CN114027196A - Tissue culture and rapid propagation culture medium combination and culture method of cercis fascicularis - Google Patents
Tissue culture and rapid propagation culture medium combination and culture method of cercis fascicularis Download PDFInfo
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Abstract
The invention discloses a tissue culture rapid propagation culture medium combination of a cluster redbud and a culture method. Relates to the technical field of tissue culture. The method comprises the following steps: selection, treatment and disinfection of explants, sterile bud induction and proliferation and rooting culture. In the invention, the culture medium A, B is alternately used for subculture proliferation and rooting culture; the culture medium A can not only induce aseptic buds, but also be used for subculture multiplication and rooting culture, and can synchronously perform subculture multiplication and seedling strengthening and rooting in the same culture medium to form seedlings once; the culture medium B is also used for subculture proliferation and rooting culture, and is used for one-time seedling formation; the culture medium A, B is used alternatively to increase the multiplication coefficient, the subculture multiplication and strong seedling rooting period is 24d, and the special culture rooting time is saved. The multiplication coefficient reaches 6.9, 2-5 roots are rooted, the root length is 2-4 cm, and the rooting rate reaches more than 98%. The rooting tissue culture seedling does not need to be hardened, and the survival rate of direct transplantation reaches 99 percent.
Description
Technical Field
The invention relates to the technical field of tissue culture, in particular to a tissue culture rapid propagation medium combination and a culture method of cercis fascicularis.
Background
The jungle Chinese redbud (Cercis chinensis Bunge) is also named as full red, the leguminous Chinese redbud belongs to the genus Cercis, deciduous shrubs are bloomed twice a year, the flowering period is 2-4 months and 8-10 months, the flowering period is long, the flowers are firstly bloomed, the leaves are secondly bloomed, the flower color is bright, the sprouting property is strong, and the jungle Chinese redbud is a good variety of the Chinese redbud. The jungle cercis can be used for greening and beautifying courtyards, green lands of parks, two sides of roads and the like, can be used for potted plant ornamental, has resistance to chlorine gas and strong dust retention capacity, can be used as a medicine for peels, fruits, trees and flowers, is a symbol of family fullness and deep bone and meat, has high economic, ecological and landscape utilization values and has wide application prospect in flowers and sea.
At present, the tissue culture and rapid propagation research of the cercis caespitiformis is rarely reported in China. The cuttage of the clump purple bramble is difficult to root, the survival rate is low, the cuttage mainly depends on the grafting propagation, the propagation speed is slow, the characteristics of seedling scarcity and uneven quality level are presented, and the demand of popularizing excellent varieties is difficult to meet. In addition, the conventional induction medium, the subculture medium, the multiplication medium and the rooting medium have different components and are long in cultivation time.
Therefore, the problem to be solved by those skilled in the art is how to provide a culture medium composition and a culture method for tissue culture of cercis fascicularis.
Disclosure of Invention
In view of the above, the present invention provides a culture medium composition for tissue culture and rapid propagation of cercis fascicularis and a culture method thereof. The tissue culture mode is adopted for propagation, the propagation speed is high, the quality of the seedlings is good, the genetic genes are stable, and the transplanting viability is high. The tissue culture propagation technology is the optimal technology for realizing the mass propagation and industrial production of the cercis fascicularis.
In order to achieve the purpose, the invention adopts the following technical scheme:
a culture medium composition for tissue culture and rapid propagation of Cercis junci comprises culture medium A and culture medium B; medium a included the following ingredients: KNO3 950mg/L、NH4NO3 825mg/L、KH2PO4 200～255mg/L、CaCl22H2O 220mg/L、MgSO47H2O 185mg/L、FeSO47H2O 41.7mg/L、Na2-EDTA 56mg/L、KI 0.83mg/L、MnSO4·4H2O 22.3mg/L、ZnSO47H2O 8.6mg/L、CuSO45H2O 0.025mg/L、CoCl26H2O 0.025mg/L、H3BO36.2mg/L、NaMoO42H2O0.25 mg/L, inositol 100mg/L, VB1 0.4mg/L、VB60.5mg/L、VB30.5mg/L, 2mg/L of glycine, 30000mg/L of sucrose, 3700mg/L of agar, 0.3-0.5 mg/L, IBA 0.3.3-0.5 mg/L of 6-BA, 1mg/L of triacontanol and 20mg/L of activated carbon 200mg/L, L-cysteine;
culturingThe base B comprises the following components: KNO3 950mg/L、NH4NO3 825mg/L、KH2PO4 200～255mg/L、CaCl22H2O 220mg/L、MgSO47H2O 185mg/L、FeSO47H2O 41.7mg/L、Na2-EDTA 56mg/L、KI 0.83mg/L、MnSO4·4H2O 22.3mg/L、ZnSO47H2O 8.6mg/L、CuSO45H2O 0.025mg/L、CoCl26H2O 0.025mg/L、H3BO3 6.2mg/L、NaMoO42H2O0.25 mg/L, inositol 100mg/L, VB10.4mg/L、VB6 0.5mg/L、VB30.5mg/L, 2mg/L of glycine, 30000mg/L of sucrose, 3700mg/L, TDZ 0.3.3-0.5 mg/L, IBA 0.3.3-0.5 mg/L of agar, 1mg/L of triacontanol and 200mg/L, L-cysteine of activated carbon.
Further, the pH of the medium A, B was adjusted to 5.8 conventionally.
The invention also provides a tissue culture and rapid propagation culture method by utilizing the tissue culture and rapid propagation culture medium combination of the jungle Chinese redbud, which is characterized by comprising the following steps:
(1) selection, treatment and disinfection of explants:
taking the tender branches of the arbuscule as explants, reserving the base parts of petioles with the length of 4-5 mm, cutting the tender branches into stem sections with the length of 5.5-6.5 cm and 3-5 axillary buds, cleaning the stem sections, and then using 0.1% HgCl2Adding 2 drops of Tween, sterilizing for 10min, and washing;
(2) sterile bud induction:
removing cuts at two ends of an explant and a base part of a petiole, cutting a stem section of 5.5-6.5 cm into two stem sections with 2-3 axillary buds respectively, and placing the stem sections in the culture medium A prepared in the claim 1; culturing conditions are as follows: the temperature is 18-22 ℃, the air humidity is 50-80%, the illumination intensity is 700-1500 lx, and the illumination time is 12 h.d-1The culture room is cultured for 10 days, then the culture room is moved to the temperature of 23-27 ℃, the air humidity of 50-80 percent, the illumination intensity of 2000-3000 lx and the illumination time of 14 h.d-1Culturing for 10-12 days in the culture room to obtain sterile buds;
(3) subculture proliferation and rooting culture:
taking aseptic buds, selecting axillary buds with vigorous growth state, cutting the axillary buds into 2.5-3.5 cm stem segments, and placing the stem segments in the culture medium A or B prepared according to claim 1; the culture conditions are as follows: the temperature is 23-28 ℃, the air humidity is 50-80%, the illumination intensity is 2000-3000 lx, and the illumination time is 14 h.d-1(ii) a Carrying out proliferation and rooting culture for 24d, cutting axillary bud stem segments from part of seedlings, carrying out subculture proliferation and rooting culture, and transplanting the rest rooted seedlings;
the culture medium A, B is used alternately for subculture and proliferation.
Further, the current-year semi-lignified twig of the jungle redbud is taken as an explant in the step (1).
Has the advantages that: 4d after inoculation, the axillary buds begin to sprout, and 7d, the axillary buds begin to extend; and after 22 days of inoculation, the height of the buds is 3-5 cm.
When the subculture proliferation is continuously carried out, 0.5mg/L of TDZ is used for replacing 0.5mg/L of 6-BA at intervals of 1 generation, the proliferation coefficient can be improved, and after four times of subculture proliferation, the proliferation coefficient reaches 6.9.
Further, the step (2) removes: the sterilized explants were clamped by holding the forceps with the left hand, and the cut at both ends of the explants and the cut at the base of the petiole was cut with the scissors with the right hand.
Preferably: cleaning in step (1): placing the stem segments in tap water and flowing water for washing for 15-20 min, soaking in 1% detergent solution for 5min, lightly scrubbing the surfaces of the stem segments by using a soft brush, and washing the stem segments by using the tap water; washing: washing with sterile water for 3-4 times, and draining for later use.
According to the technical scheme, compared with the prior art, the invention discloses a tissue culture rapid propagation medium combination and a culture method of the cercis fasciatus, compared with the prior art: the rapid propagation method provided by the invention alternately uses the culture medium A, B for subculture propagation and rooting culture; the culture medium A can not only induce aseptic buds, but also be used for subculture multiplication and rooting culture, and can synchronously perform subculture multiplication and seedling strengthening and rooting in the same culture medium to form seedlings once; the culture medium B can be used for subculture proliferation and rooting culture, and can synchronously perform subculture proliferation and seedling strengthening and rooting in the same culture medium for one-time seedling formation; the culture medium A, B is used alternatively to increase the multiplication coefficient, the subculture multiplication and strong seedling rooting period is 24d, and the special culture rooting time is saved. The multiplication coefficient reaches 6.9, 2-5 roots are rooted, the root length is 2-4 cm, and the rooting rate reaches more than 98%. The rooting tissue culture seedlings do not need to be hardened, the survival rate of direct transplantation reaches 99%, the problems of scarcity of seedlings of the clump redbud seedlings, uneven quality level, limitation of seasons and climate on propagation and the like are solved, and a good technical platform is provided for scale propagation and industrial production;
the disinfection effect, the inductivity, the multiplication coefficient, the rooting rate and the like are all superior to those of the conventional tissue culture technology. Explants were sterilized at 0.1% HgCl22 drops of Tween is added, so that the disinfection effect is good, the pollution phenomenon is avoided, and the induction rate of aseptic buds reaches 92.1 percent. The proper amount of the non-ionic surfactant Tween is added, so that the solubility of the disinfectant can be increased, the disinfectant can fully contact with explants, and the sterilization effect is improved; when the concentration of the Tween is too low, the effect of enhancing the solubility of the venom is not obvious, and when the concentration is too high, explants are easy to be damaged;
the low-concentration inorganic salt is more suitable for the growth of the cercis caespitosa. But KH is properly added into the culture medium for tissue culture and rapid propagation of the cercis fascicularis2PO4The growth vigor of the tissue culture seedlings is better;
the concentration of the ferric salt is 1.5 times of that of the MS culture medium, and the concentration of the ferric salt and the MS culture medium are obviously different. The iron salt is an inorganic salt substance and plays an important role in chlorophyll synthesis, embryo formation, bud differentiation and seedling growth;
TDZ can better promote bud differentiation and growth, the TDZ and 6-BA are alternately used to have a synergistic effect, and when successive subculture proliferation is carried out, 0.5mg/L of TDZ is used to replace 0.5mg/L of 6-BA at intervals of 1 generation, so that the proliferation coefficient can be improved, but continuous independent use of TDZ can inhibit bud point elongation;
triacontanol, activated carbon, L-cysteine, in combination, produce a synergistic effect. Triacontanol can enhance photosynthesis, transport and accumulation of nutrient substances, and promote rooting, stem and leaf growth; the activated carbon can blacken the culture medium, is beneficial to rooting, and the adsorption effect of the activated carbon can reduce the influence of some harmful substances; the L-cysteine can promote wound healing and prevent aging and browning; the combination of triacontanol 1.0mg/L and active carbon 200mg/L, L-cysteine 20mg/L is added into the culture medium, which is very favorable for promoting the induction of aseptic buds, the subculture multiplication and the rooting of strong seedlings;
the tissue culture seedling can be transplanted without hardening the seedling, and the transplanting survival rate is as high as 99%. The method is obviously different from the conventional method that the tissue culture seedlings can be transplanted only by hardening the seedlings.
Detailed Description
The following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention discloses a tissue culture rapid propagation medium combination of a cluster redbud and a culture method.
Example 1
Preparing a culture medium:
the tissue culture and rapid propagation culture medium A of the cluster Chinese redbud is used for sterile bud induction. The culture medium A is packaged and sterilized in autoclave at 121 deg.C for 20min, and then kept stand and cooled for use, and the components and contents (mg/L) of the substances are as shown in the following table 1:
TABLE 1
| Composition (I) | Content (wt.) | Composition (I) | Content (wt.) |
| KNO3 | 950 | Inositol | 100 |
| NH4NO3 | 825 | VB1 | 0.4 |
| KH2PO4 | 255 | VB6 | 0.5 |
| CaCl2·2H2O | 220 | VB3 | 0.5 |
| MgSO4·7H2O | 185 | Glycine | 2 |
| FeSO4﹒7H2O | 41.7 | Sucrose | 30000 |
| Na2-EDTA | 56 | Agar-agar | 3700 |
| KI | 0.83 | 6-BA | 0.5 |
| MnSO4·4H2O | 22.3 | IBA | 0.3 |
| ZnSO4·7H2O | 8.6 | Triacontanol | 1 |
| CuSO4·5H2O | 0.025 | Activated carbon | 200 |
| CoCl2·6H2O | 0.025 | L-cysteine | 20 |
| H3BO3 | 6.2 | PH | 5.8 |
| NaMoO4·2H2O | 0.25 |
Subculture proliferation and rooting culture, alternately using culture medium A and culture medium B, subpackaging culture medium B, sterilizing at 121 deg.C for 20min, standing, and cooling to obtain the final product with the following components and contents (mg/L) as shown in Table 2:
TABLE 2
The culture method comprises the following steps:
selection, treatment and disinfection of explants:
collecting annual semi-lignified twigs of the bramble redbud as explants in sunny days, removing leaves (reserving petiole base parts with the length of 4-5 mm), cutting into stem sections with the length of 5.5-6.5 cm and 3-5 axillary buds, placing running water for flushing for 20min, soaking in 1% detergent solution for 5min, lightly scrubbing the surfaces of the stem sections with a soft brush, and flushing with the running water. The pretreated explants were placed on a clean bench and washed with 0.1% HgCl2And (4) sterilizing the mixture with 2 drops of Tween for 10min, washing the mixture with sterile water for 4-5 times, and draining the water for later use. The stem section of the explant cannot be cut too short during disinfection, so that the incision area of the explant damaged by disinfectant is reduced as much as possible.
Sterile bud induction:
clamping the sterilized explant by a left-hand forceps, shearing two ends of the explant and a base cut of a petiole by a right-hand scissors, then shearing 5.5-6.5 cm stem sections into two stem sections with two axillary buds respectively, and then inoculating the stem sections onto a tissue culture rapid propagation medium A of the jungle gypsy. When the explant is cultured, firstly, the temperature is 18 ℃, the air humidity is 50%, the illumination intensity is 700lx, and the illumination time is 12 h.d-1Culturing in the culture room for 10 days, moving to 23 deg.C, air humidity of 50%, illumination intensity of 2000lx, and illumination time of 14 h.d-1The culture chamber of (3) for 10 d. 4d after inoculation, the axillary buds begin to sprout, and 7d, the axillary buds begin to extend; and after 22 days of inoculation, the height of the buds is 3-5 cm, the buds are strong and grow well.
The induction rate of the aseptic buds reaches 92 percent.
Subculture proliferation and rooting culture:
and lightly clamping sterile buds by using a forceps, selecting axillary buds with vigorous growth state by using scissors, cutting the axillary buds into 2.5-3.5 cm long stems, vertically inserting the stems into a wide-mouth glass bottle filled with a culture medium A, and performing subculture proliferation and rooting culture. The temperature of the culture room is 23 ℃, the air humidity is 50%, the illumination intensity is 2000lx, and the illumination time is 14 h.d-1。
Synchronously culturing for 10 days by proliferation and rooting, wherein the height of a bud is 3.5-4.5 cm, and growing white fine roots; culturing for 17 days, wherein the height of the buds is 5.0-6.0 cm, and the length of the roots is 2-3 cm; culturing for 24 days, wherein the height of the buds is 6.1-7.0 cm; 2-5 roots are grown, the root length is 2-4 cm, the rooting rate reaches 98%, the leaves are extended, and the color is emerald green, so that complete and robust plants are formed. At the moment, axillary bud stem segments are cut from one part of bottle seedlings to carry out subculture proliferation and rooting culture (the culture medium A, B is used alternately to have a synergistic effect), and the other part of rooting bottle seedlings are transplanted to a seedling culture shed.
The multiplication coefficient of the 4 th generation reaches 6.8.
Transplanting rooted seedlings:
the prepared rooting bottle seedlings can be directly transplanted into a seedling raising shed without hardening seedlings. Selecting a seedling culture hole tray as a substrate container, taking out the rooting bottle seedlings from a culture bottle during transplanting, washing off the residual culture medium on the root system by using clear water, and directly transplanting the seedlings into the substrate sterilized by using chlorothalonil. The volume ratio of the transplanting matrix is light matrix: perlite 4: 1. the seedling raising shed is attentive to heat preservation and moisture preservation, and can be subjected to conventional management after 14 days. The survival rate of the tissue culture seedling transplantation reaches 99 percent.
Example 2
The medium was prepared as in example 1.
Selection, treatment and disinfection of explants:
collecting annual semi-lignified twig of cercis chinensis as an explant in sunny weather, removing leaves (reserving petiole base parts with the length of 4-5 mm), cutting into stem sections with the length of 5.5-6.5 cm and 3-5 axillary buds, placing running water for flushing for 20min, soaking in 1% detergent solution for 5min, brushing the surface of the stem sections with a soft brush gently,and is washed clean with tap water. The pretreated explants were placed on a clean bench and washed with 0.1% HgCl2And (4) sterilizing the mixture with 2 drops of Tween for 10min, washing the mixture with sterile water for 4-5 times, and draining the water for later use. The stem section of the explant cannot be cut too short during disinfection, so that the incision area of the explant damaged by disinfectant is reduced as much as possible.
Sterile bud induction:
clamping the sterilized explant by a left-hand forceps, shearing two ends of the explant and a base cut of a petiole by a right-hand scissors, then shearing 5.5-6.5 cm stem sections into two stem sections with two axillary buds respectively, and then inoculating the stem sections onto a tissue culture rapid propagation medium A of the jungle gypsy. When the explant is cultured, firstly the temperature is 20 ℃, the air humidity is 70%, the illumination intensity is 1250lx, and the illumination time is 12 h.d-1Culturing in the culture room for 10 days, transferring to a temperature of 25 deg.C, air humidity of 70%, illumination intensity of 2500lx, and illumination time of 14 h.d-1The culture chamber of (3) is cultured for 11 d.
4d after inoculation, the axillary buds begin to sprout, and 7d, the axillary buds begin to extend; and after 22 days of inoculation, the height of the buds is 3-5 cm, the buds are strong and grow well. The induction rate of the aseptic buds reaches 92 percent.
Subculture proliferation and rooting culture:
and lightly clamping sterile buds by using a forceps, selecting 2.5-3.5 cm-long axillary buds in a vigorous growth state by using a pair of scissors, vertically inserting the axillary buds into a wide-mouth glass bottle filled with a culture medium B, and performing subculture proliferation and rooting culture. The temperature of the culture room is 25 deg.C, the air humidity is 70%, the illumination intensity is 2500lx, and the illumination time is 14 h.d-1。
Synchronously culturing for 10 days by proliferation and rooting, wherein the height of a bud is 3.5-4.5 cm, and growing white fine roots; culturing for 17 days, wherein the height of the buds is 5.0-6.0 cm, and the length of the roots is 2-3 cm; culturing for 24 days, wherein the height of the buds is 6.1-7.0 cm; 2-5 roots are grown, the root length is 2-4 cm, the leaves are extended and are emerald green in color, and complete and robust plants are formed. At the moment, axillary bud stem segments are cut from one part of bottle seedlings to carry out subculture proliferation and rooting culture (the culture medium A, B is used alternately to have a synergistic effect), and the other part of rooting bottle seedlings are transplanted to a seedling culture shed. See Table 6 for the proliferation coefficients of generations 1-4.
The procedure for transplanting rooted seedlings was the same as in example 1.
Example 3
Preparing a culture medium:
the tissue culture and rapid propagation culture medium A of the cluster Chinese redbud is used for sterile bud induction. The culture medium A needs to be sterilized in an autoclave at 121 ℃ for 20min after being subpackaged, and then is kept stand and cooled for standby, and the contained substance components and the content (mg/L) are shown in the following table 3:
TABLE 3
Subculture proliferation and rooting culture, alternately using culture medium A and culture medium B, subpackaging culture medium B, sterilizing at 121 deg.C for 20min, standing, and cooling to obtain the final product with the following components and contents (mg/L) as shown in Table 4:
TABLE 4
| Composition (I) | Content (wt.) | Composition (I) | Content (wt.) |
| KNO3 | 950 | Inositol | 100 |
| NH4NO3 | 825 | VB1 | 0.4 |
| KH2PO4 | 255 | VB6 | 0.5 |
| CaCl2·2H2O | 220 | VB3 | 0.5 |
| MgSO4·7H2O | 185 | Glycine | 2 |
| FeSO4﹒7H2O | 41.7 | Sucrose | 30000 |
| Na2-EDTA | 56 | Agar-agar | 3700 |
| KI | 0.83 | TDZ | 0.5 |
| MnSO4·4H2O | 22.3 | IBA | 0.5 |
| ZnSO4·7H2O | 8.6 | Triacontanol | 1 |
| CuSO4·5H2O | 0.025 | Activated carbon | 200 |
| CoCl2·6H2O | 0.025 | L-cysteine | 20 |
| H3BO3 | 6.2 | PH | 5.8 |
| NaMoO4·2H2O | 0.25 |
Selection, treatment and disinfection of explants:
collecting annual semi-lignified twig of Cercis chinensis Franch as explant in sunny day, and removing leaf (reserving leaf)4-5 mm long petiole base), cutting into stem segments with length of 5.5-6.5 cm and 3-5 axillary buds, placing the stem segments in running water for flushing for 20min, soaking in 1% detergent solution for 5min, lightly brushing the surfaces of the stem segments by using a soft brush, and flushing the stem segments by using the running water. The pretreated explants were placed on a clean bench and washed with 0.1% HgCl2And (4) sterilizing the mixture with 2 drops of Tween for 10min, washing the mixture with sterile water for 4-5 times, and draining the water for later use. The stem section of the explant cannot be cut too short during disinfection, so that the incision area of the explant damaged by disinfectant is reduced as much as possible.
Sterile bud induction:
clamping the sterilized explant by a left-hand forceps, shearing two ends of the explant and a base cut of a petiole by a right-hand scissors, then shearing 5.5-6.5 cm stem sections into two stem sections with two axillary buds respectively, and then inoculating the stem sections onto a tissue culture rapid propagation medium A of the jungle gypsy. When the explant is cultured, firstly, the temperature is 22 ℃, the air humidity is 80%, the illumination intensity is 1500lx, and the illumination time is 12 h.d-1Culturing in the culture room for 10 days, moving to a temperature of 27 deg.C, air humidity of 80%, illumination intensity of 3000lx, and illumination time of 14 h.d-1The culture chamber of (2) for 12 d. 4d after inoculation, the axillary buds begin to sprout, and 7d, the axillary buds begin to extend; and after 22 days of inoculation, the height of the buds is 3-5 cm, the buds are strong and grow well. The induction rate of the aseptic buds reaches 92 percent.
Subculture proliferation and rooting culture:
and lightly clamping sterile buds by using a forceps, selecting 2.5-3.5 cm-long axillary buds in a vigorous growth state by using a pair of scissors, vertically inserting the axillary buds into a wide-mouth glass bottle filled with a culture medium B, and performing subculture proliferation and rooting culture. The temperature of the culture room is 28 ℃, the air humidity is 80%, the illumination intensity is 3000lx, and the illumination time is 14h d-1. Synchronously culturing for 10 days by proliferation and rooting, wherein the height of a bud is 3.5-4.5 cm, and growing white fine roots; culturing for 17 days, wherein the height of the buds is 5.0-6.0 cm, and the length of the roots is 2-3 cm; culturing for 24 days, wherein the height of the buds is 6.1-7.0 cm; 2-5 roots are grown, the root length is 2-4 cm, the leaves are extended and are emerald green in color, and complete and robust plants are formed. At this time, axillary bud stem segments are cut from a part of the bottle seedlings to perform subculture proliferation and rooting culture (the culture medium A, B is used alternately to have synergistic effect), and the other part is subjected to subculture proliferation and rooting cultureTransplanting the rooting bottle seedlings to a seedling raising shed.
The growth coefficient of the 1 st generation was 5.0, the growth coefficient of the 2 nd generation was 5.4, the growth coefficient of the 3 rd generation was 6.5, and the growth coefficient of the 4 th generation was 6.9.
The procedure for transplanting rooted seedlings was the same as in example 1.
Comparative experiment 1
Compared with example 1, the only difference is that 0.1% HgCl is selected2Or 0.1% HgCl2Adding 1-3 drops of Tween as a disinfectant, and designing different treatment methods. Each group treated 12 vials, which were repeated 3 times. The observation is recorded every 4 days, the pollution rate and the sterile bud induction rate are counted after 10 days, and the data are shown in a table 5:
TABLE 5
As can be seen from Table 5, the optimal method for sterilizing the explants of Cercis fascicularis is 0.1% HgCl2Adding 2 drops of Tween, sterilizing for 10min, and adding 2 drops of Tween to prevent pollution and obtain sterile bud induction rate of 92.1%. Tiny villi grow on the explant of the cercis fascicularis, and bacteria are easy to grow on the villi, so that the thorough disinfection is difficult. The proper amount of the non-ionic surfactant Tween is added, so that the solubility of the disinfectant can be increased, the disinfectant can fully contact with explants, and the sterilization effect is improved; however, when the concentration of tween is too low, the effect of enhancing the solubility of the venom is not obvious, and when the concentration is too high, the explant is easily damaged.
Comparative experiment 2
The main difference from example 2 lies in the different methods of use of cytokinins, see in particular table 6:
TABLE 6
TDZ is a high-efficiency cytokinin and can better promote bud differentiation and growth. Table 6 shows that the alternating use of TDZ and 6-BA has a synergistic effect. When subculture proliferation is continuously carried out, 0.5mg/L of TDZ is used for replacing 0.5mg/L of 6-BA at intervals of 1 generation, the proliferation coefficient can be improved, but continuous single use of TDZ can inhibit bud point elongation.
Comparative experiment 3
The main difference from example 2 is the effect of different additive combinations on proliferation and rooting of strong seedlings, see in particular Table 7
TABLE 7
Wherein, the components and contents (mg/L) of the MS culture medium are shown in the following table 8:
TABLE 8
| Composition (I) | Content (wt.) | Composition (I) | Content (wt.) |
| KNO3 | 1650 | Inositol | 100 |
| NH4NO3 | 1900 | VB1 | 0.4 |
| KH2PO4 | 170 | VB6 | 0.5 |
| CaCl2·2H2O | 440 | VB3 | 0.5 |
| MgSO4·7H2O | 370 | Glycine | 2 |
| FeSO4﹒7H2O | 27.8 | Sucrose | 30000 |
| Na2-EDTA | 37.3 | Agar-agar | 8000 |
| KI | 0.83 | PH | 5.8 |
| MnSO4·4H2O | 22.3 | ||
| ZnSO4·7H2O | 8.6 | ||
| CuSO4·5H2O | 0.025 | ||
| CoCl2·6H2O | 0.025 | ||
| H3BO3 | 6.2 | ||
| NaMoO4·2H2O | 0.25 |
As shown in Table 7, on the one hand, the low concentration inorganic salt is more suitable for the growth of the cercis fascicularis, and on the other hand, the KH is properly increased in the culture medium for tissue culture and rapid propagation of the cercis fascicularis2PO4The growth vigor of the tissue culture seedlings is better. This is achieved byIn addition, triacontanol can enhance photosynthesis, transport and accumulation of nutrient substances, and promote rooting, stem and leaf growth; the activated carbon can blacken the culture medium, is beneficial to rooting, and the adsorption effect of the activated carbon can reduce the influence of some harmful substances; the L-cysteine can promote wound healing and prevent aging and browning; the combined use of triacontanol, activated carbon, L-cysteine produces a synergistic effect. The combination of triacontanol 1.0mg/L, activated carbon 200.0mg/L and L-cysteine 20.0mg/L is added into the culture medium, which is very favorable for promoting the induction of aseptic buds, the subculture proliferation and the rooting of strong seedlings.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (3)
1. A culture medium combination for tissue culture and rapid propagation of cercis fascicularis is characterized by comprising a culture medium A and a culture medium B; the culture medium A comprises the following components: KNO3 950 mg/L、NH4NO3 825 mg/L、KH2PO4200～255mg/L、CaCl22H2O 220mg/L、MgSO47H2O 185mg/L、FeSO47H2O 41.7mg/L、Na2-EDTA 56mg/L、KI 0.83mg/L、MnSO4·4H2O 22.3mg/L、ZnSO47H2O 8.6mg/L、CuSO45H2O 0.025mg/L、CoCl26H2O 0.025mg/L、H3BO3 6.2 mg/L、NaMoO4 2H2O0.25 mg/L, inositol 100mg/L、VB1 0.4 mg/L、VB6 0.5 mg/L、VB30.5mg/L, 2mg/L of glycine, 30000mg/L of sucrose, 3700mg/L of agar, 0.3-0.5 mg/L, IBA 0.3.3-0.5 mg/L of 6-BA, 1mg/L of triacontanol and 20mg/L of activated carbon 200mg/L, L-cysteine;
the culture medium B comprises the following components: KNO3 950 mg/L、NH4NO3 825 mg/L、KH2PO4200～255mg/L、CaCl22H2O 220mg/L、MgSO47H2O 185mg/L、FeSO47H2O 41.7mg/L、Na2-EDTA 56mg/L、KI 0.83mg/L、MnSO4·4H2O 22.3mg/L、ZnSO47H2O 8.6mg/L、CuSO45H2O 0.025mg/L、CoCl26H2O 0.025mg/L、H3BO3 6.2 mg/L、NaMoO42H2O0.25 mg/L, inositol 100mg/L, VB1 0.4 mg/L、VB6 0.5 mg/L、VB30.5mg/L, 2mg/L of glycine, 30000mg/L of sucrose, 3700mg/L, TDZ 0.3.3-0.5 mg/L, IBA 0.3.3-0.5 mg/L of agar, 1mg/L of triacontanol and 200mg/L, L-cysteine of activated carbon.
2. A tissue culture and rapid propagation culture method by using the tissue culture and rapid propagation culture medium combination of the cercis fascicularis according to claim 1, which is characterized by comprising the following steps:
(1) selection, treatment and disinfection of explants:
taking the tender branches of the arbuscule as explants, reserving the base parts of petioles with the length of 4-5 mm, cutting the tender branches into stem sections with the length of 5.5-6.5 cm and 3-5 axillary buds, cleaning the stem sections, and then using 0.1% HgCl2Adding 2 drops of Tween, sterilizing for 10min, and washing;
(2) sterile bud induction:
removing cuts at two ends of an explant and a base part of a petiole, cutting a stem section of 5.5-6.5 cm into two stem sections with 2-3 axillary buds respectively, and placing the stem sections in the culture medium A prepared in the claim 1; culturing conditions are as follows: the temperature is 18-22 ℃, the air humidity is 50-80%, the illumination intensity is 700-1500 lx, and the illumination time is 12 h.d-1Culturing in the culture chamber for 10d, and moving to the temperature23-27 ℃, the air humidity is 50-80%, the illumination intensity is 2000-3000 lx, and the illumination time is 14 h.d-1Culturing for 10-12 days in the culture room to obtain sterile buds;
(3) subculture proliferation and rooting culture:
taking aseptic buds, selecting axillary buds with vigorous growth state, cutting the axillary buds into 2.5-3.5 cm stem segments, and placing the stem segments in the culture medium A or B prepared according to claim 1; the culture conditions are as follows: the temperature is 23-28 ℃, the air humidity is 50-80%, the illumination intensity is 2000-3000 lx, and the illumination time is 14 h.d-1(ii) a Carrying out proliferation and rooting culture for 24d, cutting axillary bud stem segments from part of seedlings, carrying out subculture proliferation and rooting culture, and transplanting the rest rooted seedlings;
the subculture proliferation and rooting culture alternately use the medium A, B.
3. The tissue culture and rapid propagation culture method of the arbutus rosette as claimed in claim 2, wherein the cleaning in the step (1): placing the stem segments in tap water and flowing water for washing for 15-20 min, soaking in 1% detergent solution for 5min, lightly scrubbing the surfaces of the stem segments by using a soft brush, and washing the stem segments by using the tap water; the flushing: washing with sterile water for 3-4 times, and draining for later use.
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