A color calla lily regeneration tissue culture medium and a regeneration tissue culture method

Abstract

The application belongs to the technical field of plant propagation cultivation, and particularly relates to a regeneration tissue culture medium and regeneration tissue culture method of colored calla lily. The application provides a regeneration tissue culture medium of colored calla lily, wherein the tissue culture medium comprises an induction medium, a bud strengthening culture medium and a balling and rooting culture medium. In the tissue culture medium and the tissue culture method provided by the application, sterile young ball thin slices are selected as explants, a MS culture medium is used as a basic culture medium, proper concentrations of 6-BA, NAA and carbon nanotubes are added, and proper temperature and illumination are used, so that the proliferation and differentiation of the sterile young ball thin slices of the colored calla lily are induced, the propagation coefficient of the colored calla lily is improved, and a large number of tissue culture seedlings of the colored calla lily with consistent growth are obtained.

Description

Technical Field

[0001] This invention belongs to the field of plant propagation and cultivation technology, specifically relating to a tissue culture medium and a regeneration tissue culture method for colored calla lilies. Background Technology

[0002] Currently, the industrialized production of colored calla lily bulbs relies on rapid tissue culture propagation. A common method involves using sterile bulblets with buds as explants. After disinfection and sterilization, adventitious buds are induced, followed by proliferation, and then strong bud, seedling, and rooting culture to obtain test-tube seedlings. With the completion of the yellow calla lily genome sequencing, verification of the gene function of colored calla lilies is urgently needed. Establishing a regeneration system based on callus tissue is a prerequisite for conducting genetic research on colored calla lilies. However, Chinese patent CN201910992841.8, "A Culture Medium for Inducing Callus Production in Colored Calla Lilies and Its Application in Establishing a Colored Calla Lily Regeneration System," provides a method for inducing callus production using colored calla lily bulblets as explants to establish a regeneration system, but it suffers from problems such as a long induction period and low induction rate and proliferation coefficient.

[0003] Therefore, there is an urgent need to improve the regeneration culture medium for colored calla lilies and establish a regeneration system for colored calla lilies in order to shorten the induction period and increase the induction rate and propagation coefficient. Summary of the Invention

[0004] The purpose of this invention is to provide a tissue culture medium and a regeneration tissue culture method for colored calla lilies. By using the tissue culture medium provided by this invention with sterile bulb slices of colored calla lilies as explants, the regeneration of colored calla lilies can be carried out efficiently and rapidly, thereby increasing the propagation coefficient of colored calla lilies.

[0005] To address the above problems, the present invention provides the following technical solution:

[0006] This invention provides a tissue culture medium for the regeneration of colored calla lilies, the tissue culture medium comprising an induction medium, a bud-strengthening medium, and a bulb-forming and rooting medium;

[0007] The induction medium uses MS medium as the basic medium and also includes: 0.5–4.0 mg / L 6-BA, 30 g / L sucrose and 6.5–7.0 g / L agar;

[0008] The bud-strengthening medium uses MS medium as the basic medium and also includes: 1.0–2.5 mg / L 6-BA, 0–0.5 mg / L NAA, 0.5–2.0 mg / L carbon nanotubes, 40–60 g / L sucrose and 6.5–7.0 g / L agar;

[0009] The bulb-forming and rooting medium uses MS medium as the basic medium and also includes: 0.5–2.0 mg / L NAA, 0.5–2.0 mg / L carbon nanotubes, 40–60 g / L sucrose and 6.5–7.0 g / L agar.

[0010] This invention provides a method for tissue culture of clustered shoots of colored calla lilies. The method uses the induction culture medium described in the above-mentioned technical solution and includes the following steps:

[0011] Sterile bulblets of colored calla lilies were inoculated onto an induction medium for induction culture to obtain clustered shoots.

[0012] Preferably, the length and width of the sterile granule sheet are both 0.3-0.8 cm, and the thickness is 0.1-0.2 cm.

[0013] Preferably, the induction culture is a dark culture; the induction culture time is 30-35 days; and the induction culture temperature is 25±1℃.

[0014] This invention provides a method for the regeneration of colored calla lilies using tissue culture. The method employs the aforementioned bud-strengthening culture medium and bulb-forming and root-promoting culture medium, and includes the following steps:

[0015] The clustered buds described in the above technical solution are transferred to a bud-strengthening culture medium for bud-strengthening culture to obtain clustered seedlings;

[0016] The clump-forming seedlings were transferred to a bulb-forming and rooting medium for bulb-forming and rooting culture to obtain tissue culture seedlings.

[0017] Preferably, the bud strengthening culture is a light culture; the bud strengthening culture time is 30-35 days; the light culture time is 12-16 hours / day; and the light culture intensity is 800-1200 lx.

[0018] Preferably, the bulb-forming and rooting culture is a light culture, the light culture time is 12-16 h / d, the light culture intensity is 800-1200 lx, and the bulb-forming and rooting culture time is 30-35 days.

[0019] Preferably, the clustered buds include clustered bud blocks, and the diameter of the clustered bud blocks is 1.5 to 2.0 cm.

[0020] Preferably, the temperature for both the bud-strengthening culture and the bulb-forming and rooting culture is 25±1℃.

[0021] Preferably, after obtaining the tissue culture seedlings, the process also includes hardening off and transplanting.

[0022] The beneficial effects of the present invention: The present invention provides a tissue culture medium for the regeneration of colored calla lilies, wherein the tissue culture medium includes an induction medium, a bud strengthening medium, and a bulb formation and rooting medium;

[0023] The induction medium uses MS medium as the basic medium and also includes: 0.5–4.0 mg / L 6-BA, 30 g / L sucrose and 6.5–7.0 g / L agar;

[0024] The bud-strengthening culture uses MS medium as the basic medium, which also includes: 1.0–2.5 mg / L 6-BA, 0–0.5 mg / L NAA, 0.5–2.0 mg / L carbon nanotubes, 40–60 g / L sucrose and 6.5–7.0 g / L agar;

[0025] The bulb-forming and rooting medium uses MS medium as the basic medium and also includes: 0.5–2.0 mg / L NAA, 0.5–2.0 mg / L carbon nanotubes, 40–60 g / L sucrose and 6.5–7.0 g / L agar.

[0026] In the tissue culture medium, the added NAA (naphthaleneacetic acid) and 6-BA (6-benzylaminopurine) stimulate cell division, promote rooting and shoot formation, and induce the formation of clustered shoots in colored calla lilies. Carbon nanotubes have a shoot-strengthening effect, promoting the differentiation and rooting of clustered shoots in colored calla lilies. The combined action of 6-BA, NAA, and carbon nanotubes facilitates the induction of the formation, proliferation, differentiation, and rooting of clustered seedlings of colored calla lilies. The results of the examples show that the tissue culture medium of this invention can obtain a large number of tissue-cultured colored calla lily seedlings, increasing the propagation coefficient of colored calla lilies. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0028] Figure 1 This is a slice of sterile bulblets of Calla Lily 'Jingcai Yangguang' (Golden Color Sunshine) from Example 1.

[0029] Figure 2 This is the 10th day of induction of aseptic pupae shoots from the thin slices of Calla Lily 'Jingcai Yangguang' in Example 1;

[0030] Figure 3 This is the situation on day 15 of induction of aseptic pupae from the colored calla lily 'Black Hole' on Example 2. Detailed Implementation

[0031] This invention provides a tissue culture medium for the regeneration of colored calla lilies, the tissue culture medium comprising an induction medium, a bud strengthening culture medium, and a bulb formation and rooting medium;

[0032] The induction medium uses MS medium as the basic medium and also includes: 0.5-4.0 mg / L 6-BA, 30 g / L sucrose and 6.5-7.0 g / L agar.

[0033] The induction medium of this invention uses MS medium as the basal medium and further includes: 0.5–5.0 mg / L 6-BA, 30 g / L sucrose, and 6.5–7.0 g / L agar. The concentration of 6-BA in the induction medium of this invention is 0.5–5.0 mg / L, preferably 1–3 mg / L, and more preferably 2 mg / L. The concentration of agar in the induction medium is 6.5–7.0 g / L, preferably 6.7 g / L. The 6-BA added in this invention can promote the formation of clustered shoots and improve the induction rate of clustered shoots.

[0034] In this invention, the pH value of the induction culture medium is preferably 5.75 to 5.85, more preferably 5.80.

[0035] The bud-enhancing culture medium of this invention uses MS medium as the basic medium and further includes: 1.0–2.5 mg / L 6-BA, 0–0.5 mg / L NAA, 0.5–2.0 mg / L carbon nanotubes, 40–60 g / L sucrose, and 6.5–7.0 g / L agar. NAA may or may not be added to the bud-enhancing culture medium; when NAA is added, the concentration of NAA in the bud-enhancing culture medium of this invention is >0 and ≤0.5 mg / L, preferably 0.1–0.4 mg / L, more preferably 0.2–0.3 mg / L. The concentration of 6-BA in the bud-enhancing culture medium is 1.0–2.5 mg / L, preferably 1.3–2 mg / L, more preferably 1.5 mg / L. The concentration of carbon nanotubes in the bud-enhancing culture medium is 0.5–2.0 mg / L, preferably 1.3–1.8 mg / L, more preferably 1.5 mg / L. The concentration of sucrose in the bud-strengthening culture medium is 40–60 g / L, preferably 45–55 g / L, and more preferably 50 g / L. The concentration of agar in the bud-strengthening culture medium is 6.5–7.0 g / L, preferably 6.8 g / L. In this invention, 6-BA, NAA, and carbon nanotubes work together to promote the proliferation of clustered seedlings of colored calla lilies and shorten the bud-strengthening culture time.

[0036] In this invention, the pH value of the bud-strengthening culture medium is preferably 5.75 to 5.85, more preferably 5.80.

[0037] The bulb-forming and rooting culture medium of the present invention uses MS medium as the basic culture medium and further includes: 0.5-2.0 mg / L NAA, 0.5-2.0 mg / L carbon nanotubes, 40-60 g / L sucrose and 6.5-7.0 g / L agar.

[0038] The concentration of NAA in the bulb-forming and rooting medium of this invention is 0.5–2.0 mg / L, preferably 1.0–1.7 mg / L, and more preferably 1.5 mg / L. The concentration of carbon nanotubes in the bulb-forming and rooting medium is 1.0–2.0 mg / L, preferably 1.1–1.6 mg / L, and more preferably 1.4 mg / L. The concentration of sucrose in the bulb-forming and rooting medium is 40–60 g / L, preferably 45–55 g / L, and more preferably 50 g / L. The concentration of agar in the bulb-forming and rooting medium is 6.5–7.0 g / L, and more preferably 6.7 g / L. This invention, at appropriate concentrations of NAA and carbon nanotubes, can complete the bulb-forming and rooting of colored calla lilies in one step, obtaining a large number of tissue culture seedlings and shortening the seedling cultivation time.

[0039] In this invention, the pH value of the bulb-forming and rooting culture medium is preferably 5.75 to 5.85, more preferably 5.80.

[0040] The present invention does not have any special limitations on the source of the MS culture medium, 6-BA, NAA, carbon nanotubes, sucrose and agar, and conventional commercial products can be used.

[0041] The carbon nanotubes described in this invention are preferably any one of single-walled carbon nanotubes, multi-walled carbon nanotubes, and carboxylated multi-walled carbon nanotubes; from a cost perspective, multi-walled carbon nanotubes are preferred.

[0042] This invention provides a method for tissue culture of clustered shoots of colored calla lilies. The method uses the induction culture medium described in the above-mentioned technical solution and includes the following steps:

[0043] Aseptic bulbils of colored calla lilies were inoculated onto an induction medium for induction culture to obtain clustered shoots.

[0044] This invention involves inoculating sterile offsets of colored calla lilies onto an induction medium for induced culture to obtain clustered shoots. This invention does not have specific requirements for the variety of colored calla lilies; conventional varieties can be used. In the embodiments of this invention, the colored calla lily 'Jingcai Yangguang' and / or the colored calla lily 'Heidong' are preferably used for effect verification. This invention preferably uses sterile offsets of colored calla lilies as explants for seedling cultivation.

[0045] In this invention, the sterile bulblets are preferably sterile bulblets from sterile seedlings of colored calla lilies that have already formed bulbs in tissue culture flasks; the diameter of the sterile bulblets is preferably 0.8–1.5 cm, more preferably 1.0–1.3 cm. The sterile bulblets are preferably cut into thin slices and then inoculated onto an induction medium for induction culture to obtain clustered shoots. The length and width of the sterile bulblet slices are preferably 0.3–0.8 cm, more preferably 0.3–0.4 cm, and the thickness of the sterile bulblet slices is preferably 0.1–0.2 cm, more preferably 0.1 cm. The method for preparing the sterile bulblet slices preferably includes: removing all leaves and petioles from the sterile seedling bulblets, and then transversely cutting the sterile bulblets to obtain sterile bulblet slices.

[0046] This invention utilizes sterile spore slices for cultivation, eliminating the need for cleaning and sterilization steps and simplifying the operation process.

[0047] Existing technologies for rapid propagation of colored calla lilies via tissue culture mostly use offshoots as explants to induce callus formation and establish a regeneration system. However, these methods suffer from problems such as long induction cycles, low induction rates, and low proliferation coefficients. This invention uses aseptic offshoots of colored calla lilies as explants for rapid propagation through tissue culture, establishing a highly efficient and rapid propagation technology system for colored calla lilies, and providing technical support for the large-scale production of colored calla lily bulbs.

[0048] After obtaining sterile bulblets, the present invention preferably inoculates the sterile bulblets of colored calla lily onto an induction culture medium for induction culture to obtain clustered shoots.

[0049] In this invention, the temperature for induction culture is preferably 24–26°C, more preferably 25°C. The induction culture is preferably conducted in the dark; the induction culture time is preferably 30–35 days, more preferably 30–33 days, and more preferably 32 days. The sprouting of buds from the tuber slices on the 7th day of induction culture indicates rapid germination and growth of the explants. The clustered buds preferably include clustered bud blocks; the diameter of the clustered bud blocks is preferably 1.5–2.0 cm. The induction culture time in this invention is calculated from the inoculation of sterile bulb slices into the induction medium until clustered bud blocks with a diameter of 1.5–2.0 cm are obtained.

[0050] This invention provides a method for the regeneration of colored calla lilies using tissue culture. The method employs the bud-strengthening culture medium and the bulb-forming and rooting culture medium described in the above-mentioned technical solution, and includes the following steps:

[0051] The clustered buds obtained by the above-mentioned clustered bud tissue culture method are transferred to a bud strengthening culture medium for bud strengthening culture to obtain clustered seedlings;

[0052] The clump-forming seedlings were transferred to a bulb-forming and rooting medium for bulb-forming and rooting culture to obtain tissue culture seedlings.

[0053] After obtaining the clustered buds, the present invention preferably separates the clustered buds from the sterile bulb slices and transfers them to a bud-strengthening culture medium for bud-strengthening culture to obtain clustered seedlings.

[0054] In this invention, the temperature for the vigorous bud cultivation is preferably 24–26°C, more preferably 25°C. The vigorous bud cultivation is preferably conducted under light; the duration of the light cultivation is preferably 12–16 h / d, more preferably 14–16 h / d, and more preferably 16 h / d; the intensity of the light cultivation is preferably 800–1200 lx, more preferably 1000–1100 lx. The vigorous bud cultivation period is preferably 30–35 days, more preferably 30–33 days. The vigorous bud cultivation period in this invention is calculated from the start of the transfer of the clustered bud blocks to the vigorous bud cultivation until the formation of clustered seedlings. The height of the clustered seedlings is preferably 3–6 cm, more preferably 4.5 cm.

[0055] After obtaining the clump-forming seedlings, the clump-forming seedlings are divided to obtain single clump-forming seedlings. In this invention, the single clump-forming seedlings are transferred to a bulb-forming and rooting medium for bulb-forming and rooting culture to obtain tissue culture seedlings. The preferred temperature for bulb-forming and rooting culture in this invention is 24–26℃, more preferably 25℃. In this invention, the bulb-forming and rooting culture is preferably light culture; the preferred light culture time is 12–16 h / d, more preferably 14–16 h / d, more preferably 16 h / d; the preferred light intensity is 800–1200 lx, more preferably 1000–1100 lx. The preferred culture time for bulb-forming and rooting culture in this invention is 30–35 days, more preferably 30–32 days.

[0056] The bulb-forming and rooting culture time described in this invention is calculated from the time of transfer to the bulb-forming and rooting culture medium until sterile bulblets with a diameter of 0.5–0.8 cm, a height of 6.0–9.0 cm, and 3–5 roots are obtained from the tissue culture seedlings of colored calla lilies. This invention completes the bulb-forming and rooting culture in one step, shortening the seedling cycle of colored calla lilies.

[0057] After obtaining the tissue culture seedlings, the present invention performs hardening and transplanting of the tissue culture seedlings.

[0058] The bulb circumference of the tissue culture seedlings described in this invention is preferably 0.5–0.8 cm, more preferably 0.6 cm. The root system of the test-tube seedlings described in this invention is preferably 3–5, more preferably 4. The height of the test-tube seedlings described in this invention is preferably 6.0–9.0 cm, more preferably 7.0 cm.

[0059] The test-tube seedlings were hardened off and transplanted after meeting three conditions: the diameter of the sterile bulbs was 0.5-0.8 cm, the height was 6.0-9.0 cm, and there were 3-5 roots, in order to improve the survival rate of the tissue culture seedlings.

[0060] After obtaining the tissue culture seedlings, the present invention preferably performs hardening-off and transplanting on the seedlings. In the present invention, the hardening-off is preferably completed indoors, the hardening-off temperature is preferably the temperature for the growth of colored calla lilies, and the hardening-off time is preferably 4-6 days, more preferably 5-6 days. In the present invention, when hardening off the seedlings, the caps of the tissue culture bottles containing the rooted seedlings are loosened, and the bottles are first placed in the buffer room of the tissue culture room for 2-3 days, and then placed under natural diffused light for 2-3 days.

[0061] In this invention, during transplanting, the test-tube seedlings, after being hardened off, are preferably washed of their roots and then transplanted into a substrate for cultivation. The substrate for transplanting and cultivation preferably includes peat moss, perlite, and garden soil. The preferred volume ratio of peat moss, perlite, and garden soil in this invention is (1-2):(1-2):(1-2). Before transplanting, the substrate is preferably disinfected. This invention preferably uses an 800-1000 times dilution of a broad-spectrum fungicide for disinfection, more preferably 800 times. The preferred broad-spectrum fungicide in this invention is chlorothalonil.

[0062] This invention does not have any special limitations on the source and specifications of the seedling trays used in the transplanting and cultivation process; conventional products are acceptable. The preferred specifications of the seedling trays used in this invention are 10 rows × 10 columns. After transplanting, sufficient water should be applied to help the roots establish, followed by normal water and fertilizer management.

[0063] This invention uses sterile bulblets of colored calla lily as explants. The sterile bulblets are cut into thin slices to induce the production of clustered buds. After bud strengthening and rooting culture, sterile seedlings are obtained, establishing a high-efficiency and rapid propagation technology system for colored calla lily, providing technical support for the large-scale production of colored calla lily bulblets.

[0064] The innovation of this invention lies in using sterile bulblets of colored calla lilies as explants to induce the formation of clustered shoots. Through shoot strengthening culture, in-vitro bulb formation culture, and rooting culture, a rapid propagation technology system is established, providing technical support for the establishment of a large-scale tissue culture rapid propagation system for colored calla lily seedlings. The advantages of this invention are that the sterile bulblets of colored calla lilies induce a high rate and rapid occurrence of clustered shoots, and the shoots are relatively robust. In-vitro bulb formation and rooting culture can be carried out without the need for shoot strengthening or with only one shoot strengthening cycle, thereby completing the cultivation of tissue culture seedlings, shortening the seedling growth cycle, saving seedling costs, and also greatly advancing the corresponding molecular biology research process.

[0065] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0066] Example 1: Establishment of a rapid propagation technology system for the yellow calla lily variety 'Jingcai Yangguang'

[0067] (1) Obtaining explants. Sterile bulbils of 'Jingcai Yangguang' sterile seedlings that had completed bulb formation in the bottle were used as explants. The diameter of the sterile bulbils was about 0.8 to 1.5 cm.

[0068] (2) Induction of clustered shoots. Sterile bulblets with a diameter of about 0.8 to 1.5 cm were placed on an inoculation tray. All leaves were removed. The sterile bulblets were then cut into small pieces (i.e., sterile bulblet slices) with a length and width of about 0.8 cm and a thickness of about 0.2 cm. The sterile bulblet slices were then inoculated onto the shoot induction medium for induction culture.

[0069] The bud induction medium consisted of MS medium as the basal medium, with the addition of 3.0 mg / L 6-BA, 30 g / L sucrose, and 7.0 g / L agar; the pH of the induction medium was 5.8.

[0070] Induction culture was conducted in the dark. The induction culture temperature was 25±1℃. After 5 days of induction culture, the explants began to sprout, forming a layer of pale yellow, frosty callus tissue on the surface. After another week of culture, green clustered shoots sprouted around the aseptic bulblets. The total induction culture time was 30 days. Clustered shoots were obtained after the induction culture was completed.

[0071] (3) Sprout cultivation. The clustered buds are cut off from the sterile bulb slices and transferred to the sprout cultivation medium for sprout cultivation.

[0072] The bud-strengthening medium consisted of MS medium as the basal medium, supplemented with 2.0 mg / L 6-BA, 0.5 mg / L NAA, 0.5 mg / L multi-walled carbon nanotubes, 50 g / L sucrose, and 6.7 g / L agar; the pH of the bud-strengthening medium was 5.8.

[0073] The temperature for bud cultivation was 25℃, the light intensity was 1200 lx, and the light duration was 16 h / d. After 35 days of cultivation, the clustered buds grew into small seedlings with a height of approximately 6.5 cm.

[0074] (4) In-bottle bulb formation and rooting culture. In-bottle bulb formation and rooting culture are completed in one step. The clustered seedlings are divided into individual seedlings. The individual seedlings are then transferred to bulb formation and rooting medium for in-bottle bulb formation and rooting culture (i.e., bulb formation and rooting culture).

[0075] The composition of the bulb-forming and rooting medium is as follows: MS medium is used as the basic medium, and 2.0 mg / L NAA, 0.5 mg / L multi-walled carbon nanotubes, 60 g / L sucrose and 7.0 g / L agar are added; the pH of the bulb-forming and rooting medium is 5.8.

[0076] The culture temperature for bulb formation and rooting was 25±1℃, the light intensity was 1200lx, and the photoperiod was 16h / d. After 35 days of culture, individual seedlings grew into sterile bulbs with a diameter of about 0.8cm, a height of about 8.0cm, and 3-5 roots in test tubes.

[0077] (5) Hardening off and transplanting. Hardening off was carried out indoors, specifically as follows: after loosening the caps of the tissue culture bottles obtained from bulb-rooting culture, they were placed in the buffer room of the tissue culture room for 2 days, then moved into the indoor room and placed under natural diffused light for another 3 days, for a total hardening off of 5 days. After hardening off, the test-tube seedlings were removed with tweezers, the culture medium was washed off the roots, and they were transplanted into 10×10 seedling trays. The cultivation substrate consisted of peat moss, perlite, and garden soil, with a volume ratio of 2:2:1. The cultivation substrate was disinfected with 800 times diluted broad-spectrum fungicide chlorothalonil before use. Normal water and fertilizer management was carried out after transplanting.

[0078] Example 2: Establishment of a rapid propagation technology system for colored calla lily 'Black Hole' tissue culture

[0079] (1) Obtaining explants. Sterile bulbils of 'black hole' sterile seedlings that have completed bulb formation in the bottle were used as explants. The diameter of the sterile bulbils was about 0.8 to 1.5 cm.

[0080] (2) Induction of clustered shoots. Place sterile bulblets with a diameter of about 0.8 to 1.5 cm on an inoculation tray, remove all leaves, and longitudinally cut the sterile bulblets into small pieces with a length and width of about 0.5 cm and a thickness of about 0.1 cm (i.e., sterile bulblet slices). The sterile bulblet slices are then inoculated onto callus induction medium for induction culture.

[0081] The bud induction medium consisted of MS medium as the basal medium, with the addition of 2.0 mg / L 6-BA, 30 g / L sucrose, and 6.9 g / L agar; the pH of the induction medium was 5.8.

[0082] Induction culture was conducted in the dark. After 3 days of induction culture, the explants began to sprout, forming a layer of pale yellow, frosty callus tissue on their surface. After 7 days of induction culture, a layer of pale yellow callus tissue formed on the surface of the sections. After another 10 days of culture, green shoot clusters sprouted around the sections. The induction culture period was 33 days, and clustered shoots were obtained after the induction culture was completed.

[0083] (3) Shoot Growth Culture. The clustered shoots were separated from the sterile bulblet slices and transferred to a shoot growth culture medium. The medium consisted of MS as the basic medium, supplemented with 40 g / L sucrose, 6.8 g / L agar, 1.5 mg / L 6-BA, 0.5 mg / L NAA, and 1.0 mg / L multi-walled carbon nanotubes. The culture temperature was 25 ± 1℃, the light intensity was 1200 lx, and the photoperiod was 16 h / d. After 32 days of culture, the clustered shoots grew into small seedlings approximately 5.8 cm in height.

[0084] (4) Intra-vial bulb formation and rooting culture (i.e., bulb-rooting culture). Intra-vial bulb formation and rooting culture were completed in one step. After dividing the clump of seedlings into individual plants, individual plantlets were obtained. The individual plantlets were transferred to bulb-rooting medium for intra-vial bulb formation and rooting culture. The composition of the bulb-rooting medium was: MS basic medium, supplemented with 50 g / L sucrose, 6.9 g / L agar, 1.0 mg / L NAA, and 2.0 mg / L multi-walled carbon nanotubes. The culture temperature was 25 ± 1℃, the light intensity was 1200 lx, and the photoperiod was 16 h / d. After 35 days of culture, the individual plantlets grew into sterile bulbs with a diameter of 0.6 cm, a height of about 7.0 cm, and 3-5 roots in test tubes.

[0085] (5) Hardening off and transplanting. Hardening off was carried out indoors, specifically as follows: after loosening the caps of the tissue culture bottles obtained from bulb-rooting culture, they were placed in the buffer room of the tissue culture room for 3 days, then moved into the indoor room and placed under natural diffused light for another 3 days, for a total hardening off of 6 days. After hardening off, the test-tube seedlings were removed with tweezers, the culture medium was washed off the roots, and they were transplanted into 10×10 seedling trays. The cultivation substrate was peat moss: perlite and garden soil, with a volume ratio of peat moss, perlite and garden soil of 2:2:1. The cultivation substrate was disinfected with a 900-fold dilution of broad-spectrum fungicide, chlorothalonil, before use. Normal water and fertilizer management was carried out after transplanting.

[0086] Examples 3-8 and Comparative Examples 1-2

[0087] Examples 3-8 and Comparative Examples 1-2 were only induced cultured, with the same steps (1) and (2) as in Example 1. The only difference was the composition of the induction medium. The specific composition of the induction medium is shown in Table 1.

[0088] Example 7 will be used as an example for illustration, wherein the specific steps of Example 7 are as follows:

[0089] Example 7

[0090] (1) Obtaining explants. Sterile bulbils of 'Jingcai Yangguang' sterile seedlings that had completed bulb formation in the bottle were used as explants. The diameter of the sterile bulbils was about 0.8 to 1.5 cm.

[0091] (2) Induction of clustered shoots. Sterile bulblets with a diameter of about 0.8 to 1.5 cm were placed on an inoculation tray. All leaves were removed. The sterile bulblets were then cut into small pieces (i.e., sterile bulblet slices) with a length and width of about 0.8 cm and a thickness of about 0.2 cm. The sterile bulblet slices were then inoculated onto the shoot induction medium for induction culture.

[0092] The bud induction medium consisted of MS medium as the basal medium, with the addition of 3.5 mg / L 6-BA, 30 g / L sucrose, and 7.0 g / L agar; the pH of the induction medium was 5.8.

[0093] Induction culture was conducted in the dark. The induction culture temperature was 25±1℃. After 5 days of induction culture, the explants began to sprout, forming a layer of pale yellow, frosty callus tissue on the surface. After another week of culture, green shoot clusters sprouted around the aseptic bulblets. The total induction culture time was 30 days. After the induction culture was completed, clustered shoots were obtained.

[0094] The formula for calculating the bud induction rate (%) is as follows:

[0095] Bud induction rate (%) = Number of sterile bulblets that sprouted × 100 / Total number of inoculated bulblets

[0096] Table 16 - Effects of BA concentration on bud induction rate of the yellow calla lily cultivar 'Jingcai Yangguang'

[0097]

[0098]

[0099] Examples 9-16 and Comparative Examples 3-13

[0100] Examples 9-16 and Comparative Examples 3-13 underwent only bud strengthening culture, following the same steps (1)-(3) as in Example 1, with the only difference being the composition of the bud strengthening culture medium. The specific composition of the bud strengthening culture medium is shown in Table 2. The bud strengthening culture time for Examples 9-16 and Comparative Examples 3-13 was 30 days. The clustered buds were cut and separated from the sterile bulblets and transferred to the bud strengthening culture medium at a height (i.e., seedling height) of 1.5 cm. The seedling height and leaf number data measured after 30 days of bud strengthening culture are shown in Table 3.

[0101] Table 2. Composition of the bud-strengthening culture medium for Examples 1-2, Examples 9-16, and Comparative Examples 3-13.

[0102]

[0103]

[0104] Table 3. Effects of NAA concentration in the bud-strengthening medium on bud growth of the yellow calla lily cultivar 'Jingcai Yangguang'

[0105] serial number Seedling height / cm Number of leaves / blades Comparative Example 3 2.0 2 Comparative Example 4 2.5 2 Comparative Example 5 2.8 2 Comparative Example 6 3.0 2 Example 9 4.5 3 Example 10 5.0 3 Example 11 5.5 3 Comparative Example 11 3.5 3 Example 12 4.8 4 Example 2 5.8 4 Example 13 6.2 4 Example 1 6.5 4 Comparative Example 12 4.0 4 Example 14 5.8 4 Example 15 6.5 4 Example 16 7.5 4 Comparative Example 13 4.6 4 Comparative Example 7 4.0 3 Comparative Example 8 4.2 3 Comparative Example 9 3.9 3 Comparative Example 10 4.1 3

[0106] Examples 17-18 and Comparative Examples 14-15

[0107] Examples 17-18 and Comparative Examples 14-15 only underwent in-bottle bulb formation and rooting culture, following the same steps (1)-(4) as in Example 1. The only difference was the composition of the bulb formation and rooting culture medium, as shown in Table 4. The bulb formation and rooting culture time for Examples 17-18 and Comparative Examples 14-15 was 30 days. The diameter of the sterile bulbs and the number of roots were measured after 30 days of bulb formation and rooting culture, and the data are shown in Table 4. It can be seen that when the NAA concentration is 0.5-2.0 mg / L, the diameter of the sterile bulbs is large and the number of roots is high.

[0108] Table 4. Effects of NAA concentration on the enlargement of sterile bulbs of the yellow calla lily cultivar 'Jingcai Yangguang'

[0109]

[0110] Comparative Examples 16–20

[0111] Comparative samples of 16-20 tissue samples were subjected to induction culture. The induction culture steps were the same as in Example 7, the only difference being the basal medium of the induction medium. The specific composition of the induction medium is shown in Table 5. According to Table 5, MS as the basal medium resulted in the best tissue culture effect.

[0112] Table 5. Effects of basic culture medium on the bud induction rate of sliced ​​Calla Lily 'Jingcai Yangguang'

[0113]

[0114]

[0115] In summary, the technical solution of this invention enables a highly efficient and rapid propagation system for colored calla lilies, and also allows for exploration of its transgenic technology system. This invention uses sterile bulblets as explants to establish a rapid propagation system for colored calla lilies based on sterile bulblet slices, providing technical support for the propagation and genetic transformation of colored calla lilies using tissue culture methods, and laying the foundation for its industrial application.

[0116] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A tissue culture medium for the regeneration of colored calla lilies, characterized in that, The tissue culture medium includes induction medium, bud strengthening medium, and bulb formation and rooting medium; The induction medium was MS medium + 0.5~4.0 mg / L 6-BA + 30 g / L sucrose + 6.5~7.0 g / L agar; The bud-strengthening medium consisted of MS medium + 1.0~2.5 mg / L 6-BA + 0~0.5 mg / L NAA + 0.5~2.0 mg / L carbon nanotubes + 40~60 g / L sucrose + 6.5~7.0 g / L agar; The bulb-forming and rooting medium consisted of MS medium + 0.5~2.0 mg / L NAA + 0.5~2.0 mg / L carbon nanotubes + 40~60 g / L sucrose + 6.5~7.0 g / L agar; The explants for the regeneration of colored calla lilies are sterile bulblets.

2. A method for tissue culture of clustered shoots of colored calla lilies, characterized in that, The method for cultivating clustered shoots uses the induction medium described in claim 1 and includes the following steps: Sterile bulblets of colored calla lilies were inoculated onto an induction medium for induction culture to obtain clustered shoots.

3. The method for tissue culture of clustered buds according to claim 2, characterized in that, The aseptic pellet sheet has a length and width of 0.3~0.8cm and a thickness of 0.1~0.2cm.

4. The method for tissue culture of clustered buds according to claim 2, characterized in that, The induction culture is a dark culture; the induction culture time is 30~35 days; the induction culture temperature is 25±1℃.

5. A method for regenerating colored calla lilies using tissue culture, characterized in that, The regeneration tissue culture method uses the bud-strengthening culture medium and bulb-forming and rooting culture medium described in claim 1, and includes the following steps: The clustered buds obtained by the clustered bud tissue culture method according to any one of claims 2 to 4 are transferred to the bud strengthening culture medium for bud strengthening culture to obtain clustered seedlings; The clump-forming seedlings were transferred to a bulb-forming and rooting medium for bulb-forming and rooting culture to obtain tissue culture seedlings.

6. The regeneration tissue culture method according to claim 5, characterized in that, The bud strengthening culture is a light culture; the bud strengthening culture time is 30-35 days; the light culture time is 12-16 hours / day; and the light culture intensity is 800-1200 lx.

7. The regeneration tissue culture method according to claim 5, characterized in that, The bulb-forming and rooting culture is a light culture, the light culture time is 12~16h / d, the light culture intensity is 800~1200lx, and the bulb-forming and rooting culture time is 30~35d.

8. The regeneration tissue culture method according to claim 5, characterized in that, The clustered buds include clustered bud blocks, the diameter of which is 1.5~2.0cm.

9. The regeneration tissue culture method according to claim 5, characterized in that, The temperature for both the bud-strengthening culture and the bulb-forming and rooting culture was 25±1℃.

10. The regeneration tissue culture method according to claim 5, characterized in that, After obtaining the tissue culture seedlings, the process also includes hardening off and transplanting.

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