Method | |
| CN115088615B (en) | Method for improving doubling efficiency of corn haploid by using glutathione | |
| CN116875729A (en) | Method for obtaining aromatic butterfly orchid excellent tissue culture explant |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |