A virus inhibition medium for tissue culture detoxification of chayote and a method for tissue culture detoxification of chayote

Abstract

The present application belongs to the field of plant tissue culture detoxification technology, and particularly relates to a virus inhibition medium for Chayote tissue culture detoxification and a Chayote tissue culture detoxification method. The present application adds morpholinium hydrochloride as a virus inhibitor into a plant tissue culture medium to pretreat Chayote stem tips, which can inhibit virus replication and has little influence on the growth of Chayote tissue culture materials. On this basis, the stem tips treated by the virus inhibitor are subjected to proliferation culture, and Chayote detoxification seedlings can be obtained after multiple subculture (pretreatment and proliferation culture). Compared with the existing Chayote tissue culture detoxification methods of stem tip culture and heat treatment, the Chayote detoxification method of pretreatment and proliferation culture by the virus inhibition medium in the present application has high success rate, is easy to operate and short in cycle, can provide high-quality detoxification seedlings for the Chayote industry, and ensures the benign development of the Chayote planting industry.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture detoxification technology, specifically relating to a virus inhibition culture medium for detoxification of chayote tissue culture and a method for detoxification of chayote tissue culture. Background Technology

[0002] Chayote (Sechium edule (Jacq.) Swartz) is a perennial herbaceous vine belonging to the genus Sechium in the family Cucurbitaceae. In Guizhou, China, the cultivation area of ​​chayote exceeds 100,000 mu (approximately 6,667 hectares), forming a distinctive mountain industry. Viral diseases pose a significant threat to chayote cultivation. Although chayote primarily reproduces through seed, the large size of the seeds fails to prevent the spread of viruses.

[0003] Currently, the main viruses that infect chayote include Squash leaf curl Philippine virus, Pumpkin yellow mosaic Malaysia virus, Loofa yellow mosaic virus, and Mestayellow vein mosaic virus. To mitigate the harm of viruses to chayote, existing research primarily focuses on virus-free seedlings to provide the chayote industry with high-quality, virus-free seedlings, thus ensuring the healthy development of chayote cultivation.

[0004] Existing plant tissue detoxification techniques generally include shoot tip culture and heat treatment. However, during shoot tip culture, due to the extremely small size of the shoot tip, the culture is difficult and the tiny shoot tips are hard to survive in chayote tissue culture, resulting in a low success rate. High-temperature treatment has a long detoxification cycle and a low success rate. Summary of the Invention

[0005] The purpose of this invention is to provide a virus-inhibiting culture medium for detoxification of chayote tissue culture and a method for detoxification of chayote tissue culture. The virus-inhibiting culture medium can inhibit plant viruses while having minimal impact on the growth of chayote tissue culture materials. It is also easy to operate, has a high success rate, and a short cycle.

[0006] This invention provides the application of morpholine guanidine hydrochloride in the detoxification of chayote tissue culture.

[0007] The present invention also provides a virus-inhibiting culture medium for detoxification of chayote tissue culture, comprising plant tissue culture medium and morpholine guanidine hydrochloride.

[0008] Preferably, the final concentration of morpholine guanidine hydrochloride in the virus inhibition medium is 5-30 mg·L. -1 .

[0009] Preferably, the plant tissue culture medium includes MS liquid culture medium.

[0010] The present invention also provides a combined culture medium for detoxification of chayote tissue culture, comprising a proliferation medium and the virus inhibition medium described in the above technical solution.

[0011] Preferably, the proliferation medium is based on MS medium and further includes 0.1 mg·L⁻¹. -1 TDZ, 0.1 mg·L -1 It contains NAA, 2 wt.% sucrose and 0.7 wt.% agar.

[0012] The present invention also provides the application of the virus-inhibiting culture medium or the combined culture medium described in the above-mentioned technical solutions in the detoxification of chayote tissue culture.

[0013] This invention also provides a method for detoxifying chayote tissue culture, comprising the following steps:

[0014] 1) Chayote stem tips were inoculated into virus-inhibiting medium and cultured in the dark to obtain stem tips treated with virus inhibitors;

[0015] 2) The shoot tips treated with the virus inhibitor were inoculated into a proliferation medium for proliferation culture to obtain clustered shoots;

[0016] 3) Cut a 0.5-1.5cm stem tip from the highest bud of the clustered buds and repeat steps 1)-2) for subculture to obtain virus-free chayote seedlings;

[0017] The virus inhibition culture medium is the virus inhibition culture medium described in the above technical solution.

[0018] Preferably, the number of subcultures is 3-5 times.

[0019] Preferably, the conditions for the dark culture include: a temperature of 25°C, a time of 1 day, and a shaking speed of 150 rpm;

[0020] The conditions for the proliferation culture include: a temperature of 24℃, a time of 25 days, a light intensity of 2000 lx, and a light duration of 12 h / d.

[0021] Beneficial effects:

[0022] This invention provides the application of morpholine guanidine hydrochloride in chayote tissue culture for virus elimination. Specifically, it provides a virus-inhibiting culture medium for chayote tissue culture virus elimination, comprising plant tissue culture medium and morpholine guanidine hydrochloride. This invention involves adding morpholine guanidine hydrochloride as a virus inhibitor to the plant tissue culture medium for chayote shoot tip culture. This inhibits virus replication while having minimal impact on the growth of the chayote tissue culture material. Based on this, virus-inhibited shoot tips are then propagated and cultured repeatedly (repeated virus inhibitor treatment and propagation culture) to obtain virus-free chayote seedlings. Compared with existing methods of chayote tissue culture virus elimination using shoot tip culture and heat treatment, the virus-inhibiting culture medium of this invention provides a high success rate, is easy to operate, and has a short cycle. It can provide high-quality virus-free seedlings for the chayote industry, ensuring the healthy development of chayote cultivation. Detailed Implementation

[0023] This invention provides the application of morpholine guanidine hydrochloride in the detoxification of chayote tissue culture. In this invention, the morpholine guanidine hydrochloride is preferably used as a virus inhibitor in the detoxification of chayote tissue culture.

[0024] This invention also provides a virus-inhibiting culture medium for detoxification of chayote tissue culture, comprising plant tissue culture medium and morpholine guanidine hydrochloride. The plant tissue culture medium of this invention preferably comprises plant tissue liquid culture medium, more preferably MS liquid culture medium; this invention does not specifically limit the source of the MS liquid culture medium, and commercially available MS liquid culture medium from conventional channels in the art or prepared according to its standard formulation can be used. The concentration of morpholine guanidine hydrochloride in the virus-inhibiting culture medium of this invention is preferably 5-30 mg·L⁻¹. -1 More preferably 10-25 mg·L -1 More preferably 15-20 mg·L -1 .

[0025] This invention also provides a combined culture medium for virus detoxification of chayote tissue culture, comprising a proliferation medium and the virus inhibition medium described in the above-mentioned technical solution. The proliferation medium of this invention is preferably based on MS medium and further comprises 0.1 mg·L⁻¹. -1 TDZ, 0.1 mg·L -1 The medium contains NAA, 2 wt.% sucrose, and 0.7 wt.% agar. The present invention does not specifically limit the amount of proliferation medium and virus inhibition medium in the combined culture medium; adjustments can be made as needed.

[0026] This invention also provides the application of the virus-inhibiting culture medium or the combined culture medium described in the above-mentioned technical solutions in the detoxification of chayote tissue culture. In the application described in this invention, the virus types used in the detoxification of chayote tissue culture preferably include, but are not limited to, Squash leafcurl Philippinesvirus, Pumpkin yellow mosaic Malaysia virus, Loofa yellowmosaic virus, and Mesta yellow vein mosaic virus.

[0027] Specifically, the present invention also provides a method for detoxifying chayote tissue culture, comprising the following steps:

[0028] 1) Chayote stem tips were inoculated into virus-inhibiting medium and cultured in the dark to obtain stem tips treated with virus inhibitors;

[0029] 2) The shoot tips treated with the virus inhibitor were inoculated into a proliferation medium for proliferation culture to obtain clustered shoots;

[0030] 3) Cut a 0.5-1.5cm stem tip from the highest bud of the clustered buds and repeat steps 1)-2) for subculture to obtain virus-free chayote seedlings;

[0031] The virus inhibition culture medium is the virus inhibition culture medium described in the above technical solution.

[0032] This invention involves inoculating chayote stem tips into a virus-inhibiting medium and then culturing them in the dark to obtain virus-inhibit-treated stem tips. The stem tips used in this invention are preferably the top stem tips of chayote obtained through asexual propagation, and the length of the stem tips is preferably 0.5-1.5 cm, more preferably 1 cm; in practical operation, a stem tip length of approximately 1 cm is preferred for easier handling; the stem tips preferably have 1-2 nodes. The process of inoculating the chayote into the virus-inhibiting medium is performed under aseptic conditions. The preferred temperature for the dark culture is 25℃; the preferred time is 1 day; and the preferred shaking speed is 150 rpm.

[0033] After obtaining the virus-free stem tips, the present invention inoculates the virus-free stem tips into a proliferation culture medium for proliferation culture to obtain clustered buds. The preferred temperature for the proliferation culture of the present invention is 24℃; the preferred time is 25 days; the preferred light intensity is 2000 lx; ​​and the preferred photoperiod is 12 h / d. After the proliferation culture of the present invention, the stem tips of chayote can grow to 3-4 cm, and axillary buds will emerge, forming clustered buds.

[0034] After obtaining the clustered buds, the present invention cuts a 0.5-1.5cm stem tip from the highest bud of the clustered buds and repeats the dark culture and proliferation culture for subculture to obtain virus-free chayote seedlings. The length of the stem tip is preferably 1cm; the stem tip is subjected to dark culture and proliferation culture in sequence as one subculture, and the number of subcultures is preferably 3-5 times.

[0035] After obtaining the virus-free chayote seedlings, the present invention preferably inoculates the virus-free chayote seedlings into a rooting medium for rooting and seedling strengthening culture to obtain virus-free chayote seedlings. The rooting medium of the present invention preferably uses 1 / 2 MS as the basal medium and further includes 0.2 mg·L⁻¹. -1 The NAA is more preferably prepared using 1 / 2 MS as the basal medium, with 0.2 mg·L⁻¹ added to the 1 / 2 MS. -1 The NAA (Natural Acid) of the present invention. The conditions for rooting and seedling cultivation include a temperature of 24℃; a time preferably of 25 days; a light intensity preferably of 2000 lx; ​​and a photoperiod preferably of 12 h / d.

[0036] This invention involves culturing chayote shoot tips in a plant tissue culture medium using morpholine guanidine hydrochloride as a virus inhibitor. This inhibits virus replication while having minimal impact on the growth of the chayote tissue culture material. Based on this, virus-free chayote seedlings can be obtained by repeatedly subculturing the virus-free shoot tips (treatment with the virus-inhibiting medium + proliferation culture of clustered shoots). Compared with existing methods of virus removal through shoot tip culture and heat treatment, the virus removal method using the virus-inhibiting medium of this invention has a high success rate, is convenient to operate, and has a short cycle. It can provide high-quality virus-free seedlings for the chayote industry, ensuring the healthy development of chayote cultivation.

[0037] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1

[0039] A method for virus removal from chayote tissue culture, comprising the following steps:

[0040] 1. Preparation of culture medium:

[0041] 1) Preparation of detoxification solution:

[0042] MS liquid culture medium, pH 5.4, sterilized at 121℃ for 20 min, then cooled for later use.

[0043] Morpholine guanidine hydrochloride (Sigma, 99%, 278610, purchased from a reagent company). Prepare a 5 mg / L solution using purified water.-1 The stock solution was filtered through a sterile 0.22 μm microporous membrane and then added to sterile MS liquid culture medium to prepare a solution with a final concentration of 15 mg·L⁻¹. -1 The treatment solution of morpholine guanidine hydrochloride was dispensed into 100mL sterile Erlenmeyer flasks, 30mL per flask.

[0044] 2) Preparation of bud proliferation medium: TDZ and NAA were added to MS medium (containing 2% sucrose and 0.7% agar). The final concentrations of TDZ and NAA in MS medium were 0.1 mg·L⁻¹. -1 and 0.1 mg·L -1 The prepared bud proliferation medium was dispensed into 100mL Erlenmeyer flasks, 30mL per flask, and sterilized at 121℃ for 20min. After cooling, it was ready for use.

[0045] 2. Plant materials: Cut the top 1cm of the stem tip (with 1-2 stem nodes) of the sterile seedling of chayote asexual propagation (for obtaining asexual lines, refer to Wang Lanlan, Li Yurong, Chen Zhilin, et al. Research on rapid propagation technology of chayote stem segment tissue culture [J]. Chinese Vegetables, 2023, 36(9):30-35. for the optimal culture medium at each stage).

[0046] 3. Steps for virus detoxification through chayote tissue culture:

[0047] 1) In a clean bench, place the stem tips into the above detoxification solution, with 12 stem tips in each bottle of solution. Incubate at 24°C in the dark, shaking at 150 rpm for 1 day.

[0048] 2) Remove the stem tips, blot dry the culture medium with sterile filter paper, and inoculate them onto the bud proliferation medium. Inoculate 3 stem tips per bottle, for a total of 4 bottles. Incubate at 24℃, 2000 lx, 12h light for 25 days.

[0049] 3) Cut a 1cm stem tip from the highest bud of each explant and repeat steps 1)-2) for 5 subcultures. For each subculture, only the 1cm stem tip from the highest clustered bud of each explant is cut for subculture; the remaining material is used for leaf testing. Regularly observe the stem tip growth; if contamination is found, promptly transfer the material to a different bottle to remove the contaminated material.

[0050] Example 2

[0051] Chayote tissue culture detoxification was performed using the steps described in Example 1, with the only difference being that the concentration of morpholine guanidine hydrochloride in the detoxification solution was 30 mg·L⁻¹. -1 .

[0052] Example 3

[0053] Chayote tissue culture detoxification was performed using the steps described in Example 1, with the only difference being that the concentration of morpholine guanidine hydrochloride in the detoxification solution was 5 mg·L⁻¹. -1 .

[0054] Example 4

[0055] Effects of different types and concentrations of viral inhibitors on shoot tip growth

[0056] Lentinan (1% lentinan aqueous solution purchased from Taobao, 100mL of Baiyang lentinan pesticide registration number: PD20121979) was added to MS liquid medium to prepare solutions containing 5, 10, 20, and 30 mg / L of lentinan. -1 The treatment solution was dispensed into 100mL Erlenmeyer flasks, 30mL per flask, sterilized at 121℃ for 20min, and cooled for later use.

[0057] Morpholine guanidine hydrochloride (Sigma, 99%, 278610, purchased from a reagent company). Prepare a 5 mg / L solution using purified water. -1 The stock solution was filtered through a sterile 0.22 μm microporous membrane and then added to high-temperature sterilized MS liquid culture medium to prepare solutions containing morpholine guanidine hydrochloride at concentrations of 5, 10, 20, and 30 mg / L. -1 The treatment solution was dispensed into sterile 100mL Erlenmeyer flasks for later use.

[0058] Triazole nucleoside (100mg / capsule ribavirin capsule purchased from a pharmacy). Dissolve in purified water to a concentration of 5mg / L. -1 The stock solution was filtered through a sterile 0.22 μm microporous membrane and then added to high-temperature sterilized MS liquid culture medium to prepare solutions containing 5, 10, 20, and 30 mg / L of triazole nucleoside. -1 The treatment solution was dispensed into sterile 100mL Erlenmeyer flasks for later use.

[0059] Cut the top 1cm stem tip (with 1-2 nodes) of asexually propagated chayote. Aseptically place the tip into the treatment solution containing the virus inhibitor and the control MS liquid medium, 12 stem tips per bottle. Incubate at 24°C in the dark with shaking at 150 rpm for 1 day. Remove the stem segments, blot dry with sterile filter paper, and inoculate onto the bud proliferation medium (same as in Example 1), 3 stem tips per bottle.

[0060] Chayote stem tips treated with various concentrations of lentinan and morpholine guanidine hydrochloride showed normal growth. Except for very minor contamination, all inoculated stem tips grew to 3-4 cm in height after approximately 25 days of cultivation in the proliferation medium (step 2), with bright green leaves. However, stem tips treated with triazole nucleoside all exhibited browning, with the degree of browning increasing with increasing concentration (20, 30 mg / L). -1The treated stem tips showed signs of complete stem tip death; therefore, lentinan and morpholine guanidine hydrochloride were selected for subsequent experiments.

[0061] Comparative Example 1

[0062] Chayote tissue culture detoxification was performed using the steps described in Example 1, with the only difference being that morpholine guanidine hydrochloride in the detoxification solution was replaced with lentinan, and the concentration of lentinan was 5 mg·L⁻¹. -1 .

[0063] Comparative Example 2

[0064] Chayote tissue culture detoxification was performed using the steps described in Comparative Example 1, the only difference being that the concentration of lentinan in the detoxification solution was 15 mg·L⁻¹. -1 .

[0065] Comparative Example 3

[0066] Chayote tissue culture detoxification was performed using the steps described in Comparative Example 1, the only difference being that the concentration of lentinan in the detoxification solution was 30 mg / L. -1 .

[0067] Comparative Example 4

[0068] Chayote tissue culture detoxification was performed using the steps in Example 1, the only difference being that the detoxification solution did not contain morpholine guanidine hydrochloride, but was only MS liquid culture medium.

[0069] Results statistics:

[0070] The results of subculture in Examples 1-3 and Comparative Examples 1-4 were observed, virus detected, and statistically analyzed, as shown in Table 1.

[0071] The methods for virus detection are as follows:

[0072] Based on the previous collection of chayote stem segments with leaves from multiple locations in Guizhou, materials with viral disease characteristics such as patchy and twisted patterns were selected. After being flash-frozen in liquid nitrogen, the samples were stored at -80℃. Total RNA was extracted from the samples, libraries were constructed, and virus species were investigated using small RNA deep sequencing. The results of viral database comparison of the sequencing data showed that the plant viruses carried by diseased chayote plants were mainly single-stranded DNA viruses. Therefore, this invention selected the Philippine squash leafroll virus, which has the highest abundance. Reference: Neoh ZY, Lai HC, Lin CC, et al. Genetic diversity and geographic distribution of cucurbit-infecting begomoviruses in the Philippines[J]. Plants,2023,12(2):272.(https: / / doi.org / 10.3390 / plants12020272). Primer pair SLCuPV-1-SPAC 5'-GCTTTAKMWATAATWARRGARGAACT-3' (SEQ ID NO.1) and SLCuPV-1-SPAF5'-CAGGTTGTRGTTGAACATGATG-3'(SEQ ID NO.2) (K=G,T;M=A,C;R=A,G;W=A,T) were tested at an annealing temperature of 47.5℃.

[0073] Samples that had not undergone tissue culture for virus elimination tested positive, showing an 850bp band, indicating that the explants used contained Philippine squash leafroll virus. Treated tissue culture seedlings tested negative, failing to amplify the 850bp band, and were therefore virus-free.

[0074] Table 1. Effects of Virus Inhibitor Type, Concentration, and Number of Subcultures on Detoxification Efficacy

[0075] MS 0 / 12 0 / 12 0 / 12 0 / 12 0 / 12 <![CDATA[MS + Lentinan 5mg.L -1 > 0 / 12 0 / 12 0 / 12 0 / 12 0 / 12 <![CDATA[MS + Lentinan 15mg.L -1 > 0 / 12 0 / 12 0 / 9 0 / 9 0 / 9 <![CDATA[MS + Lentinan 30mg.L -1 > 0 / 12 0 / 12 0 / 12 0 / 10 0 / 9 <![CDATA[MS + Moroxydine Hydrochloride 5mg.L -1 > 0 / 12 0 / 12 0 / 12 1 / 12 2 / 12 <![CDATA[MS + Moroxydine Hydrochloride 15mg.L -1 > 0 / 12 0 / 12 3 / 12 5 / 11 6 / 11 <![CDATA[MS + Moroxydine Hydrochloride 30mg.L -1 > 0 / 12 0 / 12 3 / 12 3 / 12 5 / 12

[0076] Note: MS in Table 1 represents MS liquid culture medium; the data in Table 1 are negative explants / inoculated explants in the same example during virus detection. If contamination occurs during subculture, the number of explants will decrease accordingly.

[0077] Calculations based on the data in Table 1 show that, after five subcultures, neither Comparative Example 4 nor the lentinan treatment (Comparative Examples 1-3) yielded virus-free plants. (5 mg·L⁻¹) -1 Treatment with morpholine guanidine hydrochloride resulted in virus-free plants at the fourth subculture, but the virus-free rate was low, only 8.3%, at 15 mg·L⁻¹. -1 and 30 mg·L -1Treatment with morpholine guanidine hydrochloride resulted in virus-free plants after the third subculture, with a virus elimination rate of 25%. The virus elimination rate increased continuously with each subculture, reaching 15 mg / L. -1 After five subcultures, the detoxification rate reached a maximum of 55%. (30 mg / L) -1 After multiple subcultures, some shoot tips treated with morpholine guanidine hydrochloride exhibited vitrification.

[0078] Comparative Example 5

[0079] The method for virus elimination through micro-shoot tip culture is as follows:

[0080] Under a dissecting microscope, ten stem tips of 0.2 mm, 0.5 mm, 1 mm, and 2 mm were aseptically cut from asexual chayote seedlings and inoculated onto a bud proliferation medium. Except for the 2 mm stem tips, which had a 30% survival rate, all the smaller stem tips turned brown and died. The buds obtained from the 2 mm stem tip culture were tested for viruses using the same method as above, and the results were positive. Virus elimination was not achieved.

[0081] Micro-stem tip culture is a traditional and effective method for obtaining virus-free seedlings, but the cut stem tips should be as small as 0.2-0.5 mm. Its success rate depends on the genotype of the plant material and an efficient tissue culture system, which has become a bottleneck restricting the success of this virus-free technology. At present, the chayote tissue culture system cannot support the growth of such small stem tips.

[0082] Example 5

[0083] A method for virus removal from chayote tissue culture, comprising the following steps:

[0084] 1. Preparation of culture medium:

[0085] 1) Preparation of detoxification solution:

[0086] MS liquid culture medium, pH 5.4, sterilized at 121℃ for 20 min, then cooled for later use.

[0087] Morpholine guanidine hydrochloride (Sigma, 99%, 278610, purchased from a reagent company). Prepare a 5 mg / L solution using purified water. -1 The stock solution was filtered through a sterile 0.22 μm microporous membrane and then added to sterile MS liquid culture medium to prepare a solution with a final concentration of 15 mg·L⁻¹. -1 The treatment solution of morpholine guanidine hydrochloride was dispensed into 100mL sterile Erlenmeyer flasks, 30mL per flask.

[0088] 3) Preparation of bud proliferation medium: TDZ and NAA were added to MS liquid medium (containing 2% sucrose and 0.7% agar). The final concentrations of TDZ and NAA in the MS liquid medium were 0.1 mg·L⁻¹. -1and 0.1 mg·L -1 The prepared bud proliferation medium was dispensed into 100mL Erlenmeyer flasks, 30mL per flask, and sterilized at 121℃ for 20min. After cooling, it was ready for use.

[0089] 4) Preparation of rooting medium: Add NAA to 1 / 2 MS (MS medium with half the content of macro and micro elements, 30 g / L sucrose, and 7 g / L agar). The final concentration of NAA in 1 / 2 MS is 0.2 mg·L⁻¹. -1 The prepared rooting medium was dispensed into 100mL Erlenmeyer flasks, 30mL per flask, and sterilized at 121℃ for 20min. After cooling, it was ready for use.

[0090] 2. Plant material: Cut the top 1cm of the stem tip (with 1-2 stem nodes) of asexually propagated chayote sterile seedlings;

[0091] 3. Steps for virus detoxification through chayote tissue culture:

[0092] 1) In a clean bench, place the stem tips into the above detoxification solution, with 12 stem tips in each bottle of solution. Incubate at 24°C in the dark, shaking at 150 rpm for 1 day.

[0093] 2) Remove the stem tips, blot dry the culture medium with sterile filter paper, and inoculate them onto the bud proliferation medium. Inoculate 3 stem tips per bottle, for a total of 4 bottles. Incubate at 24℃, 2000 lx, 12h light for 25 days.

[0094] 3) Cut a 1cm stem tip from the highest bud of each explant and repeat steps 1)-2) for 5 subcultures. Each time, only the 1cm stem tip from the highest clustered bud of each explant is cut for subculture. After 3 generations, some leaves are taken for virus testing, yielding 3 negative samples. For the negative samples, the clustered buds are divided and subcultured onto a clustered bud proliferation medium. After 25 days of culture, leaves are taken again for virus testing, which is still negative. Thus, virus-free clustered buds of chayote are obtained.

[0095] 4) Cut the clustered buds into single buds, inoculate them onto rooting medium, and culture at 4℃, 2000 lx, 12h light for 25 days to obtain rooted, complete virus-free plants.

[0096] From the above embodiments, it can be concluded that the tissue culture detoxification method described in this invention can obtain detoxified chayote seedlings, and after rooting culture, complete detoxified plants can be obtained. The detoxification success rate is high, and the method is simple and easy to operate.

[0097] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detoxifying chayote tissue culture, characterized in that, Includes the following steps: 1) Chayote stem tips were inoculated into virus-inhibiting medium and cultured in the dark to obtain stem tips treated with virus inhibitors; 2) The shoot tips treated with the virus inhibitor were inoculated into a proliferation medium for proliferation culture to obtain clustered shoots; the proliferation medium was MS medium as the basal medium, supplemented with 0.1 mg·L⁻¹ -1 TDZ, 0.1 mg·L -1 It contains NAA, 2 wt.% sucrose and 0.7 wt.% agar; 3) Cut a 0.5-1.5cm stem tip from the highest bud of the clustered buds and repeat steps 1)-2) for subculture 3-5 times to obtain virus-free chayote seedlings; The virus inhibition medium is based on MS liquid medium with the addition of morpholine guanidine hydrochloride. The final concentration of morpholine guanidine hydrochloride in the virus inhibition medium is 5, 15, or 30 mg·L⁻¹. -1 ; The virus used in the chayote tissue culture for virus removal was Philippine squash curl virus.

2. The method according to claim 1, characterized in that, The conditions for the dark culture include: a temperature of 25°C, a time of 1 day, and a shaking speed of 150 rpm. The conditions for the proliferation culture include: a temperature of 24℃, a time of 25 days, a light intensity of 2000 lx, and a light duration of 12 h / d.