Rapid propagation method for tissue regeneration culture of garden euphorbia herb

Abstract

The invention discloses a rapid propagation method for tissue regeneration culture of garden euphorbia, and belongs to the technical field of agricultural biology. According to the method, euphorbia hirta leaves are used as explants, hormone types and proportion key factors involved in the processes of callus induction, callus differentiation, rooting induction and the like are screened and optimized, a rapid propagation system for tissue regeneration culture of euphorbia hirta is successfully constructed, vegetative propagation is achieved, and the tissue regeneration rate of euphorbia hirta is increased. And a large number of garden euphorbia regenerated seedlings are obtained in a short time. The method is easy to operate and control, short in planting period, high in survival rate of regenerated plants and beneficial to popularization and large-scale planting, the current situation that garden euphorbia depends on wild resources is changed, and a theoretical basis and a new method are provided for rapidly obtaining a large number of garden euphorbia plants.

Description

Technical Field

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

[0002] Euphorbia pulcherrima is an annual herbaceous plant of the Euphorbiaceae family, mainly distributed in tropical and subtropical regions. Its morphology is upright and grows upward, and the stem height can reach 50 cm, which is slender and hairy, and the leaves are opposite and oval or oblong lanceolate, with serrated edges and dark upper surfaces. In my country, Euphorbia pulcherrima, as a traditional Chinese medicinal material, has the effects of clearing away heat, promoting dampness, reducing swelling, and detoxifying. It can cure diseases such as lung abscess, breast abscess, dysentery, diarrhea, pyrexia, hematuria, eczema, tinea pedis, itchy skin, furunculosis, tooth ulcer, and has a good therapeutic effect on the symptoms of postpartum oligolactosis. However, the collection of Euphorbia pulcherrima is all from the wild at present, and the material collection is subject to regional and climatic restrictions, which prompts its utilization rate to be low. The method of tissue culture 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 large flying grass 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 buds and rooting in the tissue regeneration culture of large flying grass are established and optimized, and a rapid propagation method for tissue regeneration culture of large flying grass is successfully constructed. The method has a high proliferation coefficient, is easy and controllable to operate, and a large number of flying grass 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 the leaves of the large flying grass as explants, inoculating the 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] Inoculate the callus tissue of the successfully induced callus 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] Inoculating the differentiated callus tissue induced to sprout into 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 in 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 condition is: 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 naphthylacetic acid + 3 mg / L 6-benzylaminopurine + 2 mg / L gibberellin + 30 g / L sucrose.

[0013] Preferably, the conditions for inducing sprouting are: 25°C, light intensity of 10,000 lux, 8h light / 16h 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, 8h light / 16h dark, and culture for 30 days.

[0016] Preferably, after the adventitious buds take root, the root length grows to 3-5 cm and the plant height is 4-6 cm, the seedlings are transplanted and hardened; the substrate for transplanting and hardening the seedlings is prepared by 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 seeds of Sphagnum sphagnum and inoculating them with MS solid culture medium, and culturing them under the conditions of 28°C, a light intensity of 15000 lux, and a light time of 16h light / 8h dark; the MS solid culture medium is: MS liquid culture medium + 30g / L sucrose + 6g agar.

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

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

[0020] The invention obtains the regenerated seedlings of the giant hyssop by optimizing the rapid propagation conditions of the regenerated tissue, changes the current situation that the giant hyssop relies on wild resources, and provides a new method for the large-scale planting of the 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

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

[0023] Figure 2 It is the statistical diagram of callus induction of different explants;

[0024] Figure 3 The three callus phenotypes induced by tender 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 compact callus differentiation; wherein 1-24 correspond to the treatment groups in Table 1;

[0026] Figure 5 The phenotypes of the compact callus at different stages of differentiation; 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 The figures are the rooting phenotypes of adventitious buds induced by different concentrations of NAA; A represents the rooting phenotype of adventitious buds induced by 0 mg / L NAA, B represents the rooting phenotype of adventitious buds induced by 0.1 mg / L NAA, C represents the rooting phenotype of adventitious buds induced by 0.3 mg / L NAA, and D represents the rooting phenotype of adventitious buds induced by 0.5 mg / L NAA;

[0028] Figure 7 It is a statistical chart of rooting rate of adventitious roots induced by different concentrations of NAA;

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

[0030] Fig. 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing a particular embodiment 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 scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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, the specific steps are as follows:

[0038] 1. Obtaining sterile seedlings

[0039] Collect 30 to 50 mature seeds of wild-type giant sedge, wipe off the impurities on the surface, place them in a 1.5 mL centrifuge tube, add 50% chlorine-containing disinfectant, and rotate at 50 r / min for 5 minutes. Then pour out the disinfectant in the clean bench and wash the disinfected seeds with sterile ddH 2O wash 5 to 7 times until the water is clean and transparent. Finally, use a 10mL pipette to sow the seeds on the MS medium, and place them in a constant temperature and light incubator at 28°C and 10,000 lux of light intensity, 16 hours of light / 8 hours of darkness. Healthy seeds will germinate within 3 to 7 days. After the sterile seedlings grow for a period of time, their leaves, stems and roots can be taken 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, the root segments, stems, and leaves were selected and cut into about 0.5 cm × 0.5 cm in size with a sterilized scalpel in the clean bench, and inoculated into 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 placed in a constant temperature and light incubator under dark conditions at 28°C for culture. During the culture period, the callus induction was observed every 7 days, and subculture was performed every 14 days.

[0042] like Figure 1 and Figure 2 As shown in the figure, after 21 days of induction, the callus occurrence time of the three types of explants was 5 to 7 days, the callus induction rate was 100%, and there was no significant difference in the callus state. Considering that the plants of the flying grass used for callus induction are small, compared with roots and stems, leaves are easier to obtain and have the largest number. Therefore, considering the difficulty of obtaining the three types of explants and the callus induction conditions, leaves were selected as explants for subsequent callus induction experiments.

[0043] 3. Callus induction

[0044] Leaves were used as explants and inoculated onto MS medium to screen for the best hormone concentration ratio for callus induction, wherein the MS medium contained 0, 0.5 and 1 mg / L of naphthylacetic acid (NAA), 0, 0.5, 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; 10 leaf explants were inoculated into each 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 3As shown in Table 1, among the 24 treatments set up in the callus induction experiment, except for treatment No. 1 without hormone addition and treatment No. 2 with only 6-BA added, the callus induction rate of other treatments was 100%, and three different states of callus were obtained: 4 treatments obtained water-soaked callus; 16 treatments obtained crisp callus; 2 treatments obtained compact callus. Studies have reported that the state of callus tissue will affect callus differentiation. The results of callus differentiation showed that water-soaked and loose callus of Flying Grass failed to induce callus differentiation, and compact callus tissue was the best form for inducing callus differentiation. Therefore, the optimal hormone ratio for inducing callus and proliferation is: 0.5mg / LNAA+1mg / L 6-BA. At the same time, in order to explore the differences in callus differentiation ability in different states, callus with obvious characteristics were selected from each state for subsequent differentiation experiments.

[0046] Table 1 Statistical table of the effects of different concentrations of hormones on callus induction of Psoralea corylifolia leaves

[0047]

[0048]

[0049] 4. Differentiation of callus

[0050] The three calli obtained above were subjected to differentiation experiment. The leaves were inoculated on MS medium containing 3% sucrose and added with corresponding hormones. The growth and development of calli were observed every 7 days during the culture period, and subcultured once every two weeks. The culture conditions were: 25℃, light intensity of 10000lux, 8h light / 16h dark. It was observed during the culture process that: ① The water-soaked callus proliferated slowly at the beginning of the differentiation process, and the volume stopped proliferating after about doubling. After 30 days of culture, the callus tissue was seriously water-soaked and the bottom was browned; ② The loose state callus showed a rapid proliferation state during the differentiation process, and the color gradually changed from white or light green in dark culture to bright yellow and green, and occasionally a small part of the area was mixed with red, showing no differentiation state; ③ The dense structure callus under the treatment of 12 hormone ratios (1-6 and 19-24) proliferated slowly during the differentiation process, and its color gradually changed from the initial white or light green to red under light, and the structure gradually became loose, and embryoids gradually developed. In addition, callus tissues treated with 12 hormone ratios (7-18) proliferated faster during differentiation and remained white or light green in color, occasionally mixed with red (e.g. Figure 4 , Figure 5and as shown in Table 2). In summary, the above results show that water-soaked callus and brittle callus tissue did not show differentiation under all hormone ratios. Further analysis of the hormone ratios of 12 types of densely structured calli with differentiation ability found that 2,4-D had no significant effect on the differentiation rate; GA3 could significantly increase the differentiation rate; with the increase of 6-BA concentration, the callus differentiation rate gradually increased, and the differentiation rate reached the highest when it reached 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 was: 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 induced to produce buds obtained above was subjected to a rooting induction treatment experiment: the 1-2 cm adventitious buds differentiated on 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 cultivation; 4 treatment concentrations of NAA were set (0, 0.1, 0.3 and 0.5 mg / L), 25°C, light intensity of 10000 lux, 8h light / 16h dark, and treatment time of 30d.

[0057] During the rooting culture process, it was observed that the adventitious buds treated with 0mg / L NAA grew slowly during the entire treatment period. After 30 days of culture, only a few buds differentiated into small main roots without lateral roots. The adventitious buds treated with 0.1mg / L NAA grew slowly in the first two weeks. After 3-4 weeks, most of the buds differentiated into main roots and lateral roots, and grew vigorously. The adventitious buds treated with 0.3mg / L NAA also grew slowly in the early stage, but there was 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. 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 NAA0.1 mg / L.

[0058] 6. Hardening and transplanting

[0059] When the rooted adventitious buds grow to 3-5cm and the plant height is about 4-6cm, transplant and harden the seedlings. First, pour the vermiculite into a glass culture dish with a diameter of 15cm, then soak the vermiculite with liquid MS culture medium without vitamins, take out the regenerated seedlings in the tissue culture bottle, wash the culture medium at the roots with tap water, plant them in the soaked vermiculite, compact the roots, cover with a heat preservation cover, and place them in the plant room for 7 days. Transplant the healthy seedlings into the seedling pot (containing 1:1 nutrient soil and vermiculite, and irrigate the soil with tap water), compact the roots, and then place the whole pot outdoors for cultivation.

[0060] like Figure 8 and Fig. 9 As shown in the results, the adventitious buds of the large flying grass can be hardened when the plant height is about 3-5 cm after rooting. 53 rooted seedlings were hardened for 7 days, and finally 42 individual plants were obtained with a survival rate of 79%. During the experiment, it was found that the regenerated seedlings of the flying grass were particularly sensitive to changes in water. Sufficient water should be ensured during hardening and the initial stage of transplanting, and a heat-insulating cover should be added to reduce the impact of temperature.

[0061] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined 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 the leaves of the large flying grass as explants, inoculating the callus induction medium to induce callus tissue; the callus induction medium comprises the following components: MS liquid medium + naphthaleneacetic acid + 6-benzylaminopurine + sucrose + agar; The successfully induced callus tissue is inoculated into a callus differentiation medium to induce budding; the callus differentiation medium comprises the following components: MS liquid medium + naphthaleneacetic acid + 6-benzylaminopurine + gibberellin + sucrose; Inoculating the differentiated callus tissue induced to sprout into a rooting medium to induce rooting; the rooting medium comprises the following components: MS liquid medium + naphthaleneacetic acid + sucrose; After rooting, the seedlings are transplanted and hardened to obtain regenerated seedlings of large flying grass.

2. The method according to claim 1, characterized in that The callus induction medium includes 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.

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

4. The method according to claim 1, characterized in that The callus differentiation medium includes components with 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.

5. The method according to claim 1, characterized in that The conditions for inducing sprouting are: 25° C., light intensity of 10,000 lux, 8 h light / 16 h dark.

6. The method according to claim 1, characterized in that The rooting medium includes components with the following concentrations: MS liquid medium+0.1 mg / L naphthaleneacetic acid+10 g / L sucrose.

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

8. The method according to claim 1, characterized in that When the adventitious buds take root, the root length grows to 3-5cm and the plant height is 4-6cm, the seedlings are transplanted and hardened; the matrix for transplanting and hardening the seedlings is made of nutrient soil and vermiculite in a volume ratio of 1:1, and the vermiculite must be soaked in MS liquid culture medium without vitamins before use.

9. The method according to claim 1, characterized in that The leaves come from sterile seedlings of Sphagnum sphagnum, which are obtained by sterilizing mature seeds of Sphagnum sphagnum and inoculating them with MS solid culture medium, and culturing them under the conditions of 28° C., a light intensity of 15,000 lux, and a light time of 16 hours of light / 8 hours of darkness; the MS solid culture medium is: MS liquid culture medium + 30 g / L sucrose + 6 g agar.

Images