阅读说明：本技术 一种适宜四倍体大白菜游离小孢子培养的花蕾筛选方法和小孢子提取方法 (Bud screening method and microspore extraction method suitable for tetraploid Chinese cabbage free microspore culture ) 是由 刘晓东 王明秋 孟川 吴芳 王玉海 牟金贵 陈占良 马蕾 于 2021-10-20 设计创作，主要内容包括：本发明涉及大白菜游离小孢子培养技术领域,公开了一种适宜四倍体大白菜游离小孢子培养的花蕾筛选方法和四倍体大白菜游离小孢子的提取方法。取四倍体大白菜花序由外向内的第三圈花蕾,其花瓣长度和花药长度之比小于等于1/2,该方法得到的花蕾进行游离小孢子的提取,小孢子处于单核靠边期,适宜四倍体大白菜游离小孢子培养,能够为四倍体大白菜游离小孢子再生植株的培育提供技术基础。(The invention relates to the technical field of free microspore culture of Chinese cabbage, and discloses a bud screening method suitable for free microspore culture of tetraploid Chinese cabbage and an extraction method of free microspore of tetraploid Chinese cabbage. Taking a third round of buds of the tetraploid Chinese cabbage from outside to inside in inflorescence, wherein the ratio of the length of petals to the length of anthers is less than or equal to 1/2, extracting free microspores from the buds obtained by the method, wherein the microspores are in a mononuclear border period, and the method is suitable for culturing the free microspores of the tetraploid Chinese cabbage and can provide a technical basis for culturing regeneration plants of the free microspores of the tetraploid Chinese cabbage.)

1. A method for screening buds suitable for culturing free microspores of tetraploid Chinese cabbage is characterized in that the buds of tetraploid Chinese cabbage are taken, the sepals of the buds are stripped, the lengths of petals and anthers of the buds are respectively measured, and the following conditions are met:

wherein L1 is the length of the petals, and L is the length of the anthers.

2. The method for screening flower buds according to claim 1, wherein the flower buds are the third circle flower buds from the outside to the inside of a tetraploid Chinese cabbage inflorescence.

3. The method of claim 1, wherein the tetraploid chinese cabbage buds are sampled at 9-11 am when the outer skin of the buds is dry.

4. The method of claim 1, wherein the tetraploid chinese cabbage flower buds are 1.5-2mm in length.

5. A method for extracting free microspores from a flower bud obtained by the method of any one of claims 1 to 4, comprising the steps of: putting the tetraploid Chinese cabbage flower buds into a culture solution, extruding the flower buds to enable microspores to be fully dissociated into the culture solution, filtering through a 300-mesh filter screen, centrifuging the obtained microspore extracting solution for 3-5 minutes at 1000-1200 revolutions, and obtaining precipitates, namely the tetraploid Chinese cabbage dissociative microspores.

6. The method for extracting free microspores according to claim 5, wherein the culture solution is B5 medium.

7. The method for extracting free microspores of claim 5 wherein the precipitate is centrifuged by washing with B5 medium.

8. The method for extracting free microspores of claim 5, wherein the tetraploid Chinese cabbage free microspores are suspended in the NLN liquid medium.

9. The method for extracting free microspores of claim 8, wherein the suspension of tetraploid Chinese cabbage free microspores is dispensed into 6cm diameter petri dishes with 5ml of liquid per dish.

10. The method for extracting free microspores as claimed in claim 5, wherein the tetraploid Chinese cabbage buds are sterilized by soaking in 0.1% mercuric chloride solution and washed with sterile water.

Technical Field

The invention relates to the technical field of free microspore culture of Chinese cabbage, in particular to a bud screening method suitable for free microspore culture of tetraploid Chinese cabbage and an extraction method of free microspore of tetraploid Chinese cabbage.

Background

The isolated microspore culture and plant regeneration of Chinese cabbage have many influencing factors, and the induction culture promotes the embryo emergence of most genotype materials or embryo emergence materials to improve the embryo emergence rate, so that a specific culture technology is needed, wherein the bud selection standard for isolated microspore culture is one of the first technologies. The development of pollen microspores of cruciferous crops is divided into a tetrad period, a mononuclear period, a binuclear period and a trinuclear period, the microspores in any period are not suitable for culture, and the mononuclear period can be subdivided into a mononuclear early period, a mononuclear middle period and a mononuclear border period; the sensitivity of pollen microspores at different stages to external stimuli is different, so that the microspores induce embryos with different results.

Microspores suitable for culture of different plants are at different stages of development. The length of the flower bud of the Chinese cabbage selected by Sato et al successfully culturing the isolated microspore of the Chinese cabbage for the first time is 2.0 mm-2.5 mm, most microspores in the flower bud with the length are in the mononuclear border stage, so the study considers that the microspores in the mononuclear border stage are most suitable for culturing the isolated microspores. Screening and extracting the microspores at the edge stage of the mononuclear, wherein the selection of the flower buds is the key. The traditional flower bud selection method only depends on microscope observation to select the flower buds, and because the size range of the flower buds is wide, time and labor are wasted, and the efficiency of extracting free microspores is low.

There are three methods for separating microspores, one is a floating culture method, i.e. the anther is inoculated on a liquid culture medium, pollen in the anther is allowed to be freely released, and then centrifugal culture is carried out; the other is a magnetic stirring method, namely, the anther is put into a triangular flask containing a certain amount of culture medium or penetrant, is placed on a magnetic stirring instrument, rotates at a low speed to enable microspores to gradually overflow along with stirring until the anther is transparent, and is centrifuged and cultured; third, the extrusion method, which is the earliest way of pollen isolation. Currently, the third method is used by most researchers in brassica plants. However, at present, research on the culture of isolated microspores (n is 10) of Chinese cabbage is carried out by multiple units at home and abroad, the extraction and culture method of the associated diploid Chinese cabbage isolated microspores is clarified, and no relevant report is made on the extraction method of the tetraploid Chinese cabbage isolated microspores.

Disclosure of Invention

Therefore, the invention provides a bud screening method suitable for culturing the tetraploid Chinese cabbage free microspore and an extraction method of the tetraploid Chinese cabbage free microspore, and aims to provide a morphological standard and normative free microspore extraction method and a basis for establishing a technical system for accurately culturing and selecting the buds of the tetraploid Chinese cabbage free microspore.

The technical scheme of the invention is as follows:

the invention provides a bud screening method suitable for tetraploid Chinese cabbage free microspore culture, which comprises the steps of taking tetraploid Chinese cabbage buds, stripping off bud sepals, respectively measuring the length of petals and the length of anthers of the buds, and meeting the following conditions:

wherein L1 is the length of the petals, and L is the length of the anthers.

Preferably, the flower bud is the third circle of flower buds of the tetraploid Chinese cabbage inflorescence from outside to inside.

Preferably, the sampling time of the tetraploid Chinese cabbage bud is 9-11 am, and the bud outer skin is dried.

Preferably, the sampling is performed during flowering of the Chinese cabbage from late 3 months to early 5 months.

Preferably, the length of the tetraploid Chinese cabbage bud is 1.5-2 mm.

Another object of the present invention is to provide a method for extracting free microspores from the flower buds obtained by the above method, comprising the steps of: sterilizing tetraploid Chinese cabbage bud, placing in B5 culture medium, squeezing bud to make microspore fully dissociate in B5 culture medium, filtering with nylon gauze, centrifuging the obtained microspore extract with low-speed centrifuge, removing supernatant, and collecting precipitate as tetraploid Chinese cabbage dissociative microspore.

Preferably, the disinfectant is 0.1% mercuric chloride (HgCl)₂) And (3) solution.

Preferably, the consumption of the extruded bud B5 culture medium is 5-8ml, and the specification of a nylon filter screen is 300 meshes.

Preferably, the filtrate is centrifuged at 1000 rpm for 5 minutes or 1200 rpm for 3 minutes, and the washing and centrifugation of the B5 medium are repeated.

Preferably, the tetraploid Chinese cabbage free microspore is added with NLN culture medium to prepare suspension, and further preferably 10ml of NLN culture medium is added.

Further, the suspension of the tetraploid Chinese cabbage free microspore is subpackaged in culture dishes with the diameter of 6cm, and each dish is 5ml of liquid. As an embodiment, the free microspores are suspended in 10ml of NLN liquid culture medium and then are distributed into 10 culture dishes with the diameter of 6cm, 1ml of suspension is added in each dish, and 4ml of NLN culture medium is added in each dish.

The invention has the beneficial effects that:

the invention provides a bud screening method suitable for tetraploid Chinese cabbage free microspore culture, which can provide morphological standard for the selection of a bud for tetraploid Chinese cabbage free microspore culture and improve the selection efficiency of the bud of tetraploid Chinese cabbage free microspore by researching the corresponding relation between the length of petals and anthers of tetraploid Chinese cabbage and the development period of microspores. Meanwhile, the invention also correspondingly provides a method for extracting the free microspore from the flower bud, and provides a technical basis for cultivating the regeneration plant of the tetraploid Chinese cabbage free microspore.

Drawings

FIG. 1: anatomical structure chart of Chinese cabbage bud;

FIG. 2: sampling position map of appearance shape of Chinese cabbage bud;

FIG. 3: identifying the morphology of the isolated microspores of the Chinese cabbage by a microscope;

FIG. 4: the effect picture of filtering the Chinese cabbage free microspores by adopting nylon gauzes with different specifications.

Detailed Description

The invention develops the cultivation research of tetraploid Chinese cabbage free microspore (n is 20) by using tetraploid Chinese cabbage resource as test material, explores the corresponding cultivation technical method and provides a tetraploid Chinese cabbage free microspore cultivation technical system different from diploid Chinese cabbage free microspore cultivation.

The invention screens buds suitable for culturing tetraploid Chinese cabbage free microspore, which comprises the following steps: taking the flower bud of the tetraploid Chinese cabbage, stripping off the sepals of the flower bud, respectively measuring the length of the petal of the flower bud and the length of the anther, and screening the flower bud which is suitable for culturing the free microspore of the tetraploid Chinese cabbage from the anatomical structure of the flower bud. FIG. 1 is a view showing the inside of a flower bud after sepals are removed, wherein L1 is the length of petals and L is the length of anthers. The optimal bud of the diploid isUnlike diploids, tetraploids sample optimal buds

The period suitable for culturing the tetraploid Chinese cabbage free microspore is the same as that of the diploid, the period is a mononuclear side-by-side period, but the appearance form sampling part is completely different. FIG. 2 is a sampling position diagram of the appearance of Chinese cabbage flower buds. Taking the buds of the diploid generally takes the buds of the second circle from the outside to the inside of inflorescence, and taking the buds of the tetraploid needs to take the buds of the third circle from the outside to the inside of inflorescence. The result proves that the flower bud suitable for culturing the free microspore of the tetraploid Chinese cabbage is smaller than that of the diploid, and the length is usually 1.5-2 mm.

As an alternative embodiment, the Chinese cabbage flower bud of the present invention is sampled during the flowering period of the Chinese cabbage from 3 late months to 5 early months. Preferably, the sampling is carried out at 9-11 am, when the outer skin of the flower buds is dry.

The invention also provides a method for extracting tetraploid free microspore from flower buds, which comprises the steps of extruding flower buds, centrifuging to extract microspore, specifically, putting a proper amount of flower buds into culture, extruding flower buds to enable the microspore to be fully dissociated into a culture medium, filtering by a filter screen, centrifuging, and obtaining precipitate, namely the Chinese cabbage free microspore. Tetraploid requires 300 mesh nylon gauze filtration because of the large microsporoid volume, which is different from the 400 mesh requirement of diploid. The extraction of the tetraploid free microspores requires that the centrifuge rotates at 1000-1200 revolutions for 3-5 minutes, optionally 1000 revolutions/5 minutes or 1200 revolutions/3 minutes, which is different from 800 revolutions/5 minutes required by diploids. Other steps, diploid and tetraploid microspores may be used in the same manner.

As an implementation mode, the number of the flower buds can be 30-35. The buds are collected by the method. The picked flower buds can be disinfected and then extracted with free microspores. Preferably, 0.1% mercuric chloride (HgCl) is added into picked flower bud of Chinese cabbage₂) Surface sterilization and sterile water washing.

In one embodiment, the culture medium is B5 culture medium. And washing the precipitate obtained by filtering with a B5 culture medium for 2-3 times.

As an embodiment, the obtained yellow precipitate, namely Chinese cabbage free microspore, is suspended in NLN liquid culture medium. The density of microspores in the spore suspension is adjusted by using a blood counting plate, preferably 0.5-1X 10⁵One per ml.

In one embodiment, the invention suspends the free microspores of 30-35 buds in 10ml of NLN liquid culture medium and shakes the microspores evenly. And (3) subpackaging the extracted free microspore suspension into 6cm culture dishes for culture, wherein each dish contains 1ml of microspore suspension, and then adding 4ml of NLN liquid culture medium to ensure that each dish contains 5ml of liquid. The existing research reports that 2ml of suspension liquid is used for culturing diploid microspores in each dish to prevent the microspores from drowning; it was also studied to put 10ml of the suspension to prevent the liquid from evaporating dry. But different from diploid, the test result of the invention shows that 2ml can be evaporated and dried to cause the tetraploid microspore to be died by drought, and 10ml is adopted to cause overflow easily and cause serious pollution when the culture medium is transferred too much, so that the invention selects 5ml of liquid per dish to culture the tetraploid free microspore optimally.

The method for extracting the tetraploid isolated microspores can provide a technical basis for culturing the regeneration plants of the tetraploid Chinese cabbage isolated microspores.

The present invention will be described in detail with reference to examples for better understanding the objects, technical solutions and advantages of the present invention, but they should not be construed as limiting the scope of the present invention.

The B5 medium, NLN liquid medium, used in the following examples was a medium conventionally known in the art and prepared according to a conventional method in the art.

Example 1

Screening buds suitable for tetraploid free microspores

Sampling is carried out when the outer surface of the flower buds are dry, at 9-11 am during the flowering period of the Chinese cabbage from 3 late month to 5 early months.

Taking inflorescence of tetraploid Chinese cabbage, and taking buds in the third circle from outside to inside. Dissecting the picked flower bud, stripping off the sepals of the flower bud, respectively measuring the length of the petals and the length of the anther of the flower bud, and taking the flower bud which meets the following conditions as the flower bud suitable for culturing the free microspore of the tetraploid Chinese cabbage:

wherein L1 is the length of the petals, and L is the length of the anthers.

Comparative example 1

During the flowering period of the Chinese cabbage from 3 late month to 5 early months, the flower buds are sampled when no dew exists on the inflorescence after 9 am. Taking inflorescence of tetraploid Chinese cabbage, and taking the second round of buds from outside to inside.

Example 2

Extraction of tetraploid free microspores

Disinfection and preservation of the culture medium: sterilizing B5 culture medium in autoclave at 120 deg.C for 20min, cooling, and storing at room temperature; the NLN liquid culture medium is sterilized by a disc filter on a super clean bench by using an autoclave, filtered and sterilized by a mixed fiber micro-filtration membrane with the aperture of 0.22 mu m and the diameter of 15cm, and stored in a refrigerator at 4 ℃.

Flower buds were picked, cracked and damaged buds removed as described in example 1 and treated with 0.1% mercuric chloride (HgCl)₂) Sterilizing the surface for 5min, and washing with sterile water for 3 times.

Placing 30-35 buds into a 15ml test tube, adding 5-8ml of high-temperature sterilized B5 culture medium, and lightly squeezing the buds with a sterilized glass rod to make the microspores fully free into the solution. The suspension was collected in a centrifuge tube, centrifuged at 1000 rpm/5 min or 1200 rpm/3 min, and the supernatant was discarded. Additional B5 medium was added and the mixture was washed 2 times for centrifugation and the supernatant was decanted to leave only a yellow precipitate (microspores). Adding 10ml NLN liquid culture medium, and shaking to obtain spore suspension with microspore density of 0.5-1 × 10⁵One per ml. The spore suspension was divided into 10 petri dishes of 6cm diameter, 1ml each, then 4ml NLN medium was added, ensuring 5ml of liquid per dish, and sealed with Parafilm.

Example 3

Cytological examination of isolated microspores

During microspore exsomatization, and early embryogenesis

Tetraploid Chinese cabbage flower buds were extracted according to the method of example 1 and comparative example 1, free microspores were extracted according to the method of example 2, stained with 0.3% magenta stain, and then the morphological change and cell division of the microspores were observed with an inverted microscope. Microspores divide into 3 lobes for an optimal period, with more than 4 lobes expired and less than 2 lobes unexpired. As shown in fig. 3: the left panel shows microspores extracted in comparative example 1, in which only 2 suitable microspores are present, indicating that the bud as a whole has expired; the right panel shows microspores extracted in example 1, and more than half of the microspores are in 3-petal stage, indicating that the buds are the best buds.

The method can screen the flower buds suitable for culturing the tetraploid Chinese cabbage free microspores, is simple and convenient, and improves the screening efficiency of the tetraploid Chinese cabbage free microspore flower buds.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.