阅读说明：本技术 一种利用龙须菜畸形芽选育种质的方法 (Method for breeding germplasm by using deformed buds of asparagus ) 是由 庞通 刘建国 薛俊瑞 于 2020-04-03 设计创作，主要内容包括：本发明公开了一种利用龙须菜畸形芽选育种质的方法,包括如下过程：采集龙须菜上生长的畸形芽,将畸形芽经机械破碎后放于海水培养基中进行培养,畸形芽自身形成脱分化的瘤状组织,瘤状组织自发分化为盘状体并形成幼苗。本发明所公开的方法可以方便的应用于龙须菜种质规模品系化育种、基因工程改造,诱导突变等需要种质纯系培养的育种方式,大大降低育种中的困难程度,缩短育种时间。(The invention discloses a method for breeding germplasm by utilizing deformed buds of asparagus, which comprises the following steps: the method comprises the steps of collecting deformed buds growing on asparagus, mechanically crushing the deformed buds, putting the crushed deformed buds into a seawater culture medium for culture, forming dedifferentiated nodular tissues by the deformed buds, and spontaneously differentiating the nodular tissues into discoid bodies to form seedlings. The method disclosed by the invention can be conveniently applied to breeding modes requiring quality pure line culture, such as asparagus germplasm scale strain breeding, genetic engineering transformation, induced mutation and the like, so that the difficulty degree in breeding is greatly reduced, and the breeding time is shortened.)

1. A method for breeding germplasm by using deformed buds of asparagus is characterized by comprising the following steps: the method comprises the steps of collecting deformed buds growing on asparagus, mechanically crushing the deformed buds, putting the crushed deformed buds into a seawater culture medium for culture, forming dedifferentiated nodular tissues by the deformed buds, and spontaneously differentiating the nodular tissues into discoid bodies to form seedlings.

2. The method for breeding germplasm of Gracilaria lemaneiformis according to claim 1, wherein the malformed sprouts are in a curled shape.

3. The method for breeding germplasm by utilizing deformed sprouts of asparagus as claimed in claim 1, wherein inorganic nitrogen and phosphorus, trace elements and antibiotics are added into the seawater culture medium.

4. The method for breeding germplasm using deformed sprouts of asparagus as claimed in claim 1, wherein the culture is carried out at 10-30 ℃ under 0-1000 μmol m illumination^-2s^-1Within the range ofThe culture is carried out.

5. The method for breeding germplasm by utilizing deformed sprouts of asparagus as claimed in claim 1, wherein the seawater culture medium comprises the following components in percentage by mass: 75mg/L of sodium nitrate, 5mg/L of monobasic sodium phosphate monohydrate, 3.16mg/L of ferric chloride hexahydrate, 4.36mg/L of disodium ethylenediamine tetraacetic acid, 0.01mg/L of copper sulfate pentahydrate, 0.23mg/L of zinc sulfate heptahydrate, 0.012mg/L of calcium chloride hexahydrate, 0.18mg/L of manganese chloride tetrahydrate, 0.07mg/L of sodium molybdate dihydrate, 10.1mg/L of vitamin B, 120.5 mu g/L of vitamin B, 0.5 mu g/L of biotin and 50mg/L of ampicillin potassium.

6. The method for breeding germplasm of Gracilaria lemaneiformis according to claim 1, wherein the salinity of the seawater culture medium is 32.

7. The method for breeding germplasm using deformed asparagus sprouts as claimed in claim 1, wherein the deformed sprouts are crushed into 1-2mm algae tissue.

8. The method for breeding germplasm using deformed asparagus sprouts as claimed in claim 7, wherein the proportion of deformed algae tissues in the seawater culture medium is 0.02 g/mL.

9. The method for breeding germplasm using deformed asparagus buds as claimed in any one of claims 1-8, wherein the deformed asparagus buds and seawater culture medium are placed in a culture dish, then placed in a light incubator, placed in the dark at 20 ℃ for 24 hours, and then placed in the light for 10 μmol m^-2s^-1The illumination time is 12 hours every day, and the water is changed every 2 weeks until the seedlings grow.

Technical Field

The invention relates to a asparagus breeding method, in particular to a method for breeding germplasm by using deformed buds of asparagus.

Background

The asparagus is the main bait of abalone and the main raw material for producing agar, and after the 981 variety stock is cultivated in the last 80 th century, the asparagus is cultivated in a nutritional reproduction mode for a long time. Most of the cultivated algae are originated from stock cultured for 20 years ago, and the part of algae is subcultured for thousands of generations from the stock, and is likely to become the old head vegetable. The asparagus can be subjected to vegetative propagation, but if the strain breeding is to be realized, diversified pure line seedlings are obtained by combining dedifferentiation and redifferentiation cultivation of callus/quarternary spores/fruit spores and pressure selection of environmental conditions, so that the method is a very effective and rapid way, but the asparagus callus is not reported in research, the amount of the quarternary spores and the number of the fruit spores are small, and the spore maturation is limited by seasons, so the strain breeding is still difficult to realize.

The current life history of asparagus is considered as shown in fig. 1, and the propagation mode of dedifferentiation and redifferentiation of tissue cells of asparagus in nature is only found in the formation and morphogenesis of quartemary spores and the formation and morphogenesis of fruit spore fertilized eggs. The normal shape of asparagus is shown in fig. 3, the normal bud is in the shape of a strip, while the abnormal bud of asparagus is shown in fig. 4, and the abnormal bud is in the shape of a roll.

Patent 201310243062.0 found through literature query shows that cultured quarterwort seedlings were obtained by using cultured quarterwort spores, but the method is to culture the seedlings by using the quarterwort germ cells to the gametophyte life history part, the quarterwort cultured seedlings can be used only in the season of waiting for spore maturation, the number of the spores of the asparagus is small, and the seedlings are not convenient to obtain somatic cells. In the patent 200410050305.X and 201510983558.0, Gracilaria lemaneiformis sections are used for tissue culture vegetative propagation, the life history part of vegetative propagation of quarterbantis in the life history is used for cultivating seedlings, and the propagation mode can realize rapid propagation but is difficult to be used for breeding. The rapid propagation is equivalent to that one maternal cell is not subjected to the continuous replication of embryonic cells, the first is likely to have replicative senescence, the second is not subjected to dedifferentiation and redifferentiation processes, and when the bioengineering operation is carried out, the embryogenic callus can not be obtained to obtain the variety pure line of seedlings, so that the opportunity of obtaining the characters required by people is reduced, and the time cost of breeding is increased.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for breeding germplasm by using deformed buds of asparagus, which can be conveniently applied to breeding modes requiring pure line culture of germplasm such as mass strain breeding, genetic engineering transformation, induced mutation and the like of the asparagus, greatly reduces the difficulty degree in breeding and shortens the breeding time.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for breeding germplasm by using deformed buds of asparagus comprises the following steps: the method comprises the steps of collecting deformed buds growing on asparagus, mechanically crushing the deformed buds, putting the crushed deformed buds into a seawater culture medium for culture, forming dedifferentiated nodular tissues by the deformed buds, and spontaneously differentiating the nodular tissues into discoid bodies to form seedlings.

In the above scheme, the malformed bud is curled.

In the scheme, inorganic nitrogen and phosphorus, trace elements and antibiotics are added into the seawater culture medium.

In the above scheme, the culture is carried out at 10-30 deg.C under illumination of 0-1000 μmol m^-2s^-1Culturing is carried out within the range.

Preferably, the seawater culture medium contains the following components in percentage by mass: 75mg/L of sodium nitrate, 5mg/L of monobasic sodium phosphate monohydrate, 3.16mg/L of ferric chloride hexahydrate, 4.36mg/L of disodium ethylenediamine tetraacetic acid, 0.01mg/L of copper sulfate pentahydrate, 0.23mg/L of zinc sulfate heptahydrate, 0.012mg/L of calcium chloride hexahydrate, 0.18mg/L of manganese chloride tetrahydrate, 0.07mg/L of sodium molybdate dihydrate, 10.1mg/L of vitamin B, 120.5 mu g/L of vitamin B, 0.5 mu g/L of biotin and 50mg/L of ampicillin potassium.

Preferably, the seawater culture medium has a salinity of 32.

In the above scheme, the malformed bud is crushed into 1-2mm algal tissue.

Preferably, the proportion of the malus bud algae tissue in the seawater culture medium is 0.02 g/mL.

Preferably, the malformed bud and the seawater culture medium are placed in a culture dish, then placed in a light incubator, placed in a dark place at 20 ℃ for 24 hours, and then placed in light for 10 mu mol m^-2s^-1The illumination time is 12 hours every day, and the water is changed every 2 weeks until the seedlings grow.

Through the technical scheme, the method for breeding germplasm by using deformed asparagus buds provided by the invention forms dedifferentiated nodular tissues by using the coiled deformed buds growing on the four-quarter sporophytes of the asparagus, and the nodular tissues are spontaneously differentiated into discoids to form seedlings.

The seedlings differentiated by the embryonal nodular tissues are pure-line diversified seedlings, and more opportunities can be provided for obtaining the required characters during pressure selection, so that dedifferentiation and redifferentiation cells can be conveniently and simply obtained, and the method is expected to be conveniently applied to breeding modes requiring germplasm pure-line culture, such as asparagus germplasm strain breeding, genetic engineering transformation, induced mutation and the like, greatly reduces the difficulty degree in breeding, and shortens the breeding time. The mode is expected to inspire the seedling growing direction of other economic seaweeds.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the conventional propagation method of Gracilaria lemaneiformis;

FIG. 2 shows the propagation method of Gracilaria lemaneiformis disclosed in the embodiment of the present invention;

FIG. 3 is a normal form of Gracilaria lemaneiformis;

FIG. 4 shows Gracilaria lemaneiformis with full and abnormal bud.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

As shown in FIG. 2, the invention provides a method for breeding germplasm by using deformed buds of asparagus, which comprises the following specific embodiments:

preparing a seawater culture medium with 75mg/L of sodium nitrate, 5mg/L of monobasic sodium phosphate monohydrate, 3.16mg/L of ferric chloride hexahydrate, 4.36mg/L of disodium ethylene diamine tetraacetate, 0.01mg/L of copper sulfate pentahydrate, 0.23mg/L of zinc sulfate heptahydrate, 0.012mg/L of calcium chloride hexahydrate, 0.18mg/L of manganese chloride tetrahydrate, 0.07mg/L of sodium molybdate dihydrate, 0.1mg/L of vitamin B1, 0.5 mu g/L of vitamin B12, 0.5 mu g/L of biotin and 32 of salinity, boiling, sterilizing and cooling to room temperature, and then adding 50mg/L of ampicillin potassium.

Collecting thallus Gracilariae with abnormal bud structure, washing with sterilized seawater, crushing into 1-2mm size algae tissue, and straightening9 ml of culture medium is added into a culture dish with the diameter of 6 cm, and the crushed algae are placed into the prepared culture medium according to the proportion of 0.02 g/ml. Then placing the culture medium in a light incubator at 20 deg.C in dark for 24 hr, and placing in a light of 10 μmol m^-2s^-1Under the environment of (1), the seedlings with the length of about 2mm can be obtained by illuminating for 12 hours every day and changing water every 2 weeks and culturing for 2 months.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.