CN110741933A - Rhododendron dauricum culture medium and plant regeneration method - Google Patents

Rhododendron dauricum culture medium and plant regeneration method Download PDF

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CN110741933A
CN110741933A CN201911153313.XA CN201911153313A CN110741933A CN 110741933 A CN110741933 A CN 110741933A CN 201911153313 A CN201911153313 A CN 201911153313A CN 110741933 A CN110741933 A CN 110741933A
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rhododendron dauricum
culture medium
rhododendron
dauricum
culture
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王丹
王福德
马盈
舒钰
赵学丽
温爱亭
王钰婷
姜璟瑜
刘霞
杨扬
马静
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FORESTRY RESEARCH INSTITUTE OF HEILONGJIANG PROVINCE
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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
    • A01H4/008—Methods for regeneration to complete plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00—Botany in general
    • A01G7/04—Electric or magnetic or acoustic treatment of plants for promoting growth
    • A01G7/045—Electric or magnetic or acoustic treatment of plants for promoting growth with electric lighting
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/14—Measures for saving energy, e.g. in green houses

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  • Developmental Biology & Embryology (AREA)
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Abstract

The invention discloses a rhododendron dauricum culture medium and a plant regeneration method, and relates to a plant culture medium and a plant regeneration method. The invention solves the problems that the seed propagation period of the rhododendron dauricum is long, the excellent characters of the parent can not be maintained, the rooting rate of the asexual cutting propagation is low, and the rhododendron dauricum is extremely easy to die. The Rhododendron dauricum basic culture medium comprises macroelements, secondary elements, trace elements, organic elements, sucrose, agar and water, wherein the pH value is 5.0-5.4; the Rhododendron dauricum starting differentiation medium, the Rhododendron dauricum propagation seedling strengthening medium and the Rhododendron dauricum rooting medium respectively comprise a Rhododendron dauricum basic medium, gibberellin and indolebutyric acid, and the culture medium is used for carrying out regeneration culture on Rhododendron dauricum plants, the differentiation period is 25-30 days, the induction rate of clustered seedlings is more than 85%, the rooting rate is high, and the seedlings are not easy to die.

Description

Rhododendron dauricum culture medium and plant regeneration method
Technical Field
The invention relates to a plant culture medium and a plant regeneration method.
Background
Firstly, due to long-term cross pollination, the heterozygosity of the woody plant is high, so that the continuation of the excellent characters of the woody plant is influenced, the excellent characters of parents cannot be completely maintained by adopting seed propagation, and the characters of offspring can be separated. If the conventional method is adopted for genetic improvement, a pure line can be obtained only through five-to-six-generation selfing, the consumed time is long, the season is limited, and the seedling raising rate is low; secondly, the seeds of the rhododendron dauricum in nature are tiny, the maturity of the seeds is not high, and the seeds are easy to die due to water loss, so that the market demand cannot be met by utilizing the offspring generated by natural reproduction, and the popularization and development of the excellent tree species are seriously hindered. Thirdly, the rooting rate of the rhododendron dauricum by the asexual cuttage method is extremely low, the root of the cuttage is easy to brown and die, and the survival rate is influenced.
Disclosure of Invention
The invention aims to solve the problems that rhododendron dauricum seeds have long propagation period, cannot maintain the excellent characters of parents, and is low in rooting rate and extremely easy to die in the asexual cuttage propagation process, and provides a rhododendron dauricum culture medium and a plant regeneration method.
A base culture medium for Rhododendron dauricum comprises macroelements, secondary elements, microelements, organic elements, sucrose and agar, wherein the pH value is 5.0-5.4;
the macroelement is NH4NO3350mg/L～450mg/L、(NH4)2SO4120mg/L～150mg/L、KNO3150mg/L～250mg/L、KH2PO4350mg/L～450mg/L、MgSO4·7H2O350 mg/L-400 mg/L and CaCl2·2H2O 420mg/L～480mg/L；
The secondary element is Na2-EDTA 35mg/L～40mg/L、FeSO4·7H2O 25mg/L～30mg/L、H3BO45mg/L～8mg/L、MnSO4·4H2O15 mg/L-20 mg/L and ZnSO4·7H2O 5mg/L～10mg/L；
The microelement is Na2MoO4·2H2O 0.1mg/L～5mg/L、CuSO4·5H2O0.01-0.05 mg/L and CoCl2·6H2O 0.01mg/L～0.05mg/L；
The organic elements are VB10.2mg/L-0.5 mg/L and 50 mg/L-150 mg/L inositol;
the sucrose accounts for 25-30 g/L, and the agar accounts for 7-8 g/L.
A rhododendron dauricum differentiation starting culture medium comprises the rhododendron dauricum basic culture medium, gibberellin and indolebutyric acid, wherein each liter of rhododendron dauricum differentiation starting culture medium contains 1 mg-2.0 mg of gibberellin and 0.2 mg-1 mg of indolebutyric acid.
A Rhododendron dauricum propagation and seedling strengthening culture medium comprises the Rhododendron dauricum basal culture medium, gibberellin and indolebutyric acid, wherein each liter of Rhododendron dauricum propagation and seedling strengthening culture medium contains 0.5-1 mg of gibberellin and 0.5-1.5 mg of indolebutyric acid.
The Rhododendron dauricum rooting culture medium consists of the Rhododendron dauricum basal culture medium, gibberellin, indolebutyric acid and active carbon, wherein each liter of Rhododendron dauricum rooting culture medium contains 0.1-0.5 mg of gibberellin, 1.0-1.5 mg of indolebutyric acid and 1-2% of active carbon by mass percent.
The method for regenerating the rhododendron dauricum plants comprises the following steps:
firstly, cleaning, disinfecting and cutting the young stems of the current-year branches of the rhododendron dauricum;
secondly, inoculating the young stems treated in the step one to the rhododendron dauricum differentiation starting culture medium, and performing illumination culture to obtain rhododendron dauricum differentiation seedlings;
thirdly, inoculating the differentiated seedling of the rhododendron dauricum to the propagation strong seedling culture medium of the rhododendron dauricum to perform illumination culture to obtain a subculture propagation strong seedling of the rhododendron dauricum;
and fourthly, inoculating the subculture multiplication strong seedlings of the rhododendron dauricum to the rhododendron dauricum rooting culture medium for illumination culture to obtain a regeneration plant of the rhododendron dauricum.
The branches of the rhododendron dauricum which are grown in the current year are collected from the middle and last ten days of 5 months to 8 last days of the month.
Inoculating the rhododendron dauricum in the second step to a differentiation starting culture medium for illumination culture for 15-20d, wherein the illumination period is 10-12 h/d, the temperature is 22-28 ℃, the relative air humidity is 70-80%, and the illumination intensity is 27 mu mol.m-2·s-1～36μmol·m-2·s-1。
The three steps are inoculated into a propagation strong seedling culture medium of rhododendron dauricum and are cultured for 30 to 35 days by illumination, the illumination period is 13 to 15 hours/d, the temperature is 22 to 28 ℃, the relative air humidity is 70 to 80 percent, and the illumination intensity is 27 mu mol.m-2·s-1～36μmol·m-2·s-1。
Inoculating the seeds in the third step to a rooting culture medium of rhododendron dauricum, and culturing the seeds for 30-45 days by illumination, wherein the illumination period is 13-15 h/d, the temperature is 22-28 ℃, the relative air humidity is 70-80%, and the illumination intensity is 27 mu mol.m-2·s-1～36μmol·m-2·s-1。
The culture medium of the invention adjusts the dosage of macroelements on the basis of MS culture medium, and simultaneously reduces NH in the macroelements4NO3、KNO3Is increased in (NH)4)2SO4In a ratio of NH4 +With NO3 -The ratio of the inorganic salt to the gibberellin is changed from 1:2 to 1:1, the content of the inorganic salt is reduced, the growth of rhododendron is facilitated, and gibberellin and indolebutyric acid are used as plant growth regulators, so that one cell is induced to differentiate, and the other cell is induced to grow. By using the inventionThe culture medium is used for culturing the stem regeneration plant of the rhododendron dauricum, the culture medium has obvious bud proliferation, is easy to induce the regeneration plant, has high rooting rate, induces adventitious buds and the regeneration plant through stem culture, shows high consistency and high yield, and can achieve the aim of quickly propagating excellent seedlings; the differentiation period is started for 15-20 days in the regeneration culture of rhododendron dauricum plants by using the culture medium, the differentiation seedling inductivity is over 85 percent, and the raw material cost is saved by over 55 percent compared with that of an MS culture medium.
The culture method of the invention is the in vitro regeneration culture of the rhododendron dauricum, greatly shortens the regeneration period of rhododendron dauricum plants, has high rooting rate and is not easy to die.
Drawings
FIG. 1 is a graph showing the results of culture in the differentiation-initiating medium in experiment 1;
FIG. 2 is a graph showing the results of the culture in the culture medium for strong seedlings in experiment 1;
FIG. 3 shows the results of the rooting medium in experiment 1;
FIG. 4 is a graph showing the results of the differentiation-initiating culture of experiment 4;
FIG. 5 is a graph showing the results of the differentiation-initiating culture of experiment 5.
Detailed Description
The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.
The first embodiment is as follows: the basal culture medium of the rhododendron dauricum comprises macroelements, secondary elements, trace elements, organic elements, cane sugar and agar, wherein the pH value is 5.0-5.4;
the macroelement is NH4NO3350mg/L～450mg/L、(NH4)2SO4120mg/L～150mg/L、KNO3150mg/L～250mg/L、KH2PO4350mg/L～450mg/L、MgSO4·7H2O350 mg/L-400 mg/L and CaCl2·2H2O 420mg/L～480mg/L；
The secondary element is Na2-EDTA 35mg/L～40、FeSO4·7H2O 25mg/L～30mg/L、H3BO45mg/L～8mg/L、MnSO4·4H2O15 mg/L-20 mg/L and ZnSO4·7H2O 5mg/L～10mg/L；
The microelement is Na2MoO4·2H2O 0.1mg/L～5mg/L、CuSO4·5H2O0.01-0.05 mg/L and CoCl2·6H2O 0.01mg/L～0.05mg/L；
The organic elements are VB10.2mg/L-0.5 mg/L and 50 mg/L-150 mg/L inositol;
the sucrose accounts for 25-30 g/L, and the agar accounts for 7-8 g/L.
The second embodiment is as follows: the rhododendron dauricum differentiation starting culture medium comprises a rhododendron dauricum basic culture medium, gibberellin and indolebutyric acid, wherein each liter of rhododendron dauricum differentiation starting culture medium contains 1 mg-2.0 mg of gibberellin and 0.2 mg-1 mg of indolebutyric acid.
The third concrete implementation mode: the Rhododendron dauricum propagation and seedling strengthening culture medium comprises a Rhododendron dauricum basic culture medium, gibberellin and indolebutyric acid, wherein each liter of Rhododendron dauricum propagation and seedling strengthening culture medium contains 0.5-1 mg of gibberellin and 0.5-1.5 mg of indolebutyric acid.
The fourth concrete implementation mode: the rhododendron dauricum rooting culture medium comprises a rhododendron dauricum basic culture medium, gibberellin, indolebutyric acid and activated carbon, wherein each liter of rhododendron dauricum rooting culture medium contains 0.1-0.5 mg of gibberellin, 1.0-1.5 mg of indolebutyric acid and 1-2% of activated carbon by mass percent.
The fifth concrete implementation mode: the method for regenerating the rhododendron dauricum plants comprises the following steps:
firstly, cleaning, disinfecting and cutting the young stems of the current-year branches of the rhododendron dauricum;
secondly, inoculating the young stems treated in the first step to a rhododendron dauricum differentiation starting culture medium of the second embodiment, and performing illumination culture to obtain rhododendron dauricum differentiation seedlings;
thirdly, inoculating the differentiated seedling of the rhododendron dauricum to the propagation strong seedling culture medium of the rhododendron dauricum in the third embodiment, and carrying out illumination culture to obtain a subculture propagation strong seedling of the rhododendron dauricum;
and fourthly, inoculating the subculture multiplication strong seedlings of the rhododendron dauricum to the rhododendron dauricum rooting culture medium in the embodiment IV, and performing illumination culture to obtain a regeneration plant of the rhododendron dauricum.
The current annual branch of rhododendron dauricum according to the embodiment is collected from the middle and last ten days of 5 months to the last ten days of 8 months.
Cutting in the first step of the embodiment: cleaning and disinfecting young stems of the raw branches, and cutting the young stems into 1-2 cm stem sections on a super clean bench.
The method of the embodiment is used for carrying out regeneration culture on the rhododendron dauricum plants, the proliferation of the rhododendron dauricum buds is obvious, the regeneration plants are easy to induce, adventitious buds and the regeneration plants are induced through stem section culture, the high consistency and the high yield are shown, and the purpose of quickly propagating excellent seedlings can be achieved. The dynamic differentiation period of the rhododendron dauricum initial plant is 15-20 days, the differentiation seedling inductivity is over 85%, and compared with an MS culture medium, the raw material cost is saved by over 55%.
The sixth specific implementation mode: the fifth embodiment is different from the fifth embodiment in that: inoculating the rhododendron dauricum to a differentiation starting culture medium for illumination culture for 15-20d, wherein the illumination period is 10-12 h/d, the temperature is 22-28 ℃, the relative air humidity is 70-80%, and the illumination intensity is 27 mu mol.m-2·s-1～36μmol·m-2·s-1(ii) a The other steps and parameters are the same as those in the fifth embodiment.
The seventh embodiment: the fifth or sixth embodiment is different from the fifth or sixth embodiment in that: inoculating the rhododendron dauricum propagation strong seedling culture medium to an illumination culture for 30-35 d, wherein the illumination period is 13-15 h/d, the temperature is 22-28 ℃, the relative air humidity is 70-80%, and the illumination intensity is 27 mu mol.m-2·s-1～36μmol·m-2·s-1(ii) a The other steps and parameters are the same as those in the fifth or sixth embodiment.
The specific implementation mode is eight: book (I)Embodiments are different from the fifth to seventh embodiments in that: inoculating the four steps to a rooting culture medium of rhododendron dauricum, and culturing the rhododendron dauricum for 30-45 days by illumination, wherein the illumination period is 13-15 h/d, the temperature is 22-28 ℃, the relative air humidity is 70-80%, and the illumination intensity is 27 mu mol.m-2·s-1～36μmol·m-2·s-1(ii) a The other steps and parameters are the same as those of the fifth to seventh embodiments.
The specific implementation method nine: the method for regenerating rhododendron dauricum plants comprises the following steps:
firstly, cleaning, disinfecting and cutting the young stems of the current-year branches of the rhododendron dauricum;
secondly, inoculating the young stems treated in the step one to a rhododendron dauricum differentiation starting culture medium, and performing illumination culture to obtain rhododendron dauricum differentiation seedlings;
thirdly, inoculating the differentiated seedling of the rhododendron dauricum to a propagation strong seedling culture medium of the rhododendron dauricum and carrying out illumination culture to obtain a subculture propagation strong seedling of the rhododendron dauricum;
and fourthly, inoculating the subculture multiplication strong seedlings of the rhododendron dauricum to a rhododendron dauricum rooting culture medium for illumination culture to obtain a regeneration plant of the rhododendron dauricum.
The branches of the rhododendron dauricum which are grown in the current year are collected from the middle and last ten days of 5 months to 8 last days of the month.
Inoculating the rhododendron dauricum in the second step to a differentiation starting culture medium for illumination culture for 15-20d, wherein the illumination period is 10-12 h/d, the temperature is 22-28 ℃, the relative air humidity is 70-80%, and the illumination intensity is 27 mu mol.m-2·s-1～36μmol·m-2·s-1(ii) a The Rhododendron dauricum differentiation starting culture medium consists of a Rhododendron dauricum basal culture medium, gibberellin and indolebutyric acid, wherein each liter of Rhododendron dauricum differentiation starting culture medium contains 1.5mg of gibberellin and 0.5mg of indolebutyric acid;
the three steps are inoculated into a propagation strong seedling culture medium of rhododendron dauricum and are cultured for 30 to 35 days by illumination, the illumination period is 13 to 15 hours/d, the temperature is 22 to 28 ℃, the relative air humidity is 70 to 80 percent, and the illumination intensity is 27 mu mol.m-2·s-1～36μmol·m-2·s-1Culture medium for propagation and seedling strengthening of Xingan rhododendronThe rhododendron dauricum propagation and seedling strengthening culture medium consists of a rhododendron dauricum basal culture medium, gibberellin and indolebutyric acid, wherein each liter of rhododendron dauricum propagation and seedling strengthening culture medium contains 1mg of gibberellin and 1mg of indolebutyric acid;
the fourth step is inoculated to a rooting culture medium of rhododendron dauricum and is cultured for 30 to 45 days by illumination, the illumination period is 13 to 15 hours/d, the temperature is 22 to 28 ℃, the relative air humidity is 70 to 80 percent, and the illumination intensity is 27 mu mol.m-2·s-1～36μmol·m-2·s-1(ii) a The Rhododendron dauricum rooting culture medium consists of a Rhododendron dauricum basal culture medium, gibberellin, indolebutyric acid and active carbon, wherein each liter of Rhododendron dauricum rooting culture medium contains 0.2mg of gibberellin, 1mg of indolebutyric acid and 2% of active carbon by mass.
The base culture medium of the rhododendron dauricum comprises macroelements, secondary elements, trace elements, organic elements, sucrose and agar, and the pH value is 5.0-5.4;
the macroelement is NH4NO3400mg/L、(NH4)2SO4132mg/L、KNO3200mg/L、KH2PO4408mg/L、MgSO4·7H2O370 mg/L and CaCl2·2H2O 440mg/L；
The secondary element is Na2-EDTA 37.8mg/L、FeSO4·7H2O 27.8mg/L、H3BO46.2mg/L、MnSO4·4H2O16.9 mg/L and ZnSO4·7H2O 8.6mg/L；
The microelement is Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O0.025 mg/L and CoCl2·6H2O0.025mg/L；
The organic elements are VB10.4mg/L and 100mg/L inositol;
the sucrose content is 25-30 g/L, and the agar content is 7-8 g/L.
In the embodiment, the starting differentiation cycle in the rhododendron dauricum plant regeneration culture is 15-20 days, the differentiation seedling inductivity is 85%, and compared with an MS culture medium, the cost is saved by more than 55% on raw materials.
Experiment 1 regeneration culture of Rhododendron dauricum plants with the culture medium of the present invention
The culture medium used in this experiment and its composition were as follows:
the Rhododendron dauricum differentiation starting culture medium consists of a Rhododendron dauricum basal culture medium, gibberellin and indolebutyric acid, wherein each liter of Rhododendron dauricum differentiation starting culture medium contains 1.5mg of gibberellin and 0.5mg of indolebutyric acid;
the propagation and seedling strengthening culture medium for the rhododendron dauricum consists of a basic culture medium for the rhododendron dauricum, gibberellin and indolebutyric acid, wherein each liter of the propagation and seedling strengthening culture medium for the rhododendron dauricum contains 1mg of gibberellin and 1mg of indolebutyric acid;
the Rhododendron dauricum rooting culture medium consists of a Rhododendron dauricum basal culture medium, gibberellin, indolebutyric acid and active carbon, wherein each liter of Rhododendron dauricum rooting culture medium contains 0.2mg of gibberellin, 1mg of indolebutyric acid and 2% of active carbon by mass.
The base culture medium of the rhododendron dauricum comprises macroelements, secondary elements, trace elements, organic elements, sucrose and agar, and the pH value is 5.0-5.4;
the macroelement is NH4NO3400mg/L、(NH4)2SO4132mg/L、KNO3200mg/L、KH2PO4408mg/L、MgSO4·7H2O370 mg/L and CaCl2·2H2O 440mg/L；
The secondary element is Na2-EDTA 37.8mg/L、FeSO4·7H2O 27.8mg/L、H3BO46.2mg/L、MnSO4·4H2O16.9 mg/L and ZnSO4·7H2O 8.6mg/L；
The microelement is Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O0.025 mg/L and CoCl2·6H2O0.025mg/L；
The organic elements are VB10.4mg/L and 100mg/L inositol;
the sucrose content is 25-30 g/L, and the agar content is 7-8 g/L.
The method for culturing the rhododendron dauricum plants comprises the following steps:
1. explant Collection
Selecting healthy and strong rhododendron dauricum plants as sampling stock plants after sunny days or dew is dry in early spring, and cutting younger stem sections and stem tips at the lower part of the stock trees as explants, wherein the size of the stem sections and the stem tips is about 10-15 cm;
2. cleaning and sterilizing explant
Firstly, cleaning the surface of the explant by using a detergent, removing dust on the surface, washing the explant by using running water for 30min, and then putting the explant into a disinfection bottle; wiping a disinfection bottle filled with explants by 75% alcohol cotton, putting the disinfection bottle into a super-clean workbench, opening the disinfection bottle filled with explants on the super-clean workbench, pouring 75% alcohol, shaking the disinfection bottle, soaking and disinfecting for 10s, pouring the alcohol, and washing the explants for 3-5 times by using sterile water; then soaking the explant for 10min by NaClO with the treatment concentration of 1%, washing the explant for 3-5 times by sterile water, soaking the explant for 10min, and then sucking water on the surface of the explant by sterile filter paper;
3. explant cutting
Cutting the explant with the sterilized surface into 1-2 cm stem segments, and inoculating the stem segments into a Rhododendron dauricum differentiation starting culture medium (GA31.5mg/L in the culture medium, and IBA is 0.5 mg/L); firstly, slightly taking down a sealing film of a culture bottle, putting the sealing film on one side, inclining a culture bottle mouth, rotating and burning the culture bottle mouth on an alcohol lamp outer flame for disinfection, and killing microorganisms on the bottle mouth; burning a gun-type forceps on an alcohol lamp for disinfection and cooling, putting the cut explants into culture bottles filled with rhododendron dauricum differentiation starting culture medium, putting 3 explants in each culture bottle, covering a sealing film after inoculation, and marking the date.
4. Culture conditions of tissue culture seedling
The temperature of the culture chamber is controlled to be 25 +/-3 ℃, the relative air humidity is controlled to be 70-80 percent, and the illumination intensity is 27 mu mol.m-2·s-1～36μmol·m-2·s-1The illumination time is 12 h/d.
5. Initiating differentiation culture
Inoculating the explant to a rhododendron dauricum differentiation starting culture medium (GA3 is 1.5mg/L and IBA is 0.5mg/L), and carrying out subculture proliferation after culturing for 15-20 days.
7. Subculture of multiplication
The explant is transferred to a rhododendron dauricum propagation culture medium (GA3 is 1mg/L and IBA is 1mg/L) for subculture for 30d-35d, the illumination period is 13/day-15 h/day, the temperature is 22-28 ℃, the relative air humidity is 70-80%, and the illumination intensity is 27 mu mol.m-2·s-1～36μmol·m-2·s-1。
8. Rooting culture
Selecting a strong differentiated seedling with the length of 5-8 cm in a culture bottle, inoculating the seedling into a rooting culture medium, culturing the seedling for 30-40 d in the rooting culture medium (GA3 is 0.2mg/L, IBA is 1.0mg/L and activated carbon is 2%), wherein the illumination period is 13-15 h/day, the temperature is 22-28 ℃, the relative air humidity is 70-80%, and the illumination intensity is 27 mu mol.m-2·s-1～36μmol·m-2·s-1(ii) a Wherein white tender roots appear at the basal part of the tissue culture seedling in the growth process, the average number of the roots is more than 3 after 30 days, and the root length is about 1.0 cm-4.0 cm.
The results of the differentiation-initiating period and the differentiation-shoot induction rate after the culture in the differentiation-initiating medium are shown in Table 1.
The results of this experiment after the culture in the activated differentiation medium are shown in FIG. 1 (fully differentiated shoot); the result after the enrichment medium culture is shown in FIG. 2 (the Rhododendron dauricum bud is obviously proliferated); the results after the rooting medium culture are shown in FIG. 3; as can be seen from FIGS. 1 to 3, the culture medium of the invention has good effect of inducing cluster differentiation of rhododendron dauricum, obvious proliferation of rhododendron dauricum buds, easy induction of regeneration plants and high rooting rate, i.e. the culture medium of the invention can start differentiation of adventitious buds and regeneration plants through stem section culture, shows high consistency and high yield, and can realize rapid propagation of excellent rhododendron dauricum seedlings.
Experiment 2 starting differentiation culture of Rhododendron dauricum plants with a ZT (zeatin) -containing medium
The culture medium used in this experiment and its composition were as follows:
test comparison rhododendron initiation differentiation medium 1 consists of a rhododendron basal medium, ZT (zeatin) and indolebutyric acid, and each liter of test comparison rhododendron initiation differentiation medium 1 contains 1.5mg of ZT (zeatin) and 0.5mg of indolebutyric acid;
the base culture medium of the rhododendron dauricum comprises macroelements, secondary elements, trace elements, organic elements, sucrose and agar, and the pH value is 5.0-5.4;
the macroelement is NH4NO3400mg/L、(NH4)2SO4132mg/L、KNO3200mg/L、KH2PO4408mg/L、MgSO4·7H2O370 mg/L and CaCl2·2H2O 440mg/L；
The secondary element is Na2-EDTA 37.8mg/L、FeSO4·7H2O 27.8mg/L、H3BO46.2mg/L、MnSO4·4H2O16.9 mg/L and ZnSO4·7H2O 8.6mg/L；
The microelement is Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O0.025 mg/L and CoCl2·6H2O0.025mg/L；
The organic elements are VB10.4mg/L and 100mg/L inositol;
the content of the sucrose is 30g/L, and the content of the agar is 7 g/L;
the method for starting differentiation culture of rhododendron dauricum plants comprises the following steps:
1. explant Collection
Selecting healthy and strong rhododendron dauricum plants as sampling stock plants after sunny days or dew is dry in early spring, and cutting younger stem sections and stem tips at the lower part of the stock trees as explants, wherein the size of the stem sections and the stem tips is about 10-15 cm;
2. cleaning and sterilizing explant
Firstly, cleaning the surface of the explant by using a detergent, removing dust on the surface, washing the explant by using running water for 30min, and then putting the explant into a disinfection bottle; wiping a disinfection bottle filled with explants by 75% alcohol cotton, putting the disinfection bottle into a super-clean workbench, opening the disinfection bottle filled with explants on the super-clean workbench, pouring 75% alcohol, shaking the disinfection bottle, soaking and disinfecting for 10s, pouring the alcohol, and washing the explants for 3-5 times by using sterile water; then soaking the explant for 10min by NaClO with the treatment concentration of 1%, washing the explant for 3-5 times by sterile water, soaking the explant for 10min, and then sucking water on the surface of the explant by sterile filter paper;
3. explant cutting
Cutting the explant with the sterilized surface into 1-2 cm stem sections, and inoculating the stem sections into a test contrast rhododendron dauricum starting differentiation culture medium 1; firstly, slightly taking down a sealing film of a culture bottle, putting the sealing film on one side, inclining a culture bottle mouth, rotating and burning the culture bottle mouth on an alcohol lamp outer flame for disinfection, and killing microorganisms on the bottle mouth; burning a gun-type forceps on an alcohol lamp for disinfection and cooling, putting the cut explants into culture bottles filled with rhododendron dauricum differentiation starting culture medium, putting 3 explants in each culture bottle, covering a sealing film after inoculation, and marking the date.
4. Culture conditions of tissue culture seedling
The temperature of the culture chamber is controlled to be 25 +/-3 ℃, the relative air humidity is controlled to be 70-80 percent, and the illumination intensity is 27 mu mol.m-2·s-1～36μmol·m-2·s-1The illumination time is 12 h/d.
5. Initiating differentiation culture
Inoculating the explant into a test contrast rhododendron initiation differentiation culture medium 1, and culturing for 15-20 days to finish initiation differentiation culture.
The results of the initiation of the differentiation cycle and the induction rate of the differentiated seedling are shown in Table 1.
Experiment 3 the initiation of differentiation culture of Rhododendron dauricum plants using a 6-BA (6-benzyladenine) -containing medium
The culture medium used in this experiment and its composition were as follows:
comparative experiment Xingan rhododendron differentiation starting culture medium 2 consists of Xingan rhododendron basal culture medium, gibberellin and indolebutyric acid, and each liter of comparative experiment Xingan rhododendron differentiation starting culture medium 2 contains 1.5mg of 6-BA (6-benzyladenine) and 0.5mg of indolebutyric acid;
the base culture medium of the rhododendron dauricum comprises macroelements, secondary elements, trace elements, organic elements, sucrose and agar, and the pH value is 5.0-5.4;
the macroelement is NH4NO3400mg/L、(NH4)2SO4132mg/L、KNO3200mg/L、KH2PO4408mg/L、MgSO4·7H2O370 mg/L and CaCl2·2H2O 440mg/L；
The secondary element is Na2-EDTA 37.8mg/L、FeSO4·7H2O 27.8mg/L、H3BO46.2mg/L、MnSO4·4H2O16.9 mg/L and ZnSO4·7H2O 8.6mg/L；
The microelement is Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O0.025 mg/L and CoCl2·6H2O0.025mg/L；
The organic elements are VB10.4mg/L and 100mg/L inositol;
the content of the sucrose is 30g/L, and the content of the agar is 7 g/L;
the method for starting differentiation culture of rhododendron dauricum plants comprises the following steps:
1. explant Collection
Selecting healthy and strong rhododendron dauricum plants as sampling stock plants after sunny days or dew is dry in early spring, and cutting younger stem sections and stem tips at the lower part of the stock trees as explants, wherein the size of the stem sections and the stem tips is about 10-15 cm;
2. cleaning and sterilizing explant
Firstly, cleaning the surface of the explant by using a detergent, removing dust on the surface, washing the explant by using running water for 30min, and then putting the explant into a disinfection bottle; wiping a disinfection bottle filled with explants by 75% alcohol cotton, putting the disinfection bottle into a super-clean workbench, opening the disinfection bottle filled with explants on the super-clean workbench, pouring 75% alcohol, shaking the disinfection bottle, soaking and disinfecting for 10s, pouring the alcohol, and washing the explants for 3-5 times by using sterile water; then soaking the explant for 10min by NaClO with the treatment concentration of 1%, washing the explant for 3-5 times by sterile water, soaking the explant for 10min, and then sucking water on the surface of the explant by sterile filter paper;
3. explant cutting
Cutting the explant with the sterilized surface into 1-2 cm stem sections, and inoculating the stem sections into a Rhododendron dauricum starting differentiation culture medium 2 in a comparative test; firstly, slightly taking down a sealing film of a culture bottle, putting the sealing film on one side, inclining a culture bottle mouth, rotating and burning the culture bottle mouth on an alcohol lamp outer flame for disinfection, and killing microorganisms on the bottle mouth; burning a gun-type forceps on an alcohol lamp for disinfection and cooling, putting the cut explants into culture bottles filled with rhododendron dauricum differentiation starting culture medium, putting 3 explants in each culture bottle, covering a sealing film after inoculation, and marking the date.
4. Culture conditions of tissue culture seedling
The temperature of the culture chamber is controlled to be 25 +/-3 ℃, the relative air humidity is controlled to be 70-80 percent, and the illumination intensity is 27 mu mol.m-2·s-1～36μmol·m-2·s-1The illumination time is 12 h/d.
5. Induced differentiation culture
Inoculating the explant into a Rhododendron dauricum differentiation-initiating culture medium 2 for a contrast test, and culturing for 15-20 days to finish differentiation-initiating culture.
The results of the initiation of the differentiation cycle and the induction rate of the differentiated seedling are shown in Table 1.
TABLE 1 Effect of hormone classes on differentiation Induction
Figure BDA0002284155840000101
According to the content of the table 1, from the experimental results of experiment 1, experiment 2 and experiment 3, it can be seen that the method of the present invention has the differentiation starting period of 15-20 days, the inductivity of the differentiated seedling of rhododendron dauricum is 85%, the differentiated bud is bright green and strong, and the growth is slow by adopting a culture medium of ZT (zeatin) and 6-BA (6-benzyladenine); the culture medium is used for culturing the rhododendron dauricum stem regeneration plant, and the clustered seedlings have high inductivity, strong growth and high rooting rate and are not easy to die;
experiment 4 the differentiation culture of rhododendron dauricum plants using the existing MS medium
The culture medium used in this experiment and its composition were as follows:
contrast test rhododendron dauricum differentiation starting culture medium 3 consists of an MS culture medium, gibberellin and indolebutyric acid, and each liter of contrast test rhododendron dauricum differentiation starting culture medium 3 contains 1.5mg of gibberellin and 0.5mg of indolebutyric acid; the MS culture medium components are shown in Table 2;
TABLE 2 MS Medium composition Table
Figure BDA0002284155840000111
The method for starting differentiation culture of rhododendron dauricum plants comprises the following steps:
1. explant Collection
Selecting healthy and strong rhododendron dauricum plants as sampling stock plants after sunny days or dew is dry in early spring, and cutting younger stem sections and stem tips at the lower part of the stock trees as explants, wherein the size of the stem sections and the stem tips is about 10-15 cm;
2. cleaning and sterilizing explant
Firstly, cleaning the surface of the explant by using a detergent, removing dust on the surface, washing the explant by using running water for 30min, and then putting the explant into a disinfection bottle; wiping a disinfection bottle filled with explants by 75% alcohol cotton, putting the disinfection bottle into a super-clean workbench, opening the disinfection bottle filled with explants on the super-clean workbench, pouring 75% alcohol, shaking the disinfection bottle, soaking and disinfecting for 10s, pouring the alcohol, and washing the explants for 3-5 times by using sterile water; then soaking the explant for 10min by NaClO with the treatment concentration of 1%, washing the explant for 3-5 times by sterile water, soaking the explant for 10min, and then sucking water on the surface of the explant by sterile filter paper;
3. explant cutting
Cutting the explant with the sterilized surface into 1-2 cm stem sections, and inoculating the stem sections into a Rhododendron dauricum starting differentiation culture medium 3 in a comparative test; firstly, slightly taking down a sealing film of a culture bottle, putting the sealing film on one side, inclining a culture bottle mouth, rotating and burning the culture bottle mouth on an alcohol lamp outer flame for disinfection, and killing microorganisms on the bottle mouth; burning a gun-type forceps on an alcohol lamp for disinfection and cooling, putting the cut explants into culture bottles filled with rhododendron dauricum differentiation starting culture medium, putting 3 explants in each culture bottle, covering a sealing film after inoculation, and marking the date.
4. Culture conditions of tissue culture seedling
The temperature of the culture chamber is controlled to be 25 +/-3 ℃, the relative air humidity is controlled to be 70-80 percent, and the illumination intensity is 27 mu mol.m-2·s-1～36μmol·m-2·s-1The illumination time is 12 h/d.
5. Induced differentiation culture
Inoculating the explant into a contrast test rhododendron dauricum differentiation-initiating culture medium 3, and culturing for 15-20 days to finish differentiation-initiating culture.
The experimental results are shown in fig. 4 (incomplete organ differentiation of rhododendron dauricum), and the results of the differentiation cycle initiation and the differentiation shoot induction rate are shown in table 3.
Experiment 5 the differentiation of rhododendron dauricum plants was initiated using the existing 1/8MS medium
The culture medium used in this experiment and its composition were as follows:
comparative experiment Rhododendron dauricum differentiation-initiating culture medium 4 comprises 1/8MS culture medium, gibberellin and indolebutyric acid, and each liter of comparative experiment Rhododendron dauricum differentiation-initiating culture medium 4 contains 1.5mg of gibberellin and 0.5mg of indolebutyric acid.
The method for starting differentiation culture of rhododendron dauricum plants comprises the following steps:
1. explant Collection
Selecting healthy and strong rhododendron dauricum plants as sampling stock plants after sunny days or dew is dry in early spring, and cutting younger stem sections and stem tips at the lower part of the stock trees as explants, wherein the size of the stem sections and the stem tips is about 10-15 cm;
2. cleaning and sterilizing explant
Firstly, cleaning the surface of the explant by using a detergent, removing dust on the surface, washing the explant by using running water for 30min, and then putting the explant into a disinfection bottle; wiping a disinfection bottle filled with explants by 75% alcohol cotton, putting the disinfection bottle into a super-clean workbench, opening the disinfection bottle filled with explants on the super-clean workbench, pouring 75% alcohol, shaking the disinfection bottle, soaking and disinfecting for 10s, pouring the alcohol, and washing the explants for 3-5 times by using sterile water; then soaking the explant for 10min by NaClO with the treatment concentration of 1%, washing the explant for 3-5 times by sterile water, soaking the explant for 10min, and then sucking water on the surface of the explant by sterile filter paper;
3. explant cutting
Cutting the explant with the sterilized surface into 1-2 cm stem sections, and inoculating the stem sections into a Rhododendron dauricum differentiation starting culture medium 4 in a comparative test; firstly, slightly taking down a sealing film of a culture bottle, putting the sealing film on one side, inclining a culture bottle mouth, rotating and burning the culture bottle mouth on an alcohol lamp outer flame for disinfection, and killing microorganisms on the bottle mouth; burning a gun-type forceps on an alcohol lamp for disinfection and cooling, putting the cut explants into culture bottles filled with rhododendron dauricum differentiation starting culture medium, putting 3 explants in each culture bottle, covering a sealing film after inoculation, and marking the date.
4. Culture conditions of tissue culture seedling
The temperature of the culture chamber is controlled to be 25 +/-3 ℃, the relative air humidity is controlled to be 70-80 percent, and the illumination intensity is 27 mu mol.m-2·s-1～36μmol·m-2·s-1The illumination time is 12 h/d.
5. Induced differentiation culture
Inoculating the explant into a contrast test rhododendron dauricum differentiation-initiating culture medium 4, and culturing for 15-20 days to finish differentiation-initiating culture.
The results of the experiment are shown in FIG. 5 (weak differentiated shoot), and the results of the initiation of the differentiation cycle and the induction rate of the differentiated shoot are shown in Table 3.
TABLE 3 Effect of different basal media on differentiation Induction
Figure BDA0002284155840000131
Table 3 shows the experimental results of experiment 1, experiment 4, and experiment 5, where the differentiation induction rate of MS medium culture is only 12%, and the differentiation induction rate of 1/8MS medium is 55%, the induction rate of the start differentiation medium of the present invention is 85%, the induction rate of the differentiated seedling of rhododendron dauricum explant is inversely related to the concentration of macroelements, when the content of macroelements is large (MS medium), the explant does not differentiate or differentiates slowly, when the content is small, the plant grows weakly and is vitrified severely, and is not easy to form a complete organ, and the induction rate of MS medium on rhododendron dauricum plant is low, the differentiation effect is poor, and the effect of the present invention cannot be achieved.

Claims (8)

1. A Rhododendron dauricum basic culture medium is characterized in that the Rhododendron dauricum basic culture medium comprises macroelements, secondary elements, microelements, organic elements, sucrose and agar, and the pH value is 5.0-5.4;
the macroelement is NH4NO3350mg/L～450mg/L、(NH4)2SO4120mg/L～150mg/L、KNO3150mg/L～250mg/L、KH2PO4350mg/L～450mg/L、MgSO4·7H2O350 mg/L-400 mg/L and CaCl2·2H2O 420mg/L～480mg/L；
The secondary element is Na2-EDTA 35mg/L～40mg/L、FeSO4·7H2O 25mg/L～30mg/L、H3BO45mg/L～8mg/L、MnSO4·4H2O15 mg/L-20 mg/L and ZnSO4·7H2O 5mg/L～10mg/L；
The microelement is Na2MoO4·2H2O 0.1mg/L～5mg/L、CuSO4·5H2O0.01-0.05 mg/L and CoCl2·6H2O 0.01mg/L～0.05mg/L；
The organic elements are VB10.2mg/L-0.5 mg/L and 50 mg/L-150 mg/L inositol;
the sucrose accounts for 25-30 g/L, and the agar accounts for 7-8 g/L.
2. A Rhododendron dauricum differentiation starting culture medium is characterized by comprising the Rhododendron dauricum basic culture medium of claim 1, gibberellin and indolebutyric acid, wherein each liter of Rhododendron dauricum differentiation starting culture medium contains 1-2.0 mg of gibberellin and 0.2-1 mg of indolebutyric acid.
3. A Rhododendron dauricum propagation and seedling strengthening culture medium is characterized by comprising the Rhododendron dauricum basic culture medium of claim 1, gibberellin and indolebutyric acid, wherein each liter of Rhododendron dauricum propagation and seedling strengthening culture medium contains 0.5-1 mg of gibberellin and 0.5-1.5 mg of indolebutyric acid.
4. A Rhododendron dauricum rooting culture medium is characterized by comprising the Rhododendron dauricum basal culture medium of claim 1, gibberellin, indolebutyric acid and activated carbon, wherein each liter of Rhododendron dauricum rooting culture medium contains 0.1-0.5 mg of gibberellin, 1.0-1.5 mg of indolebutyric acid and 1-2% of activated carbon by mass percent.
5. The method for regenerating the rhododendron dauricum plant is characterized by comprising the following steps of:
firstly, cleaning, disinfecting and cutting the young stems of the current-year branches of the rhododendron dauricum;
secondly, inoculating the young stems treated in the step one to the rhododendron dauricum differentiation starting culture medium of claim 2, and culturing the young stems on light to obtain rhododendron dauricum differentiation seedlings;
thirdly, inoculating the differentiated seedling of the rhododendron dauricum into the rhododendron dauricum proliferation and strong seedling culture medium of claim 3, and carrying out illumination culture to obtain a subculture proliferation and strong seedling of the rhododendron dauricum;
and fourthly, inoculating the subculture multiplication strong seedlings of the rhododendron dauricum into the rhododendron dauricum rooting culture medium of claim 4, and performing illumination culture to obtain a regeneration plant of the rhododendron dauricum.
6. The method for plant regeneration of Rhododendron dauricum as claimed in claim 5, wherein the culture medium for photo-culturing is inoculated to the differentiation-initiating culture medium of Rhododendron dauricum in the second step for 15 d-20 d, the photo-culturing period is 10 h/d-12 h/d, the temperature is 22-28 ℃, the relative air humidity is 70-80%, and the light intensity is 27 μmol. m-2·s-1～36μmol·m-2·s-1。
7. According to claim5 the method for regenerating the rhododendron dauricum plant is characterized in that the rhododendron dauricum plant is inoculated into a propagation and strong seedling culture medium in the third step and is cultured for 30-35 d by illumination, the illumination period is 13-15 h/d, the temperature is 22-28 ℃, the relative air humidity is 70-80 percent, and the illumination intensity is 27 mu mol.m-2·s-1～36μmol·m-2·s-1。
8. The method for plant regeneration of Rhododendron dauricum according to claim 5, wherein the third step is inoculating the Rhododendron dauricum root medium to a light culture for 30-45 days with a light cycle of 13-15 h/d, a temperature of 22-28 ℃, a relative air humidity of 70-80%, and a light intensity of 27 μmol. m-2·s-1～36μmol·m-2·s-1。
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