Culture medium combination and method for constructing colored zantedeschia regeneration system by using tubers

Abstract

The invention relates to a culture medium combination and method for constructing a zantedeschia aethiopica regeneration system by tubers, the culture medium combination comprises a callus induction culture medium, a proliferation culture medium and a differentiation culture medium, the induction culture medium takes MS as a basic culture medium, and also contains 6.5-7.0 g / L of agar, 30 g / L of sucrose, 1.0-2.0 mg / L of 6-BA and 0.5-1.0 mg / L of NAA; or the culture medium also contains the following substances in concentration: 6.5-7.0 g / L of agar, 30 g / L of cane sugar, 0.5 mg / L of 6-BA and 0.8 mg / L of NAA. According to the method, the sterile bud-free tubers of the colored callalily growing to the specific period are used as the explants, the specific induction culture medium, the proliferation culture medium and the differentiation culture medium are matched to obtain the cluster buds, the callus induction rate is high, and technical support is provided for establishment of a colored callalily tissue culture rapid propagation and genetic transformation system.

Description

Technical Field

[0001] The invention belongs to the field of constructing a color calla lily regeneration system, and particularly relates to a culture medium combination and a method for constructing a color calla lily regeneration system by utilizing tubers. Background Art

[0002] At present, the industrial production method of colored calla bulbs is tissue culture and rapid propagation. The commonly used method is to use tubers with buds as explants, disinfect and sterilize them, induce the production of adventitious buds, and then obtain test tube seedlings after proliferation, strong buds, strong seedlings and rooting culture. With the completion of the genome sequencing of yellow calla, the verification of the gene function of colored calla is urgently needed. Establishing a regeneration system based on callus tissue is the prerequisite for conducting this research.

[0003] ZL201910992841.8 provides a method for inducing callus tissue and establishing a regeneration system using colored calla lily bulbs as explants. However, there are problems such as long induction cycle and low induction rate.

[0004] Finding a method to establish a callus-based colored calla lily regeneration system with high induction rate and short induction cycle has always been one of the research directions of experts in this field.

[0005] The present invention is hereby proposed. Summary of the invention

[0006] In view of the shortcomings and defects of the prior art, one of the purposes of the present invention is to provide a culture medium combination for constructing a color calla lily regeneration system using tubers. The culture medium combination is proposed based on the regeneration system of callus tissue. The culture medium combination provided by the present invention can shorten the callus induction period and improve the callus induction efficiency by cooperating with the corresponding explants.

[0007] The second object of the present invention is to provide a method for constructing a regeneration system of colored calla lily using tubers. The method uses budless tubers of colored calla lily grown to a specific size as explants, induces the production of callus tissue, and establishes its regeneration system through proliferation culture and differentiation, thereby providing technical support for the establishment of a genetic transformation system of colored calla lily and the large-scale production of seedlings, especially the establishment of a regeneration system of colored calla lily, and providing technical support for the establishment of an Agrobacterium-mediated genetic transformation system.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] The first aspect of the present invention provides a culture medium combination for constructing a color calla lily regeneration system using tubers, the culture medium combination comprising:

[0010] Callus induction medium: MS as the basic medium, further containing the following substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L, NAA 0.5-1.0 mg / L; or, MS as the basic medium, further containing the following substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 0.5 mg / L, NAA 0.8 mg / L;

[0011] Callus proliferation medium: MS as the basic medium, also contains the following concentrations of substances: agar 6.5-7.0g / L, sucrose 30g / L, 6-BA 3.0-4.0mg / L;

[0012] Differentiation medium: MS as the basic medium, also contains the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L.

[0013] Furthermore, the culture medium combination also includes:

[0014] Bud growth medium: MS-based medium, also contains the following concentrations of substances: sucrose 40-60g / L, agar 6.5-7.0g / L, NAA 0.3-0.5mg / L;

[0015] Furthermore, the culture medium combination also includes:

[0016] Rooting medium: MS is used as the basic medium, and it also contains the following substances in the following concentrations: sucrose 40-60g / L, agar 6.5-7.0g / L, and IBA 1.0-2.0mg / L.

[0017] Preferably, the concentrations of 6-BA and NAA in the callus induction medium are as follows: 6-BA 1.0-2.0 mg / L, NAA 0.8 mg / L.

[0018] Furthermore, the pH of the callus induction medium, the callus proliferation medium, the callus differentiation medium, the budding medium, and the rooting medium are all 5.75-5.85.

[0019] The second aspect of the present invention provides a method for constructing a color calla lily regeneration system using tubers, the method comprising the following steps:

[0020] S1 Selection and treatment of explants: Select sterile colored calla lily seedling tubers that have completed the balling in the bottle as explants, wherein the tuber diameter is in the range of 0.8 to 1.2 cm; first remove the leaves and buds on the tubers, and then cut them into small pieces for callus culture;

[0021] S2 callus induction culture: inoculating the small explants onto callus induction medium for induction culture, wherein the callus induction medium is based on MS and further contains the following substances at the following concentrations: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L, NAA 0.5-1.0 mg / L; or, based on MS and further contains the following substances at the following concentrations: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 0.5 mg / L, NAA 0.8 mg / L;

[0022] S3 callus proliferation culture: transferring the small explant with callus induced and cultured in step S2 to a callus proliferation medium for proliferation culture, wherein the callus proliferation medium is based on MS and further contains the following substances at the following concentrations: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 3.0-4.0 mg / L;

[0023] S4 callus differentiation culture: transferring the callus mass obtained in step S3 to a differentiation medium for bud differentiation culture to obtain clustered buds; the differentiation medium is based on MS and further contains the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L;

[0024] S5 strong bud culture: inoculating the clustered buds into a strong bud culture medium for strong bud culture;

[0025] S6 Rooting culture: The rootless seedlings obtained after the strong bud culture are subjected to rooting culture.

[0026] Furthermore, the size of the small explant is: 0.3-0.4 cm in length and width, and 0.1-0.3 cm in thickness;

[0027] And / or, the induction culture conditions are: dark culture, the number of days of dark culture is 30-40 days;

[0028] And / or, the proliferation culture conditions are: dark culture, the number of days of dark culture is 35-40 days;

[0029] and / or, obtaining a callus mass with a diameter of 1.0-1.5 cm after the proliferation culture;

[0030] And / or, the conditions of the bud differentiation culture are: light-dark alternating culture, wherein the light intensity is 800-1200Lx, the light duration is 15-17h per day, and the culture temperature is 25±1°C;

[0031] And / or, the bud differentiation culture time is 30-40 days;

[0032] And / or, the variety of the colorful calla lily is Black Hole or Jingcai Sunshine.

[0033] Furthermore, the bud-strengthening medium is based on MS and also contains the following substances at the following concentrations: sucrose 40-60 g / L, agar 6.5-7.0 g / L, NAA 0.3-0.5 mg / L;

[0034] And / or, the rooting medium is based on MS and further contains the following substances in concentrations: sucrose 40-60 g / L, agar 6.5-7.0 g / L, and IBA 1.0-2.0 mg / L.

[0035] Furthermore, the height of the rootless seedling is 5.0-7.0 cm, and the diameter of the tuber is 0.5-0.8 cm;

[0036] And / or, the conditions for the strong bud culture are: culture temperature 25±1°C, light and dark alternation culture, wherein the light intensity is 800-1200Lx, and the light time is 15-17h per day;

[0037] And / or, the time of culturing the strong buds is 30-35 days;

[0038] And / or, the rooting culture conditions are: culture temperature 25±1°C, light and dark alternating culture, wherein the light intensity is 800-1200Lx, and the light time is 15-17h per day;

[0039] And / or, the rooting culture time is 30-35 days;

[0040] And / or, the test tube plantlets obtained after the rooting culture have a height of 6.0-10.0 cm, 5-7 roots, and a tuber diameter of 0.5-0.8 cm.

[0041] Furthermore, the method further comprises:

[0042] Step S7: hardening and transplanting: hardening the test tube seedlings obtained by rooting culture and then transplanting them into a cultivation medium for cultivation. Preferably, the cultivation medium includes peat soil, perlite, and garden soil, wherein the mass ratio of peat soil:perlite:garden soil is 2:2:1.

[0043] Compared with the prior art, the present invention has the following advantages:

[0044] Firstly, the present invention uses sterile budless tubers of colored calla lily grown to a specific period (for example, the tuber diameter is about 1.0 cm) as explants, cooperates with specific callus induction medium, proliferation medium and differentiation medium to obtain clustered buds, and establishes its regeneration system. The establishment of the regeneration system provides technical support for the establishment of tissue culture rapid propagation and genetic transformation system of colored calla lily, especially provides technical support for the establishment of Agrobacterium-mediated genetic transformation system.

[0045] Secondly, the method of the present invention can improve the induction rate of callus tissue and the speed of callus tissue generation. Generally, after about 20 days of induction culture, light yellow frosty callus tissue attached to the explant can be seen, and the buds differentiated from the callus tissue are relatively strong. No bud growth is required or only one bud growth cycle is required to carry out rooting culture, thereby completing the cultivation of tissue culture seedlings, shortening the seedling cycle, saving the cost of seedling cultivation, and greatly promoting the corresponding molecular biology research process. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 These are pictures of callus induction of the Calla Lily variety 'Jing Cai Yang Guang', where (a) is a picture of the explants transplanted into the callus induction medium on the first day, and (b) is a picture of the explants transplanted into the callus induction medium on the tenth day;

[0047] Figure 2 This is a picture of the callus mass obtained after the callus tissue of the Calla Lily variety 'Jingcai Yangguang' was transferred into the proliferation medium;

[0048] Figure 3 This is a photo of the clustered buds differentiated from the callus tissue of the Calla Lily variety 'Jingcai Sunshine'. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention.

[0050] The embodiments of the present invention are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and processes are given, but the protection scope of the present invention is not limited to the following embodiments. The process parameters of the following embodiments that do not specify specific conditions are usually based on conventional conditions.

[0051] The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in the present invention.

[0052] The process parameters without specific conditions in the following examples are usually based on conventional conditions. The raw materials described in the following examples can be obtained from public commercial channels.

[0053] The first aspect of the present invention provides a culture medium combination for constructing a color calla lily regeneration system using tubers, comprising:

[0054] Callus induction medium: MS as the basic medium, and also containing the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L, NAA 0.5-1.0 mg / L, or MS as the basic medium, and also containing the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 0.5 mg / L, NAA 0.8 mg / L;

[0055] Callus proliferation medium: MS as the basic medium, also contains the following concentrations of substances: agar 6.5-7.0g / L, sucrose 30g / L, 6-BA 3.0-4.0mg / L;

[0056] Differentiation medium: MS as the basic medium, also contains the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L.

[0057] The culture medium combination of the present invention is screened for specific explants, and the sterile seedling tubers that have completed the ball in the bottle are used as explants. The above callus induction medium, proliferation medium and differentiation medium are used to efficiently induce callus, with a high induction rate, fast callus occurrence, and strong clustered buds. The 6-BA concentration in the callus induction medium should not be too high. Too high a concentration will reduce the callus occurrence rate and even reduce the callus induction rate. The 6-BA concentration should not be too low. If the 6-BA concentration is too low, it will usually lead to a low induction rate. The concentration of NAA also needs to be adapted to the 6-BA concentration, otherwise it will lead to a low induction rate. When the NAA concentration is lower than 0.5 mg / L, the induction rate is below 40%. When other cytokinins such as KT are used to replace the 6-BA of the present invention, or when other auxins such as 2,4-D are used to replace the NAA of the present invention, callus cannot occur on the surface of the explant. Preferably, the concentrations of 6-BA and NAA in the callus induction medium are as follows: 6-BA 1.0-2.0 mg / L, NAA 0.8 mg / L. The induction rate of the preferred callus induction medium can reach more than 70%.

[0058] In the regeneration system of the present invention, it is not suitable to add auxin substances to the proliferation culture medium, nor is it suitable to replace the cytokinin 6-BA with other types of cytokinins. Adding auxin or replacing the cytokinin 6-BA of the present invention with other cytokinins will lead to a significant decrease in the proliferation coefficient.

[0059] In the regeneration system of the present invention, it is not suitable to add auxin substances to the differentiation medium, as the addition of auxin will lead to a decrease in the differentiation coefficient. In addition, the concentration of 6-BA should not be too high or too low, otherwise the differentiation coefficient will be reduced.

[0060] In the regeneration system of the present invention, it is not suitable to add cytokinins to the bud-strengthening culture medium, because adding cytokinins will reduce the effect of NAA.

[0061] In the regeneration system of the present invention, for the rooting medium, the auxin IBA has a better rooting effect than other auxins, and adding 6-BA to the rooting medium will reduce the rooting rate.

[0062] After the cluster buds are differentiated from the differentiation medium, the cluster buds need to be strengthened and rooted or directly strengthened. The formula of the bud strengthening medium of the medium combination of the present invention is preferably: MS is the basic medium, and it also contains the following concentrations of substances: sucrose 40-60g / L, agar 6.5-7.0g / L, NAA0.3-0.5mg / L.

[0063] The formula of the rooting medium of the medium combination of the present invention is preferably: MS as the basic medium, and further containing the following substances in concentrations: sucrose 40-60 g / L, agar 6.5-7.0 g / L, and IBA 1.0-2.0 mg / L.

[0064] In order to ensure the culture effect, the pH of the callus induction medium, the callus proliferation medium, the callus differentiation medium, the differentiation medium and the rooting medium are all 5.75-5.85.

[0065] The second aspect of the present invention provides a method for constructing a color calla lily regeneration system using tubers, comprising the following steps:

[0066] S1 Selection and processing of explants: Select sterile tubers of Calla lily seedlings that have completed ball formation in a bottle as explants, wherein the diameter of the tubers is 0.8-1.2 cm; first remove the leaves and buds on the tubers, and then cut them into small pieces of explants for callus tissue culture; in the example of the present invention, the size of the small pieces of explants is: 0.3-0.4 cm in length and width, and 0.1-0.3 cm in thickness; the method for obtaining the sterile tubers of Calla lily seedlings that have completed ball formation in a bottle can adopt the existing technical methods for tissue culture and rapid propagation of colored Calla lily, for example, refer to steps (1)-(3) of Example 1 in the patent document with publication number CN 112042532B.

[0067] S2 callus induction culture: inoculating the small explants onto callus induction medium for induction culture, wherein the callus induction medium is based on MS and further contains substances with the following concentrations: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L, NAA 0.5-1.0 mg / L or based on MS and further contains substances with the following concentrations: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 0.5 mg / L, NAA 0.8 mg / L; in the example of the present invention, the induction culture conditions are: dark culture, and the number of days of dark culture is 30-40 days;

[0068] Specifically, a sterile seedling with a tuber (0.8-1.2 cm in diameter) is placed on an inoculation tray, all leaves and buds are removed, and the tuber is cut longitudinally into small pieces with a length and width of 0.3-0.4 cm and a thickness of about 0.2 cm, and inoculated on a callus induction medium for dark culture. After about 20 days, most of the explants sprout and form a layer of light yellow frosty callus on the surface. Continue to culture, and the callus continues to thicken. The selection of explants is closely related to the culture medium of the present invention. The culture medium combination of the present invention is suitable for using tubers that grow to a size of 0.8-1.2 cm as explants; tubers that grow too small or too large are not suitable for the culture medium of the present invention, especially when they grow too large, cell differentiation is more thorough and not easy to dedifferentiate, and the culture cycle is also relatively long, and growing to about 1 cm is the most suitable.

[0069] S3 callus proliferation culture: the small explant with callus induced by step S2 is transferred to a callus proliferation medium for proliferation culture, wherein the callus proliferation medium is based on MS and further contains the following substances at the following concentrations: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 3.0-4.0 mg / L; in the example of the present invention, after the proliferation culture, a callus mass with a diameter of 1.0-1.5 cm is obtained; the conditions of the proliferation culture are: dark culture, and the number of days of dark culture is 35-40 days;

[0070] Specifically, 35-40 days after induction culture, the explants together with the thickened callus are transferred to a proliferation medium for proliferation culture in the dark. After 35-40 days, the callus proliferates into a mass with a diameter of 1.0-1.5 cm.

[0071] S4 callus differentiation culture: the callus mass obtained in step S3 is transferred to a differentiation medium for bud differentiation culture to obtain clustered buds; the differentiation medium is based on MS and also contains the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L; in the example of the present invention, the conditions for the bud differentiation culture are: alternating light and dark culture, wherein the light intensity is 800-1200 Lx, the light duration is 15-17 h per day, and the culture temperature is 25±1°C; the bud differentiation culture time is 30-40 days.

[0072] Specifically, callus masses with a diameter of 1.0-1.5 cm are transferred to a differentiation medium for bud differentiation culture. After 30-40 days of culture, the callus differentiates into clustered buds.

[0073] S5 strong bud culture: inoculating the clustered buds into a strong bud culture medium for strong bud culture; the strong bud culture medium is preferably based on MS and further contains the following concentrations of substances: sucrose 40-60 g / L, agar 6.5-7.0 g / L, NAA 0.3-0.5 mg / L; in the example of the present invention, the conditions for the strong bud culture are: culture temperature 25±1°C, light and dark alternating culture, wherein the light intensity is 800-1200 Lx, and the light time is 15-17 h per day; the height of the rootless seedlings is 5.0-7.0 cm, and the diameter of the tubers is 0.5-0.8 cm; the time for the strong bud culture is 30-35 days;

[0074] Specifically, the clustered buds are separated from the callus tissue and transferred to a strong bud culture medium for strong bud culture. After 30-35 days of culture, the clustered buds grow into rootless seedlings with a height of 5.0-7.0 cm and a tuber diameter of 0.5-0.8 cm.

[0075] S6 Rooting culture: The rootless seedlings obtained after the strong bud culture are subjected to rooting culture, wherein the rooting culture medium is preferably based on MS and further contains the following substances at the following concentrations: sucrose 40-60 g / L, agar 6.5-7.0 g / L, IBA 1.0-2.0 mg / L; in the example of the present invention, the rooting culture conditions are: culture temperature 25±1°C, light and dark alternating culture, wherein the light intensity is 800-1200 Lx, and the light duration is 15-17 h per day; the rooting culture time is 30-35 days;

[0076] Specifically, the leaves of the rootless seedlings with a height of 5.0-7.0 cm were cut off, and only the petioles with a length of about 1.0 cm were retained, and the seedlings were transferred to the rooting medium for rooting culture. After 30-35 days of culture, the seedlings grew into test tube seedlings with a height of 6.0-10.0 cm, 5-7 roots, and a tuber diameter of 0.5-0.8 cm.

[0077] S7 Hardening and transplanting: Hardening the test tube seedlings obtained by rooting culture is then transplanted into a cultivation medium for cultivation. Preferably, the cultivation medium comprises peat soil, perlite, and garden soil, wherein the mass ratio of peat soil:perlite:garden soil is 2:2:1.

[0078] Specifically, the seedling hardening is carried out indoors. After loosening the cover of the tissue culture bottle, place it in the buffer room of the tissue culture room for 2-3 days, then move it into the indoor room and place it under natural scattered light for another 2-3 days. After the seedling hardening is completed, take out the test tube seedlings with tweezers, wash off the culture medium at the roots, and transplant them into a 10×10 plug tray. The cultivation matrix is ​​peat soil: perlite: garden soil = 2:2:1. The cultivation matrix is ​​disinfected with 800-1000 times of carbendazim before use, and then normal water and fertilizer management is carried out.

[0079] The method of the present invention is suitable for any existing calla lily varieties, and the calla lily varieties that are particularly suitable for cultivation are Jingcai Sunshine and Black Hole, which are commercialized colored calla lily varieties and can be purchased from flower sales markets or colored calla lily planting companies.

[0080] The following examples illustrate the establishment of regeneration systems for different varieties of Calla Lily.

[0081] Example 1 Establishment of the regeneration system of the yellow calla cultivar 'Jingcai Yangguang'

[0082] (1) Obtaining explants The sterile tubers of 'Jingcai Sunshine' seedlings that have completed the formation of heads in the bottle are used as explants. The tubers have grown to a diameter of about 1.0 cm.

[0083] (2) Induction of callus: Place sterile seedlings of tubers of about 1.0 cm in length on an inoculation tray, remove all leaves and buds, cut the tubers longitudinally into small pieces of about 0.3 cm in length, width and thickness of about 0.2 cm, and inoculate them on callus induction medium. The basic medium is MS, to which 7.0 g / L agar, 30 g / L sucrose, 1.0 mg / L 6-BA, 0.8 mg / L NAA are added, pH 5.75-5.85, and culture is carried out in the dark. After 3 weeks, the explants sprouted and a layer of light yellow frost-like callus formed on the surface. Continue to culture, and the callus will continue to thicken. Figure 1 (a) is a photo of the explants transplanted into the callus induction medium on the 1st day, and (b) is a photo of the explants transplanted into the callus induction medium on the 10th day. From the photo on the 10th day, it can be seen that a layer of light yellow frosty callus has formed on the surface of a small amount of explants. The callus induction rate of this embodiment refers to the induction rate shown in sequence number 16 in Table 1. A total of 50 small explants were inoculated. The induction rate in Table 1 is a statistical induction rate data of 50 small explants after callus induction culture for 30 days. The formation of a layer of light yellow frosty material on the surface of the small explants is considered to be successful in inducing callus. Induction rate = (number of successful induced samples / total number of samples 50) * 100%.

[0084] (3) Proliferation of callus After 30 days of callus induction, 30 explants with good callus induction were transferred to the proliferation medium for proliferation culture. The basic medium was MS, to which 6.7 g / L agar, 30 g / L sucrose, 3.0 mg / L 6-BA, pH 5.8 were added, and cultured in the dark. After 30 days, the callus proliferated into a mass with a diameter of 1.2-1.5 cm. The callus mass obtained after part of the callus was transferred to the proliferation medium is shown in Figure 2The callus proliferation multiple of this example refers to the proliferation coefficient shown in No. 2 in Table 3. The proliferation coefficient in Table 3 is the result of weighing statistics after 30 days of proliferation culture. The number of samples is 30. The proliferation multiple = the total weight of 30 samples after 30 days of callus proliferation culture / the total weight of 30 samples before inoculation into callus proliferation medium and after 30 days of callus induction culture.

[0085] (4) Callus differentiation 30 callus clumps with a diameter of 1.2-1.5 cm were transferred to differentiation medium for bud differentiation. The composition of the medium is: the basic medium is MS, 6.5g / L agar, 30g / L sucrose, 1.5mg / L 6-BA, pH 5.8, light intensity of 800-1200Lx, and light duration of 16h. After 30 days of culture, the callus differentiated into clustered buds. For photos of some callus differentiated into clustered buds, see Figure 3 The differentiation coefficient of this example refers to the differentiation coefficient shown in No. 3 in Table 4. The differentiation coefficient in Table 4 = the total number of buds differentiated from all sample callus tissues / the number of samples 30.

[0086] (5) Strong bud culture 30 clustered buds were separated from the callus and transferred to the strong bud culture medium for strong bud culture. The basic culture medium was MS, with 60g / L sucrose, 7.0g / L agar, and 0.5mg / L NAA added. The culture temperature was 25±1°C, the light intensity was 800-1200Lx, and the light time was 16h. After 35d of culture, the clustered buds grew into rootless seedlings with a height of about 5.0cm and a tuber diameter of about 0.5cm. The effect of the strong bud culture medium of this embodiment is represented by the seedling height, specifically referring to the seedling height shown in sequence number 3 in Table 5, and the seedling height in Table 5 = the sum of the seedling heights of all samples / the number of samples 30.

[0087] (6) Rooting culture The leaves of the rootless seedlings (30 plants in total) with a height of about 5.0 cm were cut off, and only the petioles with a length of about 0.5 cm were retained, and the seedlings were transferred to the rooting medium for rooting culture. The basic medium was MS, with 50 g / L sucrose, 7.0 g / L agar, and 1.5 mg / L IBA added. The culture temperature was 25 ± 1 ° C, the light intensity was 800-1200 Lx, and the light duration was 16 h. After 30 days of culture, the seedlings grew into test tube seedlings with a height of 8.0-10.0 cm, 5-7 roots, and a tuber diameter of 0.7 cm. The effect of the rooting medium of this embodiment is represented by the rooting rate, which is specifically referred to as the rooting rate shown in sequence number 3 in Table 6. The rooting rate (%) in Table 6 = the number of rooted seedlings * 100 / the number of seedlings inoculated, wherein the number of rooted seedlings is calculated according to the standard that the seedling height reaches 8.0-10.0 cm and the number of roots reaches 5-7.

[0088] (7) Hardening and transplanting Hardening is done indoors. Loosen the cap of the tissue culture bottle and place it in the buffer room of the tissue culture room for 3 days, then move it into the indoor room and place it under natural scattered light for another 3 days. After hardening is completed, take out the test tube seedlings with tweezers, wash off the culture medium at the roots, and transplant them into a 10×10 plug tray. The cultivation medium is peat soil: perlite: garden soil = 2:2:1 (mass ratio), and the cultivation medium is disinfected with 800-1000 times of carbendazim before use. Then carry out normal water and fertilizer management.

[0089] On the basis of Example 1, the inventors also simultaneously conducted comparative tests on a variety of other culture medium formulations, wherein the test calla lily varieties, the selection of explants, and the size of the small explants inoculated on the induction medium were the same as those in Example 1, and the concentrations of agar and sucrose in the callus induction medium, proliferation medium, differentiation medium, bud growth medium, and rooting medium were consistent with the concentrations of the corresponding culture medium in Example 1. The culture conditions and time of each culture stage were also the same as those in Example 1, except that the concentrations or types of auxins and cytokinins in each culture medium were adjusted. The calculation method of the effect data of the culture medium at each stage was the same as the calculation method of the effect data of the corresponding stage in Example 1, as follows:

[0090] (1) Callus induction under different callus induction media:

[0091] (1-1) Experiments with different concentrations of 6-BA and NAA, with two factors and four levels of 6-BA concentrations of 0.5 mg / L, 1.0 mg / L, 1.5 mg / L, 2.0 mg / L and NAA concentrations of 0.3 mg / L, 0.5 mg / L, 0.8 mg / L, 1.0 mg / L, were conducted in an orthogonal design. The results are shown in Table 1 below, where the combination of 6-BA 1.0 mg / L and NAA 0.8 mg / L, i.e., sequence number 16, is the callus induction culture in Example 1 of the present invention. Generally, a hormone combination with an induction rate higher than 60% is ideal for callus induction. From the data in Table 1, it can be seen that when the hormone combination in the callus induction medium is 6-BA 1.0-2.0 mg / L and NAA 0.5-1.0 mg / L, the induction rate is above 46%.

[0092] Table 1 Effects of 6-BA and NAA concentrations on callus induction rate

[0093] Serial number Number of explants 6-BA(mg / L) NAA(mg / L) Induction rate (%) 1 50 1.0 0.3 20.00 2 50 1.5 1.0 54.00 3 50 0.5 0.8 54.00 4 50 0.5 0.3 26.00 5 50 2.0 0.3 40.00 6 50 1.0 1.0 60.00 7 50 1.5 0.3 34.00 8 50 0.5 1.0 46.00 9 50 2.0 1.0 46.00 10 50 1.5 0.8 70.00 11 50 1.0 0.5 56.00 12 50 0.5 0.5 40.00 13 50 2.0 0.8 72.00 14 50 2.0 0.5 56.00 15 50 1.5 0.5 66.00 16 50 1.0 0.8 86.00

[0094] (1-2) Experiments on the combination of different types of cytokinins and auxins, 6-BA or KT was selected as the cytokinin, and 2,4-D was selected as the auxin to replace the NAA of the present invention. For detailed combinations, concentrations and induction rates, see Table 2.

[0095] Table 2 Effects of different hormones on callus induction rate

[0096] Serial number Number of explants Cytokinin (mg / L) Auxin (mg / L) Induction rate (%) 1 50 BA1.0 2,4-D1.0 0 2 50 KT 0.05 2,4-D 3.0 0 3 50 KT0.05 NAA0.5 0 4 50 0 2,4-D 3.0 0 5 50 0 2,4-D 2.0+NAA0.2 0

[0097] As can be seen from Table 2, the selection of cytokinin and auxin plays a key role in the callus induction of the explants of the present invention. Replacing auxin with 2,4-D or replacing part of NAA with 2,4-D will result in failure to induce callus.

[0098] (2) Proliferation culture under different callus proliferation medium: Proliferation medium with different concentrations of 6-BA was set up, and a control experiment with the addition of auxin NAA was also set up. The concentrations of 6-BA and NAA in the proliferation medium of each group of test examples are shown in Table 3, wherein the medium formula shown in sequence number 2 is the proliferation medium formula of Example 1. The proliferation coefficient of the proliferation medium of each group of tests is shown in Table 3.

[0099] Table 3 Effects of different concentrations of 6-BA and NAA on callus proliferation

[0100]

[0101]

[0102] It can be seen from Table 3 that 6-BA concentrations higher than 4 mg / L or lower than 3 mg / L will significantly reduce the proliferation coefficient. In addition, adding auxin NAA to the proliferation culture medium system of the present invention will lead to a poor proliferation effect.

[0103] (3) Differentiation culture under different differentiation medium: Differentiation medium with different concentrations of 6-BA was set up, and a control experiment with the addition of auxin NAA was also set up. The concentrations of 6-BA and NAA in the differentiation medium of each group of test examples are shown in Table 4, wherein the medium formula shown in sequence number 3 is the differentiation medium formula of Example 1. The differentiation coefficient of the differentiation medium of each group of tests is shown in Table 4.

[0104] Table 4 Effects of 6-BA concentration and NAA concentration on callus differentiation

[0105] Serial number Number of explants 6-BA concentration (mg / L) Auxin NAA (mg / L) Differentiation coefficient 1 30 0.5 0 8 2 30 1.0 0 30 3 30 1.5 0 45 4 30 2.0 0 48 5 30 2.5 0 13 6 30 0.5 0.5 4 7 30 1.0 0.5 8 8 30 1.5 0.5 5 9 30 2.0 0.5 7 10 30 2.5 0.5 11

[0106] It can be seen from Table 4 that 6-BA concentrations higher than 2 mg / L or lower than 1 mg / L will significantly reduce the differentiation coefficient. In addition, adding auxin NAA to the differentiation medium system of the present invention will lead to a poor differentiation effect.

[0107] (4) Bud cultivation under different bud-strengthening culture media: bud-strengthening culture media with different concentrations of auxin NAA were set up, and a control test of whether 6-BA was added was also set up. The concentrations of 6-BA and NAA in the bud-strengthening culture media of each group of experiments are shown in Table 5, wherein the culture medium formula shown in sequence number 3 is the bud-strengthening culture medium formula of Example 1. The seedling height of the bud-strengthening culture medium of each group of experiments is shown in Table 5.

[0108] Table 5 Effects of 6-BA concentration and NAA concentration on bud growth

[0109]

[0110]

[0111] As can be seen from Table 5, adding the auxin NAA alone and controlling its concentration to 0.3-0.5 mg / L is beneficial to strong buds, and the seedling height is significantly higher than the comparative test with other concentrations; in addition, adding the cytokinin 6-BA to the bud-strengthening culture medium system will significantly reduce the seedling height.

[0112] (5) Rooting culture under different rooting media: Rooting media with different auxins and whether 6-BA was added were set. The concentrations of IBA, NAA and 6-BA in the rooting media of each group of test examples are shown in Table 6, where the medium formula shown in sequence number 3 is the rooting medium formula of Example 1. The rooting rate of the rooting medium of each group of tests is shown in Table 6.

[0113] Table 6 Effects of IBA, NAA and 6-BA on rooting

[0114]

[0115]

[0116] As can be seen from Table 6, replacing the auxin IBA in the rooting medium of the present invention with NAA will significantly reduce the rooting rate; and the rooting rate will also decrease after adding 6-BA.

[0117] Example 2 Establishment of the regeneration system of the colorful calla lily 'Black Hole'

[0118] (1) Obtaining Explants The tubers of the sterile seedlings of the 'Black Hole' plantlets that have completed the formation of heads in the bottle are used as explants. The tubers have grown to a diameter of about 1.0 cm.

[0119] (2) Induction of callus: Sterile seedlings of tubers of about 1.0 cm in length were placed on an inoculation tray, all leaves and buds were removed, and the tubers were cut longitudinally into small pieces of about 0.2 cm in length, width and thickness, and inoculated onto callus induction medium. The basic medium was MS, to which 6.9 g / L agar, 30 g / L sucrose, 2.0 mg / L 6-BA, 0.5 mg / L NAA, pH 5.8 were added, and cultured in the dark. After 23 days, the explants sprouted and formed a layer of light yellow frosty callus on the surface. Cultivation was continued, and the callus continued to thicken. The method for calculating the induction rate of callus was the same as in Example 1, and the induction rate of this example was 80%.

[0120] (3) Proliferation of callus After 30 days of callus culture, the explants together with the thickened callus were transferred to a proliferation medium for proliferation culture. The basic medium was MS, to which 6.7 g / L agar, 30 g / L sucrose, 4.0 mg / L 6-BA, pH 5.8 were added, and cultured in the dark. After 30 days, the callus proliferated into a mass with a diameter of 1.0-1.3 cm. The calculation method of the proliferation multiple was the same as in Example 1, and the proliferation multiple of this example was 4.5.

[0121] (4) Callus Differentiation Callus masses with a diameter of 1.0-1.3 cm were transferred to differentiation medium for bud differentiation culture. The components of the medium were: basic medium was MS, 6.5 g / L agar, 30 g / L sucrose, 2.0 mg / L 6-BA, pH 5.8, light intensity of 800-1200 Lx, and light duration of 16 h. After 35 days of culture, callus differentiated into clustered buds. The calculation method of differentiation coefficient was the same as that of Example 1, and the differentiation coefficient of this example was 48.

[0122] (5) Strong bud culture The clustered buds were separated from the callus and transferred to the strong bud culture medium for strong bud culture. The basic culture medium was MS, with 60 g / L sucrose, 7.0 g / L agar, and 0.3 mg / L NAA added. The culture temperature was 25 ± 1 ° C, the light intensity was 800-1200 Lx, and the light time was 16 h. After 32 days of culture, the clustered buds grew into rootless seedlings with a height of about 7.0 cm and a tuber diameter of 0.6 cm. The calculation method of the seedling height was the same as in Example 1, and the seedling height of this example was 2.5 cm.

[0123] (6) Rooting culture: The leaves of the rootless seedlings with a height of about 7.0 cm were cut off, leaving only the petioles with a length of about 0.8 cm, and then transferred to the rooting medium for rooting culture. The basic medium was MS, with 50 g / L sucrose, 7.0 g / L agar, and 2.0 mg / L IBA added. The culture temperature was 25 ± 1 ° C, the light intensity was 800-1200 Lx, and the light duration was 16 h. After 30 days of culture, the test tube seedlings grew into a height of 6.0-8.0 cm, 5-6 roots, and a tuber diameter of 0.6 cm. The calculation method of the rooting rate was the same as in Example 1. The rooting rate of this example was 93.33%.

[0124] (7) Hardening and transplanting Hardening is done indoors. Loosen the cap of the tissue culture bottle and place it in the buffer room of the tissue culture room for 3 days, then move it into the indoor room and place it under natural scattered light for another 3 days. After hardening is completed, take out the test tube seedlings with tweezers, wash off the culture medium at the roots, and transplant them into a 10×10 plug tray. The cultivation medium is peat soil: perlite: garden soil = 2:2:1 (mass ratio), and the cultivation medium is disinfected with 800-1000 times of carbendazim before use. Then carry out normal water and fertilizer management.

[0125] The embodiments described above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are within the scope of protection of the pending claims of the present invention.

Claims

1. A culture medium combination for constructing a color calla lily regeneration system using tubers, characterized in that: The culture medium composition comprises: Callus induction medium: MS as the basic medium, and also containing the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L, NAA 0.5-1.0 mg / L; or, MS as the basic medium, and also containing the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 0.5 mg / L, NAA 0.8 mg / L; Callus proliferation medium: MS as the basic medium, also contains the following concentrations of substances: agar 6.5-7.0g / L, sucrose 30g / L, 6-BA 3.0-4.0mg / L; Differentiation medium: MS as the basic medium, also contains the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L.

2. The culture medium combination according to claim 1, characterized in that: The culture medium combination also includes: Bud growth medium: MS as the basic medium, also contains the following concentrations of substances: sucrose 40-60g / L, agar 6.5-7.0g / L, NAA 0.3-0.5mg / L; And / or, rooting medium: MS as the basic medium, further containing the following concentrations of substances: sucrose 40-60 g / L, agar 6.5-7.0 g / L, IBA 1.0-2.0 mg / L.

3. The culture medium combination according to claim 1 or 2, characterized in that: The concentrations of 6-BA and NAA in the callus induction medium are as follows: 6-BA 1.0-2.0 mg / L, NAA 0.8 mg / L.

4. The culture medium combination according to claim 2, characterized in that: The pH values ​​of the callus induction medium, the callus proliferation medium, the callus differentiation medium, the budding medium and the rooting medium are all 5.75-5.

85.

5. A method for constructing a color calla lily regeneration system using tubers, characterized in that: The method comprises the following steps: S1 Selection and treatment of explants: Select sterile colored calla lily seedling tubers that have completed the balling in the bottle as explants, wherein the tuber diameter is in the range of 0.8 to 1.2 cm; first remove the leaves and buds on the tubers, and then cut them into small pieces for callus culture; S2 callus induction culture: inoculating the small explants onto callus induction medium for induction culture, wherein the callus induction medium is: based on MS, and further contains the following substances at the following concentrations: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L, NAA 0.5-1.0 mg / L; or, based on MS, and further contains the following substances at the following concentrations: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 0.5 mg / L, NAA 0.8 mg / L; S3 callus proliferation culture: transferring the small explant with callus induced and cultured in step S2 to a callus proliferation medium for proliferation culture, wherein the callus proliferation medium is based on MS and further contains the following substances at the following concentrations: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 3.0-4.0 mg / L; S4 callus differentiation culture: transferring the callus mass obtained in step S3 to a differentiation medium for bud differentiation culture to obtain clustered buds; the differentiation medium is based on MS and further contains the following concentrations of substances: agar 6.5-7.0 g / L, sucrose 30 g / L, 6-BA 1.0-2.0 mg / L; S5 strong bud culture: inoculating the clustered buds into a strong bud culture medium for strong bud culture; S6 Rooting culture: The rootless seedlings obtained after the strong bud culture are subjected to rooting culture.

6. The method according to claim 5, characterized in that The size of the small explant is: 0.3-0.4 cm in length and width, and 0.1-0.3 cm in thickness; And / or, the induction culture conditions are: dark culture, the number of days of dark culture is 30-40 days; And / or, the proliferation culture conditions are: dark culture, the number of days of dark culture is 35-40 days; and / or, obtaining a callus mass with a diameter of 1.0-1.5 cm after the proliferation culture; And / or, the conditions of the bud differentiation culture are: light-dark alternating culture, wherein the light intensity is 800-1200Lx, the light duration is 15-17h per day, and the culture temperature is 25±1°C; And / or, the bud differentiation culture time is 30-40 days; And / or, the variety of the colorful calla lily is Black Hole or Jingcai Sunshine.

7. The method according to claim 5, characterized in that The bud-strengthening medium is based on MS and also contains the following substances in concentrations: sucrose 40-60 g / L, agar 6.5-7.0 g / L, and NAA 0.3-0.5 mg / L; And / or, the rooting medium is based on MS and further contains the following substances in concentrations: sucrose 40-60 g / L, agar 6.5-7.0 g / L, and IBA 1.0-2.0 mg / L.

8. The method according to claim 7, characterized in that The height of the rootless seedlings is 5.0-7.0 cm, and the diameter of the tubers is 0.5-0.8 cm; And / or, the conditions for the strong bud culture are: culture temperature 25±1°C, light and dark alternation culture, wherein the light intensity is 800-1200Lx, and the light time is 15-17h per day; And / or, the time of culturing the strong buds is 30-35 days; And / or, the rooting culture conditions are: culture temperature 25±1°C, light and dark alternating culture, wherein the light intensity is 800-1200Lx, and the light time is 15-17h per day; And / or, the rooting culture time is 30-35 days; And / or, the test tube plantlets obtained after the rooting culture have a height of 6.0-10.0 cm, 5-7 roots, and a tuber diameter of 0.5-0.8 cm.

9. The method according to claim 5, characterized in that The method further comprises: Step S7: hardening and transplanting: hardening the test tube seedlings obtained by rooting culture and then transplanting them into a cultivation medium for cultivation.

10. The method according to claim 9, characterized in that The cultivation matrix includes peat soil, perlite and garden soil, wherein the mass ratio of peat soil:perlite:garden soil is 2:2:1.

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