Culture medium and method for preserving transparent art in hybrid orchid and application of culture medium and method

Abstract

The invention discloses a culture medium, a method and application for maintaining a transparent technology in hybrid orchid. Belongs to the technical field of plant tissue culture. According to the method disclosed by the invention, by regulating and controlling the components of the culture medium and the culture conditions, the problem of degeneration of the middle transparent technology in the traditional hybrid orchid tissue culture is effectively solved, and the middle transparent technology retention rate can reach 94.64%. The tissue culture plantlets are subjected to the stages of rhizome induction, rhizome proliferation and differentiation and seedling strengthening and rooting culture after 140-180 days, so that robust medium-transparent hybrid orchid rooted seedlings with the plant height of 8.2-9.2 cm and the root length of 3.7-4.5 cm are obtained, the quality of the tissue culture seedlings is improved, the breeding efficiency is improved, the rhizome proliferation coefficient is high, the quality is good, the strong proliferation capacity can be kept, and the method is suitable for large-scale popularization and application. An ideal culture effect is achieved, and a powerful guarantee is provided for large-scale production and market supply of the orchid industry.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and more particularly to a culture medium, method and application for maintaining transparency in hybrid orchids. Background Art

[0002] Hybrid orchids are new orchid varieties bred by hybridizing Chinese orchids with cymbidium orchids. They have the fresh fragrance of Chinese orchids and the large, colorful and long flowering period of cymbidium orchids, and have high ornamental and economic value. Leaf art usually refers to the variation of orchid leaves into different color spots at different positions, such as striped leaves, transparent leaves, yellow leaves or spotted leaves. Leaf art varieties are more attractive for viewing leaves than flowers, and their ornamental and economic value increases exponentially. Medium transparent art is a type of leaf art, which refers to the wide lines of white, yellow or other colors that run through the leaves. However, under natural conditions, the leaf art of orchids is often difficult to maintain for a long time and is easily degraded by environmental factors. Although traditional propagation methods such as division and cuttings can retain the characteristics of the mother plant, they are less efficient and not conducive to large-scale production.

[0003] Tissue culture technology can achieve mass reproduction in a short period of time by utilizing the totipotency of plant cells. In recent years, some research reports have been published on the rapid propagation of hybrid orchid leaf art mutants by tissue culture. In 2021, Song Dandan et al. (Song Dandan, Li Hanyue, Zhang Tiantian, Zeng Ruizhen, Guo Herong, Zhang Zhisheng. Effect of leaf art mutation on tissue culture rapid propagation characteristics of 'Xiaofenglan' [J]. Heilongjiang Agricultural Science, 2021, (7): 63-68, Song Dandan, Zhu Pengyu, Li Wenmin, Zu Junrui, Zeng Ruizhen, Xie Li, Guo Herong, Zhang Zhisheng. Research on tissue culture rapid propagation technology of leaf art hybrid orchid [J]. Specialty Products Research, 2021, 43(1 ):24-28) Using the hybrid orchid 'Xiao Feng Lan' and its leaf art mutants as materials, a tissue culture rapid propagation technology was established from the aspects of rhizome induction, rhizome proliferation, rhizome differentiation, rooting and strong seedlings, and test tube seedling transplanting. The results showed that leaf art mutation would reduce the tissue culture rapid propagation efficiency of 'Xiao Feng Lan' and the content of photosynthetic pigments in the leaves of test tube seedlings, and there were obvious differences in the rhizome induction rate, proliferation coefficient, average bud differentiation number, etc. of different leaf art mutants.

[0004] Therefore, developing a culture medium and a culture method that can efficiently maintain the transparency of hybrid orchids and achieve rapid reproduction is a technical problem that technicians in this field urgently need to solve. Summary of the invention

[0005] In view of this, the present invention provides a culture medium, method and application for maintaining the medium permeability of hybrid orchids. By optimizing the culture medium components, controlling the culture conditions and adopting specific subculture operations, the medium permeability of hybrid orchids can be effectively maintained and the quality and breeding efficiency of tissue culture seedlings can be improved.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A culture medium for maintaining the art of hybrid orchids, comprising a rhizome induction culture medium, a rhizome proliferation and differentiation culture medium and a seedling rooting culture medium;

[0008] The rhizome induction medium has the following composition and ratio: MS1 medium, 6-BA 1.0-2.0 mg / L, NAA 0.2-0.5 mg / L, activated carbon 0.5-1.0 g / L, white sugar 20-30 g / L and coagulant 6.0-7.0 g / L, pH 5.6-5.8;

[0009] The composition and proportion of the rhizome proliferation and differentiation medium are as follows: MS2 medium, KT 0.7-1.0 mg / L, NAA 0.3-0.5 mg / L, activated carbon 0.5-1.0 g / L, white sugar 20-30 g / L and coagulant 6.0-7.0 g / L, pH 5.6-5.8;

[0010] The composition and proportion of the seedling rooting medium are as follows: MS3 medium, 0.1-0.2 mg / L 6-BA, 0-1.0 mg / L IBA, 0-1.0 mg / L NAA, 2.0-3.0 g / L banana powder, 0.5-1.0 g / L activated carbon, 20-30 g / L white sugar and 6.0-7.0 g / L coagulant, pH 5.6-5.8.

[0011] Preferably, the composition and ratio of the MS1 medium are as follows: KNO 3 1900 mg / L, (NH 4 ) 2 SO 4 460~500mg / L, KH 2 PO 4 150~170mg / L, MgSO 4. 7H 2 O 210~250mg / L, CaCl 2. 2H 2 O440mg / L, MnSO 4. H 2 O16.9mg / L, ZnSO 4. 7H 2 O 8.6mg / L, H 3 BO 3 6.2 mg / L, KI 0.83 mg / L, Na 2 MoO 2. 2H 2 O 0.25mg / L, CoCl 2. 6H 2 O 0.025mg / L, CuSO4. 5H 2 O 0.025mg / L, FeSO 4. 7H 2 O 27.8mg / L, Na 2. EDTA 37.3mg / L, thiamine hydrochloride 1.0-2.0mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, glycine 2.0mg / L and inositol 100mg / L.

[0012] Preferably, the composition and ratio of the MS2 medium are as follows: KNO 3 950 mg / L, (NH 4 ) 2 SO 4 230~250mg / L, KH 2 PO 4 170~190mg / L、MgSO 4. 7H 2 O 160-180 mg / L, CaCl 2. 2H 2 O220mg / L, MnSO 4. H 2 O 16.9mg / L, ZnSO 4. 7H 2 O 8.6mg / L, H 3 BO 3 6.2 mg / L, KI 0.83 mg / L, Na 2 MoO 2. 2H 2 O 0.25mg / L, CoCl 2. 6H 2 O0.025mg / L, CuSO 4. 5H 2 O 0.025mg / L, FeSO 4. 7H 2 O 27.8mg / L, Na 2. EDTA 37.3mg / L, thiamine hydrochloride 0.1mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, glycine 2.0mg / L and inositol 100mg / L.

[0013] Preferably, the composition and ratio of the MS3 medium are as follows: KNO 3 950 mg / L, (NH 4 ) 2 SO 4 230~250mg / L, KH 2 PO 4170~190mg / L, MgSO 4. 7H 2 O 160-180 mg / L, CaCl 2. 2H 2 O220mg / L, MnSO 4. H 2 O 16.9mg / L, ZnSO 4. 7H 2 O 8.6mg / L, H 3 BO 3 6.2 mg / L, KI 0.83 mg / L, Na 2 MoO 2. 2H 2 O 0.25mg / L, CoCl 2. 6H 2 O0.025mg / L, CuSO 4. 5H 2 O 0.025mg / L, FeSO 4. 7H 2 O 27.8mg / L, Na 2. EDTA 37.3mg / L, thiamine hydrochloride 0.1mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 1.0-2.0mg / L, glycine 2.0-4.0mg / L and inositol 100mg / L.

[0014] Preferably, the coagulant is agar powder, carrageenan, or a mixture of agar powder and carrageenan in a mass ratio of 1:1.

[0015] Another object of the present invention is to provide: the use of the above-mentioned rhizome induction medium, rhizome proliferation and differentiation medium and seedling rooting medium in the transparent maintenance tissue culture of hybrid orchids.

[0016] Another object of the present invention is to provide: a method for maintaining the cultivation of hybrid orchids, using the above-mentioned rhizome induction medium, rhizome proliferation and differentiation medium and seedling rooting medium to cultivate hybrid orchids, and the specific cultivation method is as follows:

[0017] (1) Rhizome induction culture: Inoculate the hybrid orchid rootless tissue culture seedlings onto the induction medium, culture them in the dark for 10 to 15 days, and then culture them under a light intensity of 1800-2000 Lux for 30 to 35 days;

[0018] (2) Rhizome proliferation and differentiation culture: After induction culture, the hybrid orchid rhizomes were cut into 1.0-1.5 cm pieces and inoculated onto rhizome proliferation and differentiation medium. After culturing under natural light for 7-10 days, they were placed under a light intensity of 1800-2000 Lux for 43-50 days to obtain seedlings.

[0019] (3) Rooting culture for strong seedlings: The differentiated seedlings obtained in step (2) are cut into single seedlings, inoculated onto a rooting culture medium for strong seedlings, cultured under natural light for 7 to 10 days, and then cultured under a light intensity of 2000-2500 Lux for 53 to 60 days to obtain rooted seedlings.

[0020] Preferably, the temperature of the rhizome induction culture in step (1) is 23-27°C, and the illumination time is 10-12 h / d; the temperature of the rhizome proliferation and differentiation culture in step (2) is 23-27°C, and the illumination time is 10-12 h / d; the temperature of the seedling rooting culture in step (3) is 23-27°C, and the illumination time is 12-14 h / d.

[0021] Preferably, the rhizomes used for rhizome proliferation and differentiation culture in step (2) are rhizomes that are entirely light yellow or yellow.

[0022] Preferably, the single seedling in step (3) is a seedling larger than 3.0 cm, with a full base and an open leaf tip.

[0023] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0024] The tissue culture method and culture medium group for maintaining the medium-transparent art of hybrid orchids provided by the present invention effectively solve the problem of medium-transparent art degradation in traditional tissue culture by regulating the culture medium components and culture conditions, and the medium-transparent art maintenance rate can reach 94.64%. It takes 140 to 180 days for the tissue culture seedlings to undergo rhizome induction, rhizome proliferation and differentiation, and strong seedling rooting culture stages, thereby obtaining strong medium-transparent art hybrid orchid rooting seedlings with a plant height of 8.2 to 9.2 cm and a root length of 3.7 to 4.5 cm, which not only improves the quality of the tissue culture seedlings, but also improves the breeding efficiency, the rhizome proliferation coefficient is high and the quality is good, and a strong proliferation ability can be maintained, achieving a relatively ideal culture effect, and providing a strong guarantee for the large-scale production and market supply of the orchid industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0026] Figure 1 For: Leaf art pictures of ‘Zhongtou Ziyanshi’ and ‘C6117T’;

[0027] Figure 2 It is: the rhizome material for rhizome proliferation and differentiation in Example 5. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The following examples use hybrid orchids 'Zhongtou Ziyanshi' and 'C6117T' as experimental materials. The 'Zhongtou Ziyanshi' is a new hybrid orchid strain of the Molan type, and the leaves have light yellow broad lines running through the leaves. The above-mentioned 'Zhongtou Ziyanshi' is the 'Zhongtou Ziyanshi' (No. K21-3) disclosed in the non-patent document "Cloning of hybrid orchid ChCAO gene and its expression characteristics in leaf art strains" by Lin Rongyan et al., and is now held by the Crop Research Institute of Fujian Academy of Agricultural Sciences (Fujian Germplasm Resources Center). In order to meet the requirements of patent disclosure sufficiency, it can be provided to the public within 20 years from the application date.

[0030] 'C6117T' is a new hybrid orchid of the Chunlan type, with broad yellow lines running through the leaves ( Figure 1 ) to further verify the effect of the culture medium and culture method of the present invention. It is now held by the Crop Research Institute of Fujian Academy of Agricultural Sciences (Fujian Germplasm Resources Center) and can be provided to the public within 20 years from the date of application in order to meet the requirements of patent disclosure sufficiency.

[0031] Example 1

[0032] A method for maintaining the medium-transparent art of a hybrid orchid 'Zhongtou Ziyanshi' comprises the following steps:

[0033] (1) Rhizome induction culture: The rootless tissue culture seedlings of 'Zhongtou Ziyanshi' were inoculated on the rhizome induction culture medium for culture. The culture medium composition was as follows: MS1 medium + 6-BA 2.0 mg / L + NAA 0.2 mg / L + activated carbon 1.0 g / L + sugar 30 g / L + coagulant 7.0 g / L, pH 5.6-5.8;

[0034] The composition and dosage of MS1 medium are KNO 3 1900 mg / L, (NH 4 ) 2 SO 4 460mg / L, KH 2 PO 4 170 mg / L, MgSO 4. 7H 2 O 250mg / L, CaCl2. 2H 2 O 440mg / L, MnSO 4. H 2 O 16.9mg / L, ZnSO 4. 7H 2 O8.6mg / L, H 3 BO 3 6.2 mg / L, KI 0.83 mg / L, Na 2 MoO 2. 2H 2 O 0.25mg / L, CoCl 2. 6H 2 O 0.025mg / L, CuSO 4. 5H 2 O 0.025mg / L, FeSO 4. 7H 2 O 27.8mg / L, Na 2. EDTA 37.3mg / L, thiamine hydrochloride 2.0mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, glycine 2.0mg / L, inositol 100mg / L.

[0035] The culture conditions were controlled at a temperature of 25±2°C, and when just inoculated, they were placed in a dark culture room for 10 days, after which the light intensity was 1800-2000 lux and the illumination time was 12h / d.

[0036] Through observation and analysis, we know that the results of rhizome induction culture are: culture period is 40 to 50 days, and the induction rate is 96.67%.

[0037] (2) Rhizome proliferation and differentiation culture: The rhizomes of the hybrid orchid "Zhongtou Ziyanshi" after induction culture (the whole rhizomes are light yellow) were cut into 1.0-1.5 cm and inoculated into proliferation and differentiation medium for culture. The medium composition is as follows: MS2 medium + KT 0.7 mg / L + NAA 0.5 mg / L + activated carbon 1.0 g / L + white sugar 30 g / L + coagulant 6.0 g / L, pH 5.6-5.8;

[0038] The composition and dosage of MS2 medium are KNO 3 950 mg / L, (NH 4 ) 2 SO 4 230mg / L, KH 2 PO 4 190 mg / L, MgSO 4. 7H 2 O 180mg / L, CaCl 2. 2H2 O 220mg / L, MnSO 4. H 2 O 16.9mg / L, ZnSO 4. 7H 2 O8.6mg / L, H 3 BO 3 6.2 mg / L, KI 0.83 mg / L, Na 2 MoO 2 . 2H 2 O 0.25mg / L, CoCl 2 . 6H 2 O 0.025mg / L, CuSO 4 . 5H 2 O 0.025mg / L, FeSO 4. 7H 2 O 27.8mg / L, Na 2. EDTA 37.3mg / L, thiamine hydrochloride 0.1mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, glycine 2.0mg / L, inositol 100mg / L.

[0039] The culture condition is controlled at a temperature of 25±2℃, and when just inoculated, it is placed in the culture room without turning on the culture rack light tube, that is, cultured under natural light for 7 days, and then the light intensity is 1800~2000lx, and the lighting time is 12h / d.

[0040] Through observation and analysis, the results of rhizome proliferation and differentiation culture were as follows: the culture period was 50-60 days, the proliferation coefficient was 4.52, the seedling rate was 93.33%, and the mid-stage permeability retention rate was 92.84%.

[0041] (3) Seedling rooting culture: The differentiated seedlings of 'Zhongtou Ziyanshi' were cut into single seedlings and inoculated into seedling rooting medium for culture. The medium composition was as follows: MS3 medium + 6-BA 0.1 mg / L + NAA 1.0 mg / L + banana powder 2.0 g / L + activated carbon 0.5 g / L + sugar 20 g / L + coagulant 7.0 g / L, pH 5.6-5.8;

[0042] The composition and dosage of MS3 medium are as follows: KNO 3 950 mg / L, (NH 4 ) 2 SO 4 230mg / L, KH 2 PO 4 190mg / L, MgSO 4. 7H2 O 180mg / L, CaCl 2. 2H 2 O 220mg / L, MnSO 4. H 2 O 16.9mg / L, ZnSO 4 . 7H 2 O 8.6mg / L, H 3 BO 3 6.2 mg / L, KI 0.83 mg / L, Na 2 MoO 2. 2H 2 O 0.25mg / L, CoCl 2. 6H 2 O 0.025mg / L, CuSO 4. 5H 2 O0.025mg / L, FeSO 4. 7H 2 O 27.8mg / L, Na 2. EDTA 37.3mg / L, thiamine hydrochloride 0.1mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 1.5mg / L, glycine 3.0mg / L, inositol 100mg / L.

[0043] The culture conditions are controlled at a culture temperature of 25±2°C, and when just inoculated, the culture rack lamps are not turned on and the culture is cultured under natural light for 7 days. Thereafter, the light intensity is 2000-2500lx and the illumination time is 12h / d.

[0044] Through observation and analysis, the results of the seedling rooting culture are as follows: the culture period is 60 to 70 days, and complete plants with a plant height of 8.5 to 9.2 cm and a root length of 3.7 to 4.2 cm are obtained, and the rooting rate is above 98.0%.

[0045] Example 2

[0046] A method for cultivating the hybrid orchid 'Zhongtou Ziyanshi' to maintain its medium-transparent appearance:

[0047] It is basically the same as Example 1, except that the formulas of the culture media are different.

[0048] The composition of the rhizome induction medium is as follows: MS1 medium + 6-BA 1.0 mg / L + NAA 0.5 mg / L + activated carbon 1.0 g / L + white sugar 30 g / L + coagulant 6.0 g / L, pH 5.6-5.8;

[0049] The composition and dosage of MS1 medium are KNO 3 1900 mg / L, (NH4 ) 2 SO 4 480mg / L, KH 2 PO 4 160 mg / L, MgSO 4. 7H 2 O 230mg / L, CaCl 2. 2H 2 O 440mg / L, MnSO 4. H 2 O 16.9mg / L, ZnSO 4. 7H 2 O8.6mg / L, H 3 BO 3 6.2 mg / L, KI 0.83 mg / L, Na 2 MoO 2. 2H 2 O 0.25mg / L, CoCl 2. 6H 2 O 0.025mg / L, CuSO 4. 5H 2 O 0.025mg / L, FeSO 4. 7H 2 O 27.8mg / L, Na 2. EDTA 37.3mg / L, thiamine hydrochloride 1.5mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, glycine 2.0mg / L, inositol 100mg / L.

[0050] The composition of the rhizome proliferation and differentiation medium is as follows: MS2 basic medium + KT 1.0 mg / L + NAA 0.3 mg / L + activated carbon 0.5 g / L + white sugar 30 g / L + coagulant 7.0 g / L, pH 5.6-5.8;

[0051] The composition and dosage of MS2 medium are KNO 3 950 mg / L, (NH 4 ) 2 SO 4 240mg / L, KH 2 PO 4 180 mg / L, MgSO 4. 7H 2 O 170mg / L, CaCl 2. 2H 2 O 220mg / L, MnSO 4. H 2 O 16.9mg / L, ZnSO 4. 7H2 O8.6mg / L, H 3 BO 3 6.2 mg / L, KI 0.83 mg / L, Na 2 MoO 2. 2H 2 O 0.25mg / L, CoCl 2. 6H 2 O 0.025mg / L, CuSO 4. 5H 2 O 0.025mg / L, FeSO 4. 7H 2 O 27.8mg / L, Na 2. EDTA 37.3mg / L, thiamine hydrochloride 0.1mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, glycine 2.0mg / L, inositol 100mg / L.

[0052] The composition of the seedling rooting medium is as follows: MS3 medium + 6-BA 0.2mg / L + NAA 1.0mg / L + banana powder 2.0g / L + activated carbon 1.0g / L + white sugar 30g / L + coagulant 6.0g / L, pH 5.6-5.8;

[0053] The composition and dosage of MS3 medium are as follows: KNO 3 950 mg / L, (NH 4 ) 2 SO 4 240mg / L, KH 2 PO 4 180mg / L, MgSO 4. 7H 2 O 170mg / L, CaCl 2. 2H 2 O 220mg / L, MnSO 4. H 2 O 16.9mg / L, ZnSO 4 . 7H 2 O 8.6mg / L, H 3 BO 3 6.2 mg / L, KI 0.83 mg / L, Na 2 MoO 2. 2H 2 O 0.25mg / L, CoCl 2. 6H 2 O 0.025mg / L, CuSO 4. 5H 2O0.025mg / L, FeSO 4. 7H 2 O 27.8mg / L, Na 2. EDTA 37.3mg / L, thiamine hydrochloride 0.1mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 2.0mg / L, glycine 4.0mg / L, inositol 100mg / L.

[0054] Through observation and analysis, it was found that the culture period for rhizome induction was 40 to 50 days, and the induction rate reached 90.83%; the culture period for rhizome proliferation and differentiation was 50 to 60 days, the proliferation coefficient was 4.83, the seedling rate was 88.33%, and the medium-term permeability retention rate was 90.66%; the culture period for strong seedlings and rooting was 60 to 70 days, and complete plants with a plant height of 8.4 to 8.8 cm and a root length of 3.9 to 4.3 cm were obtained, and the rooting rate was above 98.0%.

[0055] Example 3

[0056] A method for cultivating the hybrid orchid 'Zhongtou Ziyanshi' to maintain its medium-transparent appearance:

[0057] It is basically the same as Example 1, except that the formulas of the culture media are different.

[0058] The composition of the rhizome induction medium is as follows: the induction medium is MS1 medium + 6-BA 2.0 mg / L + NAA 0.5 mg / L + activated carbon 0.5 g / L + sugar 20 g / L + coagulant 6.5 g / L, pH 5.6-5.8; the composition and dosage of each component of MS1 medium is KNO 3 1900 mg / L, (NH 4 ) 2 SO 4 500 mg / L, KH 2 PO 4 150 mg / L, MgSO 4. 7H 2 O 210mg / L, CaCl 2. 2H 2 O 440mg / L, MnSO 4. H 2 O16.9mg / L, ZnSO 4. 7H 2 O 8.6mg / L, H 3 BO 3 6.2 mg / L, KI 0.83 mg / L, Na 2 MoO 2. 2H 2 O0.25mg / L, CoCl 2. 6H2 O 0.025mg / L, CuSO 4. 5H 2 O 0.025mg / L, FeSO 4. 7H 2 O27.8mg / L, Na 2. EDTA 37.3mg / L, thiamine hydrochloride 1.0mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, glycine 2.0mg / L, inositol 100mg / L.

[0059] The composition of the rhizome proliferation and differentiation medium is as follows: MS2 basic medium + KT 0.8 mg / L + NAA 0.5 mg / L + activated carbon 1.0 g / L + sugar 20 g / L + coagulant 6.5 g / L, pH 5.6-5.8; the composition and dosage of each component of MS2 medium are KNO 3 950 mg / L, (NH 4 ) 2 SO 4 250 mg / L, KH 2 PO 4 170mg / L, MgSO 4. 7H 2 O 160mg / L, CaCl 2. 2H 2 O220mg / L, MnSO 4. H 2 O 16.9mg / L, ZnSO 4. 7H 2 O 8.6mg / L, H 3 BO 3 6.2 mg / L, KI 0.83 mg / L, Na 2 MoO 2. 2H 2 O 0.25mg / L, CoCl 2. 6H 2 O 0.025mg / L, CuSO 4. 5H 2 O 0.025mg / L, FeSO 4. 7H 2 O27.8mg / L, Na 2. EDTA 37.3mg / L, thiamine hydrochloride 0.1mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, glycine 2.0mg / L, inositol 100mg / L.

[0060] The composition of the seedling rooting medium is as follows: MS3 medium + 6-BA 0.1mg / L + IBA 1.0mg / L + banana powder 3.0g / L + activated carbon 0.5g / L + sugar 20g / L + coagulant 7.0g / L, pH 5.6-5.8; the composition and dosage of each component of MS3 medium are as follows: KNO 3 950 mg / L, (NH 4 ) 2 SO 4 250 mg / L, KH 2 PO 4 170mg / L, MgSO 4. 7H 2 O 160mg / L, CaCl 2. 2H 2 O220mg / L, MnSO 4. H 2 O 16.9mg / L, ZnSO 4 . 7H 2 O 8.6mg / L, H 3 BO 3 6.2 mg / L, KI 0.83 mg / L, Na 2 MoO 2 . 2H 2 O 0.25mg / L, CoCl 2. 6H 2 O 0.025mg / L, CuSO 4. 5H 2 O 0.025mg / L, FeSO 4. 7H 2 O27.8mg / L, Na 2. EDTA 37.3mg / L, thiamine hydrochloride 0.1mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 1.0mg / L, glycine 2.0mg / L, inositol 100mg / L.

[0061] Through observation and analysis, it was found that the culture period for rhizome induction was 40 to 50 days, and the induction rate reached 94.17%; the culture period for rhizome proliferation and differentiation was 50 to 60 days, the proliferation coefficient was 4.65, the seedling rate was 91.67%, and the medium-term permeability retention rate was 94.64%; the culture period for strong seedlings and rooting was 60 to 70 days, and complete plants with a plant height of 8.2 to 8.6 cm and a root length of 3.8 to 4.1 cm were obtained, and the rooting rate was above 98.0%.

[0062] Example 4

[0063] It is basically the same as Example 1, except that the formulas of the culture media are different.

[0064] Specifically refer to the report of Song Dandan et al. (2021) "Research on tissue culture and rapid propagation technology of leaf art hybrid orchids" and make slight modifications, as follows:

[0065] The rhizome induction medium was MS+6-BA 1.5mg / L+NAA 0.5mg / L+activated carbon 0.5g / L+sugar 30g / L+carrageenan 7.0g / L, pH 5.8-6.0;

[0066] The rhizome proliferation medium is: MS+6-BA 1.5mg / L+NAA 0.5mg / L+activated carbon 0.3g / L+sugar 30g / L+carrageenan 7.0g / L, pH 5.8-6.0;

[0067] Rhizome differentiation medium: MS+6-BA 1.5mg / L+NAA 0.1mg / L+activated carbon 0.3g / L+sugar 30g / L+carrageenan 7.0g / L, pH 5.8-6.0;

[0068] The culture medium for seedling rooting is: 1 / 2MS+6-BA 0.1mg / L+NAA 0.5mg / L+activated carbon 0.5g / L+white sugar 20g / L+carrageenan 6.0g / L, pH 5.8~6.0.

[0069] The above culture medium was used to culture 'Zhongtou Ziyanshi', and the culture results were as follows:

[0070] Through observation and analysis, it was found that the culture period of rhizome induction was 40-50 days, and the induction rate was 66.67%; the culture period of rhizome proliferation was 40-50 days, and the proliferation coefficient was 2.85; the culture period of rhizome differentiation was 50-60 days, the seedling rate was 63.33%, and the mid-stage permeability retention rate was 65.83%; the culture period of seedling rooting was 60-70 days, and complete plants with a plant height of 6.5-7.0 cm and a root length of 3.5-3.9 cm were obtained, with a rooting rate of more than 95.0%.

[0071] Example 5

[0072] A method for cultivating hybrid orchids to maintain their beauty:

[0073] The same as Example 1, except that the rhizome materials used for rhizome proliferation and differentiation are different ( Figure 2 ).

[0074] During the experiment, it was found that after the rhizome induction culture of step (1), 'Zhongtou Ziyanshi' showed a bud change (return to green), and the resulting rhizome induction materials had light yellow or yellow rhizomes, and some rhizomes were yellow with green. When the whole rhizome of light yellow or yellow was selected as the material, the Zhongtou Ziyanshi's Zhongtou Art retention rate could reach 92.84%; when the selected rhizome was yellow-green, the Zhongtou Ziyanshi's Zhongtou Art retention rate was only 30.77%.

[0075] Example 6

[0076] A method for maintaining the cultivation of hybrid orchid 'C6117T':

[0077] The method is basically the same as Example 1, except that the tissue culture seedlings used for rhizome induction are 'C6117T'.

[0078] Through observation and analysis, it was found that the culture period for rhizome induction was 30-40 days, and the induction rate reached 91.67%; the culture period for rhizome proliferation and differentiation was 50-60 days, the proliferation coefficient was 4.82, the seedling rate was 90.00%, and the medium-term permeability retention rate was 90.80%; the culture period for strong seedlings and rooting was 60-70 days, and complete plants with a plant height of 8.5-9.0 cm and a root length of 4.0-4.5 cm were obtained, and the rooting rate was above 98.0%.

[0079] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0080] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A culture medium for maintaining hybrid orchids, characterized in that: It includes rhizome induction medium, rhizome proliferation and differentiation medium and seedling rooting medium; The rhizome induction medium has the following composition and ratio: MS1 medium, 1.0-2.0 mg / L 6-BA, 0.2-0.5 mg / L NAA, 0.5-1.0 g / L activated carbon, 20-30 g / L white sugar and 6.0-7.0 g / L coagulant, pH 5.6-5.8; The composition and proportion of the rhizome proliferation and differentiation medium are as follows: MS2 medium, KT 0.7-1.0 mg / L, NAA 0.3-0.5 mg / L, activated carbon 0.5-1.0 g / L, white sugar 20-30 g / L and coagulant 6.0-7.0 g / L, pH 5.6-5.8; The composition and proportion of the seedling rooting medium are as follows: MS3 medium, 0.1-0.2 mg / L 6-BA, 0-1.0 mg / L IBA, 0-1.0 mg / L NAA, 2.0-3.0 g / L banana powder, 0.5-1.0 g / L activated carbon, 20-30 g / L white sugar and 6.0-7.0 g / L coagulant, pH 5.6-5.

8.

2. The culture medium for maintaining hybrid orchid according to claim 1, characterized in that: The composition and proportion of the MS1 medium are as follows: KNO3 1900 mg / L, (NH4)2SO4 460-500 mg / L, KH2PO4 150-170 mg / L, MgSO 4. 7H2O210~250mg / L, CaCl 2. 2H2O 440mg / L, MnSO 4. H2O16.9mg / L, ZnSO 4. 7H2O 8.6mg / L, H3BO36.2 mg / L, KI 0.83mg / L, Na2MoO 2. 2H2O0.25mg / L, CoCl 2. 6H2O 0.025mg / L, CuSO 4. 5H2O 0.025mg / L, FeSO 4. 7H2O 27.8mg / L, Na 2. EDTA 37.3mg / L, thiamine hydrochloride 1.0-2.0mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, glycine 2.0mg / L and inositol 100mg / L.

3. The culture medium for maintaining hybrid orchid according to claim 1, characterized in that: The composition and proportion of the MS2 medium are as follows: KNO3950 mg / L, (NH4)2SO4230-250 mg / L, KH2PO4170-190 mg / L, MgSO 4. 7H2O160~180mg / L, CaCl 2. 2H2O 220mg / L, MnSO 4. H2O16.9mg / L, ZnSO 4. 7H2O 8.6mg / L, H3BO36.2 mg / L, KI 0.83mg / L, Na2MoO 2. 2H2O0.25mg / L, CoCl 2. 6H2O 0.025mg / L, CuSO 4. 5H2O 0.025mg / L, FeSO 4. 7H2O 27.8mg / L, Na 2. EDTA 37.3mg / L, thiamine hydrochloride 0.1mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, glycine 2.0mg / L and inositol 100mg / L.

4. The culture medium for maintaining hybrid orchid according to claim 1, characterized in that: The composition and proportion of the MS3 medium are as follows: KNO3 950 mg / L, (NH4)2SO4 230-250 mg / L, KH2PO4 170-190 mg / L, MgSO 4. 7H2O160~180mg / L, CaCl 2. 2H2O 220mg / L, MnSO 4. H2O16.9mg / L, ZnSO 4. 7H2O 8.6mg / L, H3BO36.2 mg / L, KI 0.83mg / L, Na2MoO 2. 2H2O0.25mg / L, CoCl2 . 6H2O 0.025mg / L, CuSO4 . 5H2O 0.025mg / L, FeSO4 . 7H2O 27.8mg / L, Na 2. EDTA 37.3mg / L, thiamine hydrochloride 0.1mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 1.0-2.0mg / L, glycine 2.0-4.0mg / L and inositol 100mg / L.

5. The culture medium for maintaining hybrid orchid according to claim 1, characterized in that: The coagulant is agar powder, carrageenan, or a mixture of agar powder and carrageenan in a mass ratio of 1:

1.

6. Use of the rhizome induction medium, rhizome proliferation and differentiation medium and seedling rooting medium as described in any one of claims 1 to 5 in tissue culture of hybrid orchids.

7. A method for maintaining the cultivation of hybrid orchids, characterized in that: The hybrid orchid is cultivated using the rhizome induction medium, rhizome proliferation and differentiation medium and seedling rooting medium described in any one of claims 1 to 5. The specific cultivation method is as follows: (1) Rhizome induction culture: Inoculate the hybrid orchid rootless tissue culture seedlings onto the induction medium, culture them in the dark for 10 to 15 days, and then culture them under a light intensity of 1800-2000 Lux for 30 to 35 days; (2) Rhizome proliferation and differentiation culture: The rhizomes obtained in step (1) are screened, cut into pieces of 1.0 to 1.5 cm, inoculated onto a rhizome proliferation and differentiation medium, cultured under natural light for 7 to 10 days, and then cultured under a light intensity of 1800 to 2000 Lux for 43 to 50 days to obtain differentiated seedlings; (3) Rooting culture for strong seedlings: The differentiated seedlings obtained in step (2) are cut into single seedlings, inoculated onto a rooting culture medium for strong seedlings, cultured under natural light for 7 to 10 days, and then cultured under a light intensity of 2000-2500 Lux for 53 to 60 days to obtain rooted seedlings.

8. The method for cultivating hybrid orchids by maintaining transparency according to claim 6, characterized in that: The temperature of the rhizome induction culture in step (1) is 23-27° C., and the illumination time is 10-12 h / d; the temperature of the rhizome proliferation and differentiation culture in step (2) is 23-27° C., and the illumination time is 10-12 h / d; the temperature of the seedling rooting culture in step (3) is 23-27° C., and the illumination time is 12-14 h / d.

9. The method for cultivating hybrid orchids by maintaining transparency according to claim 6, characterized in that: The rhizome in step (2) is a rhizome that is entirely light yellow or yellow.

10. The method for cultivating hybrid orchids by maintaining transparency according to claim 6, characterized in that: The single seedling in step (3) is a seedling larger than 3.0 cm, with a full base and an open leaf tip.

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