CN114667927A - Method for in vitro induction of bougainvillea spectabilis polyploidy - Google Patents
Method for in vitro induction of bougainvillea spectabilis polyploidy Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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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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- 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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Abstract
The invention provides a method for inducing bougainvillea speetabilis polyploidy in vitro, which comprises the steps of selecting diploid bougainvillea speetabilis hair leaf seeds or diploid light leaf seeds as female parents to be hybridized with diploid light leaf seeds as male parents, collecting mature bougainvillea speetabilis hybrid seeds after pollination, taking hypocotyls after inducing the seeds to germinate, pre-culturing the hypocotyls in a basic culture medium for 2-4 days, inoculating the pre-cultured hypocotyls into a differentiation culture medium to perform polyploid induction culture for 30 days, and then performing proliferation culture and rooting culture on the surviving differentiation buds in a proliferation culture medium and a rooting culture medium in sequence. The invention uses colchicine containing dimethyl sulfoxide to double-induce the bougainvillea spectabilis aseptic material, so that the chromosome of the bougainvillea spectabilis is doubled, a polyploid plant is obtained, and the breeding way of the bougainvillea spectabilis is increased. The polyploid plant not only has the advantages of strong growth vigor, rapid growth, prolonged ornamental period, strong comprehensive resistance and the like, but also can improve the plant fertility and provide a new germplasm for developing large-scale bougainvillea spectabilis crossbreeding.
Description
Technical Field
The invention relates to polyploid induction, in particular to a method for in vitro induction of bougainvillea spectabilis polyploid.
Background
Bougainvillea spectabilis, also known as phyllanthus, anthurium, etc., is a general name for ornamental plants of the genus Bougainvillea (Bougainvillea) in the family of mirabilis, nyctaceae (Nyctaginacea), native to brazil and peru et al in south america, first discovered in 1768 by the french botanicolan barbie in brazil, and then named after the name of the french seafarinacian experiencer (Louis-anthrone de Bougainvillea). With introduction and domestication of the plants, the main tropical and subtropical areas in the world now have the shadow of the plants. The bougainvillea spectabilis bracts are large, the color is as bright as flowers, the duration time is more than half a year, and the ornamental value is very high; the plants are evergreen in four seasons, the root systems are developed, and the plants are drought-resistant and barren and have good climate adaptability; the bougainvillea spectabilis branches and tendrils are strong in flexibility, good in plasticity, strong in germination capacity and extremely resistant to pruning, are wide in application and various in forms in landscaping, and are flowers in a plurality of cities such as san, Shenzhen, Zhuhai, Xiamen, Huizhou and Wuzhou. Bougainvillea spectabilis is called "tropical glory" in tropical countries due to its high popularity and widespread use in horticulture. Statistically, there are 18 species of bougainvillea, among which three elite species and one natural hybrid species of bougainvillea are highly ornamental, and these are the hairy leaf species (b.spectabilis), the bare leaf species (b.glabera), the peruvian species (b.peruviana), and the baryta species (b.xbutteriana), which are the main parents for breeding horticultural varieties. By 3 months 2021, the bougainvillea spectabilis horticulture association of great britain has registered up to 302 cultivars.
In recent years, the research reports on bougainvillea spectabilis show a growing trend at home and abroad, mainly about the research on the aspects of cultivation management, flowering phase regulation, tissue culture and the like of bougainvillea spectabilis, and the research on variety breeding is less, and mainly takes seedling selection, spontaneous bud mutation, physical and chemical mutation breeding as main points. More novel varieties with high ornamental value, growth stress resistance and other performances are obtained through variety breeding, and are always the targets pursued by breeders and the key research directions of ornamental plants. The breeding method of the new variety comprises cross breeding, mutation breeding, ploidy breeding and the like.
The bougainvillea spectabilis has the problems of low pollen activity, incompatibility in self-hybridization and the like, so that the development of large-scale hybrid breeding is restricted, and hybrid varieties are very limited; mutation breeding is mostly obtained by mutation on single characters such as flower color and leaf color, the mutation rate is lower, beneficial mutation is less, and directionality is lacked; ploidy breeding can double plant chromosomes, change variety characteristics on the genetic level, and polyploid plants often show the characteristics of multiple spots, gorgeous flower colors, long flowering period, thick leaves, enhanced resistance and the like. The ornamental value and the economic value are both generally improved. By utilizing chromosome doubling, a new plant type can be artificially created, and polyploidy is used as a parent, so that the incompatibility of distant hybridization can be overcome.
Therefore, polyploid breeding is one of the common breeding approaches for ornamental plants, and the key point is how to obtain the bougainvillea speetabilis polyploid germplasm efficiently.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for inducing bougainvillea spectabilis polyploidy in vitro.
The purpose of the invention is realized by the following technical scheme: a method for inducing bougainvillea spectabilis polyploidy in vitro comprises the steps of selecting a diploid bougainvillea spectabilis hair leaf seed or a diploid light leaf seed as a female parent and a diploid light leaf seed as a male parent for hybridization, collecting mature bougainvillea spectabilis hybrid seeds after pollination, taking an embryonic axis after inducing the seeds to germinate, pre-culturing the embryonic axis in a basic culture medium for 2-4 days, inoculating the pre-cultured embryonic axis into a differentiation culture medium for polyploid induction culture for 30 days, and then sequentially performing proliferation culture and rooting culture on the surviving differentiation buds in a proliferation culture medium and a rooting culture medium;
wherein the basic culture medium comprises MS, IBA 0.05-0.2 mg/L, 6-BA 3.0-4.0 mg/L, white sugar 25.00-35.00 g/L and carrageenan 6.50-7.00 g/L;
the differentiation culture medium is a basic culture medium added with colchicine containing dimethyl sulfoxide;
the enrichment culture medium comprises MS, 0.05-0.20 mg/L of NAA, 0.05-0.20 mg/L of 6-BA, 25.00-35.00 g/L of white sugar and 6.50-7.00 g/L of carrageenan;
the rooting medium is 1/2MS, 0.05-0.20 mg/L NAA, 0.2-0.8 g/L AC, 25.00-35.00 g/L white sugar and 6.50-7.00 g/L carrageenan.
Further, collecting mature bougainvillea spectabilis hybrid seeds after pollination for 25-30 days.
Further, the specific operations of inducing seed germination are as follows: cleaning selected seeds with a detergent, soaking and disinfecting with benzalkonium bromide, washing with high-temperature sterilized sterile water, paving the seeds in a culture box which is fully paved with paper towels at certain intervals and is fully wetted with the sterile water, and carrying out dark culture in an artificial climate box with the temperature set to be 22-24 ℃ to induce the seeds to germinate.
Further, the specific operation of taking the embryo axis is as follows: after the seeds are subjected to induced culture for 10-15 days, the seeds germinate to grow root hair, the hypocotyl length is 5-6 cm, 2 leaves are taken out when being completely unfolded, the root hair is cut off by using a sterile blade, the hypocotyl is placed in a beaker, washed by a detergent, washed by running water for 25-35 min, soaked by benzalkonium bromide for 1-2 min and then moved to an ultra-clean workbench, soaked by 75% alcohol for 30s, and washed by sterile water for 2-3 times; soaking the hypocotyl in 0.1% mercuric chloride solution for 8-10 min without shaking, fully contacting the disinfectant with the explant, taking out the material after disinfection, putting the material into a new sterile beaker, and washing with sterile water for 4-6 times for later use.
Further, the culture conditions of the pre-culture, the polyploid induction culture, the proliferation culture medium and the rooting culture medium are as follows: the culture temperature is 25 +/-2 ℃, the illumination time is 13-18 h/d, the illumination intensity is 1500-2000 Lx, and the pH of the culture medium is 5.8-6.0.
Further, the concentration of the colchicine is 0-100 mg/L, and the mass percentage content of the dimethyl sulfoxide is 1.5-3%.
Further, the concentration of the colchicine is 50mg/L, and the mass percentage of the dimethyl sulfoxide is 2%.
Further, the minimal medium comprises MS, IBA 0.1mg/L, 6-BA 2.0mg/L, white sugar 30.00g/L and carrageenan 6.80 g/L.
The invention has the following advantages: the invention provides a method for inducing bougainvillea speetabilis polyploidy in vitro, which overcomes the problems that the existing bougainvillea speetabilis have the phenomena of incompatibility in self-hybridization, low hybridization fruiting rate and the like, restricts the development of large-scale hybridization breeding, and the hybrid varieties are very limited. In addition, the colchicine is used for treating the isolated tissue for doubling, the method is used for doubling on the basis of plant tissue culture, the operation process is clear and feasible, and polyploids with the same genetic background can be obtained in batches. Ploidy breeding can double plant chromosomes, change variety characteristics on the genetic level, and the plant polyploids not only have the advantages of strong growth vigor, rapid growth, prolonged ornamental period, strong comprehensive resistance and the like, but also can improve the plant fertility, provide new germplasm for developing large-scale bougainvillea spectabilis crossbreeding, and be beneficial to obtaining new varieties with better properties.
Drawings
FIG. 1 is a schematic diagram showing the growth of hypocotyl doubling treatment with different concentrations of colchicine, in which A0 mg/L; b25 mg/L; c50 mg/L; d100 mg/L.
FIG. 2 is a schematic view showing the growth of adventitious bud strains differentiated at the induction treatment stage after proliferation culture in a proliferation medium for 30 days, in which A is a control strain; b, variant strain.
FIG. 3 is a diploid flow diagram.
FIG. 4 is a tetraploid flow chart.
Fig. 5 is a tetraploid flow chart.
Detailed Description
The invention is further described with reference to the following figures and examples, without limiting the scope of the invention to the following:
example 1: a method for inducing bougainvillea spectabilis polyploid in vitro comprises the steps of selecting a diploid bougainvillea spectabilis hair leaf seed as a female parent and a diploid light leaf seed as a male parent for hybridization, collecting mature bougainvillea spectabilis hybrid seeds after pollination for 30d, cleaning the selected seeds with a detergent, soaking and sterilizing with benzalkonium bromide, washing with high-temperature sterilized sterile water, flatly paving the seeds in a culture box which is fully wetted with sterile water and is full of tissues at certain intervals, and carrying out dark culture in an artificial climate box with the temperature of 22-24 ℃ to induce the seeds to germinate;
after induced culture for 15 days, germinating to grow root hair, enabling the hypocotyl to be 5-6 cm long, taking out 2 leaves when the leaves are completely unfolded, cutting off the root hair by using a sterile blade, placing the hypocotyl in a beaker, washing by using a detergent, washing for 35min by running water, soaking in benzalkonium bromide for 2min, then moving to a super-clean workbench, soaking for 30s by using 75% alcohol, and washing for 3 times by using sterile water; soaking hypocotyl in 0.1% mercuric chloride solution for 10min, shaking to make the disinfectant contact with explant, taking out the material after disinfection, placing in a new sterile beaker, and washing with sterile water for 6 times.
Pre-culturing hypocotyls in a basic culture medium for 4 days, inoculating the pre-cultured hypocotyls into a differentiation culture medium for polyploid induction culture for 30 days, and then sequentially performing proliferation culture and rooting culture on the surviving differentiation buds in a proliferation culture medium and a rooting culture medium;
wherein the basic culture medium comprises MS, IBA 0.2mg/L, 6-BA 4.0mg/L, white sugar 35.00g/L and carrageenan 7.00 g/L;
the differentiation culture medium is a basic culture medium added with colchicine containing dimethyl sulfoxide; the concentration of the colchicine is 20mg/L, and the mass percentage content of the dimethyl sulfoxide is 3 percent;
the enrichment medium is MS + NAA0.20mg/L +6-BA0.20mg/L + white sugar 35.00g/L + carrageenan 7.00 g/L;
the rooting medium is 1/2MS, NAA0.20mg/L, AC 0.8g/L, white sugar 35.00g/L and carrageenan 7.00 g/L.
The culture conditions of the pre-culture, the polyploid induction culture, the multiplication culture medium and the rooting culture medium are as follows: the culture temperature is 25 +/-2 ℃, the illumination time is 18h/d, the illumination intensity is 2000Lx, and the pH value of the culture medium is 6.0.
Example 2: a method for inducing bougainvillea spectabilis polyploidy in vitro comprises the steps of selecting a diploid bougainvillea spectabilis leaf seed as a female parent and a diploid bougainvillea spectabilis leaf seed as a male parent for hybridization, collecting mature bougainvillea spectabilis hybrid seeds after pollination for 25d, cleaning the selected seeds with a detergent, soaking and sterilizing with benzalkonium bromide, washing with high-temperature sterilized sterile water, flatly paving the seeds in a culture box which is fully wetted with sterile water and is full of paper towels at certain intervals, and carrying out dark culture in an artificial climate box with the temperature of 22-24 ℃ to induce the seeds to germinate;
after 10 days of induced culture, germinating to grow root hair, enabling the hypocotyl to be 5-6 cm long, taking out 2 leaves when the leaves are completely unfolded, cutting off the root hair by using a sterile blade, placing the hypocotyl in a beaker, washing by using a detergent, washing for 35min by running water, soaking in benzalkonium bromide for 1min, then moving to a super-clean workbench, soaking for 30s by using 75% alcohol, and washing for 2 times by using sterile water; soaking hypocotyl in 0.1% mercuric chloride solution for 8min, shaking to make the disinfectant contact with explant, taking out the material after disinfection, placing in a new sterile beaker, and washing with sterile water for 6 times.
Pre-culturing hypocotyls in a basic culture medium for 2 days, inoculating the pre-cultured hypocotyls into a differentiation culture medium for polyploid induction culture for 30 days, and then sequentially performing proliferation culture and rooting culture on the surviving differentiation buds in a proliferation culture medium and a rooting culture medium;
wherein the basic culture medium comprises MS, IBA0.05mg/L, 6-BA 3.0mg/L, white sugar 25.00g/L and carrageenan 6.50 g/L;
the differentiation culture medium is a basic culture medium added with colchicine containing dimethyl sulfoxide; the concentration of the colchicine is 100mg/L, and the mass percentage content of the dimethyl sulfoxide is 1.5 percent;
the enrichment culture medium is MS + NAA mg/L +6-BA 0.05mg/L + white sugar 25.00g/L + carrageenan 6.50 g/L;
the rooting medium is 1/2MS, 0.05mg/L NAA, 0.2g/L AC, 25.00g/L white sugar and 6.50g/L carrageenan.
The culture conditions of the pre-culture, the polyploid induction culture, the multiplication culture medium and the rooting culture medium are as follows: the culture temperature is 25 +/-2 ℃, the illumination time is 13h/d, the illumination intensity is 1500Lx, and the pH value of the culture medium is 5.8.
Example 3: a method for inducing bougainvillea spectabilis polyploidy in vitro comprises the steps of selecting a diploid bougainvillea spectabilis hair leaf seed as a female parent and a diploid light leaf seed as a male parent for hybridization, collecting mature bougainvillea spectabilis hybrid seeds after pollination for 28d, collecting the mature bougainvillea spectabilis hybrid seeds, cleaning the selected seeds with a detergent, soaking and sterilizing with benzalkonium bromide, washing with high-temperature sterilized sterile water, flatly paving the seeds in a culture box which is fully wetted with sterile water at certain intervals, and carrying out dark culture in an artificial climate box with the temperature of 22-24 ℃ to induce the seeds to germinate;
after 12 days of induced culture, germinating to grow root hair, enabling the hypocotyl to be 5-6 cm long, taking out 2 leaves when the leaves are completely unfolded, cutting off the root hair by using a sterile blade, placing the hypocotyl in a beaker, washing by using a detergent, washing for 30min by running water, soaking in benzalkonium bromide for 2min, then moving to a super-clean workbench, soaking for 30s by using 75% alcohol, and washing for 2 times by using sterile water; soaking hypocotyl in 0.1% mercuric chloride solution for 9min, shaking to make the disinfectant contact with explant, taking out the material after disinfection, placing in a new sterile beaker, and washing with sterile water for 5 times.
Pre-culturing hypocotyls in a basic culture medium for 3d, inoculating the pre-cultured hypocotyls into a differentiation culture medium for polyploid induction culture for 30d, and then sequentially carrying out proliferation culture and rooting culture on the surviving differentiation buds in a proliferation culture medium and a rooting culture medium;
wherein the basic culture medium comprises MS, IBA 0.1mg/L, 6-BA 2.0mg/L, white sugar 30.00g/L and carrageenan 6.80 g/L;
the differentiation culture medium is a basic culture medium added with colchicine containing dimethyl sulfoxide; the concentration of the colchicine is 50mg/L, and the mass percentage content of the dimethyl sulfoxide is 2%;
the multiplication culture medium is MS + NAA0.15mg/L +6-BA 0.08mg/L + white sugar 30.00g/L + carrageenan 6.80 g/L;
the rooting medium is 1/2MS, NAA0.1mg/L, AC 0.5g/L, white sugar 30.00g/L and carrageenan 6.80 g/L.
The culture conditions of the pre-culture, the polyploid induction culture, the multiplication culture medium and the rooting culture medium are as follows: the culture temperature is 25 + -2 deg.C, the illumination time is 15h/d, the illumination intensity is 1800Lx, and the culture medium pH is 5.9.
The following experiments illustrate the beneficial effects of the present invention:
1. the experimental method comprises the following steps: a method for inducing bougainvillea spectabilis polyploid in vitro comprises the following steps:
(1) obtaining of hybrid seeds: and in the period from 11 months to 3 months of the next year of 2020, selecting diploid hair leaf seeds as female parents, hybridizing the diploid hair leaf seeds as female parents and diploid light leaf seeds as male parents, collecting mature bougainvillea spectabilis hybrid seeds after pollination for 28 days, and selecting full seeds for later use after drying in the shade.
(2) Seed germination: cleaning the selected seeds in the step (1) by using a detergent, soaking and disinfecting by using benzalkonium bromide, washing by using high-temperature sterilized sterile water, then flatly paving the seeds in a culture box which is fully wetted by using sterile water and is covered with paper towels at certain intervals, and carrying out dark culture in an artificial climate box with the temperature set to be 22-24 ℃ so as to induce the seeds to germinate.
(3) And (3) sterilizing the surface of the explant: after the seeds are cultured for 15 days in the step (2), root hairs grow out from the seeds, hypocotyls grow to be about 5-6 cm, and 2 leaves are taken out when being completely unfolded. Cutting off root hair with a sterile blade, placing hypocotyl in a beaker, washing with detergent, washing with running water for 30min, soaking benzalkonium bromide for 1min, transferring to an ultra-clean workbench, soaking with 75% alcohol for 30s, and washing with sterile water for 2 times; soaking hypocotyl in 0.1% mercuric chloride solution for 10min, shaking to make the disinfectant contact with explant, taking out the material after disinfection, placing in a new sterile beaker, and washing with sterile water for 5 times.
(4) Pre-culturing: and (3) cutting the sterile hypocotyl obtained in the step (3) to about 0.5-1 cm by using a sterile blade, horizontally laying and inoculating the sterile hypocotyl in a MS + IBA 0.1mg/L +6-BA 2.0mg/L (30.00 g/L of white sugar and 6.80g/L of carrageenan are added without special description, and the same is used below) basic culture medium for pre-culture for 3 d.
(5) Polyploid induction: and (3) inoculating the hypocotyls pre-cultured in the step (4) into a hypocotyl differentiation medium MS + IBA 0.1mg/L +6-BA 2.0mg/L containing 0-100 mg/L (containing 2% dimethyl sulfoxide) of colchicine, counting the survival rate and the differentiation rate after 30 days, and transferring the surviving differentiation buds to a subculture medium for culture. The proliferation culture medium is: MS + NAA0.1mg/L +6-BA 1.0 mg/L.
Survival rate is survival number/number of hypocotyl under treatment x 100%
Differentiation rate ═ differentiation number/treatment number × 100%
(6) Rooting culture: inoculating the sterile seedling obtained in the step (5) on 1/2MS + NAA 0.10mg/L + AC 0.50g/L rooting culture medium to induce the differentiation of adventitious roots.
(7) Polyploid identification: and (4) cracking the young leaves grown after the rooting seedling is cultured for 60 days in the step (6) by using a WPB buffer solution, then carrying out flow cytometry ploidy detection, and calculating the polyploidy induction rate.
The polyploid inductivity is polyploid number/differentiated seedling number multiplied by 100%.
The culture temperature is 25 +/-2 ℃, the illumination time is 16h/d, and the illumination intensity is 1500-2000 Lx. The pH of the culture medium is 5.8-6.0.
2. Results of the experiment
1) Morphological feature observation
As shown in FIG. 1, it was observed that after the hypocotyls treated with colchicine were cultured for 30 days, the hypocotyls without colchicine had a good growth state, a high differentiation rate, and a large number of differentiated buds; the two ends of the hypocotyl added with colchicine are browned, some browned buds die, 25mg/L differentiated buds are stronger and higher than 50mg/L, and 100mg/L hypocotyls are not differentiated and bud, which shows that the high-concentration colchicine has the inhibiting effect on the differentiation and growth of the hypocotyl of bougainvillea glabra.
As shown in FIG. 2, the adventitious buds differentiated in the induction treatment stage are inoculated into a proliferation culture medium for proliferation culture for 30d, and then the observation shows that the differentiated plant of the hypocotyl which is not treated by colchicine has normal growth, smaller leaves and thinner leaf tips (FIG. 2A); the mutant plants treated with colchicine had enlarged leaves and slightly increased thickness (FIG. 2B). The morphology observation can preliminarily speculate that the plant generates variation.
2) Effect of colchicine treatment on hypocotyl doubling
Different ploidy flow charts are made, as shown in FIG. 3, FIG. 4 and FIG. 5, and the statistical results are shown in Table 1, and it can be seen from Table 1 that the differences between different treatments are obvious when the culture is added with colchicine culture medium with different concentrations. The survival rate and the differentiation rate of the hypocotyls are gradually reduced along with the increase of the treatment concentration and the treatment time, the survival rate and the differentiation rate of the hypocotyls are highest and respectively are 87.5 percent and 81.67 percent in a culture medium (0mg/L) without colchicine, the survival rate and the differentiation rate are gradually reduced along with the increase of the colchicine concentration, and the differentiation rate of the hypocotyls is 0 when the colchicine concentration reaches 100 mg/L. When the colchicine concentration is 25mg/L, 6 2X/4X mixploid variant strains and 1 4X variant strains are obtained, the induction rate is 6.80%, the survival rate is 62.33%, the differentiation rate is 31.50%, when the colchicine concentration is 75mg/L, 1 2X/4X mixploid variant strain and 1 4X variant strain are obtained, the induction rate is 5.00%, the survival rate is 62.33%, the differentiation rate is 31.50%, when the colchicine concentration is 50mg/L, 4 2X/4X mixploid variant strains and 4X variant strains are obtained, the induction rate is 11.11%, the survival rate is 36.50%, and the differentiation rate is 17.40%. The result shows that 50mg/L colchicine can induce hypocotyl to produce polyploid, which is the optimal inducing concentration.
TABLE 1 Effect of colchicine on hypocotyl doubling treatment
Note: different letters indicate significant differences (P <0.05)
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution of the present invention and the inventive concept within the technical scope of the present invention.
Claims (8)
1. A method for inducing bougainvillea spectabilis polyploids in vitro is characterized in that a diploid bougainvillea spectabilis hair leaf seed or a diploid light leaf seed is selected as a female parent and a diploid light leaf seed is used as a male parent to hybridize, mature bougainvillea spectabilis hybrid seed is collected after pollination, an embryonic axis is taken after the induced seed is germinated, the embryonic axis is pre-cultured in a basic culture medium for 2-4 d, the pre-cultured embryonic axis is inoculated into a differentiation culture medium for polyploidy induction culture for 30d, and then the surviving differentiation bud is subjected to proliferation culture and rooting culture in a proliferation culture medium and a rooting culture medium in sequence;
wherein the basic culture medium comprises MS, IBA 0.05-0.2 mg/L, 6-BA 3.0-4.0 mg/L, white sugar 25.00-35.00 g/L and carrageenan 6.50-7.00 g/L;
the differentiation culture medium is a basic culture medium added with colchicine containing dimethyl sulfoxide;
the enrichment culture medium comprises MS, 0.05-0.20 mg/L of NAA, 0.05-0.20 mg/L of 6-BA, 25.00-35.00 g/L of white sugar and 6.50-7.00 g/L of carrageenan;
the rooting medium is 1/2MS, 0.05-0.20 mg/L NAA, 0.2-0.8 g/L AC, 25.00-35.00 g/L white sugar and 6.50-7.00 g/L carrageenan.
2. The method for inducing the polyploid of bougainvillea spectabilis of claim 1, wherein mature bougainvillea spectabilis hybrid seeds are collected after pollination for 25-30 days.
3. The method for inducing bougainvillea spectabilis polyploid in vitro according to claim 1, wherein the specific operation of inducing seed germination is: cleaning selected seeds with a detergent, soaking and disinfecting with benzalkonium bromide, washing with high-temperature sterilized sterile water, paving the seeds in a culture box which is fully paved with paper towels at certain intervals and is fully wetted with the sterile water, and carrying out dark culture in an artificial climate box with the temperature set to be 22-24 ℃ to induce the seeds to germinate.
4. The method for inducing the polyploid of bougainvillea spectabilis of claim 1, wherein the specific operation of taking the embryonic axis is: after the seeds are subjected to induced culture for 10-15 days, the seeds germinate to grow root hair, the hypocotyl length is 5-6 cm, 2 leaves are taken out when being completely unfolded, the root hair is cut off by using a sterile blade, the hypocotyl is placed in a beaker, washed by a detergent, washed by running water for 25-35 min, soaked by benzalkonium bromide for 1-2 min and then moved to an ultra-clean workbench, soaked by 75% alcohol for 30s, and washed by sterile water for 2-3 times; soaking the hypocotyl in 0.1% mercuric chloride solution for 8-10 min without shaking, fully contacting the disinfectant with the explant, taking out the material after disinfection, putting the material into a new sterile beaker, and washing with sterile water for 4-6 times for later use.
5. The method of claim 1, wherein the conditions for the pre-culture, the polyploid induction culture, the propagation medium and the rooting medium are as follows: the culture temperature is 25 +/-2 ℃, the illumination time is 13-18 h/d, the illumination intensity is 1500-2000 Lx, and the pH of the culture medium is 5.8-6.0.
6. The method of claim 1, wherein the concentration of colchicine is 0-100 mg/L and the content of dimethyl sulfoxide is 1.5-3% by weight.
7. The method of claim 6, wherein the concentration of colchicine is 50mg/L and the content of dimethyl sulfoxide is 2% by weight.
8. The method for inducing bougainvillea spectabilis polyploid in vitro according to claim 1, 6 or 7, wherein the minimal medium is MS + IBA 0.1mg/L +6-BA 2.0mg/L + white sugar 30.00g/L + carrageenan 6.80 g/L.
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