A rapid propagation method for tissue regeneration culture of giant hyssop

Abstract

The present invention discloses a rapid propagation method for tissue regeneration and culture of scutellaria baicalensis, belonging to the field of agricultural biotechnology. This method uses scutellaria baicalensis leaves as explants and screens and optimizes the types and ratios of hormones involved in callus induction, callus differentiation, and rooting induction. A rapid propagation system for tissue regeneration and culture of scutellaria baicalensis is successfully constructed, achieving asexual reproduction and obtaining a large number of scutellaria baicalensis regenerated seedlings in a short period of time. This method is simple and easy to operate, has a short planting cycle, and a high survival rate of regenerated plants, facilitating promotion and large-scale planting. This method changes the current situation of scutellaria baicalensis relying on wild resources and provides a theoretical basis and a new method for rapidly obtaining a large number of scutellaria baicalensis plants.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, in particular to a rapid propagation method for regeneration and culture of giant hyssopus tissue. Background Art

[0002] Euphorbia pulcherrima is an annual herbaceous plant of the genus Euphorbia in the Euphorbiaceae family, mainly distributed in tropical and subtropical regions. Its morphology is upright and upward, with a stem height of up to 50 cm, slender and hairy, and its leaves are opposite, oval or oblong-lanceolate, with serrated edges and a dark upper surface. In my country, Euphorbia pulcherrima, as a traditional Chinese medicinal material, has the effects of clearing heat, promoting dampness, reducing swelling, and detoxifying. It can treat diseases such as lung abscess, mastitis, dysentery, diarrhea, stranguria, hematuria, eczema, tinea pedis, itchy skin, furuncle, and dental caries, and also has a good therapeutic effect on the symptom of postpartum lactation. However, the current collection of Euphorbia pulcherrima is all from the wild, and the material collection is subject to geographical and climatic restrictions, which prompts its low utilization rate. The tissue culture method can not only provide large-scale seedlings, but also maintain the favorable traits of excellent individuals. Summary of the Invention

[0003] The purpose of the present invention is to provide a rapid propagation method for tissue regeneration culture of giant sedge to solve the problems existing in the above-mentioned prior art. By exploring key factors such as different explant selection and different hormone ratios in each tissue culture stage, the steps of callus induction, adventitious bud and rooting in the tissue regeneration culture of giant sedge are established and optimized, and a rapid propagation method for tissue regeneration culture of giant sedge is successfully constructed. The method has a high proliferation coefficient, is simple and controllable to operate, and a large number of sedge seedlings can be obtained in a short period of time.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a rapid propagation method for tissue regeneration and culture of giant flying grass, comprising the following steps:

[0006] Using large flying grass leaves as explants, inoculating callus induction medium to induce callus tissue; the callus induction medium comprises the following components: MS liquid medium + naphthaleneacetic acid + 6-benzylaminopurine + sucrose + agar;

[0007] The callus tissue induced successfully was inoculated with a callus differentiation medium to induce budding; the callus differentiation medium comprises the following components: MS liquid medium + naphthaleneacetic acid + 6-benzylaminopurine + gibberellin + sucrose;

[0008] The differentiated callus tissue induced to sprout is inoculated with a rooting medium to induce rooting; the rooting medium comprises the following components: MS liquid medium + naphthaleneacetic acid + sucrose;

[0009] After rooting, the seedlings are transplanted and hardened to obtain regenerated seedlings of large flying grass.

[0010] Preferably, the callus induction medium comprises components with the following concentrations: MS liquid medium + 0.5 mg / L naphthaleneacetic acid + 1 mg / L 6-benzylaminopurine + 30 g / L sucrose + 6 g / L agar.

[0011] Preferably, the callus induction conditions are: constant temperature culture at 28° C. in the dark for 5-7 days.

[0012] Preferably, the callus differentiation medium comprises components in the following concentrations: MS liquid medium + 0.05 mg / L naphthaleneacetic acid + 3 mg / L 6-benzylaminopurine + 2 mg / L gibberellin + 30 g / L sucrose.

[0013] Preferably, the conditions for inducing budding are: 25° C., light intensity of 10,000 lux, 8 h light / 16 h dark.

[0014] Preferably, the rooting medium comprises components in the following concentrations: MS liquid medium + 0.1 mg / L naphthaleneacetic acid + 10 g / L sucrose.

[0015] Preferably, the root induction conditions are: 25° C., light intensity of 10,000 lux, 8 h light / 16 h dark, and culture for 30 days.

[0016] Preferably, the seedlings are transplanted and hardened when the adventitious buds take root, the root length reaches 3-5 cm, and the plant height reaches 4-6 cm. The substrate for transplanting and hardening the seedlings is prepared by mixing nutrient soil and vermiculite in a volume ratio of 1:1, wherein the vermiculite must be soaked in MS liquid culture medium without vitamins before use.

[0017] Preferably, the leaves are derived from sterile seedlings of Sphagnum sphagnum, which are obtained by sterilizing mature Sphagnum sphagnum seeds and inoculating them with MS solid culture medium, and culturing them at 28°C, a light intensity of 15,000 lux, and a lighting time of 16 hours light / 8 hours dark; the MS solid culture medium is: MS liquid culture medium + 30 g / L sucrose + 6 g agar.

[0018] The present invention discloses the following technical effects:

[0019] The present invention obtains a method most suitable for the rapid propagation of giant hyssopus by screening and optimizing key factors such as explant selection, the combination of optimal hormones for inducing callus tissue, the optimal hormone ratio for inducing callus differentiation, and the optimal concentration of naphthaleneacetic acid for inducing adventitious bud rooting. This method provides sufficient experimental basis for the establishment and optimization of a tissue regeneration system for giant hyssopus or herbaceous plants of the genus Euphorbia in the Euphorbiaceae family, greatly improves the survival rate of tissue-regenerated plants of giant hyssopus, shortens the planting cycle, and is conducive to large-scale planting.

[0020] The invention obtains regenerated seedlings of giant hyssop by optimizing the rapid propagation conditions of regenerated tissue, changes the current situation of giant hyssop relying on wild resources, and provides a new method for large-scale planting of giant hyssop. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Figures 2 and 3 are callus induction phenotypes of different explants; A represents the callus induction phenotype using roots as explants, B represents the callus induction phenotype using stems as explants, and C represents the callus induction phenotype using leaves as explants;

[0023] Figure 2 The green statistics of callus induction of different explants;

[0024] Figure 3 The three callus phenotypes induced from young leaves are shown in Figure 1. A represents water-soaked callus, B represents crispy callus, and C represents compact callus.

[0025] Figure 4 is a phenotype diagram of the differentiation of compact callus; 1-24 correspond to the treatment groups in Table 1;

[0026] Figure 5 The phenotypes of the different stages of compact callus differentiation are shown in Figure 1. A represents the embryoid phenotype at 15 days of differentiation, B represents the adventitious bud phenotype at 25 days of differentiation, C represents the 1 cm long adventitious bud phenotype at 30 days of differentiation, and D represents the adventitious bud phenotype at 40 days of differentiation.

[0027] Figure 6 Figure 2 is a phenotype diagram of adventitious bud rooting induced by different concentrations of NAA; A represents the phenotype diagram of adventitious bud rooting induced by 0 mg / L NAA, B represents the phenotype diagram of adventitious bud rooting induced by 0.1 mg / L NAA, C represents the phenotype diagram of adventitious bud rooting induced by 0.3 mg / L NAA, and D represents the phenotype diagram of adventitious bud rooting induced by 0.5 mg / L NAA;

[0028] Figure 7 The statistical graph of adventitious rooting rate induced by different concentrations of NAA;

[0029] Figure 8 This is the phenotype diagram of seedling hardening 7 days;

[0030] Figure 9 This is the transplanting phenotype after seedling training. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Example 1

[0037] A rapid propagation method for tissue regeneration culture of giant flying grass, comprising the following steps:

[0038] 1. Obtaining sterile vaccine

[0039] Collect 30 to 50 mature seeds of wild-type giant sedge, wipe off any surface impurities, place in a 1.5 mL centrifuge tube, add 50% chlorine disinfectant, and sterilize by rotating at 50 rpm for 5 minutes. Then, discard the disinfectant in a clean bench and rinse the sterilized seeds 5 to 7 times with sterile ddH2O until the water is clear. Finally, use a 10 mL pipette to sow the seeds onto MS culture medium. Place in a constant temperature and light incubator at 28°C and 10,000 lux, with a 16-hour light / 8-hour dark cycle. Healthy seeds will germinate within 3 to 7 days. Once the sterile seedlings have grown for a period of time, their leaves, stems, and roots can be harvested for tissue culture.

[0040] 2. Selection of explants

[0041] When the sterile seedlings cultured in the laboratory grew for 10 to 20 days and had 6 to 8 leaves, their root segments, stems, and leaves were selected and cut into pieces of approximately 0.5 cm × 0.5 cm in size using a sterilized scalpel in a clean bench. The pieces were then inoculated onto callus induction medium (MS liquid medium + 0.5 mg / L NAA + 1 mg / L 6-BA + 30 g / L sucrose + 6 g / L agar) for callus induction. Ten explants were inoculated per dish and cultured in a constant temperature and light incubator under dark conditions at 28°C. The callus induction was observed every 7 days during the culture period, and subculture was performed every 14 days.

[0042] like Figure 1 and Figure 2 As shown in the figure, after 21 days of induction, callus formation occurred in all three explant types between 5 and 7 days, with a 100% callus induction rate and no significant differences in callus status. Considering the relatively small size of the sedge plants used for callus induction, leaves are easier to obtain and are the most abundant compared to roots and stems. Therefore, considering the ease of obtaining the three explant types and the callus induction performance, leaves were selected as explants for subsequent callus induction experiments.

[0043] 3. Callus induction

[0044] Leaves were used as explants to be inoculated onto MS medium to screen the hormone concentration ratio with the best callus induction effect. The MS medium contained 0, 0.5 and 1 mg / L of naphthaleneacetic acid (NAA), 0, 0.5 and 1 mg / L of plant growth regulator (2,4-D), 0, 0.5, 1, 1.5 and 2 mg / L of 6-benzylaminopurine (6-BA), 3% sucrose and 0.6% agar powder. Ten leaf explants were inoculated per dish and placed in a constant temperature and light incubator under dark conditions at 28°C for 21 days.

[0045] The experimental results of callus induction on leaves using three hormone combinations at different concentrations are as follows: Figure 3 As shown in Table 1, of the 24 treatments in the callus induction experiment, with the exception of Treatment 1 (no hormone addition) and Treatment 2 (6-BA addition alone), all other treatments achieved a 100% callus induction rate. Three different callus states were obtained: four treatments produced water-soaked calluses; 16 treatments produced loose calluses; and two treatments produced compact calluses. Previous studies have reported that callus state can affect callus differentiation. Callus differentiation results showed that neither water-soaked nor loose calluses of the genus Flying Grass (Phyllostachys spp.) induced callus differentiation, while compact calluses were the optimal morphology for callus differentiation. Therefore, the optimal hormone ratio for callus induction and proliferation was determined to be: 0.5 mg / L NAA + 1 mg / L 6-BA. Furthermore, to investigate the differences in callus differentiation ability among different callus states, calluses with distinct characteristics from each state were selected for subsequent differentiation experiments.

[0046] Table 1 Statistical table of the effects of different concentrations of hormones on callus induction of large-winged grass leaves

[0047]

[0048]

[0049] 4. Callus differentiation

[0050] Differentiation experiments were conducted on the three calli obtained above. Leaves were inoculated onto MS medium containing 3% sucrose and supplemented with the appropriate hormones. Callus growth and development were observed every 7 days during the culture period, and subcultured every two weeks. Culture conditions were: 25°C, 10,000 lux, 8 hours light / 16 hours dark. Observations during the culture process revealed the following: ① Water-soaked calli initially proliferated slowly, approximately doubling in volume before ceasing proliferation. After 30 days of culture, the calli became severely water-soaked and had a browning base. ② Loose calli rapidly proliferated during differentiation, gradually changing from white or light green in the dark to bright yellow and green, with occasional small areas of red, indicating no differentiation. ③ In contrast, densely structured calli treated with 12 hormone ratios (1-6 and 19-24) proliferated slowly during differentiation. Their color gradually changed from white or light green to red in the light, and their structure became looser, eventually developing embryoids. In addition, callus tissues treated with 12 hormone ratios (7-18) proliferated faster during differentiation and remained white or light green in color, occasionally interspersed with red (e.g. Figure 4 、 Figure 5(See Table 2). In summary, the above results indicate that water-soaked calli and brittle calli did not show differentiation under all hormone ratios. Further analysis of the hormone ratios of 12 types of densely structured calli with differentiation ability revealed that 2,4-D had no significant effect on the differentiation rate; GA3 significantly increased the differentiation rate; and with increasing 6-BA concentration, the callus differentiation rate gradually increased, reaching its highest level at 3 mg / L. The differentiation rate of treatment No. 6 was significantly higher than that of the other 11 treatments, indicating that the optimal hormone ratio for the differentiation experiment is: 0.05 mg / L NAA + 3 mg / L 6-BA + 2 mg / L GA3.

[0051] Table 2 Statistical table of callus differentiation rate in three different states

[0052]

[0053]

[0054] Different letters indicate significant differences in differentiation rates among different treatments (P<0.05).

[0055] 5. Rooting induction

[0056] The differentiated callus tissue obtained above that had been induced to sprout was subjected to a rooting induction treatment experiment: the 1-2 cm adventitious buds differentiated from the callus tissue were separated from the callus tissue, the callus tissue at the base was cleaned, and the adventitious buds were inserted into MS medium containing NAA and 1% sucrose for culture; four treatment concentrations of NAA were set (0, 0.1, 0.3 and 0.5 mg / L), 25°C, light intensity of 10,000 lux, 8 h light / 16 h dark, and treatment time of 30 days.

[0057] During the rooting culture process, it was observed that: the adventitious buds treated with 0mg / L NAA grew slowly throughout the entire treatment period, and only a few buds differentiated into small main roots without lateral roots after 30 days of culture; the adventitious buds treated with 0.1mg / L NAA grew slowly in the first two weeks, and most of the buds differentiated into main roots and lateral roots after 3-4 weeks, and grew vigorously; the adventitious buds treated with 0.3mg / L NAA also grew slowly in the early stage, but showed obvious callus proliferation, and the rooting rate was significantly lower than that of the 0.1mg / L NAA treatment group; the adventitious buds treated with 0.5mg / L NAA had more obvious callus proliferation during culture, and the rooting rate was lower than that of the 0.3mg / L NAA treatment group. The results are as follows Figure 6 and Figure 7 As shown: the rooting rate of the 0.1 mg / L NAA treatment group was the highest, reaching 96.67%. The 0.1 mg / L NAA treatment group with the highest rooting rate was selected as the subsequent rooting treatment plan, and the hormone ratio was NAA 0.1 mg / L.

[0058] 6. Hardening and transplanting

[0059] Transplant and harden the seedlings when the rooted adventitious buds have grown to 3-5 cm and the plant height is about 4-6 cm. First, pour vermiculite into a 15 cm diameter glass culture dish, then soak the vermiculite with liquid MS medium without vitamins. Remove the regenerated seedlings from the tissue culture bottle, wash the culture medium around their roots with tap water, plant them in the soaked vermiculite, compact the roots, cover with an insulation cover, and place them in a plant room for 7 days. Transplant healthy seedlings into seedling pots (containing a 1:1 ratio of nutrient soil and vermiculite, and water the soil thoroughly with tap water), compact the roots, and then place the entire pot outdoors for cultivation.

[0060] like Figure 8 and Figure 9 As shown in the results, after rooting, the adventitious buds of the giant sedge are about 3 to 5 cm tall, they can be hardened. Fifty-three rooted seedlings were hardened for 7 days, resulting in 42 individual plants with a survival rate of 79%. The experiment revealed that the regenerated sedge seedlings are particularly sensitive to changes in water content. During hardening and the initial stages of transplanting, adequate water should be maintained, and a heat shield should be placed to minimize the effects of temperature.

[0061] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A rapid propagation method for tissue regeneration of giant flying grass, characterized in that: The following steps are involved: Using large flying grass leaves as explants, inoculating callus induction medium for callus induction; the callus induction medium comprises: MS liquid medium + 0.5 mg / L naphthaleneacetic acid + 1 mg / L 6-benzylaminopurine + 30 g / L sucrose + 6 g / L agar; The successfully induced callus tissue was inoculated into a callus differentiation medium to induce budding; the components of the callus differentiation medium were: MS liquid medium + 0.05 mg / L naphthaleneacetic acid + 3 mg / L 6-benzylaminopurine + 2 mg / L gibberellin + 30 g / L sucrose; The differentiated callus tissue induced to sprout is inoculated with a rooting medium to induce rooting; the rooting medium comprises: MS liquid medium + 0.1 mg / L naphthaleneacetic acid + 10 g / L sucrose; After rooting, the seedlings are transplanted and hardened to obtain regenerated seedlings of large flying grass.

2. The method according to claim 1, wherein The callus induction conditions are: constant temperature culture at 28° C. in the dark for 5-7 days.

3. The method according to claim 1, wherein The conditions for inducing budding are: 25° C., light intensity of 10,000 lux, 8 h light / 16 h dark.

4. The method according to claim 1, wherein The root induction conditions are: 25° C., light intensity of 10,000 lux, 8 h light / 16 h dark, and culture for 30 days.

5. The method according to claim 1, wherein When the adventitious buds take root, the roots grow to 3-5 cm in length, and the plants are 4-6 cm tall, they are transplanted and hardened. The substrate for transplanting and hardening the seedlings is made of nutrient soil and vermiculite in a volume ratio of 1:

1. The vermiculite must be soaked in MS liquid culture medium without vitamins before use.

6. The method according to claim 1, wherein The leaves are derived from sterile seedlings of P. truncatum, which are obtained by sterilizing mature P. truncatum seeds and inoculating them into MS solid culture medium, and culturing them at 28° C., a light intensity of 15,000 lux, and a light duration of 16 h light / 8 h dark.

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