阅读说明：本技术 一种无花果愈伤组织的培养方法 (Method for culturing fig callus ) 是由 李静 马梓翔 颜志明 郭正兵 赵庆 魏世平 张旭 于 2021-08-19 设计创作，主要内容包括：本发明公开了一种无花果愈伤组织的培养方法,包括：(1)茎段的无菌化处理：将植物材料带腋芽茎洗净、去枝条、消毒、清洗,将茎段切短,形态学下端接种于培养基上；(2)无花果无菌苗茎段培养：在遮光条件下培养,茎段边缘组织诱导出愈伤组织,将其切下置于继代培养基中培养,体积变为原来的1-2倍,颜色黄白色,备用；(3)筛选最佳的培养基和培养条件。本方法的‘玛斯义·陶芬’愈伤组织的培养方法,诱导率可达90％,为‘玛斯义·陶芬’分子生物学以及基因工程研究提供了一条新的途径。(The invention discloses a method for culturing fig callus, which comprises the following steps: (1) and (3) sterile treatment of the stem sections: cleaning axillary bud and stem of plant material, removing branch, sterilizing, cleaning, cutting stem segment, inoculating morphological lower end on culture medium; (2) culturing the stem sections of the aseptic fig seedlings: culturing under shading condition, inducing callus from stem edge tissue, cutting off the callus, culturing in subculture medium until the volume is 1-2 times of the original volume, and keeping yellow and white; (3) screening for optimal medium and culture conditions. The method for culturing the 'Masui-ceramic' callus has the inductivity of 90 percent, and provides a new way for the research of 'Masui-ceramic' molecular biology and genetic engineering.)

1. A method for culturing callus of figs is characterized in that: the method comprises the following steps:

(1) and (3) sterile treatment of the stem sections: cleaning axillary bud and stem of plant material, removing branch, sterilizing, cleaning, cutting stem segment, inoculating morphological lower end on culture medium;

(2) culturing the stem sections of the aseptic fig seedlings: culturing under shading condition, inducing callus from stem edge tissue, cutting off the callus, culturing in subculture medium until the volume is 1-2 times of the original volume, and keeping yellow and white;

(3) screening for optimal medium and culture conditions.

2. The method for culturing fig callus according to claim 1, wherein: the plant material of the step (1) is young sprout of Masiy pottery 6 moon bottom of Jiangsu province.

3. The method for culturing fig callus according to claim 1, wherein: the disinfection method of the step (1) comprises the following steps: sterilized with 75% ethanol, rinsed with sterile distilled water, then sterilized with 2% sodium hypochlorite, followed by rinsing with sterile distilled water under sterile conditions.

4. The method for culturing fig callus according to claim 1, wherein: the optimal culture medium formula in the step (3): the formula of a culture medium for inducing the callus of the fig stem segments comprises the following components:

MS +6-BA (1.0mg/L) + NAA (0.1mg/L) + sucrose (30g/L) + agar powder (8g/L), pH 5.8.

5. The method for culturing fig callus according to claim 1, wherein: the optimal culture conditions of the step (3): the stem section is a young stem section cultured for 1 month, and is shaded at 25 ℃ when callus is induced.

Technical Field

The invention relates to a fig cultivation method, in particular to a fig callus culture method.

Background

The fig fruits are rich in various amino acids and mineral elements required by human bodies, and have medicinal value, edible value and ornamental value. At present, fig research mostly focuses on the aspects of nutritive value, fruit maturity, flower bud differentiation and the like. In the aspect of molecular biology, model plants such as arabidopsis thaliana and tobacco can be selected for gene function verification, the selection of the original plant verification is more favorable for clarifying the problem, and the number of the current articles reporting fig transgene is small. Therefore, in fig molecular biology research, a new direction is urgently needed to be explored to perfect fig gene function verification. In the aspect of plant molecular mechanism research, callus is a common test material and is also a necessary material for gene transformation. The callus is obtained by using an in vitro plant explant, giving a suitable hormone environment under the condition of artificial control to differentiate to form parenchyma cells with vigorous division capacity, and inducing the parenchyma cells to differentiate to form a complete regeneration plant under the suitable condition. The callus is formed by the dedifferentiation of the explant, and is widely applied to the research of plant molecular mechanisms and genetic engineering because of the following characteristics. The callus has the main characteristics that: asexual propagation, maintaining excellent female parent character, high propagation coefficient and industrial seedling raising; secondly, single cell suspension culture can be carried out, and the protoplast is separated to carry out operations such as subcellular localization, crossbreeding and the like; thirdly, clone variation and mutant screening can be carried out; fourthly, the influence of seasons and climate is avoided by utilizing the mode of industrial production, and the required secondary metabolites are stably produced for a long time; providing easy-to-operate and store material for exogenous target gene conversion. The fruits of the figs are in the order of cryptoporus, the appearance of the fruits is not like flowers, the fruits are famous, the fruit ripening period of Jiangsu province can be prolonged from the middle and late 7 months to the upper 11 months, but the bottoms of the fruits contain fruit holes, so that the sterilization is difficult, and the fruits are not suitable for inducing callus.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a method for culturing fig callus with high success rate.

The technical scheme is as follows: the invention provides a method for culturing fig callus, which comprises the following steps:

(1) and (3) sterile treatment of the stem sections: cleaning axillary bud and stem of plant material, removing branch, sterilizing, cleaning, cutting stem segment, inoculating morphological lower end on culture medium;

(2) culturing the stem sections of the aseptic fig seedlings: culturing under shading condition, inducing callus from stem edge tissue, cutting off the callus, culturing in subculture medium until the volume is 1-2 times of the original volume, and keeping yellow and white;

(3) screening for optimal medium and culture conditions.

Further, the plant material of step (1) is young shoots of ' masy ' pottery ' 6 months bottom in Jiangsu province.

Further, the sterilization method of the step (1): sterilized with 75% ethanol, rinsed with sterile distilled water, then sterilized with 2% sodium hypochlorite, followed by rinsing with sterile distilled water under sterile conditions.

Further, the optimal culture medium formula in the step (3): the formula of a culture medium for inducing the callus of the fig stem segments comprises the following components:

MS +6-BA (1.0mg/L) + NAA (0.1mg/L) + sucrose (30g/L) + agar powder (8g/L), pH 5.8.

Further, the optimal culture conditions in the step (3): the stem section is a young stem section cultured for 1 month, and is shaded at 25 ℃ when callus is induced.

Common fig in Jiangsu province can germinate in early spring at the beginning of 4 months, axillary buds with stem segments can be picked before stumping at the bottom of 11 months, and the fig-type early harvest method has the advantages of long harvest period, strong meristematic capacity, low milk content and the like. The stem section with axillary buds is adopted as a material, aseptic fig seedlings are cultivated firstly, and then the stem section is used as the material to induce callus. Under the condition, the genotype and the physiological state of the stem segment of the experimental material can be kept highly consistent, and the experimental error is reduced. Meanwhile, the explant is prevented from being sterilized for many times, and the operation is time-consuming and labor-consuming.

The method adopts an MS culture medium as a basic culture medium, and respectively uses plant growth regulators 6-BA (0.5mg/L, 1.0mg/L and 2.0mg/L) and NAA (0.1mg/L and 0.3mg/L) in proportion, 30g/L of sucrose and 8g/L of agar powder, and the pH value is 5.8. The stem segments of the aseptic seedlings are inoculated on culture media with different hormone concentration ratios, and the influence of different hormone ratios on the callus induction of the figs is researched. And screening out the optimal culture medium formula.

In the invention, a culture medium formula suitable for inducing the callus of the fig stem segment is screened out:

MS +6-BA (1.0mg/L) + NAA (0.1mg/L) + sucrose (30g/L) + agar powder (8g/L), pH value 5.8

Preferably, the stem is a young stem cultured after 1 month. When callus was induced, the temperature was 25 ℃ with shading. The callus induction rate can reach 90% under the culture medium formula and the culture condition.

The preferable formula of the MS culture medium is as follows: as basic MS medium, KNO₃Potassium nitrate 1900mg/L, NH₄NO₃Ammonium nitrate 1650mg/L, KH₂PO₄Potassium dihydrogen phosphate 170mg/L, MgSO₄Magnesium sulfate 370mg/L, CaCl₂Calcium chloride 440mg/L, KI Potassium iodide 0.83mg/L, H₃BO₃Boric acid 6.2mg/L, MnSO₄Manganese sulfate 22.30mg/L, ZnSO₄Zinc sulfate 8.6mg/L, NaMo0₄Sodium molybdate 0.25mg/L, CuSO₄Copper sulfate 0.025mg/L, CoCl₂Cobalt chloride 0.025mg/L, EDTA-Na₂Ethylene diamine tetraacetic acid disodium 37.3mg/L, FeSO₄27.8mg/L ferrous sulfate, 100mg/L inositol, 2mg/L glycine, 0.1mg/L thiamine hydrochloride, 0.5mg/L pyridoxine hydrochloride and 0.5mg/L nicotinic acid.

6-BA: 6-BA is 6-benzylamino adenine, is a cytokinin substance, has the characteristics of high efficiency, stability, low price, easy use and the like, and is the favorite cytokinin for tissue culture people. Callus development can be induced. In this experiment, 6-BA was prepared as a 1mg/mL stock solution for use in the preparation of the culture medium, and the effect could be induced by adding 1.0 mg/L6-BA.

NAA: naphthylacetic acid, an auxin, can promote the formation of shoots and can induce callus. In this experiment, NAA was prepared as a 0.1mg/mL stock solution for use in the preparation of the medium, and the effect could be induced by adding 0.1mg/L NAA.

Sucrose: sucrose acts as an energy source substance and an osmotic regulator in a plant tissue culture medium, can induce the redifferentiation of callus in addition to energy supply, and uses analytically pure sucrose produced by industry. In this test, 30g/L of sucrose was added.

Agar powder: the main function of agar powder in the culture medium is that of fixed support. Generally, agar powder having a high purity and free from impurities is used. In this test, the concentration of agar was 8 g/L.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the culture method of the 'Masui-haofen' callus of the patent has the inductivity of 90 percent, provides a new way for molecular biology and genetic engineering research of the 'Masui-haofen', provides a new test material for genetic engineering research and gene transformation of figs, and simultaneously provides reference and reference for the culture of the callus of the figs.

Detailed Description

The method for culturing the fig callus comprises the following steps:

1 test Material

The plant material is young sprout of 6 months of Masiyi pottery in Jiangsu province.

2 test method

2.1 aseptic treatment of the Stem segments

And (4) slightly washing the stem segments with the axillary buds with distilled water, and removing the damaged branches in the transportation process. Sterilized with 75% ethanol for 30s under sterile conditions, rinsed with sterile distilled water 3-5 times to remove residual alcohol, followed by 2% sodium hypochlorite for 7min, and then 3-5 times to remove residual sodium hypochlorite with sterile distilled water. Placing the material on sterile filter paper, sucking off excessive water, gently stripping bracts on buds with sterile equipment, cutting stem segments into 1cm in length, and inoculating the morphological lower end on a culture medium.

2.2 aseptic seedling stem section culture of Ficus carica

After culturing at 25 ℃ for 30 days under the shading condition, the stem segment edge tissue can induce callus, the stem segment edge tissue is cut off and placed in a subculture medium, the volume of the stem segment edge tissue can be 1-2 times of the original volume of the stem segment edge tissue after 30 days, and the stem segment edge tissue is yellow and white and can be used for subsequent experiments.

3 Medium formulation screening Process

(1) Sterilizing the stem segment with axillary bud of Ficus carica Ma Si-Doufen to obtain aseptic seedling. The influence of plant growth regulators with different ratios on the culture of 'Masui-Taofen' callus is studied by inoculating 1-cm-long stem segments which grow for 1 month to a culture medium added with MS +6-BA (0.5mg/L, 1.0mg/L, 2.0mg/L) + NAA (0.1mg/L, 0.3mg/L) + sucrose (30g/L) + agar powder (8g/L) at a pH value of 5.8.

(2) The stem segments are inoculated on a culture medium with the pH value of 5.8 of a culture medium MS +6-BA (1.0mg/L) + NAA (0.1mg/L) + sucrose (30g/L) + agar powder (8g/L), one part is completely shaded, and the other part is used for researching the influence of illumination on the induction of the callus of the Masson haofen' under the illumination condition.

4 evaluation index of stem callus quality

The quality of the callus is good and bad, and there are several evaluation indexes:

(1) callus size

Taking the callus of stem induction culture for 30 days as a standard, measuring the sizes of the induced callus under different culture mediums and different culture conditions, wherein the callus with the diameter less than 3mm is unqualified callus, and the callus with the diameter more than 3mm is qualified.

(2) Color of callus

The callus with vigorous vitality should be yellow-white, have no water stain phenomenon, compact tissue particles and obvious surface compact layer structure.

(3) Redifferentiation ability of callus

The differentiation capacity of the callus depends on the size, the differentiation capacity is strong when the color is moderate, and the differentiated callus has higher quality.

5 results of the test

TABLE 1 Effect of different hormone ratios on the induction of callus of ` Massachusettufen ` stem segments

From the test results, it can be known that when 6-BA (1.0mg/L) + NAA (0.1mg/L) is added into the culture medium, the induced callus has moderate size, yellow-white color and strong tissue differentiation capability, and the callus induction rate reaches 90%, which is the best stem callus induction hormone proportion under the test conditions.

TABLE 2 Effect of light on the induction of callus from ` Masui-Doffen ` stem segments

Under the shading condition, the callus color and the differentiation capability obtained by inducing the Masiyi-pottery-fragrant stem segment are obviously superior to the illumination condition. Therefore, the induction of the massy-haofen callus was more effective under the shading condition.