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CN112335545A - Culture medium for cymbidium rhizome tissue culture and tissue culture method - Google Patents

Culture medium for cymbidium rhizome tissue culture and tissue culture method Download PDF

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CN112335545A
CN112335545A CN202011224451.5A CN202011224451A CN112335545A CN 112335545 A CN112335545 A CN 112335545A CN 202011224451 A CN202011224451 A CN 202011224451A CN 112335545 A CN112335545 A CN 112335545A
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cymbidium
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CN112335545B (en
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黄玮婷
方中明
黎钟懋
王思
马红春
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Guizhou University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001—Culture apparatus for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/62—Orchidaceae [Orchid family]

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Abstract

The invention belongs to the field of plant cell engineering, and particularly relates to a culture medium for cymbidium goeringii rhizome tissue culture and a tissue culture method. The culture medium for cymbidium rhizome tissue culture comprises a differentiation culture medium, wherein the differentiation culture medium is a basic differentiation culture medium added with differentiation promoting amino acids, and the differentiation promoting amino acids comprise asparagine. The scheme overcomes the technical problems of long tissue culture period and low propagation efficiency of the cymbidium goeringii. The scheme can fill the vacancy of influence of amino acid on growth of cymbidium goeringii, can directly guide tissue culture rapid propagation, provides reference for industrial seedling culture, and has important theoretical significance and practical value.

Description

Culture medium for cymbidium rhizome tissue culture and tissue culture method
Technical Field
The invention belongs to the field of plant cell engineering, and particularly relates to a culture medium for cymbidium goeringii rhizome tissue culture and a tissue culture method.
Background
Cymbidium goeringii belongs to the terrestrial species of the orchidaceae Cymbidium genus. The spring orchid has strong fragrance, elegant color, beautiful flower appearance and elegant leaf state, is a unique species in huge orchidaceae families, is also one of main groups of orchids in China, and has wide distribution, rich resources and long cultivation history. The cymbidium has high ornamental value, medicinal value and collection value, so the market demand is continuously expanded. In recent years, wild cymbidium resources are rapidly reduced due to problems of excessive mining, habitat destruction and the like, and a phenomenon of short supply and short demand occurs.
In order to protect the wild cymbidium resource and meet the market demand, the rapid cymbidium propagation approach is imperative. Because the cymbidium goeringii seeds are incompletely developed and are extremely difficult to germinate under the conventional conditions, the traditional propagation method depends on plant division, but the method needs longer time and has low propagation efficiency, and the requirement of large-scale production is difficult to meet. During the tissue culture of orchid, the root-like stem may be obtained through inducing protocorm from seed and growing protocorm. The rhizome is a special proliferation stage of Chinese orchid such as cymbidium goeringii and the like, and is also a key process for limiting the tissue culture and rapid propagation of the Chinese orchid. Subculture multiplication is carried out through the rhizome, which is the key for realizing the rapid propagation of the Chinese orchid. However, the proliferation rate of the cymbidium goeringii rhizome is slow, seedlings formed by differentiation are few, and the cymbidium goeringii rhizome is easy to brown or die during culture, which all limit the rapid proliferation of the cymbidium goeringii. Therefore, the characteristics of long tissue culture period, low proliferation and differentiation coefficients, high production cost and the like of the cymbidium goeringii severely limit the development process of the rapid propagation technology of the cymbidium goeringii.
Disclosure of Invention
The invention aims to provide a culture medium for cymbidium rhizome tissue culture, which overcomes the technical problems of long culture period and low propagation efficiency of cymbidium.
In order to solve the technical problems, the technical scheme of the invention is as follows:
the culture medium for cymbidium rhizome tissue culture comprises a differentiation culture medium, wherein the differentiation culture medium is a basic differentiation culture medium added with differentiation promoting amino acids, and the differentiation promoting amino acids comprise asparagine.
By adopting the technical scheme, the technical principle and the beneficial effects are as follows: through the screening research of amino acid, the asparagine has a remarkable promoting effect on the differentiation of the cymbidium goeringii rhizome, and can be added into a differentiation culture medium of the cymbidium goeringii rhizome to promote the tissue differentiation and accelerate the cymbidium goeringii cultivation process. The existing research shows that asparagine can chelate heavy metal ions such as cadmium, lead and the like, can assist plants to resist environmental stress, and can reduce the browning phenomenon of the cymbidium rhizome in the proliferation stage to a certain extent. The inventor finds a new property of asparagine in the research, asparagine not only has the effect of resisting browning, but also can remarkably promote the differentiation of the rhizome, and compared with other amino acids (most of which have no remarkable effect or have inhibition effect), asparagine has a better technical effect on the differentiation of the rhizome. This property of asparagine can be exploited for tissue culture of cymbidium. In addition, the asparagine is safe and nontoxic, is convenient to purchase and economical and practical, and can obviously promote the growth of explants by adding a small amount of asparagine, which is obviously higher than that of the control treatment without adding amino acid.
The culture medium is a basic culture medium added with proliferation promoting amino acid, and the proliferation promoting amino acid comprises one or two of arginine, cysteine, proline or lysine.
By adopting the technical scheme, arginine, cysteine, proline or lysine are used in the proliferation culture before the differentiation culture, so that the proliferation of the rhizome can be greatly promoted, and the biomass is expanded to form a material basis for the subsequent differentiation process. The combination of the four amino acids has synergistic effect, and can further promote the proliferation of rhizome and accelerate the breeding process.
Further, the differentiation-promoting amino acid may further include tyrosine or aspartic acid.
By adopting the technical scheme, the synergistic effect phenomenon is realized between aspartic acid and asparagine and between tyrosine and asparagine, and the effect of promoting the differentiation of cymbidium goeringii rhizomes is further enhanced. Tyrosine is an important component for synthesizing substances such as protein, pigment and the like, and the inventor researches and discovers that the tyrosine can promote the differentiation of the rhizome for the first time, and expands the application range of the tyrosine.
Further, the concentrations of asparagine, tyrosine and aspartic acid in the differentiation medium are all 0.2-2.0 mmol.L-1(ii) a The concentrations of arginine, cysteine, proline and lysine in the proliferation medium are all 0.2-2.0 mmol.L-1。
By adopting the technical scheme, asparagine, tyrosine and aspartic acid with the concentrations can effectively promote the differentiation of the cymbidium goeringii rhizome, and arginine, cysteine, proline and lysine with the concentrations can effectively promote the proliferation of the cymbidium goeringii rhizome. Wherein, 0.5-1.0 mmol.L-1Lysine and 0.5-2.0 mmol. multidot.L-1The proliferation promoting effect of cysteine is more obvious.
Further, the concentrations of asparagine, tyrosine and aspartic acid in the differentiation medium were 0.5 mmol.L, respectively-1、1.0mmol·L-1And 0.2 mmol. multidot.L-1。
By adopting the technical scheme, the concentration is the optimal concentration for promoting the differentiation of the cymbidium goeringii rhizomes.
Further, the concentrations of arginine, cysteine, proline and lysine in the growth medium were 0.5 mmol.L, respectively-1、2.0mmol·L-1、1.0mmol·L-1And 0.5 mmol. multidot.L-1。
By adopting the technical scheme, the concentration is the optimal concentration for promoting the proliferation of the cymbidium goeringii rhizomes. The inventor found that 0.2 mmol. multidot.L was used in the previous study-1Arginine treatment of cymbidium sinense has good proliferation promoting effect. However, the inventors have studied and found that 0.2 mmol. multidot.L is contained in the cymbidium goeringii-1Arginine did not promote the proliferation of cymbidium goeringii effectively, 0.5 mmol. multidot.L-1Arginine can effectively promote the proliferation of cymbidium goeringii.
Further, the basic proliferation culture medium is an MS culture medium added with basic proliferation components, and the basic proliferation components comprise 6-benzylaminopurine, naphthylacetic acid, sucrose and activated carbon; the basic differentiation medium is MS medium added with basic differentiation components including 6-benzylaminopurine, naphthylacetic acid and sucrose
By adopting the technical scheme, the MS culture medium containing 6-benzylaminopurine, naphthylacetic acid, sucrose and activated carbon can provide proper environment and nutrient substances for the proliferation of cymbidium goeringii rhizomes; and the MS culture medium containing 6-benzylaminopurine, naphthylacetic acid and sucrose can provide enough nutrients for the proliferation of the cymbidium goeringii rhizome.
Further, a method for culturing cymbidium rhizome tissues comprises the following steps of:
s1 proliferation culture step: culturing cymbidium goeringii rhizomes by using a proliferation culture medium to obtain a proliferation culture tissue;
s2 differentiation culture step: culturing the proliferation culture tissue by using a differentiation culture medium to obtain a differentiation culture tissue; the differentiation medium is a basic differentiation medium added with differentiation-promoting amino acids, and the differentiation-promoting amino acids include asparagine.
By adopting the technical scheme, the cymbidium goeringii rhizome is subjected to propagation culture, the biomass is enlarged, and the method is a material basis for subsequent differentiation culture. The explant cells are differentiated into corresponding tissues through differentiation culture, and sprouts grow out for subsequent rooting culture. In the scheme, the cymbidium goeringii rhizome is selected as the explant, inoculation is easy and not easy to pollute, the proliferation and differentiation speed is high, the proliferation rate is high, the browning rate of the explant and a culture medium is low, and the explant can grow into larger seedlings without frequent subculture.
Further, in the proliferation culture step of S1, the proliferation medium is a basal proliferation medium supplemented with proliferation promoting amino acids including arginine and cysteine, or including arginine and proline, or including arginine and lysine, or including cysteine and lysine.
By adopting the technical scheme, the combination of arginine and cysteine, the combination of arginine and proline, the combination of cysteine and lysine and the combination of arginine and lysine can effectively promote the proliferation of the cymbidium goeringii rhizome.
Further, in the proliferation culture step of S1, cymbidium goeringii rhizome is cultured for 60-90 days by using a proliferation culture medium; in the step of S2 differentiation culture, the proliferation culture tissue is cultured using a differentiation medium for 60 to 90 days.
By adopting the technical scheme, the differentiation culture and proliferation culture time can ensure that the cymbidium goeringii rhizome explants grow fully, and conditions are created for subsequent rooting culture. The combination of the amino acid and the cysteine, the combination of the arginine and the proline, the combination of the cysteine and the lysine and the combination of the arginine and the lysine which are added into the proliferation culture medium used by the method can promote the division of the rhizome cells, thereby achieving the rapid proliferation and preventing the rhizome and the culture medium from browning. The differentiation medium used contains asparagine and aspartic acid, and asparagine and tyrosine, which can promote the rapid differentiation of the rhizome into bud.
Drawings
FIG. 1 is a graph showing the results of the growth experiment of cymbidium goeringii in the experimental example.
FIG. 2 is a graph showing the results of the experiment for the differentiation of cymbidium goeringii in the experimental examples.
Detailed Description
The invention will be described in detail with reference to specific examples, in which the following reagents are used: MS medium powder (shanghai yufu biotechnology limited); NAA (Beijing Conbecs technologies, Inc.); 6-BA, sucrose (Hengxing Chemicals manufacturing Co., Ltd., Tianjin); agar (beijing congbes science and technology ltd); activated carbon (beijing congbes technologies ltd); huabao No. 1 and huabao No. 2 (shanghai constant distance biotechnology limited); various amino acids (Shanghai-derived leaf Biotech Co., Ltd.); the cymbidium rhizome used in the invention is from cell engineering laboratory of college of forestry of Guizhou university.
Example 1: preparation of culture Medium
And preparing a proliferation culture medium and a differentiation culture medium, and respectively using the proliferation culture medium and the differentiation culture medium for a cymbidium rhizome proliferation experiment and a cymbidium rhizome differentiation experiment.
The basic proliferation culture medium is MS culture medium (pH5.4) containing basic proliferation components,the proliferation promoting components and the contents thereof are respectively as follows: 1.5 mg. L-16-BA (6-benzylaminopurine), 5.0 mg. L-1NAA (Naphthylacetic acid), 30.0 g.L-1Sucrose, 7.0 g.L-1Agar, 2.0 g.L-1Activated carbon.
The basic differentiation culture medium is MS culture medium (pH5.4) containing basic differentiation components, and the growth promoting components and their contents are respectively: 5 mg. L-1 6-BA、0.2mg·L-1NAA、20.0g·L-1Sucrose, 7.0 g.L-1Agar.
The culture medium is separately added with a concentration of 0.2 mmol.L after filtration sterilization-1、0.5mmol·L-1、1.0mmol·L-1、2.0mmol·L-1The 20 amino acids (glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine, serine, threonine, glutamine, tyrosine, asparagine and cysteine, glutamic acid and aspartic acid, histidine, arginine, lysine, respectively), and combinations of amino acids, with media without the addition of the amino acids as controls.
The MS culture medium is a conventional culture medium in the prior art, and the conventional preparation method and formula are as follows (commercial MS culture medium powder can be purchased and is prepared into a 1 XMS culture medium after being dissolved by water):
preparing MS culture medium mother liquor:
20 × MS macroelements (1L): 38gKNO3，33gNH4NO3，8.8gCaCl2·2H2O，7.4gMgSO4·7H2O，3.4gKH2PO4
200 × MS trace elements (1L): 0.166gKI, 1.24gH3BO3，4.46gMnSO4·4H2O，1.72gZnSO4·7H2O，0.05gNa2MoO4·2H2O，0.005gCuSO4·5H2O，0.005gCoCl2·6H2O。
200 × MS vitamins and amino acids (1L): 20g inositol, 0.1g nicotinic acid, 0.1g pyridoxine hydrochloride (VB6), 0.02g thiamine hydrochloride (VB 1), 0.4g glycine.
200 × MS iron salt (1L): 5.56g FeSO4·7H2O，7.46gNa2EDTA·2H2O, first dissolved in 500mLddH2And O, heating, adjusting the pH value to 5.5, and fixing the volume to 1L.
Preparing a basic MS culture solution by using the prepared mother solution according to the dilution times: 1 × MS macroelements, 1 × MS microelements, 1 × MS vitamins and amino acids, and 1 × MS ferric salt, adjusting the pH to 5.4 after constant volume, and obtaining MS culture medium (1 × liquid culture medium, which needs high temperature and high pressure sterilization). The composition and content of the 1 × MS medium prepared using the commercial MS medium powder were the same as those of the above-described MS medium (1 × liquid medium) prepared using the mother liquor.
Adding other components such as plant growth hormone on the basis of the MS culture medium to respectively obtain a basic proliferation culture medium and a basic differentiation culture medium, and adding amino acid to be tested on the basic proliferation culture medium and the basic differentiation culture medium to obtain the proliferation culture medium to be tested and the differentiation culture medium to be tested for subsequent experiments.
Example 2: cymbidium goeringii rhizome proliferation experiment
Sterile, well-grown, branched rhizomes, approximately 2mm in diameter, are prepared and cut to 0.5cm length for use. Respectively transferring 0.5 cm-long cymbidium rhizome into proliferation culture medium, wherein each bottle contains 1g of cymbidium rhizome material, wiping off water and culture medium with sterile paper towel, weighing, uniformly inoculating on the surface of culture medium (proliferation culture medium to be tested), and irradiating at 25 deg.C for 2000Lx for 12 hr/day. After 60 days (in actual operation, the culture can be carried out for 60-90 days according to actual conditions), taking out the rhizomes in the bottles, wiping water and residual culture medium, weighing, recording the weight of each rhizomes on the culture medium, and counting the browning condition of the rhizomes.
Preparation of the proliferation medium to be tested (1L as an example): heating about 300ml of distilled water, adding 7g of agar when small bubbles appear, stirring uniformly, adding 30g of sucrose and 2.37g of MS culture medium powder after complete dissolution, stirring until complete dissolution, adding 1 mg/ml of plant growth regulator-11.5ml and 1 mg/ml of 6-BA mother liquor-1NAA 5.0 ml. Respectively subpackaged into orchid bottles in the amount of100 ml/bottle, 10 bottles in total. And when the culture medium is cooled to 50-60 ℃, adding amino acid mother liquor with various concentrations into the culture medium in a super clean bench, and uniformly mixing for later use.
Example 3: cymbidium goeringii rhizome differentiation experiment
1cm length of cymbidium goeringii rhizome (0.5 cm rhizome from example 2 was cultured to about 1cm length by basic multiplication medium) was transferred to differentiation medium, 18 per bottle, and cultured in culture room. At 25 deg.C, the illumination was 2000Lx for 12 hours/day. About 15 days, the rootstock begins to differentiate, about 60 days (in practice, it can be cultured for 60-90 days according to actual conditions), and the bud grows to 2-3 cm. The number of rootstocks on the medium was recorded, the number of shoots produced and the number of rootstocks browned was counted.
Preparation of test differentiation medium (1L as an example): heating about 300ml of distilled water, adding 7g of agar when small bubbles appear, stirring uniformly, adding 30g of sucrose and 2.37g of MS culture medium powder after complete dissolution, stirring until complete dissolution, adding 1 mg/ml of plant growth regulator-15.0ml and 1 mg/ml of 6-BA mother liquor-1NAA 0.2 ml. The components are respectively subpackaged into orchid bottles with the filling amount of 100 ml/bottle and 10 bottles in total. And when the culture medium is cooled to 50-60 ℃, adding amino acid mother liquor with various concentrations into the culture medium in a super clean bench, and uniformly mixing for later use.
Example 4: plant regeneration experiment
The shoots generated in example 3 were transplanted into a strong seedling medium and after 60 days of culture, it was observed whether the shoots grew into whole plants. The seedling strengthening culture medium contains 1 g.L-1Huabao No. 1, 1 g.L-1Huabao No. 2, 0.5 mg.L-1NAA、2g·L-1MS medium of AC.
For examples 2-4, the culture conditions were all at a culture temperature of 25. + -. 2 ℃ for 16h of daily light.
Experimental example 1: selection of proliferation Medium and differentiation Medium
And adding amino acid into the basic multiplication culture medium to obtain a multiplication culture medium to be detected, carrying out multiplication culture on the cymbidium goeringii rhizome according to the method in the example 2, and calculating the multiplication rate and observing the browning condition of the rhizome after the culture is finished. The increment rate is as follows: fresh weight of rhizome after 60 days of propagation culture/fresh weight of rhizome before propagation culture (3 replicates per set of experiments).
Adding amino acid into the basic differentiation medium to obtain a differentiation medium to be tested, performing differentiation culture on the cymbidium goeringii rhizome according to the method in example 2, calculating the differentiation rate and observing the browning of the rhizome after the culture is finished. The differentiation rate is: average number of shoots produced by the rhizome after 60 days of differential culture (3 replicates per set of experiments).
Different proliferation culture media to be detected and differentiation culture media to be detected are prepared, and a cymbidium rhizome proliferation experiment and a cymbidium rhizome differentiation experiment are carried out according to the methods of the embodiment 2 and the embodiment 3. At the end of the experiment, one-way ANOVA and Duncan tests were used to evaluate data significance. As shown in tables 1 to 6, the composition of the medium and the culture results were shown, and since the basic growth medium of all the growth medium to be tested was identical and the basic differentiation medium of all the differentiation medium to be tested was identical, only the kind and content of amino acid were described in tables 1 to 6. In tables 1 to 4, the blank for the proliferation experiment refers to the use of a basal proliferation medium, and the blank for the differentiation experiment refers to the use of a basal differentiation medium. The blank in Table 5 used basal proliferation medium (proliferation experiments in the same manner as in tables 1-4, and thus is not shown in the tables); the blank in Table 6 used the basal differentiation medium (differentiation experiments as in tables 1-4, and thus not shown).
Table 1: effect of Individual amino acids on proliferation and differentiation of cymbidium (nonpolar amino acids)
Figure BDA0002763184010000071
Table 2: effect of Individual amino acids on proliferation and differentiation of cymbidium (polar neutral amino acids)
Figure BDA0002763184010000072
Table 3: effect of Individual amino acids on proliferation and differentiation of cymbidium (polar acidic amino acids)
Figure BDA0002763184010000073
Table 4: effect of Individual amino acids on proliferation and differentiation of cymbidium (polar basic amino acids)
Figure BDA0002763184010000081
Table 5: experimental result of influence of combination of two amino acids on proliferation of cymbidium goeringii
Kind and amount of amino acid (mmol. L)-1) Proliferation rate Browning conditions
Cysteine 2.0, proline 1.0 2.61±0.19c Is free of
Cysteine 2.0, isoleucine 0.2 2.69±0.24c Is free of
Cysteine 2.0, asparagine 0.2 2.53±0.22cd Is free of
Cysteine 2.0, threonine 0.2 2.32±0.26d Severe severity of disease
Cysteine 2.0, glutamic acid 0.2 2.38±0.26d Light and slight
Cysteine 2.0, lysine 0.5 2.83±0.29ab Is free of
Arginine 0.5, cysteine 2.0 3.01±0.27a Is free of
Arginine 0.5, proline 1.0 3.05±0.43a Is free of
Arginine 0.5, isoleucine 0.2 2.29±0.22cd Is free of
Arginine 0.5, asparagine 0.2 2.66±0.36bc Is free of
Arginine 0.5, threonine 0.2 2.50±0.28bc Is free of
Arginine 0.5, glutamic acid 0.2 2.42±0.27c Is free of
Arginine 0.5, lysine 0.5 2.91±0.26ab Is free of
Table 6: experimental result of influence of two amino acids on cymbidium differentiation
Kind and amount of amino acid (mmol. L)-1) Rate of differentiation Browning conditions
Asparagine 0.5, tyrosine 1.0 5.34±0.25a Severe severity of disease
Asparagine 0.5, aspartic acid 0.2 5.39±0.21a Medium and high grade
Asparagine 0.5, cysteine 0.5 4.95±0.19b Medium and high grade
Tyrosine 1.0, aspartic acid 0.2 4.89±0.18b Severe severity of disease
Tyrosine 1.0, cysteine 0.5 4.67±0.15c Severe severity of disease
Aspartic acid 0.2, cysteine 0.5 4.78±0.25bc Medium and high grade
Tyrosine 1.0, aspartic acid 0.2 4.89±0.18b Severe severity of disease
In tables 1-6, the results were analyzed for significance of differences using the Duncan test, with the data being followed by lower case letters, and in the same table, the difference in significance between different lower case letters is shown, with p < 0.05.
The partial culture results are shown in FIGS. 1 and 2. Wherein, in FIG. 1, A-I represent: a: control (basal proliferation medium); b: proline 1.0 mmol. L-1(ii) a C: cysteine 2.0 mmol. L-1(ii) a D: asparagine 0.2 mmol. L-1(ii) a E: aspartic acid 2.0 mmol. L-1(ii) a F: arginine 0.5 mmol. L-1(ii) a G: lysine 0.5 mmol. multidot.L-1(ii) a H: arginine 0.5 mmol. L-1+ cysteine 2.0 mmol. L-1(ii) a I: arginine 0.5 mmol. L-1+ proline 1.0 mmol. L-1. In FIG. 2, A to F represent: a: control (basal differentiation medium); b: methionine 2.0 mmol. L-1(ii) a C: asparagine 0.5 mmol. L-1(ii) a D: asparagine 0.5 mmol. L-1+ tyrosine 1.0 mmol. L-1(ii) a E: asparagine 0.5 mmol. L-1+ aspartic acid 0.2 mmol. L-1(ii) a F: differentiation of shoots from rootstocks (see example 3 for differentiation experiments, in the figureThe bud is obtained by differentiation culture of asparagine-containing culture medium) transferred into Huabao No. 1 g.L-1+ Huabao No. 2 1 g.L-1+NAA 0.5mg·L-1+AC 2g·L-1The effect of strengthening seedlings is achieved after the culture medium is used for culturing for 60 days.
Aiming at the cymbidium goeringii rhizome proliferation culture experiment, referring to tables 1-5, cysteine, arginine, proline and lysine are added into a basic proliferation culture medium, so that the proliferation of the cymbidium goeringii rhizome can be obviously improved under a certain concentration, and the browning degree can be reduced. Arginine and proline are combined, arginine and cysteine are combined, arginine and lysine are combined, the proliferation rate of the cymbidium goeringii rhizome can be improved better, and arginine, proline, cysteine and any amino acid in lysine have a synergistic effect. The weight of the rhizome on the culture medium added with arginine and proline is averagely increased by 3.05 times compared with that of the rhizome inoculated before 60 days, and the culture medium has no browning phenomenon; while the weight of the rootstock in the control culture medium is only increased by 1.35 times on average compared with that of the rootstock inoculated before 60 days, and the culture medium has slight browning phenomenon.
As shown in FIG. 1, 1.0 mmol. multidot.L-1Proline (FIG. 1B), 2.0 mmol. multidot.L-1Cysteine (FIG. 1C) and 0.2 mmol. multidot.L-1Asparagine (fig. 1D) significantly promoted rhizome growth. 2.0 mmol. L-1Aspartic acid is not suitable for addition to the rhizome propagation medium, and excessive use results in restricted growth of the rhizome and browning of the rhizome (FIG. 1E). Arginine content of 0.5 mmol. L-1The growth of the rhizome was significantly promoted (fig. 1F), but as the arginine concentration increased, the promotion was reduced and the rhizome appeared brown. Lysine in 0.5 mmol. multidot.L-1Growth of the rhizome may also be promoted (FIG. 1G). Arginine 0.5 mmol. L-1And proline 1.0 mmol. multidot.L-1Combined use (FIG. 1I), and arginine 0.5 mmol. L-1And cysteine 2.0 mmol. multidot.L-1The combined use (FIG. 1H) can promote the growth of cymbidium root-like stem more effectively.
Referring to tables 1-4 and 6, tyrosine, aspartic acid and asparagine all have promoting effects on cymbidium rhizome differentiation, but all cause browning to different degrees in cymbidium rhizome differentiation culture experiments. The combination of asparagine and tyrosine and the combination of asparagine and aspartic acid has a synergistic effect and can further promote the differentiation of cymbidium goeringii rhizome. The number of differentiated shoots per rhizome on the medium supplemented with asparagine and aspartic acid was 5.39 on average, and the medium was moderately browned. While the number of buds per rhizome on the control medium was 2.73, the rhizome was also green and the medium was slightly browned.
As shown in FIG. 2, 2.0 mmol. multidot.L-1Methionine inhibits the differentiation of the rhizome (fig. 2B, non-polar amino acids are not suitable as additives to the rhizome differentiation medium). 0.5 mmol. L-1Asparagine (fig. 2C) significantly promoted rootstock differentiation. D: 0.5 mmol. L-1Asparagine and 1.0 mmol. multidot.L-10.5 mmol. L of tyrosine-1Asparagine and 0.2 mmol. multidot.L-1The combination of aspartic acid (FIG. 2E) has better promoting effect on the differentiation of the rhizome.
In addition, when the shoots obtained after the differentiation culture of example 3 were transferred to a strong seedling medium, all shoots developed to form whole plants, which indicates that the shoots differentiated from the rootstock according to the present method can be regenerated to whole plants. As shown in FIG. 2F, the sprouts obtained by differentiation culture in the asparagine-containing medium were transferred to a strong seedling medium and cultured to obtain whole plants.
The foregoing is merely an example of the present invention and common general knowledge in the art of designing and/or characterizing particular aspects and/or features is not described in any greater detail herein. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

Claims (10)

1. A culture medium for cymbidium rhizome tissue culture characterized by: the cell culture medium comprises a differentiation medium, wherein the differentiation medium is a basic differentiation medium added with differentiation promoting amino acids, and the differentiation promoting amino acids comprise asparagine.
2. The medium for cymbidium tuberosum tissue culture according to claim 1, characterized in that: the culture medium is a basic proliferation culture medium added with proliferation promoting amino acid, and the proliferation promoting amino acid comprises one or two of arginine, cysteine, proline or lysine.
3. The medium for cymbidium tuberosum tissue culture according to claim 2, characterized in that: the differentiation-promoting amino acid further includes tyrosine or aspartic acid.
4. The medium for cymbidium tuberosum tissue culture according to claim 3, characterized in that: the concentrations of asparagine, tyrosine and aspartic acid in the differentiation medium are all 0.2-2.0 mmol.L-1(ii) a The concentrations of arginine, cysteine, proline and lysine in the proliferation medium are all 0.2-2.0 mmol.L-1。
5. The medium for cymbidium goeringii rhizome tissue culture according to claim 4, characterized in that: the concentrations of asparagine, tyrosine and aspartic acid in the differentiation medium were 0.5 mmol.L, respectively-1、1.0mmol·L-1And 0.2 mmol. multidot.L-1。
6. The medium for cymbidium goeringii rhizome tissue culture according to claim 5, characterized in that: the concentrations of arginine, cysteine, proline and lysine in the proliferation medium were 0.5 mmol.L, respectively-1、2.0mmol·L-1、1.0mmol·L-1And 0.5 mmol. multidot.L-1。
7. The medium for cymbidium tuberosum tissue culture according to claim 6, characterized in that: the basic proliferation culture medium is an MS culture medium added with basic proliferation components, and the basic proliferation components comprise 6-benzylaminopurine, naphthylacetic acid, sucrose and active carbon; the basic differentiation medium is an MS medium added with basic differentiation components, and the basic differentiation components comprise 6-benzylaminopurine, naphthylacetic acid and sucrose.
8. A method for culturing cymbidium rhizome tissues is characterized in that: comprises the following steps in sequence:
s1 proliferation culture step: culturing cymbidium goeringii rhizomes by using a proliferation culture medium to obtain a proliferation culture tissue;
s2 differentiation culture step: culturing the proliferation culture tissue by using a differentiation culture medium to obtain a differentiation culture tissue; the differentiation medium is a basic differentiation medium added with differentiation-promoting amino acids, and the differentiation-promoting amino acids include asparagine.
9. The method for cymbidium goeringii rhizome tissue culture according to claim 8, wherein: in the proliferation culture step of S1, the proliferation medium is a basal proliferation medium supplemented with proliferation promoting amino acids including arginine and cysteine, or including arginine and proline, or including arginine and lysine, or including cysteine and lysine.
10. The method for cymbidium goeringii rhizome tissue culture according to claim 9, wherein: in the S1 proliferation culture step, the cymbidium rhizome is cultured for 60-90 days by using a proliferation culture medium; in the step of S2 differentiation culture, the proliferation culture tissue is cultured using a differentiation medium for 60 to 90 days.
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Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4818693A (en) * 1983-05-19 1989-04-04 Plant Genetics, Inc. Methods and materials for enhanced somatic embryo regeneration in the presence of auxin
CN102668982A (en) * 2012-05-04 2012-09-19 四川农业大学 Tissue culture method of mature wheat embryo
CN102835311A (en) * 2012-08-14 2012-12-26 江苏丘陵地区南京农业科学研究所 Method for culturing cattleya hybrida tissues
CN103229721A (en) * 2013-05-15 2013-08-07 聊城大学 Tissue culture propagation method of Gynura formosana
CN105210863A (en) * 2014-06-09 2016-01-06 谢炯 A kind of tissue culture and rapid propagation method of rhizoma Gastrodiae
CN107736246A (en) * 2017-11-04 2018-02-27 王衡 Qing Chuan gastrodia elata f. glauca tissue culture medium (TCM) and tissue culture mating system
CN107873525A (en) * 2017-12-28 2018-04-06 武汉生物工程学院 A kind of Chinese cymbidium quick breeding method for tissue culture
CN109042342A (en) * 2018-11-01 2018-12-21 翁源县天下泽雨农业科技有限公司 A kind of method for tissue culture of Chunlan
CN110338059A (en) * 2019-07-19 2019-10-18 华南农业大学 A method for improving the proliferation and differentiation efficiency of Chinese orchid rhizomes
CN110547200A (en) * 2019-09-30 2019-12-10 李传传 Marigold pollen differentiation medium and differentiation culture method
CN111727879A (en) * 2019-07-11 2020-10-02 中国科学院植物研究所 A kind of medium and method for propagating oil peony

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4818693A (en) * 1983-05-19 1989-04-04 Plant Genetics, Inc. Methods and materials for enhanced somatic embryo regeneration in the presence of auxin
CN102668982A (en) * 2012-05-04 2012-09-19 四川农业大学 Tissue culture method of mature wheat embryo
CN102835311A (en) * 2012-08-14 2012-12-26 江苏丘陵地区南京农业科学研究所 Method for culturing cattleya hybrida tissues
CN103229721A (en) * 2013-05-15 2013-08-07 聊城大学 Tissue culture propagation method of Gynura formosana
CN105210863A (en) * 2014-06-09 2016-01-06 谢炯 A kind of tissue culture and rapid propagation method of rhizoma Gastrodiae
CN107736246A (en) * 2017-11-04 2018-02-27 王衡 Qing Chuan gastrodia elata f. glauca tissue culture medium (TCM) and tissue culture mating system
CN107873525A (en) * 2017-12-28 2018-04-06 武汉生物工程学院 A kind of Chinese cymbidium quick breeding method for tissue culture
CN109042342A (en) * 2018-11-01 2018-12-21 翁源县天下泽雨农业科技有限公司 A kind of method for tissue culture of Chunlan
CN111727879A (en) * 2019-07-11 2020-10-02 中国科学院植物研究所 A kind of medium and method for propagating oil peony
CN110338059A (en) * 2019-07-19 2019-10-18 华南农业大学 A method for improving the proliferation and differentiation efficiency of Chinese orchid rhizomes
CN110547200A (en) * 2019-09-30 2019-12-10 李传传 Marigold pollen differentiation medium and differentiation culture method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
黄玮婷等: "不同有机添加物及柠檬酸钠组合对春兰根状茎增殖的影响", 《安徽农业大学学报》 *

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