Efficient berry tissue culture system and method for enriching secondary metabolites thereof

Abstract

The invention relates to the field of plant tissue culture, in particular to an efficient berry tissue culture system and a secondary metabolite enrichment method thereof.According to the efficient culture system, a WPM culture medium serves as a basic culture medium, and an organic additive embedded with microcapsules and an exogenous growth regulator are added; the organic additive and the exogenous growth regulator embedded by the microcapsule comprise 0 to 4 mg / L of ZT, 0 to 3.5 mg / L of 2, 4-D, 5 to 60 g / L of cane sugar, 25 to 200 g / L of potato juice, 25 to 200 g / L of coconut juice and 25 to 200 g / L of banana juice. The method can effectively induce berry explants to generate calluses, and can realize enrichment of plant polyphenols.

Description

Technical Field

[0001] The invention relates to the field of plant tissue culture, in particular to a berry tissue efficient culture system and a method for enriching secondary metabolites thereof. Background Art

[0002] Berries are a type of fruit with unique flavor and high nutritional value. They contain polyphenols, carotenoids, vitamins and other nutrients, and have physiological effects such as antioxidant and anti-inflammatory. Therefore, berries have become a hot ingredient for green and healthy diets. With the gradual development of berry processing and the high-value utilization of berry resources, how to enrich the secondary metabolites in berries and thus enhance the application value of berries in food has become a research hotspot.

[0003] Plant secondary metabolites such as quercetin, anthocyanins and other polyphenolic compounds are organic compounds that usually accumulate in leaves, fruits, seeds and other organs of plants. They have multiple functional activities such as hypoglycemic, anti-oxidant, hypolipidemic, anti-fatigue, anti-tumor, cancer prevention, anti-aging, etc., and are widely used in food, medicine, cosmetics and other fields. The yield of secondary metabolites obtained by traditional cultivation methods is limited and affected by factors such as climate and environment, requiring a large amount of human, material and financial resources. Summary of the invention

[0004] The purpose of the invention is to overcome the deficiencies of the prior art and to provide a berry tissue efficient culture system and a method for enriching secondary metabolites thereof.

[0005] In order to achieve the above object, the technical solution specifically adopted by the present invention is as follows:

[0006] A highly efficient berry tissue culture system, wherein the culture medium is based on WPM culture medium, and organic additives and exogenous growth regulators are added, wherein the organic additives and exogenous growth regulators include: 0-4 mg / L ZT (such as 0 mg / L, 0.5 mg / L, 1 mg / L, 1.5 mg / L, 2 mg / L, 2.5 mg / L, 3 mg / L, 3.5 mg / L, 4 mg / L), 0-3.5 mg / L 2,4-D (such as 0mg / L, 0.5mg / L, 1mg / L, 1.5mg / L, 2mg / L, 2.5mg / L, 3mg / L, 3.5mg / L), 5-60g / L sucrose (such as 5g / L, 10g / L, 20g / L, 30g / L, 40g / L, 50g / L, 60g / L), 25-200g / L potato juice (such as 25g / L, 50g / L, 75g / L, 100g / L, 125g / L, 150g / L, 175g / L, 200g / L), 25-200g / L coconut juice (such as 25g / L, 50g / L, 75g / L, 100g / L, 125g / L, 150g / L, 175g / L, 200g / L), 25-200g / L banana juice (such as 25g / L, 50g / L, 75g / L, 100g / L, 125g / L, 150g / L, 175g / L, 200g / L).

[0007] Preferably, the organic additives and exogenous growth regulators include: 0.5-2.5 mg / L ZT, 0.3-1.5 mg / L 2,4-D, 10-50 g / L sucrose, and 50-150 g / L potato juice.

[0008] In the present invention, the berry callus tissue is leaves and stem segments of blueberry, strawberry and raspberry.

[0009] In the present invention, the ZT, 2,4-D, sucrose and potato juice are embedded in the WPM culture medium after being encapsulated in microcapsules, thereby realizing the slow release of organic additives and exogenous growth regulators to provide the nutrients required in the growth process of callus tissue.

[0010] In the present invention, the microcapsules use sodium alginate and gelatin as embedding agents, and adopt a complex coacervation method to embed organic additives and exogenous growth regulators, and then mix the embedded organic additives and exogenous growth regulators into the culture medium. Specifically, the process includes the following steps:

[0011] S1. Sodium alginate and gelatin are used as embedding agents, and organic additives and exogenous growth regulators are added in a core wall mass ratio of 1:2, and 1 mol / L hydrochloric acid is added to adjust the pH to 4 to solidify the mixture;

[0012] S2, adding 1 mol / L sodium hydroxide to adjust the pH to 5-5.5, and then adding 5% calcium chloride and stirring continuously to solidify the microcapsules, removing the supernatant after standing and stratifying, washing the lower layer with water, drying, and grinding to obtain a microcapsule additive;

[0013] S3. Mix the microcapsule additive into the WPM culture medium and evenly distribute it in the WPM culture medium.

[0014] In the present invention, in the embedding agent, the mass ratio of gelatin to sodium alginate is 1-8:1, and can be 1:1 or 2:1 or 3:1 or 4:1 or 5:1 or 6:1 or 7:1 or 8:1.

[0015] Preferably, in the embedding agent, the mass ratio of gelatin to sodium alginate is 4:1.

[0016] In the present invention, the WPM culture medium comprises: 4-8g / L agar, 556mg / L calcium nitrate, 400mg / L ammonium nitrate, 170mg / L potassium dihydrogen phosphate, 370mg / L magnesium sulfate, 900mg / L potassium sulfate, 96mg / L calcium chloride, 22.4mg / L manganese sulfate, 8.6mg / L zinc sulfate, 0.25mg / L sodium molybdate, 0.25mg / L copper sulfate, 0.5mg / L nicotinic acid, 27.8mg / L ferrous sulfate, 100mg / L inositol, 2mg / L glycine, 1mg / L nicotinic acid thiamine, 0.5mg / L pyridoxine hydrochloride, and 37.3mg / L sodium ethylenediaminetetraacetic acid.

[0017] The present invention also provides a method for enriching secondary metabolites of berry tissues, which uses the above-mentioned berry tissue efficient culture system to realize the culture of berry callus tissue. During the culture, the pH value of the culture medium is 5.0-5.4, the berry callus tissue is cultured in the dark, the culture temperature is 25°C, and the culture time is 20-30 days.

[0018] The present invention has the following characteristics and beneficial effects:

[0019] The invention can effectively induce berry explants to generate callus tissue and can achieve the enrichment of plant polyphenols. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a picture of callus induction on berry leaves.

[0021] Figure 2 This is a picture of callus tissue induced from berry leaves under a stereomicroscope.

[0022] Figure 3 This is a picture of callus induction in berry stem segments.

[0023] Figure 4This is a picture of callus tissue induced from berry stem segments under a stereomicroscope. DETAILED DESCRIPTION

[0024] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0025] The steps not described in detail in the present invention are all routine operations in the art, and the materials not recorded in detail are all routine materials in the art.

[0026] Example 1: Callus induction culture of leaves and stem segments of Hokuriku blueberry

[0027] 1. Culture medium preparation

[0028] 1.1. Microcapsule additives

[0029] Sodium alginate and gelatin are used as embedding agents in a mass ratio of 4:1, and organic additives and exogenous growth regulators are added in a core-wall mass ratio of 1:2, and 1 mol / L hydrochloric acid is added to adjust the pH to 4 to solidify the microcapsules; then, 1 mol / L sodium hydroxide is added to adjust the pH to 5-5.5, 5% calcium chloride is added and stirred continuously to solidify the microcapsules, and the microcapsules are allowed to stand for stratification, the supernatant is removed, the lower layer is washed with water, dried, and ground to obtain a microcapsule additive;

[0030] Among them, the dosage of organic additives and exogenous growth regulators in each group is:

[0031] ZT (0.5-2.5 mg / L ZT), 2,4-D (0.3-1.5 mg / L 2,4-D), sucrose (10-50 g / L sucrose), and potato juice 50 g / L.

[0032] 1.2 Callus induction medium

[0033] The pH of the callus induction medium of the present invention is 5.4, WPM medium is used as the basic medium, 8 g / L agar, the above-mentioned encapsulated organic additives and exogenous growth regulators are added, and a single factor experiment is designed.

[0034] (1) Optimization of callus induction medium for leaves and stem segments

[0035] 1) Effects of different concentrations of cytokinin ZT on the induction rate of blueberry leaves and stems

[0036] The experiment set up the following concentration gradient: 0.5mg / L, 1mg / L, 1.5mg / L, 2mg / L, 2.5mg / L, 3mg / L. At the same time, each culture medium was added with 50g / L potato juice, 0.5mg / L 2,4-D, and 20g / L sucrose. The callus induction rate of explants under different concentrations was compared.

[0037] 2) Effects of different concentrations of auxin 2,4-D on the induction rate of blueberry leaves and stems

[0038] The experiment set up the following concentration gradient: 0mg / L, 0.3mg / L, 0.5mg / L, 0.7mg / L, 1mg / L. At the same time, each culture medium was added with 50g / L potato juice, 1.5mg / L ZT, and 20g / L sucrose. The callus induction rate of explants under different concentrations was compared.

[0039] 2) Effects of different sucrose concentrations on the induction rate of blueberry leaves and stems

[0040] The experiment set up the following concentration gradient: 10g / L, 20g / L, 30g / L, 40g / L, 50g / L. At the same time, each culture medium was added with 50g / L potato juice, 1.5mg / L ZT, and 0.5mg / L 2,4-D. The callus induction rate of explants under different concentrations was compared.

[0041] 2. Acquisition and processing of blueberry leaf explants

[0042] Select healthy berry sterile tissue culture seedlings, cut off the upper stem segment with sterilized tissue culture scissors, cut off the young leaves on a sterilized culture dish with sterilized scissors, remove the petioles, and place them in sterile water for later use.

[0043] The use of berry sterile tissue culture seedlings in this step is to omit the explant disinfection treatment step, improve efficiency, increase the sterile effect, and promote continuous production. The present invention can also use strong and tender berry leaves to obtain sterile berry leaves through conventional disinfection for subsequent experimental operations.

[0044] 3. Callus Induction

[0045] (1) Treatment of sterile seedling leaves

[0046] 1) With the back side (abaxial side) of the leaf facing upward, cut the leaf with scissors and make several cuts on the leaf to better promote the induction of callus tissue.

[0047] 2) Place the leaves with the back side facing up on the culture medium in the culture dish, with the front side of the leaves fully in contact with the culture medium, leaving a gap of 0.5 side between the leaves to provide the leaves with sufficient growth space.

[0048] (2) Treatment of sterile seedling stem segments

[0049] 1) Cut off the vigorous stem segments and make more cuts on them to better promote the induction of callus tissue.

[0050] 2) Spread the stem segments flat on the culture medium in the culture dish, and make sure that the stem segments are in full contact with the culture medium, leaving a gap of 0.5 side between the stem segments to provide the stem segments with sufficient growth space.

[0051] (3) Label and seal the culture dish, place it in a 25°C tissue culture room, and culture in the dark for 20-30 days.

[0052] Using sterile blueberry seedlings as explants, 10 culture dishes for each culture medium were used to observe the callus induction and growth of the explant leaves and stem segments, the time of callus emergence, the state of callus, the color of callus, and the callus induction rate.

[0053] The berry leaves and stem segments with callus tissue cultured in the callus induction medium after the culture in step (3) were observed using a stereo fluorescence microscope.

[0054] 4. Determination of polyphenol content in leaf and stem callus

[0055] The polyphenol content of leaves and stem callus was calculated by the standard curve: Y = 2.0056X + 0.1015, R = 0.9979.

[0056] like Figure 1-4 It can be seen that the combination of ZT 1.5mg / L+2,4-D 0.5mg / L+sucrose 40g / L+potato juice 50g / L as hormone culture has significantly better callus induction rate indicators for leaves and stem segments, and can effectively induce berry explants to form callus tissue. The polyphenol content is the highest, with the polyphenol content in leaf callus being 96mg / g; the polyphenol content in stem callus being 103mg / g.

[0057] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A highly efficient berry tissue culture system, wherein the culture medium is based on WPM culture medium, and organic additives and exogenous growth regulators are added, characterized in that: The organic additives and exogenous growth regulators include: 0-4 mg / L ZT, 0-3.5 mg / L 2,4-D, 5-60 g / L sucrose, 25-200 g / L potato juice, 25-200 g / L coconut juice, and 25-200 g / L banana juice.

2. A berry tissue efficient culture system as claimed in claim 1, characterized in that: The berry callus tissue is the leaves and stem segments of blueberry, strawberry and raspberry.

3. A berry tissue efficient culture system as claimed in claim 1, characterized in that: The ZT, 2,4-D, sucrose and potato juice are embedded in the WPM culture medium after being encapsulated in microcapsules.

4. A berry tissue efficient culture system as claimed in claim 3, characterized in that: The microcapsule uses sodium alginate and gelatin as embedding agents and adopts a composite coagulation method to embed organic additives and exogenous growth regulators.

5. A berry tissue efficient culture system as claimed in claim 4, characterized in that: The embedding process includes the following steps: S1. Sodium alginate and gelatin are used as embedding agents, and organic additives and exogenous growth regulators are added in a core wall mass ratio of 1:2, and 1 mol / L hydrochloric acid is added to adjust the pH to 4 to solidify the mixture; S2. Add 1 mol / L sodium hydroxide to adjust the pH to 5-5.5, and then add 5% calcium chloride and stir continuously to solidify the microcapsules. After standing and stratifying, remove the supernatant, wash the lower layer with water, dry it, and grind it to obtain a microcapsule additive.

6. A berry tissue efficient culture system as claimed in claim 4, characterized in that: In the embedding agent, the mass ratio of gelatin to sodium alginate is 1-8:

1.

7. The berry tissue efficient culture system according to claim 5, characterized in that: In the embedding agent, the mass ratio of gelatin to sodium alginate is 4:

1.

8. The berry tissue efficient culture system according to claim 1, characterized in that: The WPM culture medium comprises: 4-8 g / L agar, 20-30 g / L sucrose, 556 mg / L calcium nitrate, 400 mg / L ammonium nitrate, 170 mg / L potassium dihydrogen phosphate, 370 mg / L magnesium sulfate, 900 mg / L potassium sulfate, 96 mg / L calcium chloride, 22.4 mg / L manganese sulfate, 8.6 mg / L zinc sulfate, 0.25 mg / L sodium molybdate, 0.25 mg / L copper sulfate, 0.5 mg / L nicotinic acid, 27.8 mg / L ferrous sulfate, 100 mg / L inositol, 2 mg / L glycine, 1 mg / L nicotinic acid thiamine, 0.5 mg / L pyridoxine hydrochloride, and 37.3 mg / L sodium ethylenediaminetetraacetic acid.

9. A method for enriching secondary metabolites of berry tissues, characterized in that: The berry callus tissue is cultured by using the berry tissue efficient culture system as described in any one of claims 1 to 8.

10. The method for enriching secondary metabolites of berry tissues according to claim 9, characterized in that: The pH value of the culture medium is 5.0-5.4, the berry callus tissue culture is dark culture, the culture temperature is 25° C., and the culture time is 20-30 days.

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