阅读说明：本技术 木薯再生体系的建立方法 (Method for establishing cassava regeneration system ) 是由 牛俊乐 黄斌政 黄斌 麦馨允 杨郑州 朱正杰 于 2021-09-01 设计创作，主要内容包括：本发明公开了木薯再生体系的建立方法,包括：步骤一、获取木薯带腋芽茎段,利用第一培养基和第二培养基进行初代培养和继代增殖培养,至长出无菌叶片,所述第一培养基包括MS培养基和6-BA,所述第二培养基包括6-BA和NAA；步骤二、取步骤一得到的无菌叶片,利用第三培养基进行愈伤组织培养,所述第三培养基包括MS培养基、Picloram和CuSO-(4)；步骤三、取步骤二得到的愈伤组织,利用第四培养基诱导分化出芽,所述第四培养基包括MS培养基、6-BA和AgNO-(3)。本发明能够为木薯品种改良过程中目的基因的导入奠定理论及技术基础。(The invention discloses a method for establishing a cassava regeneration system, which comprises the following steps: the method comprises the following steps of firstly, obtaining a cassava stem section with axillary buds, and carrying out primary culture and secondary multiplication culture by utilizing a first culture medium and a second culture medium until sterile leaves grow out, wherein the first culture medium comprises an MS culture medium and 6-BA, and the second culture medium comprises 6-BA and NAA; step two, taking the sterile leaves obtained in the step one, and performing callus culture by using a third culture medium, wherein the third culture medium comprises an MS culture medium, Picloram and CuSO 4 (ii) a Step three, taking the callus obtained in the step two, and inducing differentiation and sprouting by utilizing a fourth culture medium, wherein the fourth culture medium comprises an MS culture medium, 6-BA and AgNO 3 . The invention can lay a theorem and technical foundation for the introduction of the target gene in the cassava variety improvement process.)

1. The method for establishing the cassava regeneration system is characterized by comprising the following steps:

the method comprises the following steps of firstly, obtaining a cassava stem section with axillary buds, and carrying out primary culture and secondary multiplication culture by utilizing a first culture medium and a second culture medium until sterile leaves grow out, wherein the first culture medium comprises an MS culture medium and 6-BA, and the second culture medium comprises 6-BA and NAA;

step two, taking the sterile leaves obtained in the step one, and performing callus culture by using a third culture medium, wherein the third culture medium comprises an MS culture medium, Picloram and CuSO₄；

Step three, taking the callus obtained in the step two, and inducing differentiation and sprouting by utilizing a fourth culture medium, wherein the fourth culture medium comprises an MS culture medium, 6-BA and AgNO₃。

2. The method of establishing a cassava regeneration system according to claim 1, further comprising:

before the primary culture, the axillary bud stem is sterilized, wherein the sterilization comprises the sequential use of 75% alcohol and 0.1% HgCl₂And (6) processing.

3. The method for establishing a cassava regeneration system according to claim 1, wherein the concentration of 6-BA in the first medium is 1.5mg/L, and the concentrations of 6-BA and NAA in the second medium are 0.5mg/L and 0.05mg/L, respectively.

4. The method for establishing a cassava regeneration system according to claim 1, in which in step two, after 7 days of dark culture, light culture is performed for 40 days, and Picloram and CuSO are added to the third medium₄The concentrations of (A) were 11.0mg/L and 0.2mg/L, respectively.

5. Such asThe method for establishing a cassava regeneration system according to claim 1, wherein in the third step, after 5 days of dark culture, light culture is performed for 40 days, and 6-BA and AgNO are performed in the fourth medium₃The concentrations of (A) were 0.1mg/L and 5.0mg/L, respectively.

6. Method for establishing a cassava regeneration system according to claim 1 or 5, wherein the fourth medium further comprises activated carbon and coconut water;

the preparation method of the activated carbon comprises the following steps: cutting coconut shells, soaking in sodium hydroxide solution, filtering, washing with water, carbonizing in a muffle furnace, cooling, soaking in ethanol, adding zinc oxide, irradiating with ultrasonic wave, filtering, and drying to obtain the active carbon.

7. The method for establishing the cassava regeneration system according to claim 1, wherein in the first step, the second step, the third step and the fourth step, the temperature is kept at 25-28 ℃, and the conditions of the light culture include a light time of 12-14 h/d and a light intensity of 1500-2000 lx.

8. The method for establishing a cassava regeneration system according to claim 1, wherein the first medium, the second medium, the third medium, and the fourth medium have a pH of 5.3 to 6.3.

Technical Field

The present invention relates to plant tissue culture technology. More specifically, the invention relates to a method for establishing a cassava regeneration system.

Background

Cassava is widely planted worldwide due to the advantages of strong tolerance, high yield and the like, and especially in many tropical regions, cassava is a main food source for local residents. The cassava has low requirements on soil and fertilizers, and the main limitation on the production of the cassava is adversity and pest and disease damage. The breeding of a plurality of novel cassava varieties with good resistance is an ideal method for solving the problems, but the traditional breeding method is difficult to implement in the cassava variety breeding process due to the poor characteristics of cassava germplasm resources, the complexity of genetic background, low pollen fertility, difficult regulation and control of flowering phase, difficult pairing of target combination and the like. Therefore, the breeding goal can only be achieved by establishing a cassava regeneration system and realizing the basis of the introduction of related genes.

Disclosure of Invention

The invention aims to provide a method for establishing a cassava regeneration system, which can lay a theoretic theory and a technical foundation for introducing target genes in the cassava variety improvement process.

To achieve these objects and other advantages and in accordance with the purpose of the invention, as embodied and broadly described herein, there is provided a method for establishing a cassava regeneration system, comprising: the method comprises the following steps of firstly, obtaining a cassava stem section with axillary buds, and carrying out primary culture and secondary multiplication culture by utilizing a first culture medium and a second culture medium until sterile leaves grow out, wherein the first culture medium comprises an MS culture medium and 6-BA, and the second culture medium comprises 6-BA and NAA; step two, taking the sterile leaves obtained in the step one, and performing callus culture by using a third culture medium, wherein the third culture medium comprises an MS culture medium, Picloram and CuSO₄(ii) a Step three, taking the callus obtained in the step two, and inducing differentiation and sprouting by utilizing a fourth culture medium, wherein the fourth culture medium comprises an MS culture medium, 6-BA and AgNO₃。

Further, still include: before the primary culture, the axillary bud stem is sterilized, wherein the sterilization comprises the sequential use of 75% alcohol and 0.1% HgCl₂And (6) processing.

Further, the concentration of 6-BA in the first culture medium is 1.5mg/L, and the concentration of 6-BA and NAA in the second culture medium is 0.5mg/L and 0.05mg/L respectively.

Further, in the second step, after dark culture for 7 days, light culture is performed for 40 days, and Picloram and CuSO are added in the third medium₄Respectively at a concentration of 11.0mg/LAnd 0.2 mg/L.

Further, in the third step, after dark culture for 5 days, illumination culture is carried out for 40 days, and 6-BA and AgNO are contained in the fourth culture medium₃The concentrations of (A) were 0.1mg/L and 5.0mg/L, respectively.

Further, the fourth medium further comprises activated carbon and coconut water; the preparation method of the activated carbon comprises the following steps: cutting coconut shells, soaking in sodium hydroxide solution, filtering, washing with water, carbonizing in a muffle furnace, cooling, soaking in ethanol, adding zinc oxide, irradiating with ultrasonic wave, filtering, and drying to obtain the active carbon.

Further, in the first step, the second step, the third step and the fourth step, the temperature is kept at 25-28 ℃, and the illumination culture conditions comprise illumination time of 12-14 h/d and illumination intensity of 1500-2000 lx.

Further, the pH of the first culture medium, the second culture medium, the third culture medium and the fourth culture medium is 5.3-6.3.

The invention at least comprises the following beneficial effects:

the method comprises the steps of culturing the stem segments with the axillary buds by using a first culture medium and a second culture medium to obtain sterile leaves, and then culturing the callus by using a third culture medium and a fourth culture medium, wherein the induction rate of the callus can reach 100%, and the bud differentiation rate of the callus can reach more than 35%.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

Example 1:

the establishment method of the cassava regeneration system comprises the following steps: the method comprises the following steps of firstly, obtaining a cassava stem section with axillary buds, and carrying out primary culture and secondary multiplication culture by utilizing a first culture medium and a second culture medium until sterile leaves grow out, wherein the first culture medium comprises an MS culture medium and 6-BA, and the second culture medium comprises 6-BA and NAA; step two, taking the sterile leaf obtained in the step one, cutting the sterile leaf into small blocks with the side length of 3-5 mm, and performing callus culture by using a third culture medium, wherein the third culture medium comprises an MS culture medium, Picloram and CuSO₄(ii) a Step three, taking the callus obtained in the step two, and inducing differentiation and sprouting by utilizing a fourth culture medium, wherein the fourth culture medium comprises an MS culture medium, 6-BA and AgNO₃。

Before the primary culture, the axillary bud stem is sterilized, wherein the sterilization comprises the sequential use of 75% alcohol and 0.1% HgCl₂The treatment was for 30 seconds and 5 min. The concentration of 6-BA in the first culture medium is 1.5mg/L, and the concentration of 6-BA and NAA in the second culture medium are 0.5mg/L and 0.05mg/L respectively. In the second step, after dark culture for 7 days, illumination culture is carried out for 40 days, and Picloram and CuSO are added in the third culture medium₄The concentrations of (A) were 11.0mg/L and 0.2mg/L, respectively. In the third step, after dark culture for 5 days, illumination culture is carried out for 40 days, and 6-BA and AgNO are added in the fourth culture medium₃The concentrations of (A) were 0.1mg/L and 5.0mg/L, respectively. The fourth culture medium also comprises activated carbon and coconut milk, the dosage of the activated carbon is 0.3 percent of the mass of the fourth culture medium, and the volume ratio of the coconut milk to the fourth culture medium is 1: 120; the preparation method of the activated carbon comprises the following steps: taking coconut shells, chopping, soaking in a sodium hydroxide solution, filtering, washing with water, putting into a muffle furnace for carbonization at the carbonization temperature of 250-300 ℃, cooling, soaking in ethanol, adding zinc oxide, irradiating with ultrasonic waves, filtering, and drying to obtain the activated carbon, wherein the zinc oxide accounts for 10% of the total mass of the activated carbon. In the first step, the second step, the third step and the fourth step, the temperature is kept at 26 ℃, and the conditions of the illumination culture comprise the illumination time of 12h/d and the illumination intensity of 1800 lx. The first, second, third, and fourth media have a pH of 5.8.

Comparative example 1:

replacing Picloram with CTK and NAA, the rest parameters are completely the same as in example 1, and the process is also completely the same.

Comparative example 2:

the activated carbon was replaced with commercially available activated carbon, and the remaining parameters were exactly the same as in example 1, and the process was also exactly the same.

And (3) testing:

cassava axillary bud stems were treated using the methods of example 1, comparative example 1 and comparative example 2, respectively, with at least 30 groups tested per example or comparative example. And (3) counting the callus induction rate and callus differentiation rate, wherein the counting results are respectively shown in tables 1 and 2. The callus induction rate is the total number of the small pieces/small pieces forming the callus × 100%, and the callus differentiation rate is the total number of the differentiated callus/callus × 100%.

TABLE 1 callus induction results

TABLE 2 callus differentiation results

	Number of calli	Number of differentiated calli	Differentiation rate of callus
				Example 1	35 blocks	13 blocks	37％
Comparative example 1	32 blocks	0 block	0％
				Comparative example 2	31 blocks (B)	6 blocks	19％

As can be seen from tables 1 and 2, the callus induction rate of the application can reach 100%, the callus differentiation rate can reach 37%, and a foundation can be laid for the cassava variety improvement process, for example, when a target gene is transferred into cassava cells, subsequent tissue culture is performed. Replacing Picloram with CTK and NAA does not have great influence on the callus induction rate, but the generated callus is hardly differentiated, and after the activated carbon of the application is replaced by the commercial activated carbon, the callus differentiation rate is greatly reduced, so that the activated carbon of the application can provide better differentiation conditions for the callus.

The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. The application, modification and variation of the method for establishing the cassava regeneration system of the present invention will be apparent to those skilled in the art.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is therefore not limited to the details given herein and to the embodiments shown and described without departing from the generic concept as defined by the claims and their equivalents.