CN115428731A - Method for rapidly inducing roots through tide-type in-vitro cutting of bougainvillea spectabilis and application of method - Google Patents
Method for rapidly inducing roots through tide-type in-vitro cutting of bougainvillea spectabilis and application of method Download PDFInfo
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- 241001316288 Bougainvillea spectabilis Species 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 40
- 238000005520 cutting process Methods 0.000 title claims abstract description 29
- 238000000338 in vitro Methods 0.000 title claims abstract description 23
- 230000001939 inductive effect Effects 0.000 title claims description 26
- 238000003976 plant breeding Methods 0.000 claims abstract description 32
- 230000006698 induction Effects 0.000 claims abstract description 25
- 238000002360 preparation method Methods 0.000 claims abstract description 12
- 238000009395 breeding Methods 0.000 claims abstract description 9
- 230000001488 breeding effect Effects 0.000 claims abstract description 7
- 239000001963 growth medium Substances 0.000 claims description 72
- 238000005286 illumination Methods 0.000 claims description 30
- 230000035755 proliferation Effects 0.000 claims description 29
- 230000001954 sterilising effect Effects 0.000 claims description 26
- 239000007788 liquid Substances 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 241000234295 Musa Species 0.000 claims description 16
- 235000018290 Musa x paradisiaca Nutrition 0.000 claims description 16
- 239000007787 solid Substances 0.000 claims description 15
- 241000219475 Bougainvillea Species 0.000 claims description 13
- 239000011324 bead Substances 0.000 claims description 13
- 239000011521 glass Substances 0.000 claims description 13
- 238000011081 inoculation Methods 0.000 claims description 13
- 238000004659 sterilization and disinfection Methods 0.000 claims description 13
- 238000005406 washing Methods 0.000 claims description 12
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 10
- 229930006000 Sucrose Natural products 0.000 claims description 10
- 239000005720 sucrose Substances 0.000 claims description 10
- 238000002791 soaking Methods 0.000 claims description 9
- 239000008223 sterile water Substances 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 238000011010 flushing procedure Methods 0.000 claims description 7
- 229920001817 Agar Polymers 0.000 claims description 5
- ZKQDCIXGCQPQNV-UHFFFAOYSA-N Calcium hypochlorite Chemical class [Ca+2].Cl[O-].Cl[O-] ZKQDCIXGCQPQNV-UHFFFAOYSA-N 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 5
- 230000000249 desinfective effect Effects 0.000 claims description 5
- 239000012154 double-distilled water Substances 0.000 claims description 5
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 5
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 claims description 5
- 238000007654 immersion Methods 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 238000009835 boiling Methods 0.000 claims description 4
- 238000010276 construction Methods 0.000 claims description 4
- 235000013399 edible fruits Nutrition 0.000 claims description 2
- 238000011177 media preparation Methods 0.000 claims description 2
- 229960002523 mercuric chloride Drugs 0.000 claims description 2
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 2
- 239000008399 tap water Substances 0.000 claims description 2
- 235000020679 tap water Nutrition 0.000 claims description 2
- 241000196324 Embryophyta Species 0.000 abstract description 4
- 238000012136 culture method Methods 0.000 abstract 1
- 238000009630 liquid culture Methods 0.000 description 5
- 239000003086 colorant Substances 0.000 description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 3
- 241000219469 Nyctaginaceae Species 0.000 description 2
- 235000016787 Piper methysticum Nutrition 0.000 description 2
- 240000007653 Pometia tomentosa Species 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
Classifications
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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
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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
- A01H4/002—Culture media for tissue culture
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- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a rapid root induction method of tide type in-vitro cutting of bougainvillea spectabilis and application thereof, the method sequentially comprises early-stage preparation, explant (stem section in-vitro) induction, transitional observation culture and plant breeding equipment culture, the rapid root induction method of the bougainvillea spectabilis in-vitro cutting is completed by breeding in a mode of intermittently immersing plants, high rooting rate can be obtained in a short time, the culture period of the bougainvillea spectabilis is shortened, and the quality of the cultured bougainvillea spectabilis is high; the method is simple to operate and full-automatic, and saves the consumption of human resources; meanwhile, the use of culture vessels can be reduced, the labor cost and the culture cost are saved, the low-cost culture method is realized, and the method is suitable for industrial popularization.
Description
Technical Field
The invention relates to the technical field of agricultural production, in particular to a method for rapidly inducing roots through tide-type in-vitro cutting of bougainvillea spectabilis and application thereof.
Background
Bougainvillea spectabilis (Bougainvillea ssp.), also called triangular flower, leafflower, bougainvillea trifoliata, kava, and the like, are woody vine flowers of Nyctaginaceae (Nyctaginaceae) and kava (Bougainvillea), are widely cultivated in garden greenbelts in tropical and subtropical regions of the world, and become indispensable special landscape resources in the regions. The bougainvillea spectabilis introduced in China has a history of nearly 150 years, and has the advantages of high ornamental value, long flowering phase, large flower quantity, less plant diseases and insect pests, extensive cultivation management and the like. Bougainvillea spectabilis is widely applied to green lands in cities in south China, such as Fujian, guangdong, guangxi, hainan, yunnan, guizhou and the like, and plays an important role in enriching the colors of the green lands of gardens. As the flowers bloom continuously in all seasons and blossom luxuriantly, the bougainvillea spectabilis is favored by people, the bougainvillea spectabilis is promoted to be the market flowers in cities such as Shenzhen, xiamen, zhuhai, mitsui, north sea and the like. Bougainvillea spectabilis in northern China cannot live through winter in open field, and is mostly planted in a pot culture mode in a courtyard or a balcony. Bougainvillea spectabilis varieties are different in plant type, leaf color, flower type, flower color and the like, and the plant type is a dwarf type (tower type) and a vine shrub type variety; the leaf color is full green and the flower leaves (the flower leaves have gold edges, silver edges, gold cores and the parts of scattered gold); dividing the flower type or the flower growing density into a single petal flower and a double petal flower; the basic colors of the bougainvillea spectabilis comprise purple, red, pink, orange, yellow and white according to the color classification, various colors with different shades are derived from the series, and red and white double-color varieties and the like are added to form rich bougainvillea spectabilis variety groups.
The first introduction of bougainvillea spectabilis in 1872 in China, and introduced into China by British people and horses and RogerTaiwanThus extending to the continents whereChina also successively introduced bougainvillea speetabilis varieties from Japan, singapore, australia, thailand, philippines, etc. With the expansion of international communication, the number of bougainvillea spectabilis varieties and bougainvillea spectabilis varieties in China is still increasing. However, as the breeding work starts late, most new and excellent bougainvillea spectabilis varieties still mainly depend on introduction, the varieties cultivated independently are few, and form great contrast with the industrial scale, and the development of bougainvillea spectabilis industry in China is seriously influenced. Therefore, a key technical method for quickly inducing roots by using the intelligent tide type in-vitro cutting of bougainvillea speetabilis is needed to meet the requirement of bougainvillea speetabilis cultivation.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for quickly inducing roots by tidal in vitro cutting of bougainvillea spectabilis, which is reliable in implementation, convenient in operation, low in implementation cost, and high in bougainvillea spectabilis breeding yield, and an application thereof.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
a method for inducing roots by cutting bougainvillea speetabilis in vitro comprises the following steps:
(1) Completing early-stage preparation according to preset requirements, wherein the early-stage preparation comprises culture medium preparation and breeding environment construction;
(2) Inducing the explant of bougainvillea spectabilis to obtain an effective seedling;
(3) Carrying out transitional observation culture on the effective seedlings;
(4) Inoculating the effective seedlings after transitional observation culture into plant breeding equipment for culture so as to complete the in vitro cutting induction root of the bougainvillea spectabilis.
As a preferred embodiment, step (1) of the present invention preferably includes:
1) Preparing a solid culture medium:
taking 1/2MS culture medium as a basic culture medium, and then adding the following components in final concentration:
NAA 0.2-0.8 mg/L, sucrose concentration 20-40 g/L, banana juice 40-60 g/L, agar powder 4-8 g/L;
adjusting the pH value of a culture medium system to 5.8-6.2, and finally completing the preparation of a solid culture medium for later use after boiling, subpackaging and sterilizing treatment;
2) Preparing a liquid proliferation culture medium:
taking 1/2MS culture medium as a basic culture medium, and then adding the following components in final concentration:
NAA 0.1-0.8 mg/L,6-BA 2.0-8.0 mg/L, sucrose concentration 20-40 g/L, banana juice 20-60 gL;
adjusting the pH value of a culture medium system to 5.8-6.2, and finally completing the preparation of a liquid proliferation culture medium for later use after boiling, subpackaging and sterilizing treatment;
3) Plant breeding equipment sterilization
Assembling plant breeding equipment, and then sterilizing the plant breeding equipment to complete the construction of a breeding environment for later use;
wherein, the banana juice in the step 1) and the step 2) is banana homogenate or banana extracting solution.
As a preferred embodiment, step (2) of the present invention preferably includes:
1) Selecting and sterilizing materials: taking out the explants of bougainvillea speetabilis, washing the explants with running water, soaking the explants in supernatant of saturated bleaching powder for 10min, then placing the explants in tap water to drip for 0.5h, then flushing the explants with double distilled water for 2 to 3 times, disinfecting the explants in an ultra-clean workbench for 30s by using alcohol with the volume fraction of 75 percent, then adding 0.1wt percent hypochlorous acid solution to treat the explants for 10 to 13min, flushing the explants with sterile water for 3 to 4 times, and finally absorbing the surface water by using disinfected filter paper for inoculation;
2) And (3) induction culture: inoculating the treated explant into a sterilized bud induction culture medium which is a solid culture medium; the culture conditions were: the temperature of the culture room is 23 +/-2 ℃, the illumination time is 12h/d, the illumination intensity is 1000-15001 x, and the culture time is 25-30 d;
3) And (3) proliferation culture: cutting the well-grown buds obtained by induction culture into 2-3cm long, inoculating in a liquid proliferation culture medium for proliferation induction culture to obtain effective seedlings; the culture conditions are as follows: the illumination time is 12h/d, the illumination intensity is 1500-20001x, and the proliferation culture time is 20-25d.
As a preferred embodiment, step (3) of the present solution preferably includes: the effective seedlings are firstly inoculated into a liquid multiplication culture medium for observation and culture, the inoculation density is 10-70 g/L, and the culture time is 3-5 d.
As a preferred embodiment, the explant in step (2) of the present scheme is preferably a bougainvillea spectabilis sterile seed or a bougainvillea spectabilis sterile seedling stem section tissue slice.
Preferably, the explant is a bougainvillea spectabilis sterile seed which is subjected to sterilization treatment, and the sterilization method comprises cleaning and disinfection.
As a preferred embodiment, the method for washing the bougainvillea spectabilis sterile seeds according to the present invention preferably comprises: selecting plump bougainvillea spectabilis fruit pods, soaking seeds for 15-30 s in 60-80% of anhydrous ethanol in an aseptic environment, and cleaning for 1-4 times by using sterile water; then soaking the mixture for 10 to 20min by 0.05 to 0.15 percent of mercuric chloride, and cleaning the mixture for 3 to 7 times by sterile water.
Based on the above, the invention also provides a method for rapidly inducing roots by tidal in vitro cutting of bougainvillea spectabilis, which comprises the above method, wherein the step (4) comprises the following steps: inoculating the effective seedlings cultured by transitional observation in the step (3) into plant breeding equipment for intermittent submerged culture;
wherein the inoculation density of the effective seedlings is 10-70 g/L;
in addition, the immersion frequency of the effective seedlings is 0-30 min/0-12 h, and the culture time is 20-60 d; the culture conditions were: the illumination intensity is 1000-3000 lx, the illumination is 0-24 h/d, and the temperature is 10-30 ℃.
As a preferred embodiment, in step (4), the plant breeding device comprises a growth disc for inoculating the effective seedlings, wherein a layer of glass beads is paved in the growth disc, and the diameter of the glass beads is 1-10 mm.
Based on the above, the invention also provides a bougainvillea spectabilis breeding method, which comprises the method for rapidly inducing roots by tide type in vitro cutting.
By adopting the technical scheme, compared with the prior art, the invention has the beneficial effects that:
(1) The proliferation rate is high: when effective seedlings are bred in plant breeding equipment, liquid culture is intermittently immersed and cultured, a liquid culture medium is intermittently contacted with plant tissue culture seedlings by using pressure generated by sterile air, and the advantages of maximized gas exchange of solid culture and full utilization of liquid culture nutrients are combined, so that the multiplication rate of bougainvillea speetabilis culture is 10-15 times that of common solid culture and 5-7 times that of liquid culture.
(2) Degree of automation is high, the amount of labour is little: this scheme is through the scheme of effective seedling of intermittent type submergence liquid culture, can utilize outside mechanical system to assist, reaches intelligent, full-automatic formula control, and whole proliferation culture process only needs inoculation once, need not to change the culture solution, has saved the operation such as filling washing of frequent switching and big group's tissue culture bottle in batches, great reduction the amount of labour, reduced manufacturing cost.
(3) The bougainvillea spectabilis is good quality: compared with the traditional solid culture, the method reduces the subculture times, the mutation rate and the pollution risk, and fully exchanges gas in the culture process.
Detailed Description
The present invention will be described in further detail with reference to examples. It is to be noted that the following examples are only illustrative of the present invention, and do not limit the scope of the present invention. Likewise, the following examples are only some examples, not all examples, and all other examples obtained by those skilled in the art without any inventive work are within the scope of the present invention.
Example 1
The method for rapidly inducing roots by tidal in vitro cutting of bougainvillea spectabilis comprises the following steps:
(1) Early preparation
1) Preparing a solid culture medium: the formula of the solid culture medium is 1/2MS basic culture medium, which also comprises 0.2mg/L of NAA, 20g/L of sucrose concentration, 40g/L of banana homogenate and 4g/L of agar powder, and the pH value of the prepared culture medium system is adjusted to 5.8, and the culture medium system is boiled, subpackaged and sterilized for later use;
2) Preparing a liquid proliferation culture medium: the formula of the liquid multiplication culture medium is 1/2MS basic culture medium, the liquid multiplication culture medium further comprises 0.1mg/L of NAA, 2.0 mg/L of 6-BA, 20g/L of sucrose concentration and 20gL of banana homogenate, the pH value of the prepared culture medium system is adjusted to 5.8, and after the culture solution is prepared, the culture medium is boiled, subpackaged and sterilized for later use;
3) Plant breeding equipment sterilization: assembling plant breeding equipment, putting the glass beads into the plant breeding equipment in the assembling process, and then wrapping and sterilizing; sterilizing for later use;
(2) Inducing the stem section in vitro to obtain effective seedling
1) Selecting and sterilizing materials: taking out aseptic seeds of bougainvillea spectabilis, washing the aseptic seeds with running water, soaking the aseptic seeds in supernatant of saturated bleaching powder for 10min, dripping the aseptic seeds in running water for 0.5h, flushing with double distilled water for 2 times, disinfecting the aseptic seeds with 75% alcohol by volume fraction in an ultraclean workbench for 30s, adding 0.1wt% hypochlorous acid solution for treating the aseptic seeds for 10min, washing the aseptic seeds with sterile water for 3 times, and inoculating the aseptic seeds after absorbing surface moisture with disinfection filter paper;
2) And (3) induction culture: inoculating the treated seeds into a bud induction culture medium which is sterilized at high temperature and high pressure; the culture conditions are as follows: the temperature of the culture room is 21 ℃, the illumination time is 12h/d, the illumination intensity is 10001x, and the culture time is 25d;
3) And (3) proliferation culture: cutting the well-grown bud obtained by induction culture into 2cm length, inoculating in proliferation culture medium for proliferation induction culture with illumination time of 12h/d, illumination intensity of 15001x, and proliferation culture time of 20d.
(3) Culture by transitional observation
Inoculating the high-quality effective seedlings screened in the step (2) into a liquid multiplication culture medium for observation and culture, wherein the inoculation density is 10g/L, and the culture time is 3d;
(4) Plant breeding facility culture
Inoculating the effective seedlings in the step (3) into intermittent submerged plant breeding equipment, wherein the inoculation density is 10g/L; the immersion frequency is 10min/6h, and the culture time is 20d; the culture conditions are as follows: the illumination intensity is 1000lx, the illumination is 6h/d, and the temperature is 10 ℃. A layer of glass beads is laid in a growth disc of the plant breeding device in a parallel mode, and the diameter of each glass bead is 1mm.
Example 2
The method for rapidly inducing roots by tidal in vitro cutting of bougainvillea spectabilis comprises the following steps:
(1) Preliminary preparation
1) Preparing a solid culture medium: the formula of the solid culture medium is 1/2MS basic culture medium, which also comprises 0.8mg/L of NAA, 40g/L of sucrose concentration, 60g/L of banana homogenate and 8g/L of agar powder, and the pH value of the prepared culture medium system is adjusted to 6.2, and the prepared culture medium system is boiled, subpackaged and sterilized for later use;
2) Preparing a liquid proliferation culture medium: the formula of the liquid multiplication culture medium is 1/2MS basic culture medium, the liquid multiplication culture medium further comprises 0.8mg/L of NAA, 8.0mg/L of 6-BA, 40g/L of sucrose concentration and 60gL of banana homogenate, the pH value of the prepared culture medium system is adjusted to 6.2, and after the culture solution is prepared, the culture medium is boiled, subpackaged and sterilized for later use;
3) Plant breeding equipment sterilization: assembling plant breeding equipment, putting the glass beads into the plant breeding equipment in the assembling process, and then wrapping and sterilizing; sterilizing for later use;
(2) Inducing the stem section in vitro to obtain effective seedling
1) Selecting and sterilizing materials: taking out aseptic seeds of bougainvillea spectabilis, washing the aseptic seeds with running water, soaking the aseptic seeds in supernatant of saturated bleaching powder for 10min, dripping the aseptic seeds in running water for 0.5h, flushing with double distilled water for 3 times, disinfecting the aseptic seeds with 75% alcohol by volume fraction in an ultraclean workbench for 30s, adding 0.1wt% hypochlorous acid solution for treating the aseptic seeds for 13min, washing the aseptic seeds with sterile water for 4 times, and inoculating the aseptic seeds after absorbing surface moisture with disinfection filter paper;
2) And (3) induction culture: inoculating the treated seeds into a bud induction culture medium sterilized at high temperature and high pressure; the culture conditions are as follows: the temperature of the culture room is 25 ℃, the illumination time is 12h/d, the illumination intensity is 15001x, and the culture time is 30d;
3) And (3) proliferation culture: cutting the well-grown buds obtained by induction culture into 3cm in length, inoculating the buds into a proliferation culture medium for proliferation induction culture, wherein the illumination time is 12h/d, the illumination intensity is 20001x, and the proliferation culture time is 25d.
(3) Culture by transitional observation
Inoculating the high-quality effective seedlings screened in the step (2) into a liquid multiplication culture medium for observation and culture, wherein the inoculation density is 70g/L, and the culture time is 5d;
(4) Plant breeding facility culture
Inoculating the effective seedlings in the step (3) into intermittent submerged plant breeding equipment, wherein the inoculation density is 70g/L; the immersion frequency is 30min/12h, and the culture time is 60d; the culture conditions are as follows: the illumination intensity is 3000lx, the illumination is 24h/d, and the temperature is 30 ℃. A layer of glass beads is paved in a growth disc of the plant breeding device in a parallel mode, and the diameter of each glass bead is 10mm.
Example 3
The method for rapidly inducing roots by tidal in vitro cutting of bougainvillea spectabilis comprises the following steps:
(1) Early preparation
1) Preparing a solid culture medium: the formula of the solid culture medium is 1/2MS basic culture medium, which also comprises NAA0.2 mg/L, sucrose concentration 40g/L, banana homogenate 40g/L and agar powder 8g/L, the pH value is adjusted to 5.8 after the culture medium system is prepared, and the mixture is boiled, subpackaged and sterilized for standby;
2) Preparing a liquid proliferation culture medium: the formula of the liquid multiplication culture medium is 1/2MS basic culture medium, the liquid multiplication culture medium also comprises 0.8mg/L NAA, 2.0 mg/L6-BA, 40g/L sucrose concentration and 20gL banana homogenate, the pH value is adjusted to 6.2 after the culture medium system is prepared, and the liquid multiplication culture medium is boiled, subpackaged and sterilized for later use after the culture solution is prepared;
3) Plant breeding equipment sterilization: assembling plant breeding equipment, putting the glass beads into the plant breeding equipment in the assembling process, and then wrapping and sterilizing; sterilizing for later use;
(2) Inducing the stem section in vitro to obtain effective seedling
1) Selecting and sterilizing materials: taking out aseptic seeds of bougainvillea spectabilis, washing the aseptic seeds with running water, soaking the aseptic seeds in supernatant of saturated bleaching powder for 10min, dripping the aseptic seeds in running water for 0.5h, flushing with double distilled water for 3 times, disinfecting the aseptic seeds with 75% alcohol by volume fraction in an ultraclean workbench for 30s, adding 0.1wt% hypochlorous acid solution for treating the aseptic seeds for 10min, washing the aseptic seeds with sterile water for 4 times, and inoculating the aseptic seeds after absorbing surface moisture with disinfection filter paper;
2) And (3) induction culture: inoculating the treated seeds into a bud induction culture medium which is sterilized at high temperature and high pressure; the culture conditions are as follows: the temperature of the culture room is 21 ℃, the illumination time is 12h/d, the illumination intensity is 15001x, and the culture time is 25d;
3) And (3) proliferation culture: cutting the well-grown bud obtained by induction culture into 3cm length, inoculating into proliferation culture medium for proliferation induction culture with illumination time of 12h/d, illumination intensity of 15001x, and proliferation culture time of 25d.
(3) Culture by transitional observation
Inoculating the high-quality effective seedlings screened in the step (2) into a liquid multiplication culture medium for observation and culture, wherein the inoculation density is 10g/L, and the culture time is 5d;
(4) Plant breeding facility culture
Inoculating the effective seedlings in the step (3) into intermittent submerged plant breeding equipment, wherein the inoculation density is 10g/L; the immersion frequency is 12min/6h, and the culture time is 60d; the culture conditions are as follows: the illumination intensity is 1000lx, the illumination is 4h/d, and the temperature is 30 ℃. A layer of glass beads is paved in a growth disc of the plant breeding device in a parallel mode, and the diameter of each glass bead is 10mm.
The above description is only a part of the embodiments of the present invention, and not intended to limit the scope of the present invention, and all equivalent devices or equivalent processes performed by the present invention or directly or indirectly applied to other related technical fields are included in the scope of the present invention.
Claims (10)
1. A method for inducing roots by cutting bougainvillea spectabilis in vitro is characterized by comprising the following steps:
(1) Completing early-stage preparation according to preset requirements, wherein the early-stage preparation comprises culture medium preparation and breeding environment construction;
(2) Inducing the explant of bougainvillea spectabilis to obtain an effective seedling;
(3) Carrying out transitional observation culture on the effective seedlings;
(4) Inoculating the effective seedlings after transitional observation and culture to plant breeding equipment for culture so as to complete the in vitro cutting induction root of the bougainvillea spectabilis.
2. The method for inducing roots by cutting bougainvillea speetabilis in vitro according to claim 1, wherein the step (1) comprises:
1) Preparing a solid culture medium:
taking 1/2MS culture medium as a basic culture medium, and then adding the following components in final concentration:
NAA 0.2-0.8 mg/L, sucrose concentration 20-40 g/L, banana juice 40-60 g/L, agar powder 4-8 g/L;
adjusting the pH value of a culture medium system to 5.8-6.2, and finally completing the preparation of a solid culture medium for later use after boiling, subpackaging and sterilizing treatment;
2) Preparing a liquid proliferation culture medium:
taking 1/2MS culture medium as a basic culture medium, and then adding the following components in final concentration:
NAA 0.1-0.8 mg/L,6-BA 2.0-8.0 mg/L, sucrose concentration 20-40 g/L, banana oil 20-60 gL;
adjusting the pH value of a culture medium system to 5.8-6.2, and finally completing the preparation of a liquid proliferation culture medium for later use after boiling, subpackaging and sterilizing treatment;
3) Plant breeding equipment sterilization
Assembling plant breeding equipment, and then sterilizing the plant breeding equipment to complete the construction of a breeding environment for later use;
wherein, the banana juice in the step 1) and the step 2) is banana homogenate or banana extracting solution.
3. The method for inducing roots through the cutting of bougainvillea speetabilis in vitro according to claim 1, wherein the step (2) comprises:
1) Selecting and sterilizing materials: taking out the explant of bougainvillea spectabilis, washing the explant with running water, soaking the explant in a supernatant of saturated bleaching powder for 10min, then placing the explant in tap water to drip for 0.5h, flushing with double distilled water for 2-3 times, disinfecting the explant in an ultraclean workbench with 75% alcohol by volume fraction for 30s, then adding 0.1wt% hypochlorous acid solution to treat the explant for 10-13 min, flushing with sterile water for 3-4 times, and finally absorbing surface moisture with disinfected filter paper for inoculation;
2) And (3) induction culture: inoculating the treated explant into a sterilized bud induction culture medium which is a solid culture medium; the culture conditions were: the temperature of the culture room is 23 +/-2 ℃, the illumination time is 12h/d, the illumination intensity is 1000-15001 x, and the culture time is 25-30 d;
3) And (3) proliferation culture: cutting the well-grown buds obtained by induction culture into 2-3cm long, inoculating in a liquid proliferation culture medium for proliferation induction culture to obtain effective seedlings; the culture conditions were: the illumination time is 12h/d, the illumination intensity is 1500-20001x, and the proliferation culture time is 20-25d.
4. The method for inducing roots by cutting bougainvillea spectabilis ex vivo according to claim 1, wherein the step (3) comprises: the effective seedlings are firstly inoculated into a liquid multiplication culture medium for observation and culture, the inoculation density is 10-70 g/L, and the culture time is 3-5 d.
5. The method for inducing roots by cutting bougainvillea spectabilis ex vivo according to claim 3, wherein the explant in the step (2) is a sterilized seed of bougainvillea spectabilis or a tissue slice of a cross section of a stem section of a sterilized seedling of bougainvillea spectabilis.
6. The method for inducing roots by cutting bougainvillea spectabilis ex vivo according to claim 5, wherein the explant is a sterilized seed of bougainvillea spectabilis, and the sterilization method comprises washing and sterilization.
7. The method for inducing roots through the cuttage in vitro of the bougainvillea speetabilis as claimed in claim 6, wherein the method for washing the sterile seeds of the bougainvillea speetabilis is as follows: selecting plump bougainvillea spectabilis fruit pods, soaking seeds in 60-80% of anhydrous alcohol for 15-30 s in a sterile environment, and cleaning with sterile water for 1-4 times; then soaking the mixture for 10 to 20min by 0.05 to 0.15 percent of mercuric chloride, and cleaning the mixture for 3 to 7 times by sterile water.
8. A method for rapidly inducing roots by tidal ex vivo cutting of bougainvillea spectabilis, comprising the method of any one of claims 1 to 7, wherein the step (4) comprises: inoculating the effective seedlings cultured by transitional observation in the step (3) into plant breeding equipment for intermittent submerged culture;
wherein the inoculation density of the effective seedlings is 10-70 g/L;
in addition, the immersion frequency of the effective seedlings is 0-30 min/0-12 h, and the culture time is 20-60 d; the culture conditions were: the illumination intensity is 1000-3000 lx, the illumination is 0-24 h/d, and the temperature is 10-30 ℃.
9. The method for rapidly inducing roots by tidal isolated cutting of bougainvillea spectabilis according to claim 1, wherein in step (4), the plant breeding device comprises a growth disc for inoculating the effective seedlings, a layer of glass beads is paved in the growth disc, and the diameter of the glass beads is 1-10 mm.
10. A bougainvillea spectabilis breeding method is characterized in that: which comprises the method for rapidly inducing roots by tidal ex vivo cutting of bougainvillea spectabilis as claimed in claim 8 or 9.
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