CN118077574A - Method for cultivating polyploid crape myrtle by high-temperature induction of 2n gametes - Google Patents
Method for cultivating polyploid crape myrtle by high-temperature induction of 2n gametes Download PDFInfo
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Abstract
The invention provides a method for cultivating polyploid crape myrtle by high-temperature induction of 2n gametes, which comprises the following specific steps: 1) Determining a high-temperature treatment period through the meiosis process of male and female gametes of crape myrtle; 2) Performing high-temperature treatment on the crape myrtle inflorescences according to the determined period, and inducing to form 2n gametes; 3) Hybridizing the 2n female gametes and the 2n male gametes with the crape myrtle variety to obtain a hybrid offspring; 4) Ploidy identification is carried out on the obtained filial generation. The crape myrtle 2n gamete is successfully obtained by the method, 38 triploid and 2 tetraploid crape myrtle new germplasm are cultivated, and the polyploid yield of the 2n female gamete is 2.94% and 2.91% respectively. The method can also be used for the research of plant polyploid induction technology.
Description
Technical Field
The invention relates to the field of plant breeding, in particular to a method for cultivating polyploid crape myrtle by high-temperature induction of 2n gametes.
Background
Lagerstroemia indica is an important summer flowering ornamental plant in China, but the problems of poor cold resistance, poor insect resistance and the like exist at present, and the application range of the Lagerstroemia indica is affected. At present, the crape myrtle breeding is mainly performed in traditional breeding modes such as cross breeding and selective breeding, but the crape myrtle wild distribution is the most north at the juncture of Hubei and Henan, and the cold resistance of the crape myrtle cannot be improved through the traditional breeding means. Polyploid plants tend to have stronger environmental adaptation than diploid plants, and induction of polyploid may be an effective method of improving stress resistance in plants.
The traditional crape myrtle multi-body induction method mostly adopts chemical agents to treat seeds, growing points, cluster buds or callus tissues and the like, has low induction efficiency and is difficult to obtain homozygous polyploid. At present, three tetraploid crape myrtle varieties 'four seas' are obtained in China, namely 'four seas' are obtained in China, and 'purple' and 'silver butterfly' are obtained in China, and are all asexual polyploid varieties. Sexual polyploidization can transfer the heterozygosity of parents to the greatest extent, and the proportion of obtained homozygous polyploids is high, but no successful report exists in crape myrtle at present. Therefore, a sexual polyploidy crape myrtle polyploidy breeding technology system is established, and an efficient technology is provided for cultivating crape myrtle polyploidy varieties.
Disclosure of Invention
The invention aims to provide a method for cultivating polyploid crape myrtle by high-temperature induction of 2n gametes.
In order to achieve the aim of the invention, in a first aspect, the invention provides a method for cultivating polyploid crape myrtle by high-temperature induction of 2n gametes, when the diameter of flower buds on crape myrtle inflorescences is 5.1mm-5.6mm, the flower buds are subjected to high-temperature treatment at 42-48 ℃ for 4-8 hours, and plants are naturally grown until the flowers are opened under open field conditions after the treatment; and then, taking the crape myrtle subjected to high-temperature treatment as a parent, taking another crape myrtle variety which is not subjected to high-temperature treatment as another parent, carrying out forward and reverse crossing to obtain a filial generation, and carrying out ploidy identification on the filial generation to obtain the polyploid crape myrtle.
Further, when the diameter of the flower bud on the crape myrtle inflorescence is 5.1mm-5.6mm, carrying out high-temperature treatment on the flower bud at 44 ℃ for 6 hours, and naturally growing the plant under the open field condition after the treatment until the flower is opened; and then pollinating another crape myrtle variety which is not subjected to high temperature treatment by using 2n pollen of the flower blooming on the 11 th day after the high temperature treatment, so as to obtain a filial generation.
Further, when the diameter of the flower bud on the crape myrtle inflorescence is 5.1mm-5.6mm, carrying out high-temperature treatment on the flower bud at 46 ℃ for 8 hours, and naturally growing the plant under the open field condition after the treatment until the flower is opened; and then pollinating the crape myrtle flowers which are opened on the 8 th day after the high-temperature treatment by using the normal pollen of another crape myrtle variety which is not subjected to the high-temperature treatment, so as to obtain filial generation.
Further, the plants are normally maintained after pollination, and the maintenance conditions are as follows: the temperature is 30-35 ℃, the illumination intensity is 50000-70000lx, and the illumination duration is 13 h/day.
Further, the high temperature treated crape myrtle variety may be 'velvet' crape myrtle.
Further, another parent may be crape myrtle, scarlet ' or ' DALLAS RED ', or the like, of the Jiujiu island.
Further, the illumination intensity of the crape myrtle flower bud subjected to high temperature treatment is 1500-2000lx, preferably 2000lx.
Further, the polyploid is a triploid or a tetraploid.
In a second aspect, the invention provides the use of the method in the cultivation of new crape myrtle varieties.
In a third aspect, the invention provides the use of the method in plant polyploid studies.
The object of the invention can be further achieved by the following technical measures.
The invention provides a method for cultivating new triploid crape myrtle germplasm by treating flower buds at high temperature to induce 2n gametes and pollinating crape myrtle, which comprises the following specific steps: 1) Determining a high-temperature treatment period through the meiosis process of male and female gametes of crape myrtle; 2) Performing high-temperature treatment on the crape myrtle inflorescences according to the determined period, and inducing to form 2n gametes; 3) Hybridizing the 2n female gametes and the 2n male gametes with the crape myrtle variety to obtain a hybrid offspring; 4) Ploidy identification is carried out on the obtained filial generation. By using the method provided by the invention, crape myrtle 2n gametes are successfully obtained, 38 triploid and 2 tetraploid crape myrtle new germplasm are cultivated, and the polyploid yields of 2n female gametes and male gametes are respectively 2.94% and 2.91%. The method can also be used for the research of plant polyploid induction technology.
Specifically, the invention provides a method for obtaining polyploid crape myrtle by high-temperature induction of 2n gametes, which comprises the steps of carrying out paraffin section observation and chromosome tabletting observation on the development process of female and male gametes of crape myrtle 'velvet' before the induction of 2n gametes; when the diameter of the bud on the inflorescence is 5.1mm-5.6mm, respectively carrying out high-temperature treatment at 44 ℃ for 6h (inducing 2n pollen) and at 46 ℃ for 8h (inducing 2n embryo sac), and naturally growing the plant under open field condition after the treatment until the flower is opened; pollinating the crape myrtle variety by using 2n pollen of the flower which is opened on the 11 th day after high-temperature treatment, pollinating the velvet flower which is opened on the 8 th day after high-temperature treatment by using normal crape myrtle pollen, and obtaining a filial generation; mature seeds are collected in 11 months, sowing is carried out in 3 months, and ploidy identification is carried out on filial generation after seedling emergence.
The method comprises the steps of collecting buds on a crape myrtle variety velvet, measuring the diameters of the buds, anthers and the length of ovaries by using a vernier caliper, sampling from the buds with the diameters of 4.5mm until the buds reach 7.0mm (4.5 mm, 4.6mm and 4.7mm … …), observing the color of the anthers, then respectively placing the anthers and the ovaries into a centrifuge tube filled with FAA fixing liquid, vacuumizing by using a vacuum pump to enable the fixing liquid to quickly permeate into the ovaries, and then placing the ovaries into a refrigerator at the temperature of 4 ℃ for fixing for more than 24 hours.
In the method, the development progress of the female gamete and the male gamete of velvet is observed by paraffin sections and chromosome tabletting respectively.
In the method, when the diameter of the bud of velvet is 5.1-5.6 mm, the length of anther is 0.8-1.2 mm, the anther is light yellow green, and the diameter of ovary is 1.5-2.0 mm, the megasporocyte and microsporocyte are undergoing meiosis, and the optimal period for inducing 2n female gametes and male gametes is the moment. Because the specific development period of female and male gametes cannot be accurately judged through the size of the flower buds, the development process of flowers which are opened on the same day is assumed to be the same, and therefore flowers on different days after induction are used as important basis for judging the chromosome doubling optimum period of the crape myrtle gametes.
The specific steps of the induction method are that plants with the diameters of most flower buds of 5.1mm-5.6mm on inflorescences are selected, placed into a climatic chamber, the temperature is set at 42 ℃, 44 ℃, 46 ℃, 48 ℃ and the illumination intensity is 2000lx, the treatment is respectively carried out for 4 hours, 6 hours and 8 hours, 12 treatment combinations are set, and the crape myrtle is induced at high temperature. After the treatment, the plants are placed under open field conditions to grow naturally until the flowers are opened, and the flowers which are opened on the same day are marked.
The method is characterized in that pollen is collected at about 7 am, dyed and pressed into tablets, and observed and counted under an optical microscope. With untreated pollen grain diameter as a control, 10 fields were randomly observed and the 2n pollen induction rate was counted.
The optimal doubling treatment condition of the chromosome of the crape myrtle microspores of velvet is determined to be 44 ℃ for 6 hours, and the induction rate of 2n pollen of the flowers opening on 11 days after treatment is 66.03%.
In the method, 2n pollen is used for pollinating the Lagerstroemia speciosa 'Qianli snow' and 'DALLAS RED' of Lagerstroemia speciosa; pollinating velvet after high-temperature induction of 2n embryo sacs by using crape myrtle pollen of the Jiujiu island.
Further, the method comprises the following steps:
(1) The hybridization affinity evaluation was performed by designing forward and reverse crosses using a velvet not subjected to high temperature treatment as a parent, and using banaba, scarlet snow, DALLAS RED as another parent.
(2) And (3) at the time of 5-12 months in 2022, collecting pollen of flowers opened on the 11 th day after the optimal induction combination (treatment for 6h at 44 ℃) to pollinate Lagerstroemia indica and Lagerstroemia indica varieties 'Qian-Cheng snow' and 'DALLAS RED', and normally maintaining plants after pollination.
(3) And (5) at month 8 of 2022 and 5-12, collecting the pollen of Lagerstroemia speciosa in the Jiujiu island, pollinating the flower of velvet treated at high temperature, and normally maintaining the plant after pollination.
(4) Mature hybrid fruits were collected in 11 middle 2022 and seeds were removed for dry storage.
(5) Sowing in 2023 month 1, putting into artificial air Hou Shi (temperature 25 deg.C, light intensity 2000lx, illumination 12 h/day), after seedling grows 4 th pair of true leaves, collecting fresh leaves, and collecting stem tip, and confirming ploidy of the initially-screened plants by chromosome counting method.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
According to the invention, the relation between the development process of female gametes and male gametes of velvet and the diameter of flower buds is observed through paraffin section and chromosome section, and when the diameter of the flower buds is 5.1mm-5.6mm, large and microsporocyte cells are in meiosis, which is the optimal period for induction, so that the induction efficiency of 2n gametes is greatly improved.
Secondly, the invention discovers that the high temperature above 46 ℃ leads to the reduction of the powder scattering rate of anthers and even no powder scattering; treatment at 48℃for 8 hours can lead to bud abscission.
And thirdly, the optimal condition of doubling the chromosome of the crape myrtle microspores of velvet is found to be 44 ℃ for 6 hours, and the induction rate of 2n pollen of the flowers opening on 11 days after treatment is 66.03%.
(IV) the invention finds that the highest yield of the triploid of the flowers which are opened on 8 th day after the high-temperature treatment of velvet is pollinated by using normal crape myrtle pollen and treated for 8 hours at 46 ℃ is 10.74%.
Fifth, the crape myrtle polyploid plant is obtained through the sexual polyploid method for the first time, and a new technology is provided for crape myrtle high-efficiency polyploid breeding.
Drawings
FIG. 1 shows the development process of male gametes of crape myrtle, velvet according to the invention. Wherein A-D respectively represent sporophore cell, microsporocyte, microsporotetrad and pollen grains.
FIG. 2 shows the development process of female gametes of crape myrtle, velvet according to the invention. Wherein A-I respectively represent spore original cells, megasporocyte, megasporozoites, megasporotetrads, functional megaspores, mononuclear embryo sacs, dinuclear embryo sacs, tetranuclear embryo sacs and mature egg cells.
FIG. 3 shows n pollen and 2n pollen of the 'velvet' crape myrtle of the present invention. Wherein A, B represents haplotype pollen and 2n pollen, respectively.
FIG. 4 is a flow cytometry plot of diploid, triploid and tetraploid crape myrtle of the present invention.
FIG. 5 shows chromosomal tabletting of diploid, triploid and tetraploid crape myrtle according to the invention. Wherein A-C respectively represent diploid, triploid and tetraploid.
FIG. 6 is a polyploid crape myrtle cultivated with 2n gametes in the present invention. Wherein A-C respectively represent diploid, triploid and tetraploid crape myrtle.
Detailed Description
Aiming at the current situation that sexual polyploidy of crape myrtle is not reported successfully, the invention provides a method for sexual cultivation of polyploid crape myrtle by inducing 2n gametes at high temperature.
The invention adopts the following technical scheme:
Before inducing 2n gametes, paraffin section and chromosome slide observation are carried out on the development progress of female gametes and male gametes of crape myrtle velvet, and a proper stage of high-temperature induction is determined; when the diameter of the flower bud on the inflorescence is 5.1mm-5.6mm, respectively carrying out high-temperature treatment at 46 ℃ for 8h and at 46 ℃ for 6h on the flower bud to induce 2n male and female gametes; after high-temperature treatment, the plants naturally grow under open field conditions until flowers are opened, and the flowers which are opened on the same day are marked; pollinating the crape myrtle variety by using 2n pollen of the flower which is opened on the 11 th day after high-temperature treatment, pollinating the velvet flower which is opened on the 8 th day after high-temperature treatment by using normal crape myrtle pollen, and obtaining a filial generation; mature seeds are collected for 11 months, sowing is carried out for 1 month, and ploidy identification is carried out on filial generation after seedling emergence, so that polyploid crape myrtle is obtained.
According to the invention, the development process of female and male gametes of velvet is observed through paraffin section and chromosome flaking, and the bud diameter of the velvet of crape myrtle is found to be 5.1-5.6 mm, the anther length is 0.8-1.2 mm, the anther color is light yellow green, and when the ovary diameter is 1.5-2.0 mm, the meiosis of megasporocyte and microsporocyte is carried out, and the meiosis is the optimal stage for inducing 2n female gametes and male gametes.
In the method provided by the invention, the observation of the development process of female and male gametes of the crape myrtle of velvet comprises the following steps: collecting buds on crape myrtle variety velvet, measuring the diameter of the buds and the lengths of anthers and ovaries by using a vernier caliper, sampling from the buds with the diameter of 4.5mm until the buds with the diameter of 7.0mm (4.5 mm, 4.6mm and 4.7mm … …) are collected, observing the color of the anthers, then respectively placing the anthers and the ovaries into a centrifuge tube filled with FAA fixing liquid, vacuumizing by using a vacuum pump to enable the fixing liquid to quickly permeate into the ovaries, and then placing the ovaries into a refrigerator at the temperature of 4 ℃ for fixing for more than 24 hours.
In the method provided by the invention, the development progress of the female gamete and the male gamete of velvet is observed by paraffin sections and chromosome tabletting respectively.
The optimal period for 2n male and female gamete induction is when the diameter of crape myrtle velvet bud is 5.1mm-5.6 mm.
In the method provided by the invention, the specific development period of the male and female gametes cannot be accurately judged through the size of the flower bud, and the same development process of flowers opening on the same day is assumed, so that flowering of different days after induction is used as an important basis for judging the chromosome doubling optimum period of the crape myrtle gametes.
In the method provided by the invention, the optimal doubling treatment condition of the chromosome of the crape myrtle microspores of velvet is 44 ℃ for 6 hours, and the induction rate of 2n pollen of the flowers opening on 11 days after treatment is 66.03%.
In the method provided by the invention, velvet is taken as a parent, and crape myrtle, scarlet snow and DALLAS RED are taken as another parent.
Specifically, as a specific embodiment of the present invention, the method provided by the present invention includes the following steps:
(1) Collecting buds on crape myrtle variety velvet, measuring the diameter of the buds and the lengths of anthers and ovaries by using a vernier caliper, sampling from the buds with the diameter of 4.5mm until the buds with the diameter of 7.0mm (4.5 mm, 4.6mm and 4.7mm … …) are collected, observing the color of the anthers, then respectively placing the anthers and the ovaries into a centrifuge tube filled with FAA fixing liquid, vacuumizing by using a vacuum pump to enable the fixing liquid to quickly permeate into the ovaries, and then placing the ovaries into a refrigerator at the temperature of 4 ℃ for fixing for more than 24 hours.
(2) The development process of the female gamete and the male gamete of velvet is respectively observed by paraffin sections and chromosome tabletting.
(3) Selecting crape myrtle plants with most of flower buds of 5.1-5.6 mm diameter, setting the temperature at 42 ℃, 44 ℃, 46 ℃, 48 ℃ and the illumination intensity at 2000lx by using a climatic chamber, and continuously treating for 4h, 6h and 8h for 12 treatment combinations, and carrying out high-temperature induction on crape myrtle flower buds. After high temperature treatment, the plants are naturally grown under open field conditions until the flowers are opened, and the flowers which are opened on the same day are marked.
(4) Pollen was collected at around 7 am every day, stained and pressed into tablets, and observed and counted under an optical microscope. With untreated pollen grain diameter as a control, 10 fields were randomly observed and the 2n pollen induction rate was counted.
Optimally, the chromosome doubling condition of the 'velvet' crape myrtle microspores is 44 ℃ for 6 hours, and the induction rate of 2n pollen of the flowers opening on 11 days after treatment is 66.03 percent.
(5) And 2021, 6-9 months, designing forward and reverse crosses by taking velvet which is not subjected to high temperature treatment as a parent, taking crape myrtle in the Jiujiu island and sequin scarlet and DALLAS RED as another parent, and evaluating hybridization affinity.
(6) Pollen from the flowers that opened on day 11 after treatment with the optimal induction combination (44 ℃ for 6 h) was collected for 8 months 2022 pollinating the lagerstroemia indica variety 'sequin scarlet' and 'DALLAS RED'.
(7) And 2022, 8 months, collecting crape myrtle pollen of the Jiujiu island, and pollinating the flowers of velvet which are opened on 8 th day after being subjected to high-temperature treatment at 46 ℃ for 8 hours.
(8) Mature hybrid fruits were collected in 11 middle 2022 and seeds were removed for dry storage.
(9) Sowing in 2023 month 1, putting into artificial air Hou Shi (temperature 25 deg.C, light intensity 2000lx, illumination 12 h/day), after seedling grows 4 th pair of true leaves, collecting fresh leaves, and collecting stem tip, and confirming ploidy of the initially-screened plants by chromosome counting method.
The invention also protects the application of the method in cultivating new crape myrtle varieties and inducing plant polyploidy according to the understanding of the person skilled in the art.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available. Example 1 method for sexual cultivation of polyploid Lagerstroemia indica by high temperature induction of 2n gametes
(1) Selection of materials
In the embodiment, 3-year-old velvet crape myrtle is used as a material to induce 2n gametes, and the crape myrtle and crape myrtle varieties 'Qian-layer scarlet snow' and 'DALLAS RED' are used as hybrid parents.
(2) Determining induction period of male and female gametes
After the last ten days of 6 months of 2021, collecting buds on a crape myrtle variety velvet, measuring the diameter of the buds, anthers and the length of ovaries by using a vernier caliper, sampling from the buds with the diameter of 4.5mm until the buds with the diameter of 7.0mm (4.5 mm, 4.6mm and 4.7mm … …) are collected, observing the color of the anthers, then respectively placing the anthers and the ovaries into a centrifuge tube filled with FAA fixing liquid, vacuumizing by using a vacuum pump to enable the fixing liquid to quickly permeate into the ovaries, and then placing the ovaries into a refrigerator at the temperature of 4 ℃ for fixing for more than 24 hours.
The development process of the female gamete and the male gamete of velvet is respectively observed by paraffin sections and chromosome tabletting.
The diameter of the flower bud is 5.1-5.6mm in the whole process of large velvet and microspore meiosis, the length of anther is 0.8-1.2 mm, and the color is light yellow green; the diameter of the ovary is 1.5mm-2.0mm. The specific development period of female and male gametes cannot be accurately judged due to the fact that the flower development processes of flowers opening on the same day are the same, and therefore flowering of different days after induction is used as an important basis for judging the chromosome doubling optimum period of the crape myrtle gametes.
The development process of male gametes of 'velvet' crape myrtle is shown in figure 1. Wherein A-D respectively represent sporophore cell, microsporocyte, microsporotetrad and pollen grains.
The development process of female gametes of 'velvet' crape myrtle is shown in figure 2. Wherein A-I respectively represent spore original cells, megasporocyte, megasporozoites, megasporotetrads, functional megaspores, mononuclear embryo sacs, dinuclear embryo sacs, tetranuclear embryo sacs and mature egg cells.
(3) Optimal induction technique
Selecting crape myrtle plants with most of flower buds of 5.1-5.6 mm diameter, setting the temperature at 42 ℃, 44 ℃, 46 ℃, 48 ℃ and the illumination intensity at 2000lx by using a climatic chamber, and continuously treating for 4h, 6h and 8h for 12 treatment combinations, and carrying out high-temperature induction on crape myrtle flower buds. After high temperature treatment, the plants are naturally grown under open field conditions until the flowers are opened, and the flowers which are opened on the same day are marked. Pollen was collected at around 7 am every day, stained and pressed into tablets, observed under an optical microscope, and counted. With untreated pollen grain diameter as a control, 10 fields were randomly observed and the 2n pollen induction rate was counted.
The optimal condition for doubling the chromosome of the crape myrtle microspores of velvet' is 44 ℃ for 6 hours, and the induction rate of 2n pollen of the flowers opening on 11 days after treatment is 66.03%.
The n pollen and 2n pollen of the 'velvet' crape myrtle are shown in figure 3. Wherein A, B represents haplotype pollen and 2n pollen, respectively.
(4) Hybrid offspring acquisition
And 2022, 8 months, collecting pollen of flowers which are opened on 11 days after the treatment of the optimal induction combination (the duration of 6 hours at 44 ℃) and pollinating Lagerstroemia indica and Lagerstroemia indica varieties 'Qian-er-Cheng-Xue' and 'DALLAS RED', hybridizing 5587 flowers to obtain 319 fruits, wherein the average fruit setting rate is 5.71%, and obtaining seeds 3470. Natural pollen of crape myrtle in Jiujiu island is used for pollination of velvet after high-temperature induction, 6990 flowers are hybridized together, 2595 fruits are obtained, the average fruiting rate is 37.12%, and seeds 3582 grains are obtained.
(5) Sowing seeds
Sowing in 2023 month 1, and sowing all 3470 seeds obtained by hybridization of 2n pollen and crape myrtle variety to obtain seedling 378, wherein the seedling rate is 10.89%. After 3582 seeds obtained by hybridization of Lagerstroemia indica of Jiujiu island and 'velvet' after high-temperature induction are all sown, 985 seedlings are obtained, and the average seedling rate is 24.74%.
(6) Ploidy identification of hybrid offspring
After the seedlings grow out of the 4 th pair of true leaves, collecting plants with the fresh leaves subjected to preliminary screening ploidy change by a flow cytometry, collecting stem tips, confirming the preliminary screened plants by a chromosome counting method, and determining ploidy of the hybrid seedlings.
Pollinating the crape myrtle variety by using 2n pollen of the flower opening at the 11 th day after the high-temperature treatment for 6 hours at 44 ℃, successfully obtaining 9 triploid plants, 2 tetraploid plants and 2 polyploid yield of 2.91 percent.
Normal crape myrtle pollen is used for pollinating velvet treated at high temperature, 29 triploid strains are obtained, and the average triploid yield is 2.94%. Among them, 13 triploid comes from flowers that are opened on 8 th day after 8h treatment at 46 ℃, and the triploid yield is highest and is 10.74%.
Wherein, the flow cytometry of diploid, triploid and tetraploid crape myrtle is shown in figure 4.
Chromosomal tabletting of diploid, triploid and tetraploid crape myrtle is shown in fig. 5. Wherein A-C respectively represent diploid, triploid and tetraploid.
Polyploid crape myrtle cultivated with 2n gametes is shown in figure 6. Wherein A-C respectively represent diploid, triploid and tetraploid crape myrtle.
The method provided by the invention can obtain the crape myrtle 2n gamete, and can obtain triploid and tetraploid crape myrtle through hybridization, thereby providing a new technology for crape myrtle high-efficiency polyploid breeding.
Comparative example 1 different modes of inducing 2n gametes
In the induction of plant 2n gametes, there are three induction methods: physical, chemical and biological methods, the most widely used of which is the use of colchicine to induce plant growth points.
Compared with colchicine, the high-temperature treatment has the advantages that the effect is rapid, each part can be uniformly treated, the toxic effect is low, the induction rate can reach 66.03% at most under the condition that the flower buds are normally opened, and due to the characteristic of the inflorescence of the crape myrtle, a large number of flower buds can be treated at the same time through the high-temperature treatment, so that the induction workload can be reduced, and the induction efficiency is improved.
Comparative example 2
This comparative example provides a correspondence between the progress of female and male gamete development and the diameter of the 'velvet' flower bud.
When the diameter of the flower bud is 4.5-5.1mm, the male gamete is developed into a spore forming cell, and the female gamete is developed into a spore primordial cell; when the diameter of the flower bud is 5.1-5.6mm, the male gamete develops into microsporocyte, and the female gamete develops into megasporocyte; when the diameter of the bud is 5.6-6.0mm, the male gamete develops into mononuclear pollen, and the female gamete develops into binuclear embryo sac; when the diameter of the flower bud is larger than 6.0mm, the male gamete develops into mature pollen, and the female gamete develops into mature embryo sac.
Comparative example 3 different conditions of temperature treatment for inducing 2n pollen
The comparative example provides different temperature treatment conditions, and the obtained 2n pollen induction rate.
In the comparative example, plants with the diameters of most buds of 5.1-5.6 mm on inflorescences are selected, placed into a climatic chamber, and are set at 42 ℃, 44 ℃, 46 ℃, 48 ℃ and with the illumination intensity of 2000lx, and the continuous treatment time is 4 hours, 6 hours and 8 hours, and the crape myrtle buds are induced by 12 treatment combinations. The induction results are shown in Table 1:
TABLE 1
Comparative example 4 yield of hybrid offspring triploid of 2n female gametes induced by different temperature conditions
This comparative example provides results of the rate of triploid production of the filial offspring of the 2n female gametes induced by different temperature conditions.
(1) In the comparative example, plants with the diameters of most buds of 5.1-5.6 mm on inflorescences are selected, placed into a climatic chamber, and are set at 42 ℃, 44 ℃, 46 ℃, 48 ℃ and with the illumination intensity of 2000lx, and the continuous treatment time is 4 hours, 6 hours and 8 hours, and the crape myrtle buds are induced by 12 treatment combinations.
(2) Pollinating the 'velvet' after high-temperature induction by utilizing the crape myrtle pollen of the Jiujiu island to obtain 3582 seeds, and obtaining 985 seedlings after all sowing, wherein the average seedling rate is 24.74%.
(3) Ploidy identification is carried out on the filial generation, 29 triploid strains are obtained, and the average triploid yield is 2.94%. 13 of them were from flower buds that opened 8 days after treatment at 46℃for 8 hours, and the triploid yield of the combination was the highest, 10.74%.
The effect of inducing 2n female gametes to cultivate polyploids is shown in table 2:
TABLE 2
Comparative example 5 different hybrid combination polyploid yields
This comparative example provides results for different hybrid combination polyploid yields.
(1) 3 Species and varieties of crape myrtle are pollinated by using high-temperature induced 2n pollen, so that 11 polyploid plants are obtained, and the polyploid yield is 2.91%. Wherein, the combination of ' Qian ' red snow ' as female parent obtains offspring 238 strain, triploid 7 strain, tetraploid 2 strain, the triploid and tetraploid yield is 2.94% and 0.84% respectively; the 'DALLAS RED' is the combination of the female parent to obtain 130 offspring strains, 2 triploid strains, and the triploid yield is 1.53%; and 10 offspring plants are obtained by taking the combination of the crape myrtle of the Jiujiu island as a female parent, and no polyploid plants are obtained.
(2) And pollinating the velvet treated at high temperature by using the crape myrtle pollen of the Jiujiu island to obtain 29 triploid strains, wherein the average triploid yield is 2.94%.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method for cultivating polyploid crape myrtle by high-temperature induction of 2n gametes is characterized in that when the diameter of flower buds on crape myrtle inflorescences is 5.1-5.6 mm, the flower buds are subjected to high-temperature treatment at 42-48 ℃ for 4-8 hours, and plants are naturally grown until the flowers are opened under open field conditions after the treatment; and then, taking the crape myrtle subjected to high-temperature treatment as a parent, taking another crape myrtle variety which is not subjected to high-temperature treatment as another parent, carrying out forward and reverse crossing to obtain a filial generation, and carrying out ploidy identification on the filial generation to obtain the polyploid crape myrtle.
2. The method according to claim 1, wherein when the diameter of the flower bud on the crape myrtle inflorescence is 5.1mm-5.6mm, the flower bud is subjected to high temperature treatment at 44 ℃ for 6 hours, and the plant is naturally grown until the flower is open under open field conditions after the treatment; and then pollinating another crape myrtle variety which is not subjected to high temperature treatment by using 2n pollen of the flower blooming on the 11 th day after the high temperature treatment, so as to obtain a filial generation.
3. The method according to claim 1, wherein when the diameter of the flower bud on the crape myrtle inflorescence is 5.1mm-5.6mm, the flower bud is subjected to high temperature treatment at 46 ℃ for 8 hours, and the plant is naturally grown until the flower is open under open field conditions after the treatment; and then pollinating the crape myrtle flowers which are opened on the 8 th day after the high-temperature treatment by using the normal pollen of another crape myrtle variety which is not subjected to the high-temperature treatment, so as to obtain filial generation.
4. A method according to claim 2 or 3, wherein the plants are normally maintained after pollination, the maintenance conditions being: the temperature is 30-35 ℃, the illumination intensity is 50000-70000lx, and the illumination duration is 13 h/day.
5. The method of claim 1, wherein the high temperature treated crape myrtle is a 'velvet' crape myrtle.
6. The method of claim 1, wherein the other parent is crape myrtle, 'sequin' or 'DALLAS RED'.
7. The method according to claim 1, wherein the illumination intensity of the crape myrtle flower bud is 1500-2000lx when the crape myrtle flower bud is subjected to high temperature treatment.
8. The method of claim 1, wherein the polyploid is a triploid or a tetraploid.
9. Use of the method of any one of claims 1-8 for breeding new varieties of crape myrtle.
10. Use of the method of any one of claims 1-8 in plant polyploid studies.
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