AU2020101326A6 - Lycium ruthenicum Murr Anther Culture Medium - Google Patents

Abstract

The invention discloses a black fruit wolfberry anther induction medium. The components of the medium consist of a large number of elements, trace elements, iron salts and complexing agents, organic components, inorganic additives, plant growth regulators, physiologically active substances, carbon sources, coagulants and other additives. The culture medium of the invention has the advantages of significantly improving the differentiation rate of the anther callus of Lycium barbarum and reducing the albinism rate, the obtained black Lycium barbarum anther callus quality is high, and a large number of excellent test tube seedlings can be formed in a short period of time. The breeding process of black fruit wolfberry was greatly accelerated.

Description

AUSTRALIA

PATENTS ACT 1990

PATENT SPECIFICATION FOR THE INVENTION ENTITLED:

Lycium ruthenicum Murr Anther Culture Medium

The invention is described in the following statement:-

Lycium ruthenicum Murr Anther Culture Medium

TECHNICAL FIELD

[0001] The invention relates to the technical field of culture medium, in particular to a black fruit wolfberry anther culture medium.

BACKGROUND

[0002] Lycium barbarum Murr. (Solanaceae), Solaneae Reichb., Lyciinae Wetst., Lycium L. (Lycium L.). Black fruit wolfberry is rich in natural active ingredient anthocyanins, its mature berries rich in anthocyanins and other active substances, far higher than other colored fruits, its efficacy and nutritional value, In particular, its anti-oxidation and prevention of cardiovascular and cerebrovascular diseases have been widely recognized and accepted by the public, and it is one of the popular topics in the field of lycium barbarum breeding and pharmacodynamics research in recent years. Black fruit Lycium barbarum is highly heterozygous, genetic background is complex, pure line construction is difficult.

[0003] Chinese wolfberry is the traditional common Chinese medicine in our country, which was first published in "Shen nong ben Cao jing"that is an ancient Chinese medical book, and was listed as the top grade item, whose functions are described as "bone density increasing, anti-aging effect and increasing a person's resistance to cold and heat of the weather " Lycium barbarum fruit is also a kind of functional health food with the homology of both medicine and food , it is a commonly used Chinese medicine for tonifying liver and kidney, its color is bright red, taste sour sweet. Wolfberry fruit tastes sweet, and mild, affects liver function, keeps the kidney channel flowing, tonifies liver and kidney, improves eyesight, relieves symptom of cough, delays aging effect and so on. In recent years, with people pays more attention to health care, the processing of wolfberry becomes to be a more and more in-depth research. Traditional lycium barbarum products, such as lycium barbarum juice, lycium barbarum vinegar and lycium barbarum wine, have been developed gradually; lycium barbarum polysaccharide, flavone and seed oil extraction products, lycium barbarum dry products and lycium barbarum composite products have become the core commoditoes of lycium barbarum processing industry.

[0004] Along with the further development of the medicinal health value of wolfberry, whose cultivation area has been expanding, and it has gradually become one of the main economic tree species in Northwest China. The conventional breeding methods can not meet the needs of modern agriculture to improve the variety of Lycium barbarum, and there are few new methods to cultivate new varieties of Lycium barbarum. In that haploid breed method, the haploid plants obtain by anther culture and then doubled to produce homozygous diploid flower breed have the advantages of very early stable separation of offspring, shortening breeding period, simplifying breeding procedure, It is very important for the innovation of Lycium barbarum variety.

[0005] In the study of the cultivation of Lycium barbarum flower, Zenkteler, a Polish scholar, was the first to induce pollen plants by using Atriplex leaf wolfberry, which was distributed in southeastern Europe. Chinese wolfberry haploid breeding began in the 1980s. Gu Shurong, a researcher at the Plant Research Institute of Chinese Academy of Sciences, obtained the pollen plants of Ningxia wolfberry for the first time. The regenerated plants were obtained by suspension culture of the anther calli of Lycium barbarum. These studies laid a foundation for the discovery of influencing factors of Lycium barbarum anther culture, the acquisition of homozygous flower culture materials and mutants.

[0006] At present, although the Chinese wolfberry anther culture has made considerable progress, overall, the Chinese wolfberry anther culture technology still has the defect that the embryoid induction rate is low and the flower culture technology is not perfect hence restricting the application and development of Chinese wolfberry anther culture in genetics and breeding

SUMMARY

[0007] The invention aims to provide a Lycium ruthenicum anther induction culture medium aiming at the current low efficiency of anther induction callus in Lycium ruthenicum breeding cultivation

[0008] In order to achieve the above object, the present invention provides the following scheme:

[0009] According to a first aspect, a black fruit wolfberry anther culture medium includes the follow substances per liter of distilled

water: KNO3 600~900mg/L, NH4NO3 600~900mg/L, KH2PO4 230~

300mg/L, Ca(N03)2.4H20 330~360mg/L, MnSO4-4H20 12~25mg/L

, ZnSO4-7H20 5~15mg/L, H3B03 8~18mg/L, CuSO4-5H20 0.01~

0.03mg/L, CoCl2-6H20 0.02~0.03mg/L, 2-amino-5

carboxyvaleramide 450 ~ 550 mg / L, Na2MoO4.2H20 0.2 ~ 0.3 mg / L, methylnitrosourea 0.7 ~ 0.9 mg / L, inositol 90 ~ 110 mg / L, glycine 1.8 2.2 mg / L, alanine 0.55 ~ 0.65 mg / L, Folic acid 0.45 ~ 0.55mg / L, vitamin B10.35 ~ 0.45mg / L, vitamin B6 0.45 0.55mg / L, nicotinic acid 0.45 ~ 0.55mg / L, sucrose 23 27g / L, agar 7 8g / L, activated carbon 1.1~ 1.5g / L. Colchicine 160 220mg / L, hydrolyzed protein 0.4 ~ 0.5g/ L, coconut milk 20 ~ 25g / L.

[0010] The black wolfberry anther culture medium preferably includes

the following substances per liter of distilled water: KNO3 700~

800mg/L, NH4NO3 700~800mg/L, KH2PO4 250~280mg/L,

Ca(N03)2.4H20 340~350mg/L, MnSO4-4H20 15~20mg/L,

ZnSO4-7H20 8~12mg/L, H3B03 10~15mg/L, CuSO4-5H20 0.01~

0.03mg/L, CoCl2-6H20 0.02~0.03mg/L, 2-amino-5

carboxypentanamide 480 ~ 500mg / L, Na2MoO4.2H20 0.2 ~ 0.3mg / L, methylnitrosourea 0.7 ~ 0.9mg / L, inositol 90 ~ 110mg / L, glycine 1.8 ~

2.2mg / L. Alanine 0.55 ~ 0.65mg / L, folic acid 0.45 ~ 0.55mg / L, vitamin B10.35 0.45mg / L, vitamin B6 0.45 ~ 0.55mg / L, nicotinic acid 0.45 ~ 0.55mg /L, sucrose 24 ~ 25g / L, agar 78g / L. Activated carbon 1.3 ~ 1.5g / L, colchicine 180 ~ 200mg / L, hydrolyzed protein 0.4 ~ 0.5g / L, coconut milk 20 ~ 25g / L.

[0011] The black wolfberry anther culture medium preferably has a pH value of 5.6 to 6.0.

[0012] The time between the condensation of the medium and the inoculation of the anther callus of the black wolfberry preferably does not exceed 48 hours.

[0013] As another improvement of the present invention, the black fruit Lycium barbarum anther culture medium includes the following

substances per liter of distilled water: KNO3 700~800mg/L, NH4NO3

700~800mg/L, KH2PO4 250~280mg/L, Ca(N03)2.4H20 340~

350mg/L, MnSO4-4H20 15~20mg/L, ZnSO4-7H20 8~12mg/L,

H3B03 10~15mg/L, CuSO4-5H20 0.01~0.03mg/L, CoCl2-6H20 0.02

~0.03mg/L, 2-amino-5-carboxyvaleramide480 500mg/L, Na2MoO4.2H20 0.2 0.3 mg / L, methynitrosourea 0.7 ~ 0.9 mg / L, inositol 90 ~ 110 mg /L, glycine 1.8 ~ 2.2 mg / L, alanine 0.55 ~ 0.65 mg/ L, Folic acid 0.45 ~ 0.55mg / L, vitamin B10.35 0.45mg / L, vitamin B6 0.45 ~ 0.55mg / L, nicotinic acid 0.45 ~ 0.55mg /L, sucrose 24 25g / L, agar 7 ~ 8g / L, activated carbon 1.3 ~ 1.5g / L. Colchicine 180 200mg/ L, hydrolyzed protein 0.4 ~ 0.5g / L, coconut milk 20 ~ 25g / L.

[0014] As a further improvement of the present invention, the pH value of the black fruit Lycium barbarum anther culture medium is 5.6 to

6.0.

[0015] As a further improvement of the invention, the time between the condensation of the medium and the inoculation of the anther callus of Lycium barbarum does not exceed 48 h.

[0016] The present invention discloses the following technical effects:

[0017] The culture medium of the invention has the advantages of significantly improving the differentiation rate of the anther callus of Lycium barbarum and reducing the albinism rate, the obtained black Lycium barbarum anther callus quality is high, and a large number of excellent test tube seedlings can be formed in a short period of time. The breeding process of black fruit wolfberry was greatly accelerated.

DESCRIPTION OF THE INVENTION

[0015] Various exemplary embodiments of the invention will now be described in detail, which should not be construed as limiting the invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the invention.

[0016] It should be understood that the terms described in the present invention are merely intended to describe particular embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between intermediate values within any stated value or range of statements and between intermediate values within any other stated value or range of statements is also included within the present invention. The upper and lower limits of these smaller ranges may be included or excluded independently within the range.

[0017] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art described herein. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All documents referred to in this specification are incorporated by reference to disclose and describe methods and / or materials associated with such documents. In the event of conflict with any incorporated literature, the contents of this specification shall prevail.

[0018] Various modifications and variations may be made to specific embodiments of the specification of the invention without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments resulting from the description of the invention will be apparent to the skilled person. The specification and embodiments of the present application are merely exemplary.

[0019] The terms "containing," "including," "having," "containing," and the like used herein are all open, that is, are intended to include, but are not limited to.

[0020] The anther culture process of Examples 1 to 4 of the present invention is as follows:

[0021] In that invention, the Chinese wolfberry variety Ningqi No. 1 and black wolfberry are selected as anther donors for anther culture, the induced culture medium of the Chinese wolfberry is prepared, and anther culture is carried out with the following steps:

[0022] The results were as follows:

a. The buds were harvested and treated at low temperature

[0023] The seedlings of Ningqi 1 and Lycium barbarum in the field were collected from 9: 00 a.m. to 11: 00 a.m. at the beginning of flowering or at the end of flowering, and the buds in the middle and late stage of monocyte development were taken as test materials. The collected buds were wrapped with wet gauze and then wrapped with self-sealing plastic bag, and then placed in a refrigerator at 4°C for 2-7 days at low temperature;

[0024] (2) Preparation of culture medium

[0025] The medium components of Examples 1-4 are detailed in Examples 1-4. The anther induction medium of Lycium barbarum was prepared according to the formula of Examples 1-4, and the pH value was controlled to 5.8. After the medium is prepared, it is divided into 200 ml triangular flasks, each containing 40 ml ~ 60 ml of medium, sealed and sterilized at 121°C under high pressure steam pressure of 15 kPa for 20 minutes, and then condensed to 45 ~ 55 °C, Take it out and put it into super-clean worktable, aseptic operation, add N-methyl nitrosourea solution, shake and condense, and set aside within 48 hours;

[0026] (3) Anther inoculation

[0027] Take out the low temperature treated buds, rinse them with running water for 1~ 2 min, peel them off with tweezers, soak them in 70% alcohol for 10 s, rinse them with sterile water for 2 ~ 3 times, then soak them with 0.1% Hg + 2 drops of Tween-80 solution for 10 min, After washing 4 to 6 times with sterile water, put the bud into a sterile culture dish covered with filter paper, peel the anther with sterilized tweezers under sterile conditions, and inoculate on the induction medium prepared in step (2), Anthers inoculated with 4 ~ 6 buds per bottle;

[0028] And (4) temperature change induced culture

[0029] The medium inoculated with anthers was dark cultured at 25 27 °C for 1.5 ~ 2.5 days, then transferred to 37 ~ 39 °C for 2 ~ 3 days under high temperature and dark conditions. Then the anther callus was induced under the dark conditions of 27 ~ 29 °C and 75% 80% humidity, and the induction rate of anther callus was counted.

[0030] (5) Differentiation and rooting

[0031] After the calli grew to 2 cm in diameter, the light yellow loose calli were transferred into the differentiation medium for differentiation and culture, and the calli gradually differentiated into green seedlings. In this case, that differentiation rate of green seedling of anther callus was counted, and the green seedling with a length of more than 2.5 cm was cut off. Transfer into rooting medium (rooting medium formula: MS basic medium + 0.5g / L activated carbon + 1.0mg / L paclobutrazol), The culture conditions were as follows: Temperature 24 ~ 26 °C, humidity 70% ~ 80%, light intensity 1800 ~ 23001ux, light period 12h / 12h;

[0032] (6) Domestication and transplanting

[0033] If tube seedling grows to 3-5 leaf age, the seal membrane is opened to acclimate the seedling, after 7-10 days, the test tube seedling is taken out, the root is clean, the root is transplanted into the nutrient bowl containing the grass charcoal soil, Seedlings grew up, transplanted to the field, conventional cultivation, one month after the survival rate of transplanting statistics.

[0034] Example 1

[0035] The culture medium was prepared as follows: KNO3 600mg/L

, NH4NO3 600mg/L, KH2PO4 230mg/L, Ca(N03)2.4H20 330mg/L, MnSO4-4H20 12mg/L, ZnSO4-7H20 5mg/L, H3B03 8mg/L,

CuSO4-5H20 0.01mg/L, CoCl2-6H20 0.02mg/L, 2-amino-5

carboxypentanoamide 450mg / L, Na2MoO4-2H20 0.2mg/L, Methyl

nitrosourea 0.7mg / L, inositol 90mg / L, glycine 1.8mg / L, alanine 0.55mg /L, folic acid 0.45mg / L, vitamin B1 0.35mg / L, vitamin B6 0.45mg /L, nicotinic acid 0.45mg / L, sucrose 23g / L. Agar 7g / L, activated carbon 1.1g / L, colchicine 160mg / L, hydrolyzed protein 0.4g/ L, coconut milk 20g / L.

[0036] Example 2

[0037] The anther culture medium of Fructus Lycii was made up of

KNO3 900mg/L, NH4NO3 900mg/L, KH2PO4 300mg/L,

Ca(N03)2.4H20 360mg/L, MnSO4-4H20 25mg/L, ZnSO4-7H20

15mg/L, H3B03 18mg/L, CuSO4-5H20 0.03mg/L, CoCl2-6H20

0.03mg/L, 2-amino-5-carboxypentanamide550mg/L,Na2MoO4-2H20

0.2mg/L, methyl nitrosourea 0.9mg / L, inositol. Glycine 2.2mg / L, alanine 0.65mg / L, folate 0.55mg /L, vitamin B10.45mg / L, vitamin B6 0.55mg / L, nicotinic acid 0.55mg /L, sucrose 27g / L, agar 8g / L, activated carbon 1.5g / L, colchicine 220mg / L, Hydrolyzed protein 0.5g / L, coconut milk 25g / L.

[0038] Example 3

[0039] The anther culture medium of Fructus Lycii was made up of

KNO3 700mg/L, NH4NO3 700mg/L, KH2PO4 250mg/L,

Ca(N03)2.4H20 340mg/L, MnSO4-4H20 15mg/L, ZnSO4-7H20 8mg/L

, H3B03 10mg/L, CuSO4-5H20 0.01mg/L, CoCl2-6H20 0.02mg/L, 2 amino-5-carboxypentanamide 480mg / L, Na2MoO4-2H20 0.2mg/L, methyl nitrosourea 0.7mg / L, inositol 90mg / L, Glycine 1.8mg / L, alanine 0.55mg / L, folate 0.4mg / L, vitamin B1 0.35mg / L, vitamin B6 0.45mg / L, nicotinic acid 0.45mg / L, sucrose 24g / L, agar 7g / L, activated carbon 1.3 g / L. Colchicine 180mg / L, hydrolyzed protein 0.4g / L, coconut milk 20g / L.

[0040] Example 4

[0041] The anther culture medium of Fructus Lycii was made up of

KNO3 800mg/L, NH4NO3 800mg/L, KH2PO4 280mg/L,

Ca(N03)2.4H20 350mg/L, MnSO4-4H20 20mg/L, ZnSO4-7H20

12mg/L, H3B03 15mg/L, CuSO4-5H20 0.03mg/L, CoCl2-6H20

0.03mg/L, 2-amino-5-carboxypentanamide 500mg / L, Na2MoO4-2H20 0.3mg/L, methyl nitrosourea 0.9mg / L, inositol 0.9mg / L. Glycine 2.2mg / L, alanine 0.65mg / L, folate 0.55mg / L, vitamin B10.45mg / L, vitamin B6 0.55mg / L, nicotinic acid 0.55mg / L, sucrose 25g / L, agar 8g / L, activated carbon 1.3 g / L, colchicine 200mg / L. Hydrolyzed protein 0.5g / L, coconut milk 25g / L.

[0042] Comparative Example 1

[0043] The cultivation process of Fructus Lycii was the same as that of Examples 1-4 except that the anther induction treatment was not performed.

[0044] The results of callus induction rate, green seedling differentiation rate and transplanting survival rate of the Chinese wolfberry varieties Ningqi No. 1 and Black wolfberry of Examples 1-4 and Comparative Example 1 are shown in Table 1.

Table 1

Differentiation Induction ate rTransplant rate of green sria of anther survival callus /% plantlets of rate/% anther calli /%

Example 1 30.5 65.2 100

Example 2 28.7 63.5 99.8

29.6 64.3 100 Ningqi 1 Example 3 Example 4 32.4 68.4 100

Comparative 15.4 40.1 85 Example 1

Example 1 31.5 68.7 100

Example 2 30.9 65.9 100

Blackberry Example 3 32.4 67.4 100 wolfberry Example 4 33.3 69.1 100

Comparative 14.8 41.2 80 Example 1

[0045] From Table 1, it can be found that the anther induction medium of Lycium barbarum L. provided by the present invention can induce up to 33.3% of anther calli of Lycium barbarum variety Ningqi No. 1 and Lycium barbarum L. In that anther callus green seedling, the average differentiation rate can reach 69.1%, and the transplant survival rate can reach 100%. It can be found that the anther induction medium of the invention is very suitable for the requirement of black fruit wolfberry anther culture, It has the advantages of improving the differentiation rate of anther callus and reducing the albinism rate of Lycium barbarum L.

[0046] The above-described embodiments merely describe a preferred mode of the present invention, and are not intended to limit the scope of the present invention, and without departing from the spirit of the design of the present invention, Various modifications and improvements made by those of ordinary skill in the art to the technical solution of the present invention should fall within the scope of protection defined in the claims of the present invention.

Claims (4)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A black fruit wolfberry anther culture medium including the follow substances per liter of distilled water: KNO3 600-900mg / L, NH4NO3 600 900mg / L, KH2PO4 230-300mg / L, Ca (NO3) 2.4H20 330 ~ 360mg / L, MnSO4-4H20 12 ~ 25mg / L, ZnSO4-7H20 5 ~ 15mg / L, H3B03 8 ~18mg/L,, CuSO4-5H200.01 0.03mg/L,CoCl2-6H200.03mg/L.2

amino-5-carboxyvaleramide 450 ~ 550 mg / L, Na2MoO4-2H20 0.2 ~ 0.3 mg / L, methynitrosourea 0.7 ~ 0.9 mg / L, inositol 90 ~ 110 mg / L, glycine 1.8 ~ 2.2 mg / L, alanine 0.55 ~ 0.65 mg / L, Folic acid 0.45 ~ 0.55mg / L, vitamin BI 0.35 ~ 0.45mg / L, vitamin B6 0.45 ~ 0.55mg / L, nicotinic acid 0.45 ~ 0.55mg / L, sucrose 23 ~ 27g / L, agar 7 ~ 8g / L, activated carbon 1.1 ~ 1.5g / L. Colchicine 160 ~ 220mg / L, hydrolyzed protein 0.4 ~ 0.5g / L, coconut milk 20 ~ 25g / L.

2. The black wolfberry anther culture medium according to claim 1, characterized in that the black wolfberry anther culture medium includes the following substances per liter of distilled water: KNO3 700-800mg/L, NH4NO3 700-800mg/L, KH2PO4

250~280mg/L, Ca(NO3)2-4H20 340~350mg/L, MnSO4-4H20

15-20mg/L, ZnSO4-7H20 8-12mg/L, H3B03

10~15mg/L, CuSO4-5H20 0.01~0.03mg/L, CoCl2-6H20

0.02~0.03mg/L, 2-amino-5-carboxypentanamide 480 ~ 500mg /L,

Na2MoO4.2H20 0.2 ~ 0.3mg / L, methynitrosourea 0.7 ~ 0.9mg /L, inositol 90 ~110mg / L, glycine 1.8 ~ 2.2mg / L. Alanine 0.55 ~ 0.65mg/ L, folic acid 0.45 ~ 0.55mg / L, vitamin BI 0.35 ~ 0.45mg / L, vitamin B6 0.45 ~ 0.55mg / L, nicotinic acid 0.45 ~ 0.55mg / L, sucrose 24 ~ 25g / L, agar 78g / L. Activated carbon 1.3 ~ 1.5g / L, colchicine 180 ~ 200mg / L, hydrolyzed protein 0.4 ~ 0.5g / L, coconut milk 20 ~ 25g / L.

3. The black wolfberry anther culture medium according to claim 1, wherein the black wolfberry anther culture medium has a pH value of 5.6 to 6.0.

4. The black wolfberry anther culture medium according to claim 1, characterized in that the time between the condensation of the medium and the inoculation of the anther callus of the black wolfberry does not exceed 48 hours.