CN111084097A

CN111084097A - Method for hybridizing quinoa tissue culture seedlings under aseptic condition

Info

Publication number: CN111084097A
Application number: CN202010001441.9A
Authority: CN
Inventors: 林春; 刘正杰; 毛自朝; 董玉梅; 李玉平; 张玉敏
Original assignee: Yunnan Agricultural University
Current assignee: Yunnan Agricultural University
Priority date: 2020-01-02
Filing date: 2020-01-02
Publication date: 2020-05-01

Abstract

The invention relates to a method for hybridizing by utilizing quinoa tissue culture seedlings under aseptic conditions, which belongs to the technical field of quinoa hybridization breeding and comprises the following steps of 1) tissue culture and rapid propagation of quinoa; 2) hybridizing; 3) and (5) detecting the hybrid seeds. The invention adopts a tissue culture hybridization mode, obtains hybrid seeds in about 60 days by adjusting nutrition and illumination time, greatly shortens the growth period of quinoa hybridization, thereby achieving the purpose of rapid hybridization or selfing propagation of offspring and saving time and cost for breeding work. Moreover, the method is not limited by external environment, can be operated all the year round, and overcomes the problems of difficult emasculation, difficult hybridization of field hybridization chenopodium quinoa flowers, and the dissimilarity of the flowering phases of two chenopodium quinoa plants with different growth periods. The tissue culture bottle is a closed environment, so that pollen of other quinoa varieties can be reduced from falling onto the column head, the outcrossing rate is reduced, and purer hybrid seeds can be obtained.

Description

Method for hybridizing quinoa tissue culture seedlings under aseptic condition

Technical Field

The invention belongs to the technical field of quinoa hybridization breeding, and particularly relates to a method for hybridizing quinoa tissue culture seedlings under aseptic conditions.

Background

Chenopodium quinoa willd (Chenopodium quinoa willd)C. quinoaWilld, 2n = 36) genus Amaranthaceae (amaranthhaceae) quinoa (Chenopodioideae) genus chenopodium (Chenopodioideae) ((Willd, 2n = 36)Chenopodium) Annual herbaceous plants, the seeds of which are used as foodstuffs, are pseudocereals. The method is screened as a staple grain crop by native residents in the Tibet kark lake region of south America about 5000 years before the Yuan, is called as a 'mother of grain', and breeds the Yijia civilization. The invasion and the control of countries such as Spain in the 16 th century to south America emphasize that wheat is used as staple food, the production of quinoa is weakened, a large amount of domesticated germplasm resources are lost, the health level of local residents is reduced, and people can realize the excellent nutrition and health care value of quinoa again. In the 70's of the 20 th century, quinoa was selected as a space staple food by the American aerospace office, and dieticians called "nutrient gold" and "super grain". Quinoa is currently generally considered as an edible crop with the best life activities which can be maintained by a single crop. The grains contain nutrients such as protein, essential amino acid and the like which are superior to traditional crops such as rice, wheat and the like, and health-care components such as vitamins, terpenes, flavonoids and the like, wherein the content of starch is 58.1-64.2%, and the content of amylopectin is low (8-20%); the protein content is 12% -23%, and the gluten-free food does not contain sensitized gluten or gluten, so that the gluten-free food is the first choice staple food for most people sensitive to gluten. The grains have rich amino acid content and balanced composition, the content of the essential amino acids of human bodies such as lysine, tryptophan, histidine, methionine, phenylalanine and the like is higher than that of common cereal crops, and the content of the histidine and the tryptophan is even higher than that of soybeans. The kernel has a lipid content of about 9.5% and is mostly unsaturated fatty acid. In addition, the grains are rich in mineral elements such as calcium, magnesium, potassium, iron, copper, zinc, manganese and the like, and rich in V_C、V_E、V_B1、V_B2、V_B3、V_B6And vitamins such as folic acid; in addition, the kernel also contains dietary fiber comparable to rye, and the content of the dietary fiber is about 3.4 percent and is higher than that of rice (0.4 percent), wheat (2.7 percent) and corn (1.7 percent). Therefore, the quinoa can ensure the nutrition supply necessary for human and has the effect of promoting the health of human body.

Quinoa is a natural food source containing antioxidant phenolic acid compounds such as quercetin, kaempferol, myricetin and the like except buckwheat and barley, and the phenolic acid compounds are mainly distributed in seed coats, have slight bitterness and astringency, and can reduce the incidence rate of cancers and cardiovascular diseases as important antioxidant ingredients although the palatability is reduced. The pericarp also contains saponin compounds, has effects of resisting bacteria, resisting inflammation, inhibiting cholesterol absorption, reducing hypertension, hyperglycemia and hyperlipidemia, and protecting liver, and has good effects of preventing and treating arteriosclerosis, thrombosis, etc.

Biologically, quinoa has outstanding stress resistance and is recognized as a preferred crop capable of producing grains under the adverse environment of soil and climate. The food and agriculture organization of the united nations determines the year 2013 as the international quinoa year so as to show the importance of the quinoa and promote the global production, consumption and industrial upgrading. With the improvement of the living standard and the enhancement of health care consciousness of people in China, the requirements of people on food diversification, nutrition, safety, health and the like are continuously improved, and the increase of the planting, the cognition and the consumption of quinoa is promoted year by year. The researchers in China develop the research and development in the aspects of production, consumption, food processing and the like on the basis of the research on the introduction evaluation, the cultivation technology, the biology and the genetic breeding of the chenopodium quinoa.

Quinoa has a limited growing panicle, is a typical crop with symbiosis of amphoteric flowers and female flowers, and branches are terminated after the apical amphoteric flowers appear. Quinoa is bred in a mode of mainly selfing and secondarily anemophily and facultative outcrossing, and the characteristics of flowers of quinoa cause difficult emasculation and are not beneficial to crossbreeding. At present, although successful hybridization is reported, the demand of the chenopodium quinoa industry on new varieties cannot be totally met.

Disclosure of Invention

In order to overcome the problems in the background technology, the invention provides a method for hybridizing quinoa tissue culture seedlings under the aseptic condition, the tissue culture rapid propagation of quinoa and quinoa hybridization are organically combined, the reproductive isolation effect of sterile tissue culture seedlings formed by quinoa tissue culture in a tissue culture bottle (under the aseptic condition) is fully utilized, and the possible hybridization is completed by utilizing the higher outcrossing rate of quinoa. Through the literature search, the same public reports as the invention are not found.

In order to realize the purpose, the invention is realized by the following technical scheme:

the method for hybridizing by utilizing quinoa tissue culture seedlings under the aseptic condition comprises the following steps:

1) tissue culture and rapid propagation of quinoa: inoculating the disinfected seeds on a culture medium, culturing for 20 days to obtain chenopodium quinoa seedlings, and performing expanded propagation by taking stem sections with leaves on the chenopodium quinoa seedlings as explants;

2) and (3) hybridization: tissue culturing the stem or seedling with leaf until the chenopodium quinoa plant begins to bloom (about 40 days), inoculating the female parent chenopodium quinoa plant at the middle position of a culture bottle, inoculating the male parent plant around the female parent, and ensuring that the male parent plant is higher than the female parent plant; continuing culturing for 20 days to obtain seeds of the intermediate female parent quinoa plant;

3) detection of hybrid seeds: and detecting and verifying the grains to obtain hybrid seeds.

Further preferably, in step 1), the seed disinfection step is as follows: the quinoa seeds are cleaned, and are subjected to water bath for 3 hours at 37 ℃ by using sterile water, and the sterilization time of the quinoa seeds is 9 minutes by using 0.2% sodium hypochlorite.

Further preferably, in step 1) or step 2), the culture medium: MS + 30 g/L glucose +7 g agar +0.5 ml/L1000 ppm dwarf (CCC). Dwarfing can shorten the growth period by dwarfing the plant ontogenesis.

Further preferably, in step 1) or step 2), the culture conditions are: temperature conditions of 22 ℃, 16 hours of darkness, 8 hours of light conditions.

The invention has the beneficial effects that:

(1) the invention is not limited by external environment and can be operated all the year round. In conventional field hybridization, a large amount of different quinoa needs to be planted in the field, so that the workload is large; the method is only carried out in the extremely short time when two varieties of quinoa florescence meet each other every year, the difficulty is high, and if the hybridization is unsuccessful, the quinoa still needs to be planted again.

(2) The hybrid obtained by the method has short time. In the tissue culture bottle, the time required for hybridization can be shortened by shortening the breeding period by adjusting the nutrition and illumination time. Hybridizing in a tissue culture bottle, forming seedlings, adult plants and blooming of the quinoa seeds, and obtaining the hybrid seeds only needs about 60 days. The method greatly shortens the growth period of the chenopodium quinoa hybrid, thereby achieving the purpose of rapid hybridization or selfing breeding of offspring and saving time and cost for breeding work.

(3) The method overcomes the problem that the plant florescence of the two chenopodium quinoa plants with different growth periods in the field is not met. Tissue culture hybridization is carried out, tissue culture of tissue culture seedlings of different varieties of quinoa is carried out every week, so that quinoa in different flowering periods and different growth periods can be inoculated in a culture bottle at the same time, flowering period meeting is easy, and the success rate of hybridization is high. The tissue culture bottle is a closed environment, so that pollen of other quinoa varieties can be reduced from falling onto the column head, the outcrossing rate is reduced, and purer hybrid seeds can be obtained.

(4) The method is simple and easy to implement, and greatly solves the problems of difficult emasculation and difficult hybridization of the quinoa flowers.

Drawings

FIG. 1 is a Chenopodium quinoa seedling grown for 10 days in example 1;

wherein, A, the stem section with leaves of the quinoa wheat is inoculated with a culture medium to induce the growth condition of lateral buds; B. the peripheral red quinoa wheat is taken as a male parent, and the middle white quinoa wheat is taken as a female parent;

FIG. 2 is a quinoa plant flowering for 30-40 days in the tissue culture of example 1;

FIG. 3 shows the growth of quinoa in example 1 by crossing or selfing;

wherein, A, quinoa wheat is inoculated with flowers formed in 30 days; B. inoculating the flowers formed in 40 days on the top of the quinoa wheat; C. inoculating red quinoa for about 60 days to form seeds; D. inoculating white quinoa and red quinoa into the same culture bottle for about 40 days to form seed grains; E. white quinoa as female parent and red quinoa as male parent, inoculating possible hybrid seeds formed in about 60 days; F. and 7, seeds are formed in 80 days of lateral buds of the red gooseberry.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below to facilitate understanding of the skilled person.

Examples

In the embodiment, quinoa tissue culture seedling hybridization is carried out by taking red quinoa as a male parent and quinoa as a female parent, and the method comprises the following steps:

1) tissue culture and rapid propagation of quinoa:

a. the quinoa seeds are cleaned, and are subjected to water bath for 3 hours at 37 ℃ by using sterile water, and the sterilization time of the quinoa seeds is 9 minutes by using 0.2% sodium hypochlorite.

b. Culture medium: MS + 30 g/L glucose +7 g agar +0.5 ml/L1000 ppm dwarf (CCC); the culture conditions are as follows: temperature conditions of 22 ℃, 16 hours of darkness, 8 hours of light conditions.

c. Inoculating the disinfected seeds on a culture medium, culturing for 20 days to obtain chenopodium quinoa seedlings, and performing expanded propagation by taking stem sections with leaves on the chenopodium quinoa seedlings as explants.

2) And (3) hybridization: culturing stem segments or seedlings with leaves for about 40 days in the same culture medium and culture conditions as the step 1), inoculating the female parent quinoa plant to the middle position of a culture bottle when the tissue-cultured quinoa plant starts to bloom, inoculating the male parent quinoa plant to the periphery of the female parent, and ensuring that the male parent plant is higher than the female parent plant; and continuing culturing for 20 days, and harvesting seeds of the intermediate maternal quinoa plant.

Analysis of results

Inoculating chenopodium quinoa of different varieties, taking female parent as the middle part and male parent as the periphery, and making the standard for natural cross breeding. FIG. 1 shows a 10-day-old Chenopodium quinoa seedling.

As shown in FIGS. 2-3, in the sterile tissue culture flask, quinoa and quinoa were crossed or selfed, and about 60 days were taken to obtain the condition of the crossed or selfed seeds. The chenopodium quinoa has the top ear capable of setting seeds and the lateral ear capable of setting seeds normally. The method shortens the growth period of the hybrid quinoa, thereby achieving the purpose of rapidly hybridizing to obtain hybrid seeds and saving time for breeding work.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

1. A method for hybridizing quinoa tissue culture seedlings under aseptic conditions is characterized by comprising the following steps: the method comprises the following steps:

2) and (3) hybridization: tissue culturing stem segment or seedling with leaf until chenopodium quinoa plant begins to bloom, inoculating female parent chenopodium quinoa plant in the middle position of a culture bottle, inoculating male parent plant around the female parent, and the male parent plant needs to be higher than the female parent plant; continuing culturing for 20 days to obtain seeds of the intermediate female parent quinoa plant;

2. The method of claim 1, wherein the hybridization is performed using quinoa tissue culture seedling under aseptic conditions, and the hybridization comprises: in step 1), the seed disinfection step is as follows: the quinoa seeds are cleaned, and are subjected to water bath for 3 hours at 37 ℃ by using sterile water, and the sterilization time of the quinoa seeds is 9 minutes by using 0.2% sodium hypochlorite.

3. The method according to any one of claims 1 to 2, wherein the hybridization is carried out using a quinoa tissue culture seedling under aseptic conditions: in step 1) or step 2), the culture medium: MS + 30 g/L glucose +7 g agar +0.5 ml/L1000 ppm dwarf (CCC).

4. The method according to any one of claims 1 to 3, wherein the hybridization is carried out using a quinoa tissue culture seedling under aseptic conditions: in step 1) or step 2), the culture conditions are as follows: temperature conditions of 22 ℃, 16 hours of darkness, 8 hours of light conditions.