Method for cultivating Phalaenopsis tissue culture seedlings without carrying CymMV and ORSV

Abstract

The invention discloses a method for cultivating Phalaenopsis tissue culture seedlings that do not carry CymMV and ORSV, comprising the following steps: screening of tissue culture explants, induction of peduncle axillary buds, induction and screening of stem apex adventitious buds, induction of clustered buds, cultivation of tissue culture seedlings, and detection of tissue culture seedlings. The invention combines tissue culture propagation technology with sensitive and reliable virus detection technology to establish a reliable method for cultivating Phalaenopsis tissue culture seedlings that do not carry Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV), blocks the spread of CymMV and ORSV in the downstream production links of Phalaenopsis from the seed source, improves the quality of Phalaenopsis products, saves cultivation time, manpower and material resources, and has high cultivation efficiency.

Description

Technical Field

[0001] The invention relates to the field of plant propagation, and in particular to a method for cultivating Phalaenopsis tissue culture seedlings without carrying CymMV and ORSV. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Phalaenopsis spp. is a plant of the genus Phalaenopsis in the family Orchidaceae. It is known as the "Queen of Orchids" for its rich and bright colors, unique flower shape and long flowering period. It is an important variety of New Year flowers and has important commercial value.

[0004] Phalaenopsis plants are relatively single-plant, rarely produce tillers, and rarely can be divided during cultivation. Commercially cultivated Phalaenopsis orchids are mostly propagated by tissue culture. Long-term asexual reproduction leads to serious accumulation of viruses, resulting in serious degradation of variety characteristics and quality decline. Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are two viruses that are more serious and common in Phalaenopsis orchids, and are the two most critical viruses affecting the development of the Phalaenopsis orchid industry. The vast majority of Phalaenopsis tissue culture seedlings are infected with CymMV and ORSV alone or in combination, resulting in the subsequent production of Phalaenopsis potted plants carrying CymMV and ORSV. After Phalaenopsis plants are infected with CymMV and ORSV, yellow ring spots and striped spots will appear on the leaves, and the flowers will fade, greatly reducing the ornamental value, causing huge economic losses, and seriously restricting the development of my country's Phalaenopsis orchid industry.

[0005] The prior art discloses a detoxification and rapid propagation technology for Phalaenopsis. The method and rapid propagation approach for secondary detoxification of Phalaenopsis using physical methods combined with chemical reagents are obtained through the steps of primary culture, stem tip elongation culture, detoxification culture, seedling strengthening, rooting culture, etc. Although the method can obtain detoxification seedlings, chemical reagents are required, which have an adverse effect on the growth of detoxification seedlings and may cause plant mutations, affecting the ornamental characteristics of Phalaenopsis. Moreover, the detoxification rate of the prior art is only 90%, which shows that the tissue culture seedlings obtained by the method after the final cultivation still carry CymMV and ORSV, so that when the tissue culture seedlings are put into use, each batch of tissue culture seedlings still needs to be tested for viruses, which will be a very huge cost investment. For the tissue culture seedlings that still carry CymMV and ORSV after the test, the time, materials and manpower spent in the cultivation process are all wasted.

[0006] The construction of a method for cultivating Phalaenopsis tissue culture seedlings that do not carry CymMV and ORSV will be of great significance to the rapid and healthy development of the domestic Phalaenopsis industry. At present, there is no report in China on the cultivation method of Phalaenopsis tissue culture seedlings that do not carry CymMV and ORSV by combining tissue culture propagation technology with sensitive and reliable virus detection technology, starting from explant source screening. Summary of the invention

[0007] The purpose of the present invention is to provide a method for cultivating Phalaenopsis tissue culture seedlings that do not carry CymMV and ORSV in response to the serious problem that Phalaenopsis tissue culture seedlings currently carry CymMV and ORSV. Plant materials that do not carry CymMV and ORSV are screened layer by layer in each step for the next step of cultivation, and the tissue culture seedlings finally cultivated do not carry CymMV and ORSV.

[0008] The technical solution of the present invention is as follows:

[0009] The method for cultivating Phalaenopsis tissue culture seedlings without carrying CymMV and ORSV comprises the following steps:

[0010] Step 1: Screening of tissue culture explants

[0011] Step 1.1: Virus detection based on RT-PCR (Reverse transcription-polymerase chain reaction): Select Phalaenopsis plants with normal phenotypes, use leaves as materials, and use RT-PCR to detect CymMV and ORSV respectively;

[0012] Step 1.2: Virus detection based on qRT-PCR (Quantitative Real-time polymerase chain reaction): For plants with negative RT-PCR test results in step 1.1, take each pedicel node with healthy growth, and use qRT-PCR to test the pedicel bracts of each pedicel node for CymMV and ORSV respectively. The pedicel nodes corresponding to the pedicel bracts with a test result Cq value ≥ 35 can be used as tissue culture explants. The pedicel bracts specifically refer to the bracts covering the outer side of the axillary buds of each pedicel node. The "RT-PCR test result is negative" means that there are no virus bands in the RT-PCR electrophoresis test.

[0013] Since the distribution of viruses in different parts of Phalaenopsis plants such as roots, leaves, pedicels, etc. is different, the material of virus detection will also affect the accuracy of virus detection results. All Phalaenopsis tissue culture seedlings originate from the pedicel axillary buds inside the pedicel node, and the pedicel axillary buds are the most critical parts in tissue culture explants. The pedicel axillary buds are covered with pedicel bracts outside, and the attachment positions of the two are closely connected, and the virus carrying situation is the closest, but the tissue sample amount of pedicel bracts is very limited. This application first uses blades as materials, uses RT-PCR to detect CymMV and ORSV, and preliminary screening obtains plants with negative test results, and then takes pedicel bracts for each pedicel node used as a candidate explant to carry out qRT-PCR detection of CymMV and ORSV, and can make full use of pedicel bract materials without damaging pedicel axillary buds, give play to the value of limited pedicel bract materials, and ensure the accuracy of test results.

[0014] After the initial screening by RT-PCR, most of the plants with high virus content can be eliminated. For the plants that show no virus bands in RT-PCR, due to the limitation of naked eye vision and the sampling location during the detection process, they may not be completely free of CymMV and ORSV. The plants that show no virus bands in RT-PCR are tested again by qRT-PCR, and the pedicel bracts of each pedicel node used as candidate explants are sampled as detection materials, which can eliminate the errors caused by the difference in sampling positions and can quantitatively display the virus content, so as to avoid detection errors from the sample itself, and improve the accuracy of detection from the detection method, improve the accuracy of virus detection results as a whole, and assist in screening out tissue culture explants with extremely low virus content or no virus. As the first step in the cultivation process of tissue culture seedlings, the screening of explants significantly affects the results of subsequent cultivation. Improving the accuracy of explant screening has an important impact on obtaining better cultivation results.

[0015] Step 2: Induction of pedicel axillary buds

[0016] The tissue culture explants screened out in step 1 are disinfected and sterilized, inoculated on a pedicel axillary bud induction medium, first cultured in the dark for 2 weeks, and then inducing pedicel axillary buds in a weak light environment; the pedicel axillary bud induction medium is 1 / 2MS+2.0-3.0mg / L 6-BA+0.2mg / L NAA+30g / L potato+50ml / L coconut water+25g / L sucrose+7.0g / L agar, and the pH is 5.70.

[0017] Step 3: Induction and screening of adventitious buds at the shoot apex

[0018] Step 3.1: Use the robust axillary buds induced in step 2 to peel off the stem tips, inoculate them on an adventitious bud induction medium, culture them in the dark for 2 to 3 weeks, and then transfer them to a weak light environment for culture; the adventitious bud induction medium is 1 / 2MS+1.5-3.0mg / L TDZ+0.2mg / L NAA+30g / L potato+50ml / L coconut water+25g / L sucrose+7.0g / L agar, and the pH is 5.70.

[0019] Step 3.2: All induced adventitious buds were tested for CymMV and ORSV using the "double detection method", and the adventitious buds with double negative detection results were selected for the induction of clustered buds. The "double detection method" is to use RT-PCR and qRT-PCR to detect CymMV and ORSV at the same time. The "double negative detection result" means that there is no virus band in the electrophoresis detection of RT-PCR and there is no numerical value of the Cq value of the qRT-PCR detection result.

[0020] RT-PCR uses electrophoresis to detect the presence of virus bands to determine whether the plant carries the virus. This is a semi-quantitative test. When the virus content is too low and the fluorescence is difficult to observe with the naked eye, it does not mean that the plant does not contain the virus. qRT-PCR can quantitatively detect the virus content. The quantified data can help confirm whether the plant without virus bands in RT-PCR carries the virus. Based on the virus quantification, it can help screen out tissue culture intermediate propagation materials or tissue culture plants that meet the requirements for continued cultivation. Using two virus detection methods at the same time can cross-verify the results of virus detection, reduce the randomness of virus detection results, make the detection results more credible, and improve the success rate of cultivating tissue culture seedlings that do not carry CymMV and ORSV.

[0021] After the operations of step 1, step 2 and step 3, tissue culture intermediate propagation materials that do not carry CymMV and ORSV can be screened out. Propagation with the tissue culture intermediate propagation materials can further increase the probability that the cultivated tissue culture seedlings do not carry CymMV and ORSV.

[0022] Step 4: Induction of budding

[0023] The adventitious buds screened in step 3 are inoculated on a cluster bud induction medium to induce cluster buds in a weak light environment; the cluster bud induction medium is: 1 / 2MS+3.0-4.0mg / L 6-BA+0.2mg / L NAA+30g / L potato+50ml / L coconut water+25g / L sucrose+7.0g / L agar, with a pH of 5.70.

[0024] Step 5: Cultivation of tissue culture seedlings

[0025] The clustered buds induced in step 4 are divided into individual buds and inoculated on a tissue culture seedling medium for tissue culture seedling cultivation; the tissue culture seedling medium comprises: Huabao + 1 / 2MS + 0.1-0.2mg / L NAA + 30g / L potato + 1-2g / L activated carbon + 25g / L sucrose + 7.0g / L agar, with a pH of 5.70.

[0026] Step 6: Testing of tissue culture seedlings

[0027] 10% of the tissue culture seedlings obtained in step 5 were randomly inspected, and the leaves were used as the material. The "double detection method" was used to detect CymMV and ORSV. If the test results of the randomly inspected tissue culture seedlings were all double negative, then this batch of tissue culture seedlings could be used as tissue culture seedlings that do not carry CymMV and ORSV for subsequent potted production.

[0028] Through the above steps, RT-PCR and qRT-PCR are successively used to detect CymMV and ORSV during the explant selection process in step 1, so that explants with high virus content can be eliminated from the source, and explants with extremely low virus content or even virus-free are selected for further cultivation, thereby increasing the success probability of cultivating tissue culture seedlings that do not carry CymMV and ORSV. In step 3, the "double detection method" is used to detect CymMV and ORSV for the adventitious buds cultivated from the shoot tip, and the adventitious buds that do not carry CymMV and ORSV are selected for the next step of induction. Virus detection is performed again in the cultivation process, and a small amount of adventitious buds that may carry CymMV and ORSV are eliminated, further increasing the probability that the cultivated tissue culture seedlings do not carry CymMV and ORSV. The cultivation process of tissue culture seedlings was intervened many times with the help of virus detection. The cultivation of tissue culture intermediate propagation materials that did not meet the requirements for continued cultivation was terminated, and only the tissue culture intermediate propagation materials that met the requirements were cultivated. While ensuring that the tissue culture seedlings finally cultivated did not carry CymMV and ORSV, it avoided waste of manpower, material resources and time and improved cultivation efficiency.

[0029] According to a preferred embodiment, the weak light environment is: temperature 20-25°C, light intensity 5-10 μmol·m -2 ·s -1 , the daily lighting time is 12 hours and the relative humidity of the air is 40%.

[0030] According to a preferred embodiment, the method further includes step 7: virus detection of plants at various growth stages: CymMV and ORSV detection is performed on Phalaenopsis tissue culture seedlings, 2-month-old plants planted with tissue culture seedlings, and flowering plants obtained by the method of the present application.

[0031] According to a preferred embodiment, the detection method of CymMV and ORSV in step 7 is: 30 Phalaenopsis tissue culture seedlings, 2-month-old plants planted in tissue culture seedlings and flowering plants obtained by the method of the present application are selected respectively, and CymMV and ORSV are detected on their leaves by the "double detection method" described in step 3, and the virus-carrying rate of the plants is statistically analyzed, and the virus-carrying rate of the plants (%) = the number of plants carrying the virus / the number of plants detected × 100%.

[0032] According to a preferred embodiment, the virus-carrying plant in step 7 refers to: in the detection of CymMV and ORSV, the RT-PCR electrophoresis detection has a virus band or the qRT-PCR detection result Cq value has a numerical value.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The method for cultivating Phalaenopsis tissue culture seedlings that do not carry CymMV and ORSV can obtain Phalaenopsis tissue culture seedlings that do not carry CymMV and ORSV in batches, provide reliable high-quality seed sources for the production of downstream Phalaenopsis potted products, block the spread of CymMV and ORSV in the downstream production links of Phalaenopsis from the seed source, and improve the quality of Phalaenopsis products;

[0035] 2. The method for cultivating Phalaenopsis tissue culture seedlings that do not carry CymMV and ORSV, with the help of accurate virus detection methods and reliable steps, only requires conventional operating methods, and ultimately can batch-produce tissue culture seedlings that do not carry CymMV and ORSV. It has low operating requirements and high breeding efficiency, saving manpower, material resources and time, and provides a new idea for the cultivation of high-quality Phalaenopsis tissue culture seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The arrow indicates the pedicel bract covering the outside of the axillary bud at the pedicel node of the present application. DETAILED DESCRIPTION

[0037] The following examples are provided for a better understanding of the present invention. The test methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available. The quantitative tests in the following examples are all repeated three times, and the results are averaged.

[0038] The preparation method of the culture medium in the following examples is a conventional method, that is, after mixing the components according to their contents according to the culture medium formula, adjusting the pH to 5.70, and performing conventional disinfection and sterilization operations on the culture medium, the culture medium is prepared.

[0039] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0040] Example 1 Method for cultivating Phalaenopsis tissue culture seedlings without CymMV and ORSV

[0041] Step 1: Screening of tissue culture explants

[0042] Step 1.1: Virus detection based on RT-PCR: Select Phalaenopsis plants with normal phenotypes, use leaves as materials, and use RT-PCR to detect CymMV and ORSV respectively;

[0043] Step 1.2: Virus detection based on qRT-PCR: For plants with negative RT-PCR test results in step 1.1 (no virus bands in RT-PCR electrophoresis), take each healthy pedicel node, and use qRT-PCR to test the pedicel bracts of each pedicel node for CymMV and ORSV. The pedicel nodes corresponding to the pedicel bracts with a Cq value ≥ 35 can be used as tissue culture explants. The pedicel bracts specifically refer to the bracts covering the outer side of the axillary buds of each pedicel node, such as Figure 1 As shown by the arrow in the figure. The line segment in the figure is a scale bar with a length of 5 mm.

[0044] Step 2: Induction of pedicel axillary buds

[0045] The pedicel nodes selected in step 1 were used as tissue culture explants for disinfection and sterilization, and inoculated on the pedicel axillary bud induction medium. They were first cultured in the dark for 2 weeks and then cultured at 20°C with a light intensity of 5 μmol·m -2 ·s -1 The induction of pedicel axillary buds was carried out in a weak light environment with a light exposure time of 12 hours per day and a relative air humidity of 40%. The pedicel axillary bud induction medium was 1 / 2MS+2.0mg / L 6-BA+0.2mg / L NAA+30g / L potato+50ml / L coconut water+25g / L sucrose+7.0g / L agar, with a pH of 5.70.

[0046] Step 3: Induction and screening of adventitious buds at the shoot apex

[0047] The sturdy axillary buds induced in step 2 are used to peel the stem tips, inoculate them on the adventitious bud induction medium, and culture them in the dark for 2 to 3 weeks, and then transfer them to the weak light environment described in step 2 for culture. The induced adventitious buds are tested for CymMV and ORSV respectively using the "double detection method" consisting of RT-PCR and qRT-PCR. Adventitious buds with double negative test results (i.e., no virus bands in the electrophoresis test of RT-PCR and no numerical Cq value in the qRT-PCR test result) can be used for the induction of clustered buds. The adventitious bud induction medium is 1 / 2MS+1.5mg / L TDZ+0.2mg / L NAA+30g / L potato+50ml / L coconut water+25g / L sucrose+7.0g / L agar, with a pH of 5.70.

[0048] Step 4: Induction of budding

[0049] The adventitious buds screened in step 3 were inoculated on a cluster bud induction medium of 1 / 2MS + 3.0 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L potato + 50 ml / L coconut water + 25 g / L sucrose + 7.0 g / L agar, the pH of the medium was 5.70, and cluster buds were induced in the weak light environment described in step 2.

[0050] Step 5: Cultivation of tissue culture seedlings

[0051] The clustered buds induced in step 4 were divided into individual buds and inoculated on a medium of Huabao + 1 / 2MS + 0.1 mg / L NAA + 30 g / L potato + 1 g / L activated carbon + 25 g / L sucrose + 7.0 g / L agar for tissue culture seedling cultivation, and the pH of the medium was 5.70.

[0052] Step 6: Testing of tissue culture seedlings

[0053] The tissue culture seedlings obtained in step 5 are randomly inspected at a ratio of 10%, and the CymMV and ORSV are tested respectively using the "double detection method". If the test results are both double negative as described in step 3, then this batch of tissue culture seedlings can be used as tissue culture seedlings that do not carry CymMV and ORSV for subsequent potted production.

[0054] Step 7: Virus testing of plants at different growth stages

[0055] 30 Phalaenopsis tissue culture seedlings, 2-month-old plants planted in tissue culture seedlings and flowering plants obtained by the method of the present application were selected respectively, and the CymMV and ORSV detection of their leaves were performed using the "double detection method" described in step 3, and the virus-carrying rate of the plants was statistically analyzed, and the virus-carrying rate of the plants (%) = the number of plants carrying the virus / the number of plants tested × 100%, wherein the virus-carrying plants refer to: in the detection of CymMV and ORSV, the RT-PCR electrophoresis detection has virus bands or the qRT-PCR test result Cq value has a numerical value.

[0056] Examples 2 and 3 are methods for cultivating Phalaenopsis tissue culture seedlings without CymMV and ORSV

[0057] Embodiment 2 and embodiment 3 are further improvements on embodiment 1; the same contents will not be repeated here.

[0058] The differences between Example 2 and Example 3 and Example 1 are shown in Table 1:

[0059] Table 1 Differences between Example 2 and Example 3 and Example 1

[0060]

[0061] The virus carrying rates of Phalaenopsis tissue culture seedlings, 2-month-old plants planted with tissue culture seedlings and flowering plants in Examples 1, 2 and 3 are shown in Table 2:

[0062] Table 2 Virus carrying rate of Phalaenopsis plants at different growth stages obtained in Examples 1-3 (%)

[0063] project Example 1 Example 2 Example 3 Tissue culture seedlings 0 0 0 Planting of 2-month-old tissue culture seedlings 0 0 0 Already flowering plants 0 0 0

[0064] According to Table 2, it can be seen that CymMV and ORSV were not detected in the tissue culture seedlings cultivated by the method of the present application at all stages after cultivation. It can be seen that during the continuous growth process of the method of the present application, since all tissue culture seedlings do not carry CymMV and ORSV, they will not infect each other during the cultivation process, and thus can maintain CymMV and ORSV-free in all growth stages. The tissue culture seedlings cultivated by the method of the present application perform well.

[0065] Comparative Example 1

[0066] Comparative Example 1 shows that the tissue culture seedlings of two varieties of Phalaenopsis 'Big Pepper' and 'Blue Sapphire' purchased from commercial channels and their transplanted plants carry CymMV and ORSV.

[0067] (1) Research subjects: Phalaenopsis orchids of the cultivar 'Big Pepper' and 'Sapphire' purchased from commercial channels, as well as 2-month-old plants and flowering plants transplanted from tissue culture seedlings. Thirty plants of each cultivar at each stage were selected.

[0068] (2) Virus detection method: The leaves were tested for CymMV and ORSV using the "dual detection method" described in step 3 of the present invention, that is, RT-PCR and qRT-PCR were used simultaneously for detection.

[0069] (3) Data statistics: The virus-carrying ratio of plants of the two varieties at each growth stage was statistically analyzed, and the virus-carrying ratio of plants (%) = number of plants carrying the virus / number of plants tested × 100%, wherein the virus-carrying plants refer to: in the detection of CymMV and ORSV, the RT-PCR electrophoresis test has virus bars or the qRT-PCR test result has a numerical value of the Cq value.

[0070] (4) The virus status of Phalaenopsis orchids 'Big Pepper' and 'Blue Sapphire' purchased from commercial channels, as well as 2-month-old plants and flowering plants planted from tissue culture seedlings are shown in Table 3:

[0071] Table 3 Virus infection rate of Phalaenopsis 'Big Pepper' and 'Blue Sapphire' plants at different growth stages

[0072] project Phalaenopsis 'Big Pepper' Phalaenopsis 'Sapphire' Tissue culture seedlings 100％ 60％ Planting of 2-month-old tissue culture seedlings 100％ 63.33％ Already flowering plants 100％ 63.33％

[0073] It can be seen from Table 3 that the existing tissue culture seedlings have a high rate of virus infection. Even under the best conditions, the rate of virus infection in plants reaches 60%. In addition, during the cultivation process, due to mutual infection, the probability of carrying CymMV and ORSV will further increase.

[0074] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A method for cultivating Phalaenopsis tissue culture seedlings without CymMV and ORSV, characterized in that: The following steps are involved: Step 1: Screening of tissue culture explants Step 1.1: Virus detection based on RT-PCR: Select Phalaenopsis plants with normal phenotypes, use leaves as materials, and use RT-PCR to detect CymMV and ORSV respectively; Step 1.2: Virus detection based on qRT-PCR: For plants with negative RT-PCR test results in step 1.1, take each pedicel node with healthy growth, and use qRT-PCR to test the pedicel bracts of each pedicel node for CymMV and ORSV respectively, and select the pedicel nodes corresponding to each pedicel bract with a test result Cq value ≥35 as tissue culture explants, wherein the pedicel bract refers to the bract covering the outer side of the axillary bud of each pedicel node; Step 2: Induction of pedicel axillary buds The tissue culture explants selected in step 1 are sterilized and inoculated on a pedicel axillary bud induction medium, first cultured in the dark for 2 weeks, and then induced in a pedicel axillary bud in a weak light environment; Step 3: Induction and screening of adventitious buds at the shoot apex Step 3.1: Use the robust axillary buds induced in step 2 to peel off the stem tips, inoculate them on the adventitious bud induction medium, culture them in the dark for 2 to 3 weeks, and then transfer them to a weak light environment for culture; Step 3.2: The induced adventitious buds are tested for CymMV and ORSV using the "double detection method", and the adventitious buds with double negative detection results are selected for the induction of clustered buds; the "double detection method" is to use RT-PCR and qRT-PCR for detection at the same time; Step 4: Induction of budding The adventitious buds screened in step 3 are inoculated on a cluster bud induction medium to induce cluster buds in a weak light environment; Step 5: Cultivation of tissue culture seedlings The clustered buds induced in step 4 are divided into individual buds and inoculated on a tissue culture seedling medium to culture tissue culture seedlings; Step 6: Testing of tissue culture seedlings The tissue culture seedlings obtained in step 5 were randomly inspected and tested for CymMV and ORSV using the "double detection method". If the test results of the randomly inspected tissue culture seedlings were all double negative, then this batch of tissue culture seedlings could be used as tissue culture seedlings that do not carry CymMV and ORSV for subsequent potted production.

2. The method for cultivating Phalaenopsis tissue culture seedlings without carrying CymMV and ORSV according to claim 1, characterized in that: In the step 1.2, the "RT-PCR test result is negative" means: no virus bands are found in the electrophoresis test of RT-PCR.

3. The method for cultivating Phalaenopsis tissue culture seedlings without carrying CymMV and ORSV according to claim 1, characterized in that: In the step 3.2, the "detection result is double negative" means that: there is no virus band in the electrophoresis detection of RT-PCR and there is no numerical value in the Cq value of the qRT-PCR detection result.

4. The method for cultivating Phalaenopsis tissue culture seedlings without carrying CymMV and ORSV according to claim 1, characterized in that: The sampling rate in step 6 is 10%.

5. The method for cultivating Phalaenopsis tissue culture seedlings without carrying CymMV and ORSV according to claim 1, characterized in that: In step 2, the pedicel axillary bud induction medium is: 1 / 2MS+2.0-3.0mg / L 6-BA+0.2mg / L NAA+30g / L potato+50ml / L coconut water+25g / L sucrose+7.0g / L agar, with a pH of 5.

70.

6. The method for cultivating Phalaenopsis tissue culture seedlings without carrying CymMV and ORSV according to claim 1, characterized in that: The weak light environment is: temperature 20-25°C, light intensity 5-10 μmol·m -2 ·s -1 , the daily lighting time is 12 hours and the relative humidity of the air is 40%.

7. The method for cultivating Phalaenopsis tissue culture seedlings without carrying CymMV and ORSV according to claim 1, characterized in that: The adventitious bud induction medium is: 1 / 2MS+1.5-3.0mg / L TDZ+0.2mg / L NAA+30g / L potato+50ml / L coconut water+25g / L sucrose+7.0g / L agar, and the pH value is 5.

70.

8. The method for cultivating Phalaenopsis tissue culture seedlings without carrying CymMV and ORSV according to claim 1, characterized in that: The clustered bud induction culture medium is: 1 / 2MS+3.0-4.0mg / L 6-BA+0.2mg / L NAA+30g / L potato+50ml / L coconut water+25g / L sucrose+7.0g / L agar, and the pH value is 5.

70.

9. The method for cultivating Phalaenopsis tissue culture seedlings without carrying CymMV and ORSV according to claim 1, characterized in that: The tissue culture seedling culture medium is: Huabao + 1 / 2MS + 0.1-0.2mg / L NAA + 30g / L potato + 1-2g / L activated carbon + 25g / L sucrose + 7.0g / L agar, and the pH value is 5.70.

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