Method for disinfection and induced regeneration of malania oleifera excellent single plant explant

Abstract

The invention discloses a malania oleifera excellent single plant explant disinfection and induced regeneration method. The method comprises the following steps: S1, disinfecting terminal buds or / and axillary buds of malania oleifera; s2, inoculating the terminal buds or / and the axillary buds into an improved MS culture medium, and culturing under preset conditions to obtain pollution-free terminal buds or / and axillary buds; s3, the pollution-free terminal buds or / and axillary buds are / is inoculated into an induction culture medium and cultured under preset induction conditions, multiple shoots are obtained, and the induction culture medium is prepared from improved MS as a base and added with 6-BA, IPA, prochloraz, cysteine hydrochloride and L-arginine; s4, dividing the cluster buds into single plants, inoculating the single plants into a subculture medium, and culturing under preset culture conditions to obtain malania oleifera single plant seedlings. Compared with the prior art, the method has the advantages that terminal buds or / and axillary buds of malania oleifera are used as explant materials, a disinfection method with high survival rate and an improved MS culture medium with obvious plant vital signs are screened out, so that more pollution-free terminal buds or / and axillary buds which grow well are obtained, and tissue culture can be performed for a long time; and more materials are provided for malania oleifera tissue culture.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a method for disinfecting and inducing regeneration of superior single garlic fruit explants. Background Art

[0002] Malania oleifera, also known as Shantong fruit and Malanhou (Zhuang language), is an evergreen tree of the family Asclepiadaceae. It is a monospecific plant of the genus Malania and is only found in Yunnan and Guangxi. It is a national second-level rare and endangered protected tree species and a target of rescue and protection of a very small population in Yunnan Province. The content of nervonic acid in the kernel oil of Malania oleifera is as high as about 45%, which has a positive effect on the repair and regeneration of damaged nerve tissue.

[0003] The use of tissue culture to propagate garlic fruit is currently a relatively fast method for obtaining a single garlic fruit plant. In the prior art, Tao Junfeng and Wang Qiong studied the browning of garlic fruit tissue culture, and disclosed that the semi-lignified part of the current year near the end of the new shoot was used as an explant, and disinfected with 0.1% mercuric chloride for 6 minutes + 2% sodium hypochlorite for 10 minutes, and cultured on a 1 / 2WPM medium containing 2g / L PVP to reduce the occurrence of browning. Wang Qiong and Chen Wenyi's technique for controlling the browning of garlic fruit tissue culture disclosed that the use of 0.3% mercuric chloride to disinfect the explant and the use of a 1 / 2WPM medium containing ascorbic acid for tissue culture can make the acquisition rate of garlic fruit sterile seedlings only 51.7% at most, and the garlic fruit acquisition rate is low.

[0004] So far, there are no reports on tissue culture research using the apical buds and axillary buds of garlic fruit as explant materials. The contamination rate, survival rate and growth status of the apical buds and axillary buds of garlic fruit as explant materials are unknown.

[0005] Therefore, the present invention adopts terminal buds and axillary buds as explant materials, different disinfection methods will affect the contamination rate and survival rate of terminal buds and / or axillary buds, different culture media and culture conditions have great differences on the browning rate, induction rate and growth of garlic fruit, and different culture media may cause the transferred garlic fruit plants to brown or even die and lose growth signs within a certain transfer cycle. Suitable explant disinfection methods and culture media are selected to achieve low contamination rate, high survival rate and good growth of garlic fruit plants through tissue culture of terminal buds and axillary buds. Summary of the invention

[0006] The invention aims to provide a method for disinfecting and inducing regeneration of superior single garlic fruit explants, aiming to solve the technical problems of unclear contamination rate, survival rate and growth status of garlic fruit apical buds and axillary buds as explant materials.

[0007] To achieve the above object, the present invention provides a method for disinfecting and inducing regeneration of superior single garlic fruit explants, the method comprising:

[0008] S1, disinfection of the terminal buds and / or axillary buds of garlic fruit;

[0009] S2, inoculating the terminal buds and / or axillary buds into a modified MS medium, and culturing under preset conditions to obtain uncontaminated terminal buds and / or axillary buds;

[0010] S3, inoculating the uncontaminated apical buds and / or axillary buds into an induction medium, culturing under preset induction conditions, and obtaining clustered buds, wherein the induction medium comprises a modified MS as a base, supplemented with 6-BA, IPA, prochloraz, cysteine ​​hydrochloride, and L-arginine;

[0011] S4, dividing the clustered buds into individual plants, inoculating the individual plants into a subculture medium, and culturing them under preset culture conditions to obtain garlic fruit individual plant seedlings.

[0012] Preferably, in the above technical solution, in step S1, the step of disinfecting the apical buds and / or axillary buds of garlic fruit is specifically as follows:

[0013] S11, take the terminal buds and / or axillary buds of garlic fruits with good growth, disinfect them with 75% alcohol for 30-60 seconds, and then wash them with sterile water for 2-3 times;

[0014] S12, centrifuge at 3000-4000P / m for 5-10 minutes;

[0015] S13, disinfect with sodium hypochlorite for 5-10 min and wash with sterile water 5-8 times;

[0016] S14, disinfect with 0.1% mercuric chloride for 5-10 minutes and wash with sterile water 5-8 times.

[0017] Preferably, in the above technical solution, in step S2, the culturing under preset conditions, the preset conditions are dark culturing for 5-8 days at a culturing temperature of 26-30°C, the light intensity of the dark culturing is 300-500Lx and the illumination time is 6-8h.

[0018] Preferably, in the above technical solution, the induction medium is made of modified MS medium, 0.1-0.2 mg / L 6-BA, 0.1-0.2 mg / L IPA, 0.05-0.2 mg / L prochloraz, 0.1-0.2 mg / L cysteine ​​hydrochloride and 0.1-0.2 mg / L L-arginine.

[0019] Preferably, in the above technical solution, in step S3, the culturing is carried out under preset induction conditions, and the preset induction conditions are culturing in the dark for 5-8 days at a culture temperature of 26-30°C, and then culturing for 7-10 days at a light intensity of 1000-2000Lx and a lighting time of 12-14h.

[0020] Preferably, in the above technical solution, the subculture medium is made of modified MS medium, 0.1-0.2 mg / L 6-BA, 0.1-0.2 mg / L IPA, 0.1-0.2 mg / L cysteine ​​hydrochloride and 0.1-0.2 mg / L L-arginine.

[0021] Preferably, in the above technical scheme, the modified MS culture medium is 550 mg / L potassium nitrate, 633.3 mg / L ammonium nitrate, 146.6 mg / L calcium chloride, 56.7 mg / L potassium dihydrogen phosphate, 123.3 mg / L magnesium sulfate, 0.55 mg / L potassium iodide, 57.33 mg / L zinc sulfate, 0.83 mg / L cobalt chloride, 41.33 mg / L boric acid, 148.67 mg / L manganese sulfate, 1.67 mg / L sodium molybdate, 0.83 mg / L copper sulfate, 18.53 mg / L ferrous sulfate, 24.86 mg / L disodium ethylenediaminetetraacetic acid, 3.33 mg / L pyridoxine hydrochloride, 0.67 mg / L thiamine hydrochloride, 1.33 mg / L glycine, 3.33 mg / L nicotinic acid, and 666.67 mg / L inositol.

[0022] Preferably, in the above technical solution, the culturing is carried out under preset culturing conditions, and the preset culturing conditions are culturing in the dark for 5-8 days at a culturing temperature of 26-30°C, and then culturing in a light intensity of 1000-2000Lx and a lighting time of 12-14h.

[0023] Preferably, in the above technical solution, the dark culture is performed with a light intensity of 300-500 Lx and a lighting time of 6-8 h.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] (1) The first tissue culture study using garlic fruit apical buds and axillary buds as explant materials;

[0026] (2) providing a disinfection method with a survival rate of 91.67%, further screening out improved MS for cultivation, reducing the browning of garlic fruit plants, and improving the survival rate of uncontaminated garlic fruit apical buds and / or axillary buds, providing a material basis for garlic fruit tissue culture research and obtaining excellent garlic fruit tissue culture seedlings;

[0027] (3) The induction culture medium can improve the proliferation coefficient of garlic fruit tissue culture seedlings while ensuring the robustness of the cluster buds; the subculture culture medium can prolong the survival rate of garlic fruit plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings.

[0029] Figure 1 It is a graph of the contamination rate and survival rate of garlic fruit explants disinfected according to the AL treatment method of the present invention;

[0030] Figure 2 This is a graph showing the growth of garlic fruit explants in a basal medium of 1 / 2MS;

[0031] Figure 3 It is a graph showing the growth of garlic fruit explants with MS as the basic medium;

[0032] Figure 4 This is a graph showing the growth of garlic fruit explants whose basal medium is modified 1 / 2MS;

[0033] Figure 5 This is a graph showing the growth of garlic fruit explants whose basal medium is modified MS;

[0034] Figure 6 It is a graph showing the growth of garlic fruit explants in the basic medium B5;

[0035] Figure 7 is a graph showing the growth of garlic fruit explants in which the basic medium is N6;

[0036] Figure 8 This is a graph showing the growth of garlic fruit explants grown on WPM as the basal medium;

[0037] Fig. 9 It is a picture of robust garlic fruit clustered buds in the induction culture using the method of the present invention;

[0038] Fig.10 It is a picture of robust garlic fruit clustered buds in the induction culture using the method of the present invention;

[0039] Fig.11 It is a picture of a pair of robust garlic fruit plants in subculture using the method of the present invention;

[0040] Fig.12 This is a picture of a robust garlic fruit plant that has been subcultured for more than 30 days using the method of the present invention;

[0041] Fig.13 The present invention is used to carry out the subculture of the robust garlic fruit plant for more than 50 days. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Example 1

[0044] A method for disinfecting and inducing regeneration of superior single garlic fruit explants, the method comprising the following steps:

[0045] S1, disinfection of garlic fruit explants:

[0046] (1) Cut off the terminal buds and axillary buds of well-grown garlic fruits, wash away the dust on the surface of the explants with running tap water, then put them in a conical flask filled with saturated detergent solution for 7-10 minutes (shaking during the period), and rinse them in running tap water for about 1 hour to obtain the disinfected terminal buds and axillary buds;

[0047] (2) Treat the apical buds and axillary buds of garlic fruit according to the different disinfection methods described in Table 1, and inoculate them into MS medium without hormones, with 50 bottles inoculated for each treatment. Observe and record the contamination rate of explants treated with different disinfection methods every day.

[0048] Table 1 Different disinfection methods

[0049]

[0050]

[0051] The survival rate and contamination rate of the above garlic fruit explant disinfection screening experiment are as follows Figure 1 As shown, the calculation formulas of the contamination rate and the survival rate are as follows:

[0052] Contamination rate (%) = number of contaminated bottles / number of inoculated bottles × 100%;

[0053] Survival rate (%) = number of surviving bottles / number of inoculated bottles × 100%.

[0054] from Figure 1It can be seen that only the survival rates of axillary buds and / or terminal buds of garlic fruit treated with G, H, I, J, and K were greater than or equal to the contamination rate, and only the contamination rate of axillary buds and terminal buds of garlic fruit disinfected with K treatment was low and the survival rate was high, that is, the contamination rate was 66% and the survival rate was 91.67%; therefore, when the explant object is axillary buds or terminal buds, the best disinfection method is: disinfect with 75% alcohol for 30 seconds, then wash twice with sterile water, then place in sodium hypochlorite, disinfect for 10 minutes, wash 5 times with sterile water, and then put in a glass bottle with 0.1% mercuric chloride, disinfect for 5 minutes, and then wash 5 times with sterile water.

[0055] S2, induction and culture of garlic fruit explants:

[0056] The uncontaminated apical buds and axillary buds cultured in hormone-free MS medium for 7 days were transferred to the culture medium described in Table 2. Ten bottles were inoculated for each culture medium, and 1-2 buds were inoculated in each bottle.

[0057] Table 2. Medium composition of each treatment

[0058] Culture medium Basic culture medium 6-BA(mg / L) NAA(mg / L) A 1 / 2MS 0.2 0.2 B MS 0.2 0.2 C Improved 1 / 2MS 0.2 0.2 D Improved MS 0.2 0.2 E B5 0.2 0.2 F N6 0.2 0.2 G WPM 0.2 0.2

[0059] The formula of the improved 1 / 2MS is as follows: 275 mg / L potassium nitrate, 266.65 mg / L ammonium nitrate, 73.3 mg / L calcium chloride, 28.35 mg / L potassium dihydrogen phosphate, 61.65 mg / L magnesium sulfate, 0.275 mg / L potassium iodide, 28.665 mg / L zinc sulfate, 0.415 mg / L cobalt chloride, 20.665 mg / L boric acid, 74.335 mg / L manganese sulfate, 0.84 mg / L sodium molybdate, 0.47 mg / L copper sulfate, 9.27 mg / L ferrous sulfate, 12.43 mg / L disodium ethylenediaminetetraacetic acid, 1.665 mg / L pyridoxine hydrochloride, 0.335 mg / L thiamine hydrochloride, 0.665 mg / L glycine, 1.665 mg / L niacin, and 333.335 mg / L inositol.

[0060] The formula of the improved MS is as follows: 550 mg / L potassium nitrate, 633.3 mg / L ammonium nitrate, 146.6 mg / L calcium chloride, 56.7 mg / L potassium dihydrogen phosphate, 123.3 mg / L magnesium sulfate, 0.55 mg / L potassium iodide, 57.33 mg / L zinc sulfate, 0.83 mg / L cobalt chloride, 41.33 mg / L boric acid, 148.67 mg / L manganese sulfate, 1.67 mg / L sodium molybdate, 0.83 mg / L copper sulfate, 18.53 mg / L ferrous sulfate, 24.86 mg / L disodium ethylenediaminetetraacetic acid, 3.33 mg / L pyridoxine hydrochloride, 0.67 mg / L thiamine hydrochloride, 1.33 mg / L glycine, 3.33 mg / L niacin, and 666.67 mg / L inositol.

[0061] The above treatments were first cultured in the dark for 7 days at a culture temperature of 28 ± 2°C, and then cultured for 30 days at a light intensity of 1000-3000Lx and a light duration of 12-14h. During the culture process, the induction start of garlic fruit in the above treatments was observed, and the growth of garlic fruit explants was recorded as shown in Table 3, with each culture of 7 days as an observation cycle.

[0062] Table 3 Growth of garlic fruit explants in each treatment group

[0063]

[0064]

[0065] Therefore, when the basal culture medium is modified 1 / 2MS and modified MS, the life characteristics of garlic fruit explants are obvious, especially when the basal culture medium is modified MS, the growth of garlic fruit explants is best. The formula of modified MS culture medium is more suitable for the growth of tender leaves from the original petioles of the axillary buds or terminal buds of garlic fruit, and promotes the growth of garlic fruit plants. Too much or too little nutrients will cause the garlic fruit tissue to melanize or the plant to show no signs of growth.

[0066] A large number of garlic fruit axillary buds and / or terminal buds are treated according to the above-mentioned K disinfection method, and then inoculated into a hormone-free modified MS medium for 7 days to obtain a large number of uncontaminated terminal buds and axillary buds, and then the proliferation coefficient is used as an indicator to obtain an induction medium. The induction medium is based on the modified MS and is supplemented with 6-BA, NAA, indolepropionic acid (IPA), prochloraz, cysteine ​​hydrochloride or L-arginine.

[0067] Table 4 Induction medium

[0068]

[0069] As can be seen from Table 4 above, the induction medium based on modified MS with the addition of 6-BA, IPA, prochloraz, cysteine ​​hydrochloride and L-arginine has the best induction rate.

[0070] Subculture medium: A culture medium based on modified MS with additions of 6-BA, NAA, indolepropionic acid (IPA), cysteine ​​hydrochloride or L-arginine. After long-term cultivation of axillary buds and / or terminal buds of garlic fruit, the growth conditions of garlic fruit plants are observed, and a culture medium formula suitable for long-term cultivation of garlic fruit plants is selected as the subculture medium.

[0071] Table 5 Culture medium with different components

[0072] Culture medium 6-BA NAA IPA Cysteine ​​Hydrochloride L-Arginine A 0.2 0.2 0 0 0 B 0.2 0.2 0.2 0 0 C 0.2 0 0.2 0 0 D 0.2 0.2 0 0.2 0 E 0.2 0 0.2 0.2 0 F 0.2 0.2 0.2 0.2 0 G 0.2 0.2 0 0 0.2 I 0.2 0 0.2 0 0.2 J 0.2 0.2 0.2 0 0.2 K 0.2 0.2 0 0.2 0.2 L 0.2 0 0.2 0.2 0.2

[0073] Each culture medium was inoculated with 15 bottles, each bottle was inoculated with 1 bud, and each culture was 7 days as an observation period. The results of observing the survival of garlic fruit after induction were shown in Table 5 below. The survival standard was that the life characteristics of the plant were obvious, and the death standard was that the callus tissue was blackened or the plant was withered or the plant had no growth signs for a long time.

[0074] Table 6 Number of surviving bottles of garlic fruit plants on various culture media within a certain period

[0075]

[0076]

[0077] After 22 days of culture, all 14 bottles of garlic fruit tissue culture seedlings showed obvious plant life characteristics in the axillary buds or terminal buds of garlic fruit in the L treatment. Therefore, the modified MS was selected as the culture medium for the induction culture of garlic fruit with the addition of 6-BA, IPA, cysteine ​​hydrochloride and L-arginine.

[0078] Example 2

[0079] A method for disinfecting and inducing regeneration of superior individual garlic fruit explants, the method comprising:

[0080] S1, using a disinfection method with a survival rate-contamination rate ≥ 20% to disinfect the terminal buds and / or axillary buds of garlic fruit, wherein the disinfection method with a survival rate-contamination rate ≥ 20% has the following specific steps:

[0081] S11, take the terminal buds and / or axillary buds of garlic fruits with good growth, disinfect them with 75% alcohol for 30 seconds, and then wash them with sterile water twice;

[0082] S12, centrifuge at 3000P / m for 5 minutes;

[0083] S13, disinfect with sodium hypochlorite for 10 min and wash with sterile water 5 times;

[0084] S14, disinfect with 0.1% mercuric chloride for 5 min and wash with sterile water 5 times;

[0085] S2, inoculating the terminal buds and / or axillary buds into a modified MS medium, culturing in the dark for 7 days at a culture temperature of 26-30°C, with a light intensity of 300Lx and a light duration of 6h, to obtain uncontaminated terminal buds and / or axillary buds, wherein the formula of the modified MS medium is as follows: 550mg / L potassium nitrate, 633.3mg / L ammonium nitrate, 146.6mg / L calcium chloride, 56.7mg / L potassium dihydrogen phosphate, 123.3mg / L magnesium sulfate, 0.55mg / L Potassium iodide, 57.33mg / L zinc sulfate, 0.83mg / L cobalt chloride, 41.33mg / L boric acid, 148.67mg / L manganese sulfate, 1.67mg / L sodium molybdate, 0.83mg / L copper sulfate, 18.53mg / L ferrous sulfate, 24.86mg / L disodium ethylenediaminetetraacetic acid, 3.33mg / L pyridoxine hydrochloride, 0.67mg / L thiamine hydrochloride, 1.33mg / L glycine, 3.33mg / L niacin, 666.67mg / L inositol;

[0086] S3, inoculating the uncontaminated terminal buds and / or axillary buds into an induction medium, wherein the induction medium is made of a modified MS medium, 0.2 mg / L 6-BA, 0.2 mg / LIPA, 0.1 mg / L prochloraz, 0.2 mg / L cysteine ​​hydrochloride and 0.2 mg / L L-arginine, and culturing them in the dark for 7 days at a light intensity of 300 Lx and a light duration of 6 hours at a culture temperature of 26-30° C., and then culturing them for 7-10 days at a light intensity of 1000 Lx and a light duration of 14 hours to obtain garlic fruit clustered buds;

[0087] S4, dividing the clustered buds into individual plants, inoculating the individual plants into a subculture medium, wherein the subculture medium is made of a modified MS medium, 0.2 mg / L 6-BA, 0.2 mg / L IPA, 0.2 mg / L cysteine ​​hydrochloride and 0.2 mg / L L-arginine, and culturing at a culture temperature of 26-30°C, first in dark culture at a light intensity of 300 Lx and a light duration of 6 h for 7 days, and then in culture at a light intensity of 2000 Lx and a light duration of 14 h to obtain garlic fruit individual plant seedlings.

[0088] Example 3

[0089] A method for disinfecting and inducing regeneration of superior individual garlic fruit explants, the method comprising:

[0090] S1, using a disinfection method with a survival rate-contamination rate ≥ 20% to disinfect the terminal buds and / or axillary buds of garlic fruit, wherein the disinfection method with a survival rate-contamination rate ≥ 20% has the following specific steps:

[0091] S11, take the terminal buds and / or axillary buds of garlic fruits with good growth, disinfect them with 75% alcohol for 30 seconds, and then wash them with sterile water twice;

[0092] S12, centrifuge at 3500P / m for 6 minutes;

[0093] S13, disinfect with sodium hypochlorite for 10 min and wash with sterile water 5 times;

[0094] S14, disinfected with 0.1% mercuric chloride for 5 min and washed with sterile water 5 times;

[0095] S2, inoculating the terminal buds and / or axillary buds into a modified MS medium, culturing in the dark for 7 days at a culture temperature of 26-30°C, with a light intensity of 400Lx and a light duration of 7h, to obtain uncontaminated terminal buds and / or axillary buds, wherein the formula of the modified MS medium is as follows: 550mg / L potassium nitrate, 633.3mg / L ammonium nitrate, 146.6mg / L calcium chloride, 56.7mg / L potassium dihydrogen phosphate, 123.3mg / L magnesium sulfate, 0.55mg / L Potassium iodide, 57.33mg / L zinc sulfate, 0.83mg / L cobalt chloride, 41.33mg / L boric acid, 148.67mg / L manganese sulfate, 1.67mg / L sodium molybdate, 0.83mg / L copper sulfate, 18.53mg / L ferrous sulfate, 24.86mg / L disodium ethylenediaminetetraacetic acid, 3.33mg / L pyridoxine hydrochloride, 0.67mg / L thiamine hydrochloride, 1.33mg / L glycine, 3.33mg / L niacin, 666.67mg / L inositol;

[0096] S3, inoculating the uncontaminated terminal buds and / or axillary buds into an induction medium, wherein the induction medium is made of a modified MS medium, 0.1 mg / L 6-BA, 0.1 mg / L IPA, 0.05 mg / L prochloraz, 0.1 mg / L cysteine ​​hydrochloride and 0.1 mg / L L-arginine, and culturing them in the dark for 7 days at a light intensity of 400 Lx and a light duration of 7 hours at a culture temperature of 26-30° C., and then culturing them for 7-10 days at a light intensity of 1500 Lx and a light duration of 13 hours to obtain garlic fruit clustered buds;

[0097] S4, dividing the clustered buds into individual plants, and inoculating the individual plants into a subculture medium, wherein the induction medium is made of a modified MS medium, 0.1 mg / L 6-BA, 0.1 mg / L IPA, 0.1 mg / L cysteine ​​hydrochloride and 0.1 mg / L L-arginine, and culturing at a culture temperature of 26-30°C, first in dark culture at a light intensity of 400 Lx and a light duration of 7 h for 7 days, and then in culture at a light intensity of 1500 Lx and a light duration of 13 h to obtain garlic fruit individual plant seedlings.

[0098] Example 4

[0099] A method for disinfecting and inducing regeneration of superior individual garlic fruit explants, the method comprising:

[0100] S1, using a disinfection method with a survival rate-contamination rate ≥ 20% to disinfect the terminal buds and / or axillary buds of garlic fruit, wherein the disinfection method with a survival rate-contamination rate ≥ 20% has the following specific steps:

[0101] S11, take the terminal buds and / or axillary buds of garlic fruits with good growth, disinfect them with 75% alcohol for 30 seconds, and then wash them with sterile water twice;

[0102] S12, centrifuge at 4000P / m for 5 minutes;

[0103] S13, disinfect with sodium hypochlorite for 10 min and wash with sterile water 5 times;

[0104] S14, disinfect with 0.1% mercuric chloride for 5 min and wash with sterile water 5 times;

[0105] S2, inoculating the terminal buds and / or axillary buds into a modified MS medium, culturing in the dark for 7 days at a culture temperature of 26-30°C, with a light intensity of 500Lx and a light duration of 6h, to obtain uncontaminated terminal buds and / or axillary buds, wherein the formula of the modified MS medium is as follows: 550mg / L potassium nitrate, 633.3mg / L ammonium nitrate, 146.6mg / L calcium chloride, 56.7mg / L potassium dihydrogen phosphate, 123.3mg / L magnesium sulfate, 0.55mg / L Potassium iodide, 57.33mg / L zinc sulfate, 0.83mg / L cobalt chloride, 41.33mg / L boric acid, 148.67mg / L manganese sulfate, 1.67mg / L sodium molybdate, 0.83mg / L copper sulfate, 18.53mg / L ferrous sulfate, 24.86mg / L disodium ethylenediaminetetraacetic acid, 3.33mg / L pyridoxine hydrochloride, 0.67mg / L thiamine hydrochloride, 1.33mg / L glycine, 3.33mg / L niacin, 666.67mg / L inositol;

[0106] S3, inoculating the uncontaminated terminal buds and / or axillary buds into an induction medium, wherein the induction medium is made of a modified MS medium, 0.2 mg / L 6-BA, 0.2 mg / LIPA, 0.05 mg / L prochloraz, 0.2 mg / L cysteine ​​hydrochloride and 0.2 mg / L L-arginine, and culturing them in the dark for 7 days at a light intensity of 500 Lx and a light duration of 6 hours at a culture temperature of 26-30° C., and then culturing them for 7-10 days at a light intensity of 2000 Lx and a light duration of 12 hours to obtain garlic fruit clustered buds;

[0107] S4, dividing the clustered buds into individual plants, and inoculating the individual plants into a subculture medium, wherein the induction medium is made of a modified MS medium, 0.2 mg / L 6-BA, 0.2 mg / LIPA, 0.2 mg / L cysteine ​​hydrochloride and 0.2 mg / L L-arginine, and culturing at a culture temperature of 26-30°C, first in dark culture at a light intensity of 500 Lx and a light duration of 6 h for 7 days, and then in culture at a light intensity of 2000 Lx and a light duration of 12 h to obtain garlic fruit individual plant seedlings.

[0108] Comparative Example 1

[0109] It is basically the same as Example 2, except that the culture conditions of S2 are different.

[0110] S2, inoculate the apical buds and / or axillary buds into the modified MS medium, and culture them in the dark at a culture temperature of 26-30°C for 7 days. The dark culture is to lay opaque material on the bottom of the culture rack and cover the opaque material after placing the tissue culture bottle. After the culture is completed, take out the culture bottle and observe the plant condition.

[0111] The uncontaminated terminal buds and / or axillary buds of garlic fruits of Example 2 and Comparative Example 1 are shown in Table 7 below.

[0112] Table 7 Growth of garlic fruit explants

[0113]

[0114] Comparative Example 2

[0115] It is basically the same as Example 2, except that the culture conditions of S3 are different.

[0116] S3, inoculating the uncontaminated apical buds and / or axillary buds into an induction medium, wherein the induction medium is made of a modified MS medium, 0.1 mg / L 6-BA, 0.1 mg / L IPA, 0.05 mg / L prochloraz, 0.1 mg / L cysteine ​​hydrochloride and 0.1 mg / L L-arginine, and culturing for 7-10 days at a culture temperature of 26-30°C, a light intensity of 4000Lx and a light duration of 13h to obtain garlic fruit clustered buds.

[0117] Comparative Example 3

[0118] It is basically the same as Example 2, except that the culture medium components of S3 are different.

[0119] S3, inoculating the uncontaminated apical buds and / or axillary buds into an induction medium, wherein the induction medium is made of a modified MS medium, 0.2 mg / L 6-BA, 0.4 mg / LIPA, 0.5 mg / L prochloraz, 0.3 mg / L cysteine ​​hydrochloride and 0.5 mg / L L-arginine, and culturing the culture medium at a culture temperature of 26-30°C for 7 days in the dark at a light intensity of 400 Lx and a light duration of 7 hours, and then culturing the culture medium at a light intensity of 1500 Lx and a light duration of 13 hours for 7 days to obtain garlic fruit clustered buds.

[0120] The clustered sprouts of garlic fruit in Example 2, Comparative Example 2 and Comparative Example 3 are shown in Table 8 below.

[0121] Table 8 Garlic fruit cluster sprout situation

[0122]

[0123]

[0124] Comparative Example 4

[0125] It is basically the same as Example 2, except that the culture medium components of S4 are different.

[0126] S4, dividing the clustered buds into individual plants, inoculating the individual plants into a subculture medium, wherein the subculture medium is made of a modified MS medium, 0.2 mg / L 6-BA, 0.2 mg / L IPA, 0.2 mg / L ascorbic acid and 0.1 mg / L activated carbon, and culturing at a culture temperature of 26-30°C, first in dark culture at a light intensity of 300 Lx and a light duration of 6 h for 7 days, and then in culture at a light intensity of 2000 Lx and a light duration of 14 h to obtain garlic fruit individual plant seedlings.

[0127] Example 2 and Comparative Example 4 were inoculated with 20 bottles, each with one bud. The survival of garlic fruit plants within a certain period of time is shown in Table 9 below.

[0128] Table 9 Number of surviving bottles of garlic fruit plants on various culture media within a certain period

[0129] 21d 28d 35d 42d 49d Comparative Example 4 20 18 17 13 10 Example 2 20 20 20 19 19

[0130] The present invention can be implemented in various ways and is not limited to the embodiments described. A person skilled in the art can understand that the present invention can be implemented in other specific ways without changing the technical concept or essential features of the present invention. Therefore, it should be understood that the embodiments described above are exemplary and are not intended to limit the present invention.

Claims

1. A method for disinfecting and inducing regeneration of superior single garlic fruit explants, characterized in that: The method comprises: S1, disinfection of the terminal buds and / or axillary buds of garlic fruit; S2, inoculating the terminal buds and / or axillary buds into a modified MS medium, and culturing under preset conditions to obtain uncontaminated terminal buds and / or axillary buds; S3, inoculating the uncontaminated apical buds and / or axillary buds into an induction medium, culturing under preset induction conditions, and obtaining clustered buds, wherein the induction medium comprises a modified MS as a base, supplemented with 6-BA, IPA, prochloraz, cysteine ​​hydrochloride, and L-arginine; S4, dividing the clustered buds into individual plants, inoculating the individual plants into a subculture medium, and culturing them under preset culture conditions to obtain garlic fruit individual plant seedlings.

2. The method for sterilizing and inducing regeneration of superior individual garlic fruit explants as claimed in claim 1, characterized in that: In step S1, the steps of disinfecting the terminal buds and / or axillary buds of garlic fruit are specifically as follows: S11, take the terminal buds and / or axillary buds of garlic fruits with good growth, disinfect them with 75% alcohol for 30-60 seconds, and then wash them with sterile water for 2-3 times; S12, centrifuge at 3000-4000P / m for 5-10 minutes; S13, disinfect with sodium hypochlorite for 5-10 min and wash with sterile water 5-8 times; S14, disinfect with 0.1% mercuric chloride for 5-10 minutes and wash with sterile water 5-8 times.

3. The method for sterilizing and inducing regeneration of superior individual garlic fruit explants as claimed in claim 1, characterized in that: In step S2, the culturing is carried out under preset conditions, wherein the preset conditions are dark culturing for 5-8 days at a culturing temperature of 26-30°C, a light intensity of 300-500 Lx, and a lighting time of 6-8 h.

4. The method for sterilizing and inducing regeneration of superior individual garlic fruit explants as claimed in claim 1, characterized in that: The induction medium is prepared from a modified MS medium, 0.1-0.2 mg / L 6-BA, 0.1-0.2 mg / L IPA, 0.05-0.2 mg / L prochloraz, 0.1-0.2 mg / L cysteine ​​hydrochloride and 0.1-0.2 mg / L L-arginine.

5. The method for sterilizing and inducing regeneration of superior individual garlic fruit explants as claimed in claim 1, characterized in that: In step S3, the culture is carried out under preset induction conditions, which are culture at a culture temperature of 26-30°C, first in dark culture for 5-8 days, and then in culture at a light intensity of 1000-2000Lx and a light duration of 12-14h for 7-10 days.

6. The method for sterilizing and inducing regeneration of superior individual garlic fruit explants as claimed in claim 1, characterized in that: The subculture medium is prepared from a modified MS medium, 0.1-0.2 mg / L 6-BA, 0.1-0.2 mg / L IPA, 0.1-0.2 mg / L cysteine ​​hydrochloride and 0.1-0.2 mg / L L-arginine.

7. The method for sterilizing and inducing regeneration of superior individual garlic fruit explants as claimed in claim 1, 4 or 6, characterized in that: The modified MS culture medium comprises 550 mg / L potassium nitrate, 633.3 mg / L ammonium nitrate, 146.6 mg / L calcium chloride, 56.7 mg / L potassium dihydrogen phosphate, 123.3 mg / L magnesium sulfate, 0.55 mg / L potassium iodide, 57.33 mg / L zinc sulfate, 0.83 mg / L cobalt chloride, 41.33 mg / L boric acid, 148.67 mg / L manganese sulfate, 1.67 mg / L sodium molybdate, 0.83 mg / L copper sulfate, 18.53 mg / L ferrous sulfate, 24.86 mg / L disodium ethylenediaminetetraacetic acid, 3.33 mg / L pyridoxine hydrochloride, 0.67 mg / L thiamine hydrochloride, 1.33 mg / L glycine, 3.33 mg / L nicotinic acid, and 666.67 mg / L inositol.

8. The method for sterilizing and inducing regeneration of superior individual garlic fruit explants as claimed in claim 1, characterized in that: The culture is carried out under the preset culture conditions, and the preset culture conditions are: at a culture temperature of 26-30° C., first culture in the dark for 5-8 days, and then culture under a light intensity of 1000-2000 Lx and a light exposure time of 12-14 hours.

9. The method for sterilizing and inducing regeneration of superior individual garlic fruit explants as claimed in claim 5 or 8, characterized in that: The dark culture is performed at a light intensity of 300-500 Lx and a light duration of 6-8 h.

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