CN114711140A

CN114711140A - Establishment method of Vaccinium myrtillus callus regeneration system

Info

Publication number: CN114711140A
Application number: CN202111388155.3A
Authority: CN
Inventors: 崔富强; 宋艳平
Original assignee: Zhejiang A&F University ZAFU
Current assignee: Zhejiang A&F University ZAFU
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2022-07-08
Anticipated expiration: 2041-11-22
Also published as: CN114711140B

Abstract

The invention discloses a method for establishing a calluses regeneration system of vaccinium myrtillus, which comprises the following steps: taking tender branches of vaccinium myrtillus seedlings which grow well under the aseptic culture condition as explant materials, inoculating the explant with callus to a culture medium, and continuously culturing to generate adventitious buds; the formula of the culture medium is as follows: WPM +3% sucrose +1% agar +0.5 mg/L IBA +2.0 mg/L ZT, pH =5.2 ± 0.02; the adventitious bud grows to 2-3 cm, and is transferred to a rooting culture medium to induce rooting. The formula of the rooting medium is WPM, 2% of sucrose, 1% of agar, 0.3 mg/L of IAA, 1.0 mg/L of KT and 0.50 g/L of activated carbon, and the pH is =5.2 +/-0.02, so that the regenerated seedlings of the vaccinium myrtillus are obtained. The invention aims at the vaccinium myrtillus, and the yield of the callus and the adventitious bud is respectively 100 percent and more than 95 percent. The method only needs 2-3 months from the explant to the soil cultivation seedling, and provides technical support for clonal propagation of the vaccinium myrtillus.

Description

Establishment method of Vaccinium myrtillus callus regeneration system

Technical Field

The invention relates to the technical field of plant propagation, in particular to a method for establishing a calluses regeneration system of vaccinium myrtillus.

Background

Bilberry (latin scientific name:Vaccinium myrtillus) Also called Bell berry (Bellberry), is a family of Ericaceae (A. chinensis)Ericaceae) Vaccinium (A. macrocarpon)Vaccinium) The plant is a low shrub originally produced in northern Europe, contains more than 30 anthocyanins, is a cowberry variety containing most anthocyanins, is one of the most abundant natural sources of anthocyanins, has high economic value and medicinal value up to 37.47%, and is classified as a type 1 herbaceous plant by American herbaceous product association^［1,2］The blueberry is the blueberry with the highest price in the market nowadays, is the only blueberry variety which can be used as medicine at present, and is most commonly used for treating eye diseases^［3］. Modern medical research shows that the Vaccinium myrtillus fruits have blood sugar lowering, antiinflammatory and blood lipid reducing effects, and can promote antioxidant defense and reduce oxidative stress, so as to be used in treatment or preventionHas important effects in preventing diabetes, cancer, cardiovascular diseases and improving memory^［4-5］. Bilberry is commonly grown in wastelands, grasslands and wet conifers. Although the vaccinium myrtillus has high value, the artificial propagation is difficult, the cuttage is difficult to survive, the soil cultivation tillering propagation is realized only in a Finland laboratory, and the efficiency is low.

In the genus of cowberry, the most mature technique for clonal propagation and regeneration is blueberry. The blueberry is similar to the fruit of Vaccinium myrtillus in shape, belongs to the subgenus of Vaccinium, and comprises four major groups of Rabbit eye blueberry, highbush blueberry, half-highbush blueberry and lowbush blueberry. The commercialization degree and the planting popularization degree of the blueberries are the highest, and the existing blueberry plant callus regeneration method is developed by taking the blueberries as materials. The blueberry vegetative propagation technology greatly promotes the popularization and planting and variety improvement of the blueberries after introduction in China. 2011 the regeneration system of stem segments of Brassian blueberry Meideng as material such as Zhuyanming^［6］(ii) a Debnath and Barney, etc. in 2012 propose that an adventitious bud regeneration system of three Blueberry varieties including Cascade Huckleberry, Mountain Huckleberry and Oval-leaf blue berry is established by using lower-concentration Zeatin (ZT)^［7］. By using the system, the regeneration period of the blueberries is longer, namely 5-6 months, so that the system is not beneficial to rapid propagation of the seedlings; 2013, plum blossom red and the like research the influence of nitric oxide donor Sodium Nitroprusside (SNP) on the regeneration of three blueberry varieties^［8］(ii) a Effective method for establishing four half-high blueberry somatic embryogenesis in tissue culture by Amrita Ghosh and the like in 2018^［9］. Although regeneration systems are established for a plurality of varieties of blueberries of the vaccinium genus, the regeneration system of the vaccinium myrtillus with the highest economic value is not reported all the time.

The vaccinium myrtillus has higher economic value and research value, but the current callus regeneration system of vaccinium plants is not suitable for the vaccinium myrtillus. Finland, Norway, New Zealand and several other laboratories attempted to develop a regeneration system for Vaccinium myrtillus, all of which failed. Vaccinium myrtillus is recognized as a species of vaccinium vitis-idaea in which it is more difficult to establish a regeneration system. Although a plurality of laboratories in northern Europe screen some excellent strains from the field, large-scale propagation can not be realized due to low propagation expanding efficiency and no regeneration system, and the breeding process is greatly delayed. Therefore, a set of rapid and efficient callus regeneration system needs to be developed to provide reference for rapid propagation of improved varieties and scientific research of vaccinium myrtillus.

Reference to the literature

[1] Upton R, editor. Bilberry Fruit Vaccinium myrtillus L. Standards of Analysis, Quality Control, and Therapeutics. Santa Cruz, CA: American Herbal Pharmacopoeia and Therapeutic Compendium; 2001.

[2] Chu WK, Cheung SCM, Lau RAW, Benzie IFF. Bilberry (Vaccinium myrtillus L.). In: Benzie IFF, Wachtel-Galor S, editors. Herbal Medicine: Biomolecular and Clinical Aspects. 2nd ed. Boca Raton (FL): CRC Press/Taylor & Francis; 2011. Chapter 4.

[3] Drugs and Lactation Database (LactMed) [Internet]. Bethesda (MD): National Library of Medicine (US); 2006–. Bilberry. 2021 May 17.

[4] Zafra-Stone S, Taharat Y, Bagchi M, Chatterjee A, Vinson J.A, Bagchi D. Berry anthocyanins as novel antioxidants in human health and disease prevention. Mol Nutr Food Res. 2007;51:675–83.

[5] Erlund I, Kol R, Alfthan G, et al., editors. Favourable effects of berry consumption on platelet function, blood pressure, and HDL cholesterol. Am J Clin Nutr. 2008;87:323–31.

[6] Establishment of Zhang Cai Ling, Zhu Yan Ming, Bush blueberry regeneration system [ J ]. agricultural science and technology Communication, 2011, (09): 78-82.

[7] Debnath S C, Barney D L. Shoot Regeneration and Plantlet Formation by Cascade Huckleberry, Mountain Huckleberry, and Oval-leaf Bilberry on a Zeatin-containing Nutrient Medium [J]. HortTechnology, 2012.

[8] Plum red, shichen rock, li jian hua, yao peijian, wang smart, zhan qian, study of blueberry regeneration and physiological and biochemical effects by exogenous NO [ J ] academic newspaper of Shandong agriculture university, 2013.

[9] Ghosh A, Igamberdiev A U, Debnath S C. Thidiazuron-induced somatic embryogenesis and changes of antioxidant properties in tissue cultures of half-high blueberry plants [J]. Sci Rep, 2018, 8(1): 16978.

Disclosure of Invention

The invention provides an establishment method of a Vaccinium myrtillus callus regeneration system, and provides technical support for asexual propagation of Vaccinium myrtillus.

The specific technical scheme is as follows:

the invention provides a method for establishing a Vaccinium myrtillus callus regeneration system, which comprises the following steps:

(1) taking tender stem segments and leaves of vaccinium myrtillus seedlings which grow well on a sterile culture medium as explant materials, then cutting the tender branches into proper sizes, inoculating the cut tender branches into a callus induction culture medium, and culturing to obtain an explant containing callus;

the culture medium for inducing the callus comprises: WPM +3% sucrose +1% agar +0.50 mg/L IBA +2.0 mg/L ZT, pH =5.2 ± 0.02;

(2) transferring the explant with the callus to a fresh culture medium for culture, wherein the formula of the culture medium is as shown in (1), inducing the generation of adventitious buds, and culturing the callus for 3-5 days to generate bud-shaped growing points and then obviously visible buds within one week;

(3) and (3) transferring the adventitious bud into a rooting culture medium for rooting culture, wherein the rooting culture medium comprises the following formula: WPM +2% sucrose +1% agar +0.3 mg/L IAA +1.0 mg/L KT +0.5 g/L activated carbon, pH =5.2 ± 0.02. After hardening off, transplanting the seedlings into grass carbon soil to obtain the regenerated seedlings of the vaccinium myrtillus.

In the early stage of an experiment, a published blueberry-related tissue regeneration method is collected, and an attempt is made on a vaccinium myrtillus, and the finding that the existing blueberry regeneration system is not suitable for the vaccinium myrtillus and cannot promote the explant of the vaccinium myrtillus to grow buds is found. Therefore, the formula is searched from the beginning, the optimization of various formulas is carried out, a high-efficiency regeneration system suitable for the calluses of the vaccinium myrtillus is invented, and a new way is provided for the improved variety propagation of the vaccinium myrtillus.

In the present invention, "IBA" means indolebutyric acid; "ZT" means zeatin; "2-IP" refers to 2-isopentenyladenine; "6-BA" means 6-benzylaminoadenine; "IAA" refers to indoleacetic acid; "KT" refers to kinetin.

The explants are selected from young stems and leaves of vaccinium myrtillus seedlings which grow well under aseptic conditions. Preferably, in step (1), the culture conditions for callus induction are: culturing for 16 h at 28 deg.C for 6-9 d, and improving the induction rate of callus;

preferably, in step (1), the explant material is processed by the following steps: cutting young and tender branches into small sections of about 0.8 cm, and cutting the removed edge parts of the leaves into squares of about 0.5 cm; the induction rate of the callus is improved;

preferably, in step (1), the explant is inoculated by the following method: the leaves are spread on the surface of the culture medium, and the front faces of the leaves face downwards; the stem segments are fully contacted with the culture medium; the induction rate of the callus is improved;

preferably, in the step (2), the adventitious bud induction culture conditions are as follows: culturing at 28 deg.C for 16 h each day, and subculturing every 14 d; the induction rate of the adventitious bud is improved;

preferably, in the step (3), the rooting culture conditions are as follows: culturing at 25 deg.C for 16 hr with illumination intensity of 1000 LX every day; is favorable for improving the rooting rate.

Compared with the prior art, the invention has the following beneficial effects:

(1) by using the method, the Vaccinium myrtillus can be regenerated and survived from callus induction to soil, the whole period is only 2-3 months, and the time consumption is shorter than that of other Vaccinium plants in the prior art;

(2) the invention aims at the induction culture of adventitious buds of the vaccinium myrtillus, and can complete callus induction and adventitious bud regeneration by using the same induction culture medium, thereby simplifying the operation procedure;

(3) the yield of the callus and the adventitious bud is high (95-100%);

(4) according to the invention, the young branches and leaves of the vaccinium myrtillus can be used as explants, and the materials are easily obtained.

Drawings

FIG. 1 leaf or stem outgrowth of callus (bar =1 mm);

FIG. 2 after subculture, more pronounced shoots (bars =1 mm) grew in the callus;

FIG. 3 is a graph showing a Vaccinium myrtillus (column =1 mm) in which adventitious buds grow to about 1cm in a callus of a leaf or stem segment in an adventitious bud induction medium;

FIG. 4 shows a Vaccinium myrtillus (column =1 cm) with adventitious root length of 2cm when taken out to the outside for acclimatization;

FIG. 5 shows regenerated seedlings of Vaccinium myrtillus obtained after acclimatization.

Detailed Description

The present invention will be further described with reference to the following specific examples, which are only illustrative, but not intended to limit the scope of the present invention.

Example 1

A method for establishing a Vaccinium myrtillus callus regeneration system comprises the following specific steps:

(1) taking tender stem sections and leaves of vaccinium myrtillus seedlings which grow well on a sterile culture medium as explant materials (because the materials are the explant materials which grow in a sterile environment, sterilization and disinfection are not needed), cutting the tender stems into small sections of about 0.8 cm, cutting edge parts of the leaves into square sections of about 0.5 cm, inoculating the small sections into a callus induction culture medium for culture, flatly laying the leaves on the surface of the culture medium with the front surfaces facing downwards, placing the stem sections with nodes facing downwards, and culturing the stem sections under illumination at 28 ℃ every day for 16 hours to 9 days to obtain explants with calluses (as shown in figure 1);

the culture medium for inducing the callus comprises: WPM +3% sucrose +1% agar +0.5 mg/L IBA +2.0 mg/L ZT, pH =5.2 ± 0.02;

(2) transferring the explants with the grown callus into a fresh culture medium, culturing the explants with the formula as described in (1) under the condition of 28 ℃ for 16 h every day, inducing the generation of adventitious buds, culturing the callus for 5-8 d to generate bud-shaped growing points, then generating obviously visible buds within one week (shown in figure 2), subculturing every 14 d for about 25 d, and culturing until the adventitious buds grow to the length of about 1cm (shown in figure 3);

(3) transferring the grown adventitious bud with the length of 1cm to a rooting culture medium for culturing, performing illumination culture at 25 ℃ for 16 h every day with the illumination intensity of 1000 LX, taking out and placing in an outdoor for seedling exercising when the root length is 2cm (shown in figure 4), and obtaining a regenerated seedling of the vaccinium myrtillus (shown in figure 5);

wherein, the rooting culture medium is: WPM +2% sucrose +1% agar +0.3 mg/L IAA +1.0 mg/L KT +0.5 g/L activated carbon, pH =5.2 ± 0.02.

The experimental results are as follows: the growth cycle of the whole regeneration system is 2-3 months, wherein the induction rate of the callus can reach 100%, the induction rate of the adventitious bud can reach 95% -100%, and the rooting rate can reach 50%.

Comparative example 1

This comparative example used 2,4-D as an auxin during callus induction, and found that the obtained callus could not differentiate into shoots.

The method comprises the following specific steps:

this comparative example is exactly the same as example 1 except that 2,4-D was used in place of IBA

(1) Taking tender stem segments and leaves of vaccinium myrtillus seedlings which grow well on a sterile culture medium as explant materials (because the materials are the explant materials which grow in a sterile environment, the vaccinium myrtillus seedlings do not need to be sterilized and disinfected), cutting the tender branches into small segments of about 0.8 cm, cutting edge parts of the removed leaves into square segments of about 0.5 cm, inoculating the square segments into a culture medium for inducing callus to culture, paving the leaves on the surface of the culture medium with the front side facing downwards, fully contacting the stem segments with the culture medium, and performing illumination culture for 16 hours at 28 ℃ every day for 6-9 days to obtain callus;

wherein, the callus induction culture medium is as follows: WPM +3% sucrose +1% agar +0.5 mg/L2, 4-D +1.0 mg/L ZT, pH =5.2 ± 0.02;

(2) transferring the explant containing the callus to an adventitious bud induction culture medium for culture, and culturing for 16 h at 28 ℃ per day under illumination to induce adventitious buds;

wherein the adventitious bud induction culture medium comprises: WPM +3% sucrose +1% agar +0.5 mg/L IBA +1.0 mg/L ZT + 0.6 mg/L KT, pH =5.2 ± 0.02.

Although the induction rate of the callus is high, the callus is better in the initial growth stage and is milky white, when adventitious bud induction is carried out, the whole callus is whitened but no obvious bud point is found, and adventitious buds are not induced.

Comparative example 2

This comparative example compares the different hormone formulations in figure 4 and found that none of these formulations had a comparable regeneration efficiency to the example formulations.

The specific contents are as follows:

this comparative example is completely the same as example 1 except that the treatment with different callus and adventitious bud induction medium components was performed;

the following 4 treatments were set up according to different media compositions:

treatment 1: WPM +3% sucrose +1% agar +0.5 mg/L NAA +1.0 mg/L ZT, pH =5.2 ± 0.02;

and (3) treatment 2: WPM +3% sucrose +1% agar +0.5 mg/L IBA +0.5 mg/L KT, pH =5.2 ± 0.02;

and (3) treatment: WPM +3% sucrose +1% agar + 0.7 mg/L IBA +2.0 mg/L2-IP, pH =5.2 ± 0.02;

and (4) treatment: WPM +3% sucrose +1% agar + 0.7 mg/L IBA + 0.8 mg/L6-BA, pH =5.2 ± 0.02;

the results showed that the callus induction rates of treatment 1 and treatment 4 were 100%, that of treatment 2 was 53.85%, and that of treatment 3 was 78.6%. The calli of the treatments 1, 3 and 4 have good growth, only a small amount of adventitious buds are generated in the adventitious bud induction stage, and the induction rate is lower than 50%, wherein the calli of the treatments 1 and 4 with the adventitious buds are easy to generate red and brown phenomena in the adventitious bud induction stage. The calli of treatment 2 were more prone to browning. The browning phenomenon is not relieved when the induction of the adventitious bud is carried out, which causes the induction rate of the adventitious bud to be greatly reduced and lower than 10%, and the adventitious bud obtained by differentiation can not grow normally after being cultured for a period of time.

Claims

1. A method for establishing a Vaccinium myrtillus callus regeneration system is characterized by comprising the following steps:

(1) taking the young leaves of the vaccinium myrtillus seedlings which grow well on a sterile culture medium as explant materials, cutting the explant materials into proper sizes, inoculating the cut explant materials into a callus induction culture medium, and culturing to obtain explants with callus; the formula of the culture medium is as follows: WPM +3% sucrose +1% agar +0.50 mg/L IBA +2.0 mg/L ZT, pH =5.2 ± 0.02;

(2) transferring the explant with the callus to a fresh culture medium for culture, wherein the formula of the culture medium is as shown in (1), inducing the generation of adventitious buds, and culturing the callus for 3-5 days to generate bud-shaped growing points and then generating obviously visible buds within one week;

(3) transferring the adventitious bud into a rooting culture medium for culture, wherein the formula of the rooting culture medium is WPM +2% sucrose +1% agar +0.3 mg/L IAA +1.0 mg/L KT +0.50 g/L activated carbon, and the pH is =5.2 +/-0.02;

(4) transplanting the rooted plantlets into turfy soil to obtain regenerated seedlings of the vaccinium myrtillus.

2. The method according to claim 1, wherein in step (1), the young shoots are cut into small pieces of about 0.8 cm, and the removed edge of the leaf is cut into a square of about 0.5 cm.

3. The method for establishing the vaccinium myrtillus callus regeneration system as claimed in claim 1, wherein in the step (1), the explant inoculation mode is as follows: the leaves are laid on the surface of the culture medium with the front face facing downwards.

4. The method for establishing the Vaccinium myrtillus callus regeneration system as claimed in claim 1, wherein in the step (1), the callus induction culture conditions are as follows: culturing at 28 deg.C for 16 hr, and culturing for 6-9 days.

5. The method for establishing the calluses regeneration system of Vaccinium myrtillus as claimed in claim 1, wherein in the step (2), the conditions for the adventitious bud induction culture are as follows: after the explant grows out callus, the culture medium is changed for 5-8 days, and the culture medium is changed every 14 days later.