A method for inducing in vitro culture of taro seedlings by herbicide

Abstract

The application belongs to the technical field of plant breeding, and particularly relates to a method for inducing taro tissue culture seedlings by using herbicides. The method comprises the following steps: S1, treating explants; S2, inducing polyploidy; S3, subculture; and S4, inducing rooting. The method uses an inducer liquid medium to induce taro single buds, and then uses a separation medium to culture, so that polyploid taro single buds which are obviously morphologically changed are separated, then a subculture medium is used for subculture to obtain polyploid multiple buds, and finally a rooting medium is used for rooting culture of the taro single buds on the polyploid multiple buds, so that the taro tissue culture seedlings are finally obtained. The method is simple to operate and easy to realize. In addition, the taro tissue culture seedlings obtained by using the herbicides have high resistance to taro blight, can enrich taro germplasm resources, and promote the development of the taro industry.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant breeding, and particularly relates to a method for inducing taro tissue culture seedlings by using herbicides. BACKGROUND

[0002] Plant polyploidy breeding is widely used by plant breeders at home and abroad by using tissue culture combined with chemical reagents to induce plant polyploidy, so as to improve plant varieties and enrich germplasm resources. Plant polyploidy breeding can improve economic traits of crops, cultivate seedless fruits, and screen for apomictic growth materials. Polyploidy breeding is an effective breeding method, which can be completed in a short time, is easy to operate, and can effectively improve the utilization rate. Anti-mitotic agents are one of the main cytotoxic drugs for treating cancer, and the basic principle is to destroy the binding target of microtubular protein with many natural or synthetic molecules, thereby inhibiting the formation of spindle, preventing the movement of chromosomes to the two poles of the cell, destroying the cell cycle, and achieving the effect of doubling the chromosomes in the cell. In the field of plant breeding, common anti-mitotic agents include colchicine and some herbicides.

[0003] At present, colchicine is mainly used for polyploidy induction breeding of multiple varieties of taro, but colchicine is highly toxic and can cause lethal damage to plants, resulting in a decrease in the induction rate of polyploidy, and also endangering the health of the operator.

[0004] Flurazifop, asulam, propyzamide, pentoxazide, pendimethalin and metolachlor herbicides can be used for polyploidy induction. The patent CN103053414B discloses a method for doubling maize haploid by using herbicides and a special herbicide therefor, and the effective component of the special herbicide is any one of the following compounds: methyl amine phosphorothioate, propyzamide and flurazifop.

[0005] The patent CN107980620B discloses a composition for staining and doubling of maize haploid seedlings, which is composed of a composite doubling agent, an auxiliary agent, a detoxifying agent and a plant growth regulator; the composite doubling agent is nocodazole, methyl amine phosphorothioate, asulam, chlorobenzenamine, flurazifop, pentoxazide and pendimethalin; the auxiliary agent is dimethyl sulfoxide and ethylene glycol; the detoxifying agent is potassium dihydrogen phosphate and urea; and the plant growth regulator is ABT rooting powder No. 6.

[0006] However, the breeding objects of the above-mentioned polyploidy breeding are not taro. In the polyploidy breeding of taro varieties, there are few reports on breeding by using herbicides.

[0007] The information disclosed in this BACKGROUND section is only for the purpose of increasing the understanding of the background of the present application and should not be regarded as an acknowledgment or any form of suggestion that this information forms prior art that is publicly known. SUMMARY

[0008] The application aims to provide a method for inducing taro tissue culture seedlings by herbicides.

[0009] To achieve the above-mentioned purpose, the application provides the following technical scheme.

[0010] A method for inducing taro tissue culture seedlings by herbicides, comprising the following steps:

[0011] S1. Treating explants

[0012] Selecting a current-year and healthy taro single bud, soaking and disinfecting the taro single bud with 75% alcohol for 60-80s, then soaking and disinfecting the taro single bud with 0.1% mercury for 20-25min, washing the taro single bud with sterile water for 8-10min after taking out, and then absorbing the water on the surface of the taro single bud with sterile filter paper for standby use;

[0013] S2. Inducing polyploidy

[0014] Shaking and culturing the disinfected taro single bud in an inducing agent liquid medium for 72-96h to obtain a polyploid taro single bud; wherein the inducing agent liquid medium is: MS basic medium, 3-5μmol / L herbicide composition, and the herbicide composition is compounded by methyamamosulf and sulfometuron methyl;

[0015] The culture condition is: shaking frequency 150-200r / min, light time 12h / d, light intensity 1600-1800lx, and temperature 25℃.

[0016] S3. Subculture

[0017] Culturing the polyploid taro single bud in a separation medium for 45-60d, then separating the polyploid taro single bud which has obvious morphological variation, inoculating the polyploid taro single bud into a subculture medium, and continuously subculturing the polyploid taro single bud for 3-4 times to obtain a polyploid cluster bud.

[0018] S4. Rooting induction

[0019] When the polyploid cluster bud grows to 2-3cm, cutting the taro single bud and inoculating the taro single bud into a rooting medium to culture, and then obtaining a taro tissue culture seedling.

[0020] Preferably, in step S2, the mass ratio of methyamamosulf to sulfometuron methyl is 1.5:1.

[0021] Preferably, in step S3, the separation medium and the subculture medium are: MS basic medium, 0.2mg / L NAA, 3.0mg / L 6-BA, and 30.0g / L sucrose.

[0022] The culture condition is: light time 12h / d, light intensity 1600-1800lx, and temperature 25℃.

[0023] As preferred, in step S4, the rooting medium is: 1 / 2MS basic medium, 1.0 mg / L NAA, 2.0 mg / L ABT-6.

[0024] The culture condition is: light time 12h / d, light intensity 1600-1800lx, temperature 25℃.

[0025] Compared with the prior art, the application has the following beneficial effects:

[0026] (1) The application utilizes the liquid induction agent medium to induce taro single buds, then utilizes the separation culture medium to culture, separates out the morphologically obviously varied polyploid taro single buds, then utilizes the subculture medium to subculture to obtain polyploid multiple buds, finally utilizes the rooting medium to root culture the taro single buds on the polyploid multiple buds, and finally obtains taro tissue culture seedlings. The operation is simple and easy to realize.

[0027] (2) The herbicide composition of the application is compounded by methyldymron and benefin. Compared with the single use of methyldymron and benefin, the herbicide composition of the application can induce polyploid taro seedlings after treating the taro single buds, and has a high induction rate.

[0028] (3) The taro tissue culture seedlings induced by the herbicide show high resistance to taro blight, can enrich taro germplasm resources, and promote the development of the taro industry. DETAILED DESCRIPTION

[0029] The technical solutions of the application will be described below in a clear and complete manner. Obviously, the described embodiments are part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0030] In the embodiments, the solvent of methyldymron and benefin is dimethyl sulfoxide.

[0031] Example 1 : Induction effects of different induction agents on taro single buds

[0032] A method for inducing taro tissue culture seedlings by using herbicides, comprising the following steps:

[0033] S1. Treating explants

[0034] Select the current year and healthy "white bud taro" taro single buds, soak them in 75% alcohol for disinfection for 60s, then soak them in 0.1% mercury for disinfection for 20min, take them out, wash them with sterile water for 10min, then dry the surface moisture of the taro single buds with sterile filter paper for standby use;

[0035] S2. Polyploid induction

[0036] The sterilized taro single buds were cultured in the induction agent liquid medium for 72 h to obtain polyploid taro single buds; wherein the induction agent liquid medium was shown in Table 1.

[0037] The culture condition was: shaking frequency 200 r / min, light time 12 h / d, light intensity 1800 lx, temperature 25℃;

[0038] S3. Subculture

[0039] The polyploid taro single buds were inoculated into the separation medium for culture for 60 d, then the polyploid taro single buds with obvious morphological variation were separated out and inoculated into the subculture medium for continuous subculture for 3 times to obtain polyploid multiple buds; each subculture was for 60 d;

[0040] The separation medium and the subculture medium were: MS basic medium, 0.2 mg / L NAA, 3.0 mg / L 6-BA, 30.0 g / L sucrose;

[0041] The culture condition was: light time 12 h / d, light intensity 1800 lx, temperature 25℃;

[0042] S4. Rooting induction

[0043] When the polyploid multiple buds grew to 2 cm, the taro single buds were cut and inoculated into the rooting medium for culture to obtain taro tissue culture seedlings;

[0044] The rooting medium was: 1 / 2MS basic medium, 1.0 mg / L NAA, 2.0 mg / L ABT-6;

[0045] The culture condition was: light time 12 h / d, light intensity 1800 lx, temperature 25℃.

[0046] Table 1 Induction agent liquid medium

[0047]

[0048] 150 taro single buds were treated by each induction agent liquid medium, after obtaining the tissue culture seedlings by rooting induction, the polyploid cytological identification was carried out according to the method of Jiang H X et al., the number of doubled strains was counted, the induction rate of different induction agent liquid medium was calculated, and the results were shown in Table 2.

[0049]

[0050] Table 2 Effect of different induction agents on induction and screening of taro germplasm

[0051]

[0052] As shown in Table 3, the herbicide composition of the present application has higher induction rate on taro single bud than methylammonium phosphorodithioate and lenacil alone, and is obviously higher than colchicine. It is shown that the polyploid taro seedlings can be induced after the taro single bud is treated by the herbicide composition of the present application, and has higher induction rate.

[0053] Example 2 Identification of taro tissue culture seedlings on blight resistance

[0054] 1. Test pathogenic bacteria

[0055] Phytophthora colocasiae is isolated from taro blight samples in Liupu City, Guangxi Zhuang Autonomous Region.

[0056] 2. Test taro germplasm resources

[0057] The taro tissue culture seedlings induced and screened by the liquid culture medium of the test group and the comparison groups 1-3 in Example 2 are transplanted into the test field of Guangxi Zhuang Autonomous Region Academy of Agricultural Sciences, and the normal growth seedlings are selected as test objects after 30 days.

[0058] 2. Test method

[0059] According to the method of Mo Junjie et al., the Phytophthora colocasiae is expanded and cultured on PDA medium for 5 days, and then the 5mm-diameter fungus cake is cut by a puncher at the edge of the colony. It is inoculated on the back of the second tender leaf, and the fungus block is sealed with transparent tape. Each test material is inoculated with 5 leaf blades. After 5 days of inoculation, the lesion diameter is measured, and the average value is calculated as the standard of taro resistance to Phytophthora colocasiae. The results are shown in Table 3.

[0060] Table 3 Identification results of taro tissue culture seedlings on blight resistance

[0061]

[0062] As shown in Table 3, the taro tissue culture seedlings induced by the present application have higher resistance to taro blight.

[0063] The foregoing description of specific exemplary embodiments of the present application is intended for purposes of illustration and example only. These descriptions are not intended to limit the application in any way, and it is apparent that many modifications and variations are possible without departing from the scope of the application. The examples were chosen and described in order to explain the principles of the application and its practical application and to enable others skilled in the art to best utilize the application, various embodiments of which are realized and are admissible to various substitutions and modifications. The scope of the application is to be defined by the claims and their equivalents.

Claims

1. A method for inducing in vitro culture of taro seedlings by using herbicide, characterized in that, Includes the following steps: S1. Treatment of explants Select healthy taro sprouts from the current year, disinfect them by soaking them in 75% alcohol for 60-80 seconds, then disinfect them by soaking them in 0.1% mercuric chloride for 20-25 minutes. After taking them out, rinse them with sterile water for 8-10 minutes, and then use sterile filter paper to absorb the surface moisture of the taro sprouts for later use. S2. Polyploidy induction Disinfected taro shoots were cultured in an induction agent liquid medium with shaking for 72-96 hours to obtain polyploid taro shoots; wherein, the induction agent liquid medium was: MS basal medium and 5 μmol / L herbicide composition, wherein the herbicide composition was a mixture of methyl parathion and ammoniasulfuron, and the mass ratio of methyl parathion to ammoniasulfuron was 1.5:1; The cultivation conditions are: oscillation frequency 150-200 r / min, light duration 12 h / d, light intensity 1600-1800 lx, and temperature 25℃; S3. Subgeneration Polyploid taro buds were inoculated into isolation medium and cultured for 45-60 days. Then, polyploid taro buds with obvious morphological variations were isolated and inoculated into subculture medium for 3-4 consecutive subcultures to obtain polyploid cluster buds. S4. Rooting Induction When the polyploid clustered buds grow to 2-3 cm, cut off the taro single buds and inoculate them into rooting medium to obtain taro tissue culture seedlings.

2. The method for inducing taro tissue culture seedlings with herbicide according to claim 1, characterized in that, In step S3, the isolation medium and subculture medium are: MS basal medium, 0.2 mg / L NAA, 3.0 mg / L 6-BA, and 30.0 g / L sucrose; The cultivation conditions were: 12 hours / day of light, 1600-1800 lx of light intensity, and 25℃.

3. The method of claim 1, wherein the herbicide is applied to the taro explant in an amount of 0.1 to 1.0 mg per 1 g of the taro explant. In step S4, the rooting medium is: 1 / 2 MS basal medium, 1.0 mg / L NAA, and 2.0 mg / L ABT-6; The cultivation conditions were: 12 hours / day of light, 1600-1800 lx of light intensity, and 25℃.

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