A method for inducing in vitro regeneration of amaryllis via the direct embryogenesis pathway

Abstract

This invention discloses a method for inducing in vitro regeneration of Amaryllis through direct embryogenesis, comprising the following steps: using the bulbs of tissue-cultured aseptic seedlings of Amaryllis 'Cognac Orange Wine' as explant material, longitudinally cutting them into appropriately sized pieces to obtain Amaryllis tissue-cultured seedling bulb pieces; inoculating the obtained Amaryllis tissue-cultured seedling bulb pieces into an embryogenesis induction medium for embryogenesis induction culture; separating the obtained embryoids and inoculating them into a maturation medium for maturation culture to obtain complete regenerated plants, which, after cleaning and surface disinfection, are transplanted into seedling trays containing substrate for seedling cultivation. This invention provides a highly efficient method for year-round propagation of Amaryllis 'Cognac Orange Wine' seedlings that maintains the superior characteristics of the parent plant, providing technical support for the germplasm preservation, industrialized production of superior seedlings, and subsequent genetic improvement of Amaryllis 'Cognac Orange Wine'.

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology, and specifically to a method for inducing in vitro regeneration of amaryllis via the direct embryogenesis pathway. Background Technology

[0002] Amaryllis (Hippeastrum rutilum), also known as red hibiscus or hundred-branch hibiscus, is a perennial herbaceous bulbous flowering plant belonging to the Amaryllidaceae family. Native to Brazil, it blooms from April to May. Its flowers are brightly colored and varied, with rich petal characteristics. It symbolizes good fortune and prosperity and is commonly used in cut flower and potted plant production. It has high ornamental value and broad market application prospects.

[0003] Currently, tissue culture or bulb cutting propagation are the main methods for rapid propagation of amaryllis internationally. Plant tissue culture, as a modern biotechnology, has advantages such as low material requirements, high propagation efficiency, high propagation coefficient, no seasonal or material limitations, and the ability to achieve large-scale seedling production in a short period. Research on in vitro propagation of amaryllis using tissue culture techniques with bulbs, leaves, flower stalks, and petals as explant materials has been reported. However, the propagation process suffers from problems such as high initial culture contamination rates, long propagation cycles, low propagation efficiency, low proliferation coefficient, and high production costs, failing to meet the demands of industrialized production of valuable amaryllis seedlings. Plant embryoids refer to embryo-like structures formed under in vitro culture conditions without fertilization but having undergone embryonic development. Embryoids formed through direct embryoid regeneration exhibit bipolarity, intact embryo structure, and minimal connection with the maternal vascular system, resulting in virus-free plants. Furthermore, this method yields a large number of propagules, and the regenerated plants exhibit stable genetic traits, providing technical support for research on plant germplasm resource preservation and germplasm creation. Methods for inducing amaryllis plant regeneration through direct embryogenesis have been reported; for example, Chinese patent application CN109362567A discloses a method for inducing somatic embryos from amaryllis leaves and regenerating plants. However, this method uses leaves as explants, resulting in a long regeneration cycle, and experiments have shown it is not suitable for the in vitro regeneration of "Cognac Orange" leaves. Therefore, there is an urgent need to develop a method for inducing amaryllis in vitro regeneration through direct embryogenesis to meet the current needs for rapid industrialized propagation and variety improvement of amaryllis "Cognac Orange" seedlings. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to improve the propagation efficiency of tissue-cultured amaryllis "cognac orange wine".

[0005] The present invention solves the above-mentioned technical problems through the following technical means:

[0006] A method for inducing in vitro regeneration of amaryllis via the direct embryogenesis pathway includes the following steps:

[0007] S1. Using the bulbs of the tissue-cultured sterile seedlings of amaryllis “Cognac Orange Wine” as explant material, longitudinally cut them into appropriately sized pieces to obtain amaryllis tissue-cultured seedling bulb pieces.

[0008] S2. The bulb pieces of amaryllis tissue culture seedlings obtained in S1 were inoculated into embryoid induction medium for embryoid induction culture.

[0009] S3. After separating the embryoids obtained in S2, they are inoculated into a maturation culture medium for maturation culture to obtain complete regenerated plants. After cleaning and surface disinfection, they are transplanted into seedling trays containing substrate for seedling culture.

[0010] Preferably, in S1, the bulbs of the amaryllis "cognac orange wine" tissue culture sterile seedlings are obtained by culturing the amaryllis "cognac orange wine" sterile seedlings in a culture medium.

[0011] Preferably, in S1, the culture medium is DKW medium supplemented with 0.1-0.5 mg / L GA3, 0.5-3.0 mg / L paclobutrazol, 4.0-6.0% (w / v) sucrose, and 0.6% (w / v) agar.

[0012] Preferably, in S1, the culture time is 4 weeks.

[0013] Preferably, in S2, the induction culture time of the embryoid is 8 weeks; in S3, the maturation culture time is 3 weeks; and the seedling culture time is 6 weeks.

[0014] Preferably, in S2, the embryoid induction medium is DKW medium supplemented with 0.1-1.0 mg / L IAA, 0.2-2.0 mg / L meta-topolin, 0.01-0.2 mg / L TDZ, 2.5-3.0% (w / v) sucrose, and 0.6% (w / v) agar.

[0015] Preferably, in S3, the maturation medium is a 1 / 2 DKW medium supplemented with 3.0% (w / v) sucrose and 0.6% (w / v) agar.

[0016] Preferably, in S3, the surface disinfection includes immersion in an aqueous solution of potassium permanganate with a mass fraction of 0.05% to 0.2% for 10 to 15 minutes.

[0017] Preferably, the substrate is a mixture of Pinsbury nutrient soil and vermiculite in a volume ratio of 2-5:1-3.

[0018] Preferably, the culture conditions for induction culture of embryoids in S2 and maturation culture in S3 are: temperature 25±2℃, light intensity 2500~3000lx, and photoperiod 16 / 8h (light / dark).

[0019] Preferably, the conditions for seedling cultivation are a temperature of 20–30°C, a humidity of 75%–85%, and a light intensity of 1600–3500 lx.

[0020] The advantages of this invention are:

[0021] This invention provides a method for inducing in vitro regeneration of Amaryllis via direct embryogenesis. Using bulb segments from tissue-cultured aseptic seedlings of Amaryllis 'Cognac Orange Wine' as explants, the method involves embryogenesis induction, maturation culture, and acclimatization transplantation to ultimately obtain complete regenerated Amaryllis plants. Because it induces embryoids through direct embryogenesis, resulting in complete regenerated plants, it offers advantages such as high propagation efficiency, a large propagation coefficient, intact embryoid structure, and maintenance of stable genetic traits in the parent plant. This provides crucial technical support for the large-scale year-round production and subsequent genetic improvement of Amaryllis 'Cognac Orange Wine' seedlings. Attached Figure Description

[0022] Figure 1 The bulbs of the aseptic seedlings of Amaryllis "Cognac Orange Wine" used in Example 1 of this invention;

[0023] Figure 2 The bulb fragments of amaryllis tissue culture seedlings inoculated on embryoid induction medium in Example 1 of this invention;

[0024] Figure 3 The embryoids were initially induced from bulb segments of amaryllis tissue culture seedlings that were inoculated on embryoid induction medium and cultured for 2 weeks in Example 1 of this invention.

[0025] Figure 4 The embryoids induced from bulb segments of amaryllis tissue culture seedlings cultured on embryoid induction medium for 4 weeks in Example 1 of this invention;

[0026] Figure 5 The embryoids induced from bulb segments of amaryllis tissue culture seedlings cultured on embryoid induction medium for 6 weeks in Example 1 of this invention;

[0027] Figure 6 The embryoids induced from bulb segments of amaryllis tissue culture seedlings cultured on embryoid induction medium for 8 weeks in Example 1 of this invention;

[0028] Figure 7 The amaryllis embryoids isolated and inoculated on a mature culture medium in Example 1 of this invention;

[0029] Figure 8 The amaryllis embryoids in Example 1 of this invention were inoculated on a mature culture medium and cultured for 3 weeks.

[0030] Figure 9 The amaryllis tissue culture regenerated plant was acclimatized and transplanted for one and a half months in Example 1 of this invention. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0033] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0034] Example 1

[0035] A method for inducing in vitro regeneration of amaryllis via the direct embryogenesis pathway, the specific operation of which is as follows:

[0036] (1) Aseptic seedling bulbs of Amaryllis 'Cognac Orange' obtained by culturing aseptic seedlings of Amaryllis 'Cognac Orange' on DKW medium supplemented with 0.3 mg / L GA3, 1.5 mg / L paclobutrazol, 5.0% (w / v) sucrose, and 0.6% (w / v) agar for 4 weeks. Figure 1 () is used as explant material, and is longitudinally cut into 0.1-0.2cm pieces. 2 The bulbs of the tissue-cultured amaryllis seedlings were cut into pieces and set aside. During the culture process, the seedlings were cultured in a culture room with a temperature of 25±2℃, a light intensity of 2500~3000lx, and a photoperiod of 16 / 8h (light / dark).

[0037] (2) The amaryllis tissue culture seedling bulb pieces obtained in step (1) were inoculated into DKW medium supplemented with 0.5 mg / L IAA, 1.0 mg / L meta-topolin, 0.1 mg / L TDZ, 2.5% (w / v) sucrose, and 0.6% (w / v) agar at a temperature of 25±2℃, a light intensity of 2500-3000 lx, and a photoperiod of 16 / 8h (light / dark) for embryoid induction culture. Figure 2After two weeks of light culture, embryoids began to be induced from bulb sections of amaryllis tissue culture seedlings. Figure 3 After 4 weeks of light culture, a large number of embryoids were induced around the bulb sections of amaryllis tissue culture seedlings. Figure 4 After two more weeks of culture, the embryoid induction rate reached 95.7%, with an average of 9.3 embryoids produced per explant, achieving proliferation and showing asynchronous embryoids at the base of the bulb cuttings. Figure 5 ).

[0038] (3) Continue to culture the embryos obtained in step (2) under light for 2 weeks. Figure 6 The isolates were isolated and inoculated into 1 / 2 DKW medium supplemented with 3.0% (w / v) sucrose and 0.6% (w / v) agar, and matured in a culture room at a temperature of 25±2℃, a light intensity of 2500-3000 lx, and a photoperiod of 16 / 8h (light / dark). Figure 7 After 3 weeks of light culture, the embryoids developed into complete regenerated plants with a maturity rate as high as 98.1%, and the plants were robust. Figure 8 ).

[0039] (4) After rinsing the complete regenerated plants obtained in step (3) with running water, soak them in an aqueous solution containing 0.1% potassium permanganate for 15 minutes. Then, transplant them into seedling trays containing a mixed nutrient substrate (V Pinscher potting soil:V vermiculite = 3:1) and cultivate them in a greenhouse at a temperature of 20-30℃, a humidity of 75%-85%, and a light intensity of 1600-3500 lx. After 6 weeks of cultivation, robust regenerated plants were obtained, with a transplant survival rate of 100%. Figure 9 ).

[0040] Example 2

[0041] This example tested the effects of GA3, paclobutrazol, and sucrose concentrations on bulb formation in aseptic seedlings of *Amaryllis 'Cognac Orange'*. Aseptic seedlings of *Amaryllis 'Cognac Orange'* were transferred to DKW medium supplemented with different types and concentrations of GA3, paclobutrazol, sucrose, and 0.6% (w / v) agar, and bulb induction culture was performed under the conditions described in Example 1. After 4 weeks of light culture, the formation of *Amaryllis 'Cognac Orange'* bulbs was statistically analyzed. The study found (Table 1) that the addition of GA3, paclobutrazol, and sucrose to the medium significantly increased the bulb formation rate and bulb size of *Amaryllis 'Cognac Orange'*. Among the tested culture media, the DKW medium supplemented with 0.3 mg / L GA3, 1.5 mg / L paclobutrazol, 5.0% (w / v) sucrose, and 0.6% (w / v) agar showed the best induction effect on amaryllis 'Cognac Orange' bulbs, with a bulb formation rate of up to 100% and an average bulb diameter of 1.13 cm.

[0042] Table 1. Effects of GA3, paclobutrazol, and sucrose concentrations on bulb formation in aseptic seedlings of *Amaryllis 'Cognac Orange Wine'*.

[0043]

[0044]

[0045] Note: Data are averages. Each treatment contained 120 explants and was repeated three times.

[0046] Example 3

[0047] This example tested the effects of the type and concentration of plant growth regulators on the induction of embryoids in *Amaryllis 'Cognac Orange'*. Bulbs of *Amaryllis* tissue culture seedlings obtained in step (1) of Example 1 were cut and inoculated into DKW medium supplemented with different concentrations of IAA (0.1–1.0 mg / L), meta-topolin (0.2–2.0 mg / L), and TDZ (0.01–0.2 mg / L), as well as 2.5% (w / v) sucrose and 0.6% (w / v) agar. Embryoid induction was performed in a culture room at a temperature of 25 ± 2 °C, a light intensity of 2500–3000 lx, and a photoperiod of 16 / 8 h (light / dark). After 8 weeks of light culture, the embryoid induction rate and the average number of embryoids produced per explant were recorded. The study found (Table 2) that the addition of IAA, meta-topolin, and TDZ to the culture medium promoted the induction of embryoids from the bulbs of Amaryllis 'Cognac Orange Wine', and the induction rate of embryoids gradually increased with the increase of the concentration of the three substances in the culture medium. The best embryoid induction effect was observed in DKW medium supplemented with 0.5 mg / L IAA, 1.0 mg / L meta-topolin, 0.1 mg / L TDZ, 2.5% (w / v) sucrose, and 0.6% (w / v) agar, with an embryoid induction rate as high as 95.7%, and an average of 9.3 embryoids produced per explant.

[0048] Table 2. Effects of different types and concentrations of plant growth regulators on embryonic induction in *Amaryllis 'Cognac Orange Wine'*.

[0049]

[0050]

[0051] Note: Data are averages. Each treatment contained 120 explants and was repeated three times.

[0052] Comparative Example 1

[0053] The only difference from Example 1 is that in (1), the embryoid induction was not successful when using leaves of 4-week-old amaryllis "Cognac Orange" sterile seedlings as explant material instead of the sterile seedling bulbs.

[0054] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for inducing in vitro regeneration of amaryllis via the direct embryogenesis pathway, characterized in that: Includes the following steps: S1. Using the bulbs of the tissue-cultured aseptic seedlings of Amaryllis 'Cognac Orange Wine' as explant material, longitudinally cut them into appropriately sized pieces to obtain Amaryllis tissue-cultured seedling bulb pieces; the bulbs of the tissue-cultured aseptic seedlings of Amaryllis 'Cognac Orange Wine' were obtained by culturing the Amaryllis 'Cognac Orange Wine' aseptic seedlings in a culture medium; the culture medium was DKW medium supplemented with 0.1-0.5 mg / L GA3, 0.5-3.0 mg / L paclobutrazol, 4.0-6.0% w / v sucrose, and 0.6% w / v agar; S2. The bulb segments of the amaryllis tissue culture seedlings obtained in S1 were inoculated into the embryoid induction medium for embryoid induction culture; the embryoid induction medium was DKW medium supplemented with 0.1-1.0 mg / L IAA, 0.2-2.0 mg / L meta-topolin, 0.01-0.2 mg / L TDZ, 2.5-3.0% w / v sucrose, and 0.6% w / v agar; S3. After separating the embryoids obtained in S2, they are inoculated into a maturation medium for maturation culture to obtain complete regenerated plants. After cleaning and surface disinfection, they are transplanted into seedling trays containing substrate for seedling culture. The maturation medium is 1 / 2 DKW medium supplemented with 3.0% w / v sucrose and 0.6% w / v agar.

2. The method for inducing in vitro regeneration of amaryllis via the direct embryogenesis pathway according to claim 1, characterized in that: In S2, the induction culture time of the embryoids is 8 weeks; in S3, the maturation culture time is 3 weeks; and the seedling culture time is 6 weeks.

3. The method for inducing in vitro regeneration of amaryllis via the direct embryogenesis pathway according to claim 1, characterized in that: In S3, the surface disinfection includes immersion in an aqueous solution of potassium permanganate with a mass fraction of 0.05% to 0.2% for 10 to 15 minutes.

4. The method for inducing in vitro regeneration of amaryllis via the direct embryogenesis pathway according to claim 1, characterized in that: The substrate is a mixture of Pinscher nutrient soil and vermiculite in a volume ratio of 2-5:1-3.

5. The method for inducing in vitro regeneration of amaryllis via the direct embryogenesis pathway according to claim 1, characterized in that: The culture conditions for induction culture of embryoids in S2 and maturation culture in S3 are as follows: temperature 25±2℃, light intensity 2500~3000lx, and photoperiod 16h light / 8h dark.

6. The method for inducing in vitro regeneration of amaryllis via the direct embryogenesis pathway according to any one of claims 1-5, characterized in that: The conditions for seedling cultivation are a temperature of 20–30℃, a humidity of 75%–85%, and a light intensity of 1600–3500 lx.

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