A method for establishing a Rubus chingii callus regeneration system

Abstract

The application discloses a method for establishing a Rubus chingii Hu callus regeneration system, which comprises the following steps: taking tender leaves of Rubus chingii Hu which grow well under aseptic conditions as explant material, inoculating the explant material into a culture medium to obtain explants with callus and differentiate adventitious buds; the culture medium is MS+3% sucrose+3g / L plant gel+0.05mg / L IBA+0.5mg / L TDZ, pH=5.7-5.8; the material is subcultured into a new culture medium for culture after growing on the culture medium for 3-4 weeks, so that the regenerated buds are larger, thicker and more robust; the culture medium is WPM+3% sucrose+3g / L plant gel+0.1mg / L IBA+0.1TDZ mg / L, pH=5.2+ / -0.02; the adventitious buds are moved to a rooting culture medium for root induction when the adventitious buds grow to 2-3cm; the rooting culture medium is WPM+2% sucrose+3g / L plant gel+0.2mg / L IBA+0.05mg / L KT+0.5g / L activated carbon, pH=5.2+ / -0.02, and then Rubus chingii Hu regenerated seedlings are obtained. According to the application, the induction rates of callus, adventitious buds and roots of Rubus chingii Hu are 100%, 100% and 92.86% respectively. The method can obtain soil cultivation seedlings from explants in only 2-3 months, and also provides technical support for asexual propagation of Rubus chingii Hu.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant propagation technology, and particularly relates to a method for establishing a Rubus chingii Hu callus regeneration system. BACKGROUND

[0002] Rubus chingii Hu, also known as large horn, East China raspberry, etc., is a perennial shrub of the Rosaceae family and Rubus genus, and is also a Chinese characteristic food and medicine. The mature fruits are rich in amino acids, minerals Mn and Zn, vitamin C, carotenoids and phenols, etc., which are easily absorbed by the human body, and have the effects of improving metabolism, delaying aging and improving immunity, etc. In particular, the contents of anti-aging substance SOD and anti-cancer substance (anthraquinonic acid) are higher than those of existing cultivated and wild fruits [1] , and the ripe fruits have bright red color, good flavor, high sugar content and rich nutrition, and are a new fruit representative with both delicious taste and health care effect. In 1993, the Food and Agriculture Organization (FAO) recommended it as the third generation of health and pollution-free health care and nutrition fruit in the world [2-5] . The unripe fruits can also be used as medicine, and are a commonly used traditional Chinese medicine, according to the Compendium of Materia Medica, it has the effects of tonifying liver and kidney, astringing essence and reducing urine [6-8] , and also has immunological effects such as antibacterial, anti-inflammatory, soothing essence and calming spirit and antioxidant [7] , and is selected as a traditional Chinese medicine cultivation variety of "New Zhejiang Eight Flavors" [3,9-14] .

[0003] In the past decade, Rubus chingii Hu has been planted on a large scale in Jiangsu, Zhejiang, Anhui and Jiangxi provinces, and the area has been increasing year by year. In 2019, the planting area in Zhejiang Province reached about 8580hm 2 , accounting for half of the national planting area, and the economic benefits are significant. However, the seedling sources of Rubus chingii Hu are complex, good seeds are in short supply, the seed germination rate is low, and the branch cutting rooting is difficult, and at present, it is mainly propagated by root sprouting. The low propagation coefficient has become the main limiting factor for the development of its industry [15,16] . The establishment and optimization of plant tissue culture system is an effective way to accelerate the rapid propagation of good raspberry seeds

[14] . In the early stage, scholars have carried out preliminary research on the tissue culture of Rubus chingii Hu, and found that stem segments can be used to induce adventitious bud regeneration and proliferation [8,17-19] . The research results of Liu Jiqi

[17] show that the most suitable induction medium is MS+IBA 0.3mg / L+BA 0.5mg / L, the best subculture medium is MS+IBA 0.1mg / L+BA 1.0mg / L+GA0.05mg / L, and the average proliferation coefficient is more than 8; Pan Binrong et al. [8] and Zhu et al.

[18] The results showed that the optimal induction medium was MS+BA 1.5 mg / L+NAA 0.2-0.3 mg / L, and the optimal differentiation medium was MS+KT 2.0 mg / L+NAA 0.4-0.5 mg / L, and the proliferation reached 3.6-4.2 times. Wang Xiuluan et al.

[14] The optimal proliferation medium was determined as modified MS medium (2 times of Ca, Cu and Mn, 1 / 4 Mg and 1 / 2 Zn)+BA 0.5 mg / L+KT 1.0 mg / L+NAA 0.1 mg / L, which could effectively inhibit vitrification and promote the growth of test-tube seedlings. Although there have been many reports on the tissue culture of Rubus laciniatus, most of them used stem segments and branches as experimental materials to induce axillary bud germination and establish a tissue rapid propagation system, but there was no report on callus regeneration system.

[0004] Rubus laciniatus has high economic value, medicinal value and research value, but the callus regeneration system of Rubus is not suitable for Rubus laciniatus. The existing research on tissue culture of Rubus laciniatus basically uses stem segments and branches as materials to induce axillary bud germination to obtain Rubus laciniatus strains, and to establish a callus rapid propagation system, but this is not a tissue regeneration system, which cannot be used for rapid propagation and breeding of Rubus laciniatus, which greatly delays the breeding process of Rubus. Therefore, it is urgent to develop a rapid and efficient callus regeneration system to provide reference for the breeding and scientific research of Rubus laciniatus.

[0005] References

[0006] [1] Wang L, Chen Z, Jiang J Y, et al. Establishment of rapid propagation system of high-quality Rubus laciniatus[J]. Zhejiang Agricultural Sciences, 2013, 8: 967-70.

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[0008] [3] SM, ET. AL Y M C C L. An aqueous extract of Rubus chingii fruits protects primary rat hepatocytes against tert-butyl hydroperoxide induced oxidative stress[J]. Life Science, 2002, 72(03): 329-38.

[0009] [4]Xiao H M, Zu L B, Li S P, et al. Chemical constituents of Rubus chingii Hu[J]. Chinese Journal of Pharmaceutical, 2011, 21(03): 220-226.

[0010] [5]Yan J L, Zhou L P, Zhang L, et al. Nutritional quality and storage stability of red ripe fruits of Rubus chingii Hu[J]. Zhejiang Journal of Agricultural Sciences, 2019, 60(02): 242-4.

[0011] [6]Sheng Y B, Zhang C L, Tong P S, et al. Research on the development and utilization of Rubus chingii Hu[J]. Shaanxi Forestry Science and Technology, 2001, (04): 69-72.

[0012] [7]DING H Y. Extracts and constituents of Rubus chingii with 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity[J]. Int J Mol Sci, 2011, 12(6): 3941-9.

[0013] [8]Pan B R, Luo T K, Zhang Y X. Tissue culture technology of Rubus chingii Hu[J]. Zhejiang Journal of Agricultural Sciences, 2010, (03): 508-10.

[0014] [9]Dai Z H, Da F F, Meng W X, et al. Discussion on poisonous Chinese herbal medicines and decoction pieces in Chinese Pharmacopoeia (2015 edition) part one[J]. Chinese Journal of Traditional Chinese Medicine Science and Practice, 2017, 35(09): 2320-2.

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[10] YU G L, Z, WANG, W, ET. AL. Rubus chingii Hu: A Review of the Phytochemistry and Pharmacology[J]. Front Pharmacol, 2019, 10: 799.

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[11] CHEN Y C, Z, GUO, Q, ET. AL. Identification of Ellagitannins in the Unripe Fruit of Rubus Chingii Hu and Evaluation of its Potential Antidiabetic Activity[J]. J Agric Food Chem, 2019, 67(25): 7025-39.

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[12] SHENG J Y W, S. Q, LIU, K. H, ET. AL. Rubus chingii Hu: an overview of botany, traditional uses, phytochemistry, and pharmacology[J]. Chin J Nat Med, 2020, 18(6): 401-16.

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[13] KE H B, T, CHEN, W. New function of polysaccharide from Rubus chingii Hu: protective effect against ethyl carbamate induced cytotoxicity[J]. J Sci Food Agric, 2021, 101(8): 3156-64.

[0019]

[14] WANG Xiuluan, ZOU Yiqiao, LIU Lingling, et al. Optimization of Growth Regulators and Mineral Elements in Tissue Culture and Rapid Propagation of Rubus chingii Hu[J]. Zhejiang Journal of Agricultural Sciences, 2022, 34(07): 1431-8.

[0020]

[15] JIANG Jingyong, CHEN Zhen, LU Xiuyou, et al. Rooting and Rapid Propagation of Rubus chingii Hu[J]. Zhejiang Journal of Agricultural Sciences, 2013, (02): 145-6.

[0021]

[16] YOU Xiaqing, CHEN Hui, LI Xiaohui, et al. Study on the Phenotypic Traits and Germination Rate of Seeds and Fruits of Rubus chingii Hu from Different Provenances[J]. Southern Forestry Science, 2019, 47(03): 16-9+34.

[0022]

[17] LIU Jiqi. Research on Rapid Propagation of Rubus idaeus L. by Tissue Culture[J]. Central South Pharmacy, 2006, (06): 426-8.

[0023]

[18] WEIJUN ZHU*, SENFU XU Y C. Research on Rapid Propagation and Domestication of Wild Raspberry Plantlets[J]. Agricultural Science Technology, 2016, 17(02): 298-300.

[0024]

[19] XIE Congshou, CHEN Yonghe, YING Xuanyang, et al. Tissue Culture and Rapid Propagation of Rubus chingii Hu[J]. Fujian Journal of Agricultural Sciences, 2020, (10): 32-6. SUMMARY

[0025] The application provides a method for establishing a Rubus chingii callus regeneration system, and provides technical support for asexual propagation of the Rubus chingii.

[0026] The specific technical scheme is as follows:

[0027] The application provides a method for establishing a Rubus chingii callus regeneration system, and provides technical support for asexual propagation of the Rubus chingii.

[0028] (1) tender leaves of the Rubus chingii seedlings growing well on a sterile culture medium are used as explant materials, and then the tender leaves are cut to a proper size and inoculated into a callus induction culture medium to obtain the explants containing callus;

[0029] The callus induction culture medium is MS+3% sucrose+3g / L plant gel+0.05mg / L IBA+0.5mg / L TDZ, pH=5.7-5.8;

[0030] (2) the explants growing callus are moved to fresh culture medium for culture, after 2 weeks of dark culture, the culture medium is as in (1), the production of adventitious buds is induced, after 3-5 days of culture, the callus appears bud-like growth points, and then small buds are obviously visible within one week;

[0031] (3) the materials with small buds are moved to fresh culture medium for culture and growth, and after 12-16 days of culture, the small buds growing from the materials are thicker, larger and stronger; the culture medium used is WPM+3% sucrose+3g / L plant gel+0.1mg / L IBA+0.1TDZ mg / L, pH=5.2±0.02;

[0032] (4) the adventitious buds are moved to a rooting culture medium for rooting culture, the rooting culture medium is WPM+2% sucrose+3g / L plant gel+0.2mg / L IBA+0.05mg / L KT+0.5g / L activated carbon, pH=5.2±0.02; after seedling raising, the regenerated seedlings of the Rubus chingii are transplanted into grass carbon soil.

[0033] In the early stage of the experiment, the application collects published tissue regeneration methods related to Rubus plants, and tries the methods on the Rubus chingii, and finds that the existing regeneration systems of the Rubus plants are not suitable for the Rubus chingii and cannot promote the bud growth of the explants of the Rubus chingii. Therefore, the application gropes for the formula from the beginning and optimizes a plurality of formulas, and finally invents a high-efficiency callus regeneration system suitable for the Rubus chingii, which provides a new way for propagation of the good seed of the Rubus chingii.

[0034] In the present application, "WPM" refers to woody plant medium; "IBA" refers to indole butyric acid; "TDZ" refers to thidiazuron; "6-BA" refers to 6-benzylaminopurine; "NAA" refers to naphthalene acetic acid; and "KT" refers to kinetin.

[0035] The explant is selected from tender leaves of the Rubus palmatus seedlings which grow well under sterile conditions. Preferably, in step (1), the culture conditions for inducing callus are: dark culture at 23 DEG C per day, and culture for 14 days, which is conducive to improving the callus induction rate.

[0036] Preferably, in step (1), the explant material is treated by removing the edge portion of the leaves and cutting into a quadrilateral of about 0.8 cm*0.4, which is conducive to improving the callus induction rate.

[0037] Preferably, in step (1), the explant is inoculated by laying the leaves on the surface of the culture medium with the front of the leaves facing downward, which is conducive to improving the callus induction rate.

[0038] Preferably, in step (2), the adventitious bud induction culture conditions are: light culture for 16 hours per day at 23 DEG C, and subculture once every 14 days, which is conducive to improving the adventitious bud induction rate.

[0039] Preferably, in step (3), the regenerated bud induction culture conditions are: light culture for 16 hours per day at 23 DEG C, and culture for about 2 weeks, which is conducive to improving the survival rate of the regenerated buds.

[0040] Preferably, in step (4), the rooting culture conditions are: light culture for 16 hours per day at 23 DEG C, and the light intensity is 5000 LX-7000 LX, which is conducive to improving the rooting rate.

[0041] Compared with the prior art, the present application has the following beneficial effects:

[0042] (1) Using the present method, the Rubus palmatus is regenerated from callus induction to soil in a cycle of only 2-3 months, which is shorter in time;

[0043] (2) The present application is directed to the adventitious bud induction culture of the Rubus palmatus, and the same induction culture medium can be used to complete callus induction and adventitious bud regeneration, which simplifies the operation procedure;

[0044] (3) The present application is directed to the culture of the regenerated buds of the Rubus palmatus, and a new culture medium is found, which makes the regenerated buds grow faster, larger and thicker, shortens the time, and increases the survival possibility of the regenerated buds;

[0045] (4) The induction rates of callus, adventitious buds and roots are high, and are 100%, 100% and 92.86%, respectively;

[0046] (5) The tender leaf of Rubus pinnatus as the explant in the application is easy to obtain. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 A (column = 1mm) is the leaf with callus;

[0048] Figure 1 B (column = 1mm) is the leaf with callus after subculture, and the callus has obvious small buds;

[0049] Figure 1 C (column = 1cm) is Rubus pinnatus when the adventitious buds in the callus of the leaf grow to about 1-2cm in the adventitious bud induction medium;

[0050] Figure 1 D, E (column = 2cm) are Rubus pinnatus when the adventitious roots grow to 2cm and can be taken out to the outside for domestication;

[0051] Figure 1 F (column = 5cm) is the regenerated seedling of Rubus pinnatus obtained after domestication. DETAILED DESCRIPTION

[0052] The application will be further described below in combination with specific examples, and the following examples are only specific examples of the application, but the protection scope of the application is not limited to this.

[0053] Example 1

[0054] A method for establishing a callus regeneration system of Rubus pinnatus, and the specific steps are as follows:

[0055] (1) The tender leaf of Rubus pinnatus seedling growing well on the sterile medium is used as the explant material (since the material is the explant material growing in a sterile environment, it does not need to be sterilized and disinfected), and the tender leaf is cut into a quadrilateral of about 0.8cm*0.4cm by removing the edge part, inoculated into the callus induction medium, and the leaf is laid flat on the surface of the medium with the front face downward, and cultured in darkness at 23℃ for about 10-14d, to obtain the explant with callus (as shown in A); the callus induction medium is: MS+3% sucrose+3g / L plant gel+0.05mg / L IBA+0.5mg / L TDZ, pH=5.7-5.8; Figure 1 A shown);the callus induction medium is: MS+3% sucrose+3g / L plant gel+0.05mg / L IBA+0.5mg / L TDZ, pH=5.7-5.8;

[0056] (2) The callus growing explant is moved to fresh medium with the formula as described in (1), and cultured under light for 16h at 23℃ per day to induce the generation of adventitious buds, and after 5-7d of culture, the callus appears bud-like growth points, and then obvious small buds appear within a week (as shown in Figure 1The explants with regenerated buds are moved to fresh culture medium, and cultured under light at 23°C for 16h per day, so that the regenerated buds are thicker, larger and stronger, and cultured for 12-16d; the culture medium is WPM+3% sucrose+3g / L phytagel+0.1mg / L IBA+0.1TDZ mg / L, pH=5.2±0.02; Figure 1 The explants with regenerated buds are moved to fresh culture medium, and cultured under light at 23°C for 16h per day, so that the regenerated buds are thicker, larger and stronger, and cultured for 12-16d; the culture medium is WPM+3% sucrose+3g / L phytagel+0.1mg / L IBA+0.1TDZ mg / L, pH=5.2±0.02;

[0057] The explants with regenerated buds are moved to fresh culture medium, and cultured under light at 23°C for 16h per day, so that the regenerated buds are thicker, larger and stronger, and cultured for 12-16d; the culture medium is WPM+3% sucrose+3g / L phytagel+0.1mg / L IBA+0.1TDZ mg / L, pH=5.2±0.02;

[0058] The explants with regenerated buds are moved to fresh culture medium, and cultured under light at 23°C for 16h per day, so that the regenerated buds are thicker, larger and stronger, and cultured for 12-16d; the culture medium is WPM+3% sucrose+3g / L phytagel+0.1mg / L IBA+0.1TDZ mg / L, pH=5.2±0.02; Figure 1 The explants with regenerated buds are moved to fresh culture medium, and cultured under light at 23°C for 16h per day, so that the regenerated buds are thicker, larger and stronger, and cultured for 12-16d; the culture medium is WPM+3% sucrose+3g / L phytagel+0.1mg / L IBA+0.1TDZ mg / L, pH=5.2±0.02; Figure 1 The explants with regenerated buds are moved to fresh culture medium, and cultured under light at 23°C for 16h per day, so that the regenerated buds are thicker, larger and stronger, and cultured for 12-16d; the culture medium is WPM+3% sucrose+3g / L phytagel+0.1mg / L IBA+0.1TDZ mg / L, pH=5.2±0.02;

[0059] The experimental results show that the growth cycle of the whole regeneration system is 2-3 months, the induction rate of callus is 100%, the induction rate of adventitious buds is 100%, and the rooting rate is 92.86%.

[0060] Comparative Example 1

[0061] In the comparative example, NAA is used as the auxin in the callus induction stage, and KT is used as the cytokinin in the callus induction stage, and it is found that the obtained callus is less and cannot be differentiated into buds.

[0062] The specific steps are as follows:

[0063] The steps or treatments of the comparative example are the same as those of Example 1 except that NAA is used to replace IBA, and KT is used to replace TDZ.

[0064] (1) The tender leaves of Rubus laciniatus grown well on the sterile medium are used as the explant material (since the material is an explant material grown in a sterile environment, it does not need to be sterilized and disinfected), the tender leaves are cut into quadrilaterals of about 0.8cm*0.4cm by removing the edge parts, and inoculated into the callus induction culture medium, with the leaves flat on the surface of the culture medium and the front down, and cultured in the dark at 23°C per day, to obtain callus after 12-14d;

[0065] The callus induction medium is MS+3% sucrose+3g / L phytagel+1.0mg / L NAA+0.1mg / L KT, pH=5.7-5.8.

[0066] (2) The explant containing callus is moved to fresh induction medium for culture, the formula is shown in (1) above, 23°C light culture for 16h per day, and induction of adventitious buds;

[0067] Although the callus induction is successful, the callus is white in the initial growth stage, but the whole callus has no change during the induction of adventitious buds, and the growth is also good, but no adventitious bud is generated, only a small amount of material induces adventitious roots.

[0068] Comparative Example 2

[0069] The present comparative example compares different hormone formulas in 4, and it is found that the regeneration efficiency of these formulas is lower than that of the formula of the embodiment.

[0070] The specific content is as follows:

[0071] The present comparative example is the same as Example 1 in the remaining steps or treatments except that different callus and adventitious bud induction medium components are set;

[0072] According to different medium components, the following four treatments are set:

[0073] Treatment 1: MS+3% sucrose+3g / L phytagel+0.2mg / L NAA+0.6mg / L 6-BA, pH=5.7-5.8;

[0074] Treatment 2: WPM+3% sucrose+3g / L phytagel+0.5mg / L IBA+4.0mg / L TDZ, pH=5.2±0.02; Treatment 3: WPM+3% sucrose+3g / L phytagel+0.2mg / L TDZ+0.6mg / L 6-BA, pH=5.2±0.02;

[0075] Treatment 4: WPM+3% sucrose+3g / L phytagel+0.02mg / L TDZ+0.6mg / L 6-BA, pH=5.2±0.02;

[0076] The results showed that the callus induction rate of treatment 1, treatment 2, treatment 3 and treatment 4 were all 100%, the four treatments of raspberry materials in the induction of callus were good, however, in the induction of adventitious buds were not very prominent effect. Treatment 1 material growth condition was good, callus overall yellow, callus was more, but did not induce adventitious buds; treatment 2, treatment 3 and treatment 4 their callus grew well, treatment 2 callus yellow, callus was more, treatment 3 and treatment 4 with callus material arrangement green, but they induced callus was less. Nonetheless, they all have material differentiation of adventitious buds, treatment 2 adventitious bud induction rate was 13.51%, differentiation of bud material was not much, grew slower, adventitious buds were also less; compared with treatment 2, treatment 3 and treatment 4 performance was better, their adventitious bud induction rate was 55.81%, 51.35% respectively, the difference between the two was not much, the growth condition of adventitious buds also not much, but the overall growth state of adventitious buds was general, adventitious buds grew sparse, growth rate and growth condition was not as good as our example.

Claims

1. A method for establishing a Rubus rosifolius callus regeneration system, characterized by, The method comprises the following steps: (1) taking young leaves of Rubus chingii Hu as explant material, cutting the explant material into proper size, inoculating into a culture medium for inducing callus, and obtaining explant with callus; the culture medium is prepared by the following formula: MS+3% sucrose+3g / L plant gel+0.05mg / L IBA+0.5mg / L TDZ, pH=5.7-5.8; (2) moving the explant with callus to a fresh culture medium, and inducing adventitious bud generation, wherein the culture medium is prepared by the formula in (1); after 5-7 days of culture, the callus appears bud-like growth points, and then small buds are obviously visible within one week; (3) moving the material with small buds to a fresh culture medium for culture and growth, so that the small buds of the material grow thicker, larger and stronger; the culture medium is prepared by the following formula: WPM+3% sucrose+3g / L plant gel+0.1mg / L IBA+0.1mg / L TDZ, pH=5.2±0.02; (4) moving the small buds which grow thicker, larger and stronger to a rooting culture medium for culture, wherein the rooting culture medium is prepared by the following formula: WPM+2% sucrose+3g / L plant gel+0.2mg / L IBA+0.05mg / L KT+0.5g / L activated carbon, pH=5.2±0.02; (5) transplanting the seedlings after rooting into grass carbon soil, and obtaining regenerated cultivation seedlings of Rubus chingii Hu.

2. The method of claim 1, wherein the Rubus rosifolius callus regeneration system is established by the steps of: In step (1), the young leaves are cut into quadrilaterals with a size of 0.8cm*0.4cm by removing the edge part.

3. The method of claim 1, wherein the Rubus rosifolius callus regeneration system is established by the steps of: In step (1), the callus induction culture is carried out under the condition of 23℃ darkness every day, and the culture is carried out for 12-14 days.

4. The method of claim 1, wherein the Rubus rosifolius callus regeneration system is established by the steps of: In step (2), the adventitious bud induction culture is carried out under the condition of 23℃ light every day for 16 hours; after the explant grows callus, the culture medium is replaced after 5-7 days of culture, and the culture medium is replaced every 14 days thereafter.

5. The method of claim 1, wherein the Rubus rosifolius callus regeneration system is established by the steps of: In step (3), the culture is carried out under the condition of 23℃ light every day for 16 hours, and the culture is carried out for 12-16 days.

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