A method for disinfecting and inducing regeneration of a single excellent explant of malania oleifera

Abstract

The application discloses a method for disinfecting and inducing regeneration of a single excellent plant of Malus baccata, and comprises the following steps: S1, disinfecting the terminal bud or / and axillary bud of the Malus baccata; S2, inoculating the terminal bud or / and axillary bud into a modified MS culture medium, and culturing under preset conditions to obtain uncontaminated terminal bud or / and axillary bud; S3, inoculating the uncontaminated terminal bud or / and axillary bud into an induction culture medium, and culturing under preset induction conditions to obtain a cluster bud, wherein the components of the induction culture medium comprise a modified MS as a base, and 6-BA, IPA, prochloraz, cysteine hydrochloride and L-arginine are added; and S4, dividing the cluster bud into single plants, inoculating the single plants into a subculture medium, and culturing under preset culture conditions to obtain Malus baccata single seedlings. Compared with the prior art, the method selects a disinfection method with a high survival rate and a modified MS culture medium with obvious plant life characteristics by taking the terminal bud or / and axillary bud of the Malus baccata as explant material, so that more uncontaminated Malus baccata terminal buds or / and axillary buds which grow well can be obtained, and the tissue culture can be carried out for a long time, thereby providing more materials for the tissue culture of the Malus baccata.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant tissue culture, in particular to a method for disinfecting and inducing regeneration of excellent single explant of Malania oleifera. BACKGROUND

[0002] Malania oleifera, also known as mountain chestnut fruit and Malania, is a evergreen tree of Malania oleifera in the family Malaniaceae, which is a unique single-species plant in China. It is only found in Yunnan and Guangxi, and is a national second-class rare and endangered protected tree species. Malania oleifera is a small population rescue and protection object in Yunnan Province. The nervonic acid content in Malania oleifera kernel oil is about 45%, which has a positive promoting effect on the repair and regeneration of damaged nerve tissue.

[0003] Tissue culture propagation of Malania oleifera is currently the fastest way to obtain Malania oleifera single plants. Tao Junfeng and Wang Qiong have studied the browning of Malania oleifera tissue culture, and disclosed that the half-lignified part near the terminal end of the current year's shoot is used as an explant, and the explant is disinfected by 0.1% mercury for 6 minutes and 2% sodium hypochlorite for 10 minutes, and cultured on 1 / 2WPM medium containing 2g / L PVP, which can better reduce the generation of browning. Wang Qiong and Chen Wenyi disclosed a Malania oleifera tissue culture browning control technology, which uses 0.3% mercuric chloride to disinfect the explant, and uses 1 / 2WPM medium containing ascorbic acid for tissue culture, so that the highest obtainable rate of Malania oleifera aseptic seedlings is only 51.7%, and the obtainable rate of Malania oleifera is low.

[0004] Currently, there is no report on tissue culture research using Malania oleifera terminal buds and axillary buds as explant materials. The contamination rate, survival rate and growth status of Malania oleifera terminal buds and axillary buds as explant materials are unknown.

[0005] Therefore, the present application uses 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 in the browning rate, induction rate and growth of Malania oleifera. Within a certain transfer period, different culture media may cause the transferred Malania oleifera plants to appear browning, even death and lose growth signs. The suitable explant disinfection method and culture medium are selected to realize the Malania oleifera single plants with low contamination rate, high survival rate and good growth through tissue culture of terminal buds and axillary buds. SUMMARY

[0006] The purpose of the present application is to provide a method for disinfecting and inducing regeneration of excellent single explant of Malania oleifera, which aims to solve the technical problem of unknown contamination rate, survival rate and growth status of Malania oleifera terminal buds and axillary buds as explant materials.

[0007] To achieve the above purpose, the present application provides a method for disinfecting and inducing regeneration of excellent single explant of Malania oleifera, which comprises:

[0008] S1, sterilizing the terminal bud or / and axillary bud of Malania oleifera;

[0009] S2, inoculating the terminal bud or / and axillary bud into modified MS medium, culturing in preset conditions, obtaining uncontaminated terminal bud or / and axillary bud;

[0010] S3, inoculating the uncontaminated terminal bud or / and axillary bud into induction medium, culturing in preset induction conditions, obtaining cluster shoots, wherein the components of the induction medium include modified MS as the basis, adding 6-BA, IPA, prochloraz, cysteine hydrochloride and L-arginine;

[0011] S4, dividing the cluster shoots into single plants, inoculating the single plants into subculture medium, culturing in preset culture conditions, obtaining Malania oleifera single seedlings.

[0012] Preferably, in the above technical solution, in step S1, the step of sterilizing the terminal bud or / and axillary bud of Malania oleifera is specifically as follows:

[0013] S11, taking the terminal bud or / and axillary bud of Malania oleifera growing well, sterilizing with 75% alcohol for 30-60s, and then washing with sterile water for 2-3 times;

[0014] S12, centrifuging on a centrifuge with a rotation speed of 3000-4000P / m for 5-10 minutes;

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

[0016] S14, sterilizing with 0.1% mercuric chloride for 5-10min, and washing with sterile water for 5-8 times.

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

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

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

[0020] Preferably, in the above technical solution, the subculturing 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 solution, the modified MS 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 ethylenediaminetetraacetate, 3.33 mg / L pyridoxine hydrochloride, 0.67 mg / L thiamine hydrochloride, 1.33 mg / L glycine, 3.33 mg / L nicotinic acid, 666.67 mg / L myo-inositol.

[0022] Preferably, in the above technical solution, the culturing in the preset culturing condition is culturing in the dark for 5-8 days at a culturing temperature of 26-30°C, and then culturing for 7-10 days under light intensity of 1000-2000 Lx and light time of 12-14 hours.

[0023] Preferably, in the above technical solution, the dark culturing is culturing under light intensity of 300-500 Lx and light time of 6-8 hours.

[0024] Compared with the prior art, the application has the following beneficial effects:

[0025] (1) The tissue culture research is carried out for the first time by using Allium cepa L. top buds and axillary buds as explant materials;

[0026] (2) The disinfection method with a survival rate of 91.67% is provided, and the modified MS is further screened for culturing, so as to reduce the browning of Allium cepa L. plants and improve the survival rate of uncontaminated Allium cepa L. top buds or / and axillary buds, thereby providing material basis for Allium cepa L. tissue culture research and obtaining excellent Allium cepa L. tissue culture seedlings;

[0027] (3) The induction medium improves the proliferation coefficient of Allium cepa L. tissue culture seedlings under the condition of ensuring the robustness of the multiple shoots, and the subculturing medium prolongs the survival rate of Allium cepa L. plants. Attached Figure Description

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

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

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

[0031] Figure 3 This is a graph showing the growth of garlic fruit explants on MS basal medium;

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

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

[0034] Figure 6 This is a graph showing the growth of garlic fruit explants on a B5 basal culture medium.

[0035] Figure 7 This is a graph showing the growth of garlic fruit explants on N6 basal culture medium;

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

[0037] Figure 9 This is a diagram of robust garlic bulb shoot clusters during induction culture using the method of this invention;

[0038] Figure 10 This is a diagram of robust garlic bulb shoot clusters during induction culture using the method of this invention;

[0039] Figure 11 This is a diagram of two robust garlic bulb plants undergoing subculture using the method of this invention;

[0040] Figure 12 The image shows a robust garlic bulb plant that has been subcultured for more than 30 days using the method of this invention.

[0041] Figure 13 This is a picture of a robust garlic bulb plant that has been in subculture for more than 50 days using the method of this invention. Detailed Implementation

[0042] The technical solutions in the embodiments of this invention are clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0043] Example 1

[0044] A method for disinfecting and inducing regeneration of superior single explants of garlic fruit, 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 cloves, wash off the dust on the surface of the explant with running tap water, then place them in a triangular flask containing saturated detergent solution for 7-10 minutes (shaking during this time), and rinse them with running tap water for about 1 hour to obtain disinfected terminal buds and axillary buds.

[0047] (2) The terminal and axillary buds of garlic fruits were treated with different disinfection methods as described in Table 1 and inoculated into hormone-free MS medium, with 50 bottles inoculated for each treatment. The contamination rate of explants treated with different disinfection methods was observed and recorded daily.

[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 formulas for calculating the contamination rate and survival rate are as follows:

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

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

[0054] from Figure 1It can be seen that only G, H, I, J, K treatment of garlic fruit axillary bud and / or top bud survival rate is greater than or equal to the pollution rate, and only with K treatment method disinfection garlic fruit axillary bud and top bud pollution rate is low and the survival rate is high, that is, the pollution rate is 66% and the survival rate is 91.67%; therefore, when the explant object is axillary bud or top bud, the best disinfection method is: 75% alcohol disinfection for 30s, then washed with sterile water for 2 times, then placed in sodium hypochlorite, disinfection for 10min, then washed with sterile water for 5 times, then placed in glass bottle with 0.1% mercuric chloride, disinfection for 5min, then washed with sterile water for 5 times.

[0055] S2, garlic fruit explant induction and culture:

[0056] The 7d uncontaminated top bud and axillary bud were transferred to the medium as shown in Table 2, 10 bottles were inoculated for each medium, and 1-2 buds were inoculated for each bottle.

[0057] Table 2: Medium composition of each treatment

[0058] Medium Basal medium 6-BA (mg / L) NAA (mg / L) A 1 / 2MS 0.2 0.2 B MS 0.2 0.2 C Modified 1 / 2MS 0.2 0.2 D Modified 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 improved 1 / 2MS formula is as follows: 275mg / L potassium nitrate, 266.65mg / L ammonium nitrate, 73.3mg / L calcium chloride, 28.35mg / L potassium dihydrogen phosphate, 61.65mg / L magnesium sulfate, 0.275mg / L potassium iodide, 28.665mg / L zinc sulfate, 0.415mg / L cobalt chloride, 20.665mg / L boric acid, 74.335mg / L manganese sulfate, 0.84mg / L sodium molybdate, 0.47mg / L copper sulfate, 9.27mg / L ferrous sulfate, 12.43mg / L disodium ethylenediaminetetraacetate, 1.665mg / L pyridoxine hydrochloride, 0.335mg / L thiamine hydrochloride, 0.665mg / L glycine, 1.665mg / L nicotinic acid, 333.335mg / L inositol.

[0060] The improved MS formula 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 ethylenediaminetetraacetate, 3.33mg / L pyridoxine hydrochloride, 0.67mg / L thiamine hydrochloride, 1.33mg / L glycine, 3.33mg / L nicotinic acid, 666.67mg / L inositol.

[0061] The above treatments were first dark cultured for 7 days at a culture temperature of 28±2°C, and then cultured for 30 days at a light intensity of 1000-3000 Lx and a light time of 12-14 h. During the culture, the induction initiation of the Allium cepa fruits of the above treatments was observed, and the growth of the Allium cepa fruit explants was recorded every 7 days as an observation period, as shown in Table 3.

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

[0063]

[0064]

[0065] Therefore, when the basic medium is modified 1 / 2MS and modified MS, the life characteristics of the Allium cepa fruit explants are obvious, and especially when the basic medium is modified MS, the growth of the Allium cepa fruit explants is best. The formula of the modified MS medium is more suitable for the Allium cepa axillary bud or apical bud to grow out tender leaves, and promotes the growth of the Allium cepa plant. Too much or too little nutrient substance will cause the Allium cepa tissue to blacken or the plant to have no growth.

[0066] A large number of Allium cepa axillary buds and / or apical buds were treated by the above K disinfection method, inoculated into a modified MS medium without hormones for 7 days to obtain more apical buds and axillary buds without pollution, and then the induction medium was obtained by taking the proliferation coefficient as an index. The induction medium is based on modified MS and adds 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 rate of the induction medium based on modified MS and adding 6-BA, IPA, prochloraz, cysteine hydrochloride and L-arginine is best.

[0070] Subculture medium: the medium based on modified MS and adding 6-BA, NAA, indolepropionic acid (IPA), cysteine hydrochloride or L-arginine, after long-term culture of the Allium cepa axillary buds and / or apical buds, the growth of the Allium cepa plant was observed, and the medium formula suitable for long-term culture of the Allium cepa plant was selected as the subculture medium.

[0071] Table 5 Medium with different components

[0072] 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 medium was inoculated with 15 bottles, each with 1 bud, and each culture was observed for 7 days as a cycle. The results of the survival of the plants after induction are shown in Table 5. The standard for survival was obvious plant life, and the standard for death was blackening of the callus or withering of the plant or no growth for a long time.

[0074] Table 6 Number of bottles with survival of plants of Allium cepa in various media for a certain period

[0075]

[0076]

[0077] The axillary buds or terminal buds of Allium cepa were treated with L and cultured for 22 days. In 14 bottles, the plants showed obvious signs of life. Therefore, modified MS was selected as the base medium, and 6-BA, IPA, cysteine hydrochloride and L-arginine were added as the medium for induction of Allium cepa.

[0078] Example 2

[0079] A method for disinfecting and inducing regeneration of a single excellent plant of Allium cepa, the method comprising:

[0080] S1, disinfecting the terminal buds or / and axillary buds of Allium cepa using a disinfection method with a survival rate-contamination rate of ≥20%, wherein the disinfection method with a survival rate-contamination rate of ≥20% comprises the following steps:

[0081] S11, taking the terminal buds or / and axillary buds of Allium cepa that grow well, disinfecting them with 75% alcohol for 30 seconds, and then washing them with sterile water twice;

[0082] S12, centrifuging them in a centrifuge with a rotation speed of 3000 P / m for 5 minutes;

[0083] S13, disinfecting them with sodium hypochlorite for 10 minutes, and then washing them with sterile water five times;

[0084] S14, disinfecting them with 0.1% mercuric chloride for 5 minutes, and then washing them with sterile water five times;

[0085] S2, inoculate the apical bud or / and axillary bud into modified MS medium, and culture in dark for 7 days at a culture temperature of 26-30 DEG C, the light intensity of the dark culture is 300Lx and the light time is 6h, to obtain uncontaminated apical bud or / and axillary bud, 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 ethylenediaminetetraacetate, 3.33mg / L pyridoxal hydrochloride, 0.67mg / L thiamine hydrochloride, 1.33mg / L glycine, 3.33mg / L nicotinic acid, 666.67mg / L myo-inositol;

[0086] S3, inoculate the uncontaminated apical bud or / and axillary bud into an induction medium, the induction medium is made of modified MS medium, 0.2mg / L 6-BA, 0.2mg / L IPA, 0.1mg / L prohexadione-calcium, 0.2mg / L cysteine hydrochloride and 0.2mg / L L-arginine, and culture at a culture temperature of 26-30 DEG C, first culture in dark for 7 days at a light intensity of 300Lx and a light time of 6h, and then culture at a light intensity of 1000Lx and a light time of 14h for 7-10 days, to obtain a cluster of bulb fruits;

[0087] S4, divide the cluster into single plants, inoculate the single plants into a subculture medium, the subculture medium is made of modified MS medium, 0.2mg / L 6-BA, 0.2mg / L IPA, 0.2mg / L cysteine hydrochloride and 0.2mg / L L-arginine, and culture at a culture temperature of 26-30 DEG C, first culture in dark for 7 days at a light intensity of 300Lx and a light time of 6h, and then culture at a light intensity of 2000Lx and a light time of 14h, to obtain single seedlings of bulb fruits.

[0088] Example 3

[0089] A method for disinfecting and inducing regeneration of a single excellent explant of a bulb fruit, the method comprising:

[0090] S1, disinfect the apical bud or / and axillary bud of the bulb fruit by a disinfection method with a survival rate-contamination rate of greater than or equal to 20%, wherein the disinfection method with a survival rate-contamination rate of greater than or equal to 20% comprises the following steps:

[0091] S11, take the apical bud or / and axillary bud of the bulb fruit growing well, disinfect the apical bud or / and axillary bud with 75% alcohol for 30s, and then wash the apical bud or / and axillary bud with sterile water for 2 times.

[0092] S12, centrifuging on a centrifuge with a rotating speed of 3500P / m for 6 minutes;

[0093] S13, sterilizing with sodium hypochlorite for 10 minutes and washing with sterile water for 5 times;

[0094] S14, sterilizing with 0.1% mercury chloride for 5 minutes and washing with sterile water for 5 times;

[0095] S2, inoculating the terminal bud or / and axillary bud into modified MS medium, and culturing in dark for 7 days at a culture temperature of 26-30℃, the light intensity of the dark culture being 400Lx and the light time being 7h, to obtain uncontaminated terminal bud or / and axillary bud, 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 ethylenediaminetetraacetate, 3.33mg / L pyridoxine hydrochloride, 0.67mg / L thiamine hydrochloride, 1.33mg / L glycine, 3.33mg / L nicotinic acid, 666.67mg / L myo-inositol;

[0096] S3, inoculating the uncontaminated terminal bud or / and axillary bud into an induction medium, the induction medium being modified MS medium, 0.1mg / L 6-BA, 0.1mg / L IPA, 0.05mg / L prohexadione, 0.1mg / L cysteine hydrochloride and 0.1mg / L L-arginine, and culturing at a culture temperature of 26-30℃, first culturing in dark for 7 days at a light intensity of 400Lx and a light time of 7h, and then culturing at a light intensity of 1500Lx and a light time of 13h for 7-10 days, to obtain garlic fruit multiple shoots;

[0097] S4, dividing the multiple shoots into single plants, and inoculating the single plants into a subculture medium, the induction medium being modified MS medium, 0.1mg / L 6-BA, 0.1mg / L IPA, 0.1mg / L cysteine hydrochloride and 0.1mg / L L-arginine, and culturing at a culture temperature of 26-30℃, first culturing in dark for 7 days at a light intensity of 400Lx and a light time of 7h, and then culturing at a light intensity of 1500Lx and a light time of 13h, to obtain garlic fruit single plant seedlings.

[0098] Example 4

[0099] A method for disinfecting and inducing regeneration of a single excellent explant of Malania oleifera, comprising:

[0100] S1, disinfecting the apical bud or / and axillary bud of Malania oleifera by a disinfection method with a survival rate-pollution rate ≥ 20%, wherein the disinfection method with a survival rate-pollution rate ≥ 20% comprises the following steps:

[0101] S11, taking the apical bud or / and axillary bud of Malania oleifera growing well, disinfecting with 75% alcohol for 30s, and then washing with sterile water for 2 times;

[0102] S12, centrifuging on a centrifuge with a rotation speed of 4000P / m for 5 minutes;

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

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

[0105] S2, inoculating the apical bud or / and axillary bud into modified MS medium, and culturing in the dark at a culture temperature of 26-30℃ for 7d, wherein the light intensity of the dark culture is 500Lx and the light time is 6h, to obtain the apical bud or / and axillary bud without pollution, 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 ethylenediaminetetraacetate, 3.33mg / L pyridoxine hydrochloride, 0.67mg / L thiamine hydrochloride, 1.33mg / L glycine, 3.33mg / L nicotinic acid, 666.67mg / L inositol;

[0106] S3, inoculating the apical bud or / and axillary bud without pollution into an induction medium, wherein the induction medium is prepared by modified MS medium, 0.2mg / L 6-BA, 0.2mg / L IPA, 0.05mg / L prohexadione, 0.2mg / L cysteine hydrochloride and 0.2mg / L L-arginine, and culturing at a culture temperature of 26-30℃, first culturing in the dark for 7d at a light intensity of 500Lx and a light time of 6h, and then culturing for 7-10d at a light intensity of 2000Lx and a light time of 12h, to obtain the Malania oleifera multiple shoots;

[0107] S4. The clustered buds are divided into individual plants, and the individual plants are inoculated into a subculture medium. The induction medium is prepared by modified MS medium, 0.2 mg / L 6-BA, 0.2 mg / L LIPA, 0.2 mg / L cysteine ​​hydrochloride and 0.2 mg / L L-arginine. The plants are cultured at a temperature of 26-30℃, first in the dark at a light intensity of 500 Lx and a light duration of 6 h for 7 days, and then in the dark at a light intensity of 2000 Lx and a light duration of 12 h to obtain individual garlic seedlings.

[0108] Comparative Example 1

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

[0110] S2, inoculate the apical bud and / or axillary bud into modified MS medium and culture in the dark for 7 days at a culture temperature of 26-30℃. The dark culture is achieved by lining the bottom of the culture rack with an opaque material, placing the tissue culture bottle on it, and then covering it with the opaque material. After the culture is completed, remove the culture bottle and observe the condition of the plant.

[0111] The uncontaminated apical buds and / or axillary buds of the garlic cloves in 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 for S3 are different.

[0116] S3. The uncontaminated apical buds and / or axillary buds are inoculated into an induction medium, which is prepared by modified MS medium, 0.1 mg / L 6-BA, 0.1 mg / L IPA, 0.05 mg / L imazalil, 0.1 mg / L cysteine ​​hydrochloride and 0.1 mg / L L-arginine. The induction medium is cultured at a temperature of 26-30℃, a light intensity of 4000 Lx and a light duration of 13 h for 7-10 days to obtain garlic clove cluster 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 non-contaminated terminal buds or / and axillary buds into an induction medium prepared from modified MS medium, 0.2 mg / L 6-BA, 0.4 mg / L IPA, 0.5 mg / L prochloraz, 0.3 mg / L cysteine hydrochloride and 0.5 mg / L L-arginine, and culturing at a culture temperature of 26-30°C, first in dark for 7 days at a light intensity of 400 Lx and a light time of 7 h, and then at a light intensity of 1500 Lx and a light time of 13 h for 7-10 days, to obtain the bulbil cluster shoots.

[0120] The bulbil cluster shoots of Example 2, Comparative Example 2 and Comparative Example 3 are shown in Table 8 below.

[0121] Table 8 Bulbil cluster shoot conditions

[0122]

[0123]

[0124] Comparative Example 4

[0125] The same as Example 2, except that the medium component of S4 is different.

[0126] S4, dividing the cluster shoots into single plants, inoculating the single plants into a subculture medium prepared from 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 for 7 days at a light intensity of 300 Lx and a light time of 6 h, and then at a light intensity of 2000 Lx and a light time of 14 h, to obtain the bulbil single plant seedlings.

[0127] Example 2 and Comparative Example 4, each of which is inoculated with 20 bottles, each of which is inoculated with one bud, and the bulbil plant survival conditions within a certain period of time are shown in Table 9 below.

[0128] Table 9 Bulbil plant survival bottle number within a certain period of time

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

[0130] The present application can be implemented in various different ways and is not limited to the described embodiments, and it will be understood by those skilled in the art that the present application can be implemented by other specific ways without changing the technical idea or essential characteristics of the present application. Therefore, it should be understood that the above-described embodiments are exemplary and are not intended to limit the present application.

Claims

1. A method for disinfecting and inducing regeneration of superior single-plant explants of garlic cloves, characterized in that, The method includes: S1, disinfecting the apical bud and / or axillary buds of garlic bulbs; S2, the apical bud and / or axillary bud is inoculated into modified MS medium and cultured in the dark at a culture temperature of 26-30℃ for 5-8 days to obtain uncontaminated apical buds and / or axillary buds. The light intensity of the dark culture is 300-500 Lx and the light duration is 6-8 h. S3, the uncontaminated apical buds and / or axillary buds are inoculated into an induction medium and cultured in the dark for 5-8 days at a temperature of 26-30℃, and then cultured for 7-10 days at a light intensity of 1000-2000 Lx and a light duration of 12-14 h to obtain clustered buds. The induction medium is composed 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 imazalil, 0.1-0.2 mg / L cysteine ​​hydrochloride and 0.1-0.2 mg / L L-arginine. S4, the clustered buds are divided into individual plants, and the individual plants are inoculated into a subculture medium. They are cultured in the dark for 5-8 days at a temperature of 26-30℃, and then cultured at a light intensity of 1000-2000 Lx and a photoperiod of 12-14 h to obtain individual garlic bulb seedlings. The subculture medium is prepared from 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; wherein, The modified MS medium consisted of 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 EDTA, 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.

2. The method for disinfection and induced regeneration of superior single-plant explants of garlic fruit as described in claim 1, characterized in that, In step S1, the disinfection of the apical bud and / or axillary buds of the garlic bulb is specifically as follows: S11. Take the terminal bud and / or axillary bud of a well-grown garlic bulb, disinfect it with 75% alcohol for 30-60 seconds, and then wash it with sterile water 2-3 times. S12, centrifuge at 3000-4000 P / m for 5-10 minutes; S13, disinfect with sodium hypochlorite for 5-10 minutes, and rinse with sterile water 5-8 times; S14, disinfect with 0.1% mercuric chloride for 5-10 minutes, and rinse with sterile water 5-8 times.

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