CN113575415B - Time for inducing phalaenopsis 2n pollen and induction method - Google Patents

Abstract

The application discloses a method for determining the time for inducing phalaenopsis to generate 2n pollen by using colchicine, which comprises the following steps: (a) natural 2n pollen yield statistics; (b) 2n pollen induction comprising: selecting pollen in each meiosis period, and treating flower buds with 0.01-0.1% colchicine solution for 2n pollen induction; (c) 2n pollen rate statistics after induction; and (d) comparing the post-induction 2n pollen rate with the native 2n pollen rate, thereby determining the timing of induction of 2n pollen. Also provides a method for inducing phalaenopsis to generate 2n pollen by using colchicine. The invention determines the time for inducing 2n pollen by colchicine and the corresponding colchicine concentration, and lays a foundation for obtaining 2n pollen by polyploid induction and cultivating a new butterfly orchid variety with large and bright flowers and high ornamental value.

Description

Time for inducing phalaenopsis 2n pollen and induction method

Technical Field

The invention belongs to the technical field of polyploid breeding, and particularly relates to a time and an induction method for inducing phalaenopsis to generate 2n pollen by colchicine.

Background

Butterfly orchid is one of the most important commercial crops in the markets of potted plants and cut flowers in the world, the flower shape of the butterfly orchid is similar to butterfly, the flower color is bright and rich, and the butterfly orchid has the reputation of orchid queen. Polyploid plants generally have larger organs and stronger adaptability under adverse conditions, and polyploid orchids generally have the advantages of stout plants, bright colors, strong stress resistance and the like. In fact, the butterfly orchid varieties currently on the market are polyploidy, the number of chromosomes of the original butterfly orchid is 2n =2x =38, while the chromosomes of the cultivated species are mostly unfixed, mainly tetraploid, triploid and aneuploid. Zhuang Donggong et al (Zhuang Donggong et al, 2007) chromosome enumeration and morphological analysis of 38 butterfly orchid varieties or lines, considered tetraploids to account for 82%. In addition, most varieties are produced by interspecific hybridization or intergeneric hybridization, due to ploidy difference, excellent genes of the original diploid are difficult to transfer into commercial hybrids, the size difference of the butterfly orchid is large, most of offspring generated by the hybridization between species with different sizes of chromosomes is sterile, and the sterility of the hybrids can be overcome by inducing the doubling of the butterfly orchid.

Polyploid plants in nature are mainly attributed to the production of 2n gametes (Eng and Ho, 2019, baduel et al, 2018, van de Peer et al, 2017 can et al, 2012), which have genetic diversity and heterosis compared to somatic polyploidization (Lai et al, 2015, younis et al, 2014 brownfield and kohler,2011 raynana and jacobsen, 2003. There are more than 34 families of plants with unreduced gametes reported in the predecessors (dewit et al, 2012, sora et al, 2016), where plants such as citrus (Ahmed et al, 2020, sora et al, 2016) have successfully obtained polyploid progeny using native 2n gametes.

2n pollen induction is an important approach for polyploid breeding of plants. Plant polyploidy formation is mainly through somatic cell doubling and 2n gamete production. Because the tissue culture technology of the butterfly orchid is mature, most of the current researches are to induce explants to obtain asexual polyploidy plants by using chemical reagents such as colchicine, oryzalin and the like under the condition of tissue culture, but a large amount of chimera and aneuploid plants can be generated after induction, and the defects can be well overcome by the generation of 2n gametes.

When the predecessors explore ploidy of offspring generated by different ploidy butterfly orchids, the fact that triploid and tetraploid can be generated by hybridizing diploid and diploid, the fact that polyploid can be generated by hybridizing diploid and triploid, and the fact that polyploid and tetraploid can be generated by hybridizing tetraploid and tetraploid shows that 2n gametes are generated by hybridizing parents is proved. Zhu Jiao et al (Zhu Jiao et al, 2014) statistically measured the natural 2n pollen rates of partial diploids, triploids and tetraploids of Phalaenopsis, and found that the natural 2n pollen rate of triploid Phalaenopsis is 2.39%, and the diploids and tetraploids are 0.59% and 0.67% lower than the triploids, respectively. 2n pollen is less competitive than n pollen, and the probability of producing polyploid offspring is lower under natural conditions, so it is very necessary to increase the ratio of 2n pollen by artificial induction (Zhou Qing et al, 2020 Youinis et al, 2014.

However, in the prior art, the method adopts 2n pollen induction to obtain double plants, and cannot obtain ideal 2n pollen induction rate. In addition, the phalaenopsis has multiple varieties and complex ploidy and genetic backgrounds, and the difficulty is increased for the research of 2n pollen induction.

Therefore, in order to cultivate a new butterfly orchid germplasm with large and bright flowers and high ornamental value, an improved 2n pollen induction method is urgently needed to improve the 2n pollen induction rate of the butterfly orchid. Meanwhile, more varieties of phalaenopsis need to be researched, and a theoretical basis is provided for sexual polyploidization breeding of phalaenopsis.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method for determining the time for inducing phalaenopsis to generate 2n pollen by using colchicine and a method for inducing phalaenopsis to generate 2n pollen by using colchicine.

In a first aspect of the invention, there is provided a method of determining the timing of induction of 2n pollen production by phalaenopsis with colchicine, comprising the steps of:

(a) Natural 2n pollen rate statistics, including: counting the numbers of dyads, tritons and tetrads in mature butterfly orchid pollen and calculating the 2n pollen rate;

(b) 2n pollen induction comprising: selecting pollen of each meiosis stage, and inducing with colchicine, wherein the meiosis stage comprises microsporocyte stage, thin line stage-even line stage, pachytene stage-final transition stage, I reducing metaphase-I reducing telophase and II reducing prophase-II reducing telophase, and the inducing comprises treating flower bud with colchicine solution with concentration of 0.01% -0.1%;

(c) 2n pollen rate statistics after induction, comprising: when the flower buds reach a mature pollen state, collecting the flower buds, counting the numbers of dichotomies, tripartites and tetrads, and calculating the 2n pollen rate; and

(d) Determining the time for inducing 2n pollen: the post-induction 2n pollen rate is compared to the native 2n pollen rate to determine the timing of induction of 2n pollen.

In some embodiments, wherein step (b) comprises: the diameter of the flower buds to be treated is measured and recorded, and then the flower buds are treated (e.g. 3 days) by wrapping the cotton wool soaked with colchicine solution at a concentration of 0.01-0.1%, e.g. 0.01-0.05%. In some embodiments, wherein step (b) further comprises: and judging the meiotic period according to the measured bud diameter.

In some embodiments, said step (d) comprises determining the timing of induction of 2n pollen as thin line-even line, pachytene-terminal line, or metaphase-telase. In some embodiments, step (d) comprises performing statistics and analysis on the obtained data, comprising: analysis of variance and use of LSD multiple comparison method, where the data variation range values are all standard deviations of all the processed data values. Specifically, excel2016 is used for data analysis, SPSS2.0 is used for variance analysis, an LSD multiple comparison method is used, the data variation range value (+/-) in the table is the mean value +/-standard deviation, and when multiple comparisons are carried out on percentages, the percentages are subjected to arcsine conversion and then are analyzed.

In a second aspect, the invention provides a method for inducing phalaenopsis to produce 2n pollen by using colchicine, which comprises the following steps: selecting one of meiotic stages according to the bud diameter based on the correspondence of the bud diameter to the pollen meiotic stage, treating the bud with a colchicine solution thereby inducing 2n pollen production, wherein the meiotic stage comprises: fine line-even line, pachytene-terminal transition or metaphase of minus I-terminal phase. In some embodiments, the colchicine solution has a concentration of 0.01% to 0.1%.

In some embodiments, wherein treating the flower bud with a colchicine solution comprises: immersing absorbent cotton in colchicine solution, and wrapping the absorbent cotton soaked in colchicine solution on flower buds for treatment (such as 3 days), wherein the concentration of the colchicine solution is 0.01% -0.1%, such as 0.01-0.05%.

In some embodiments, the correspondence of bud diameter to meiosis age is established by:

(i) Measuring the bud diameter: measuring and recording the bud diameter;

(ii) Microsporocyte development period observation: (ii) collecting the flower buds of step (i), and performing chromosome tabletting analysis on the pollen therein to determine the development period of microsporocytes; and

(iii) (iii) performing statistics and analysis on the data obtained in steps (i) and (ii) to establish a correspondence between meiosis time periods and bud diameters.

In some embodiments, the 2n pollen induction is performed during thin-even line, pachytene-terminal line, or metaphase-telogen.

In a third aspect of the invention, there is provided a method for inducing moth orchid to produce 2n pollen by using colchicine, which comprises the following steps:

(a) Natural 2n pollen rate statistics, including: observing and/or counting the number of dyads, tritads and tetrads in mature phalaenopsis pollen, and calculating the 2n pollen rate;

(b) Inducing 2n pollen with colchicine, comprising: immersing absorbent cotton in a colchicine solution, and then wrapping the absorbent cotton soaked in the colchicine solution on flower buds for treatment (for example, 3 days), wherein the concentration of the colchicine solution is 0.01-0.1%; and

(c) Post-induction 2n pollen rate statistics, including: when the flower buds reach the mature pollen state, collecting the flower buds, counting the numbers of the dichotomy, the trichotomy and the tetrad, and calculating the 2n pollen rate.

In some embodiments, the method further comprises the step of (d) performing statistics and analysis on the obtained data, including: variance analysis was performed and an LSD multiple comparison method was used, where the data variation range values were the standard deviations of all the processed data values. Specifically, excel2016 is used for data analysis, SPSS2.0 is used for variance analysis, an LSD multiple comparison method is used, the data variation range value (+/-) in the table is the mean value +/-standard deviation, and when multiple comparisons are carried out on percentages, the percentages are subjected to arcsine conversion and then are analyzed.

The fourth aspect of the invention provides a method for inducing phalaenopsis to produce 2n pollen by using colchicine, which comprises the following steps:

(1) Selecting one period of meiosis periods according to the bud diameter based on the corresponding relation between the bud diameter and the pollen meiosis period for 2n pollen induction, wherein the meiosis period comprises a microspore mother cell period, a fine line period-an even line period, a pachytene period-a terminal change period, an I-reducing middle period-an I-reducing end period and an II-reducing prophase-an II-reducing end period; and

(2) Treating the flower buds with a colchicine solution at a concentration of 0.01% to 0.1% during a selected meiotic period thereby inducing 2n pollen production.

In some embodiments, wherein step (1) comprises:

(1a) Measuring the bud diameter: measuring and recording the bud diameter;

(1b) Microsporocyte development period observation: collecting the flower buds of the step (a), and carrying out chromosome tabletting analysis on the pollen in the flower buds to determine the development period of microsporocyte; and

(1c) And (3) counting and analyzing the data obtained in the steps (1 a) and (1 b), so as to establish the corresponding relation between the development period of the microsporocyte and the bud diameter.

In some embodiments, treating the flower bud with a colchicine solution in step (2) comprises: immersing absorbent cotton in colchicine solution, and wrapping the absorbent cotton soaked in colchicine solution on flower buds for 3 days, wherein the concentration of the colchicine solution is 0.01-0.1%, such as 0.01-0.05%.

In some embodiments, the flower buds are treated with colchicine solution when the pollen is in the thin-even line phase, the thick line phase-terminal phase, or the minus I mid-minus I end phase.

In some embodiments of the various aspects of the invention, the concentration of the colchicine solution is 0.01-0.05%, 0.02-0.05%, 0.03-0.05%, 0.04-0.05%, 0.02-0.1%, 0.05-0.1%, 0.03-0.07%, 0.04-0.06% or 0.01%, 0.05%, 0.1%, e.g., 0.05%.

In some embodiments of the various aspects of the present invention, in steps (a) and (c), the 2n pollen rate is calculated using the formula: 2n pollen rate = (2Dy + Tr)/(2Dy +3Tr + 4Te), wherein Dy is the number of dyads, tr is the number of triads, and Te is the number of tetrads.

In some embodiments of the various aspects of the invention, in steps (a) and (c), the 2n pollen rate statistics comprise:

fixing the bud in Carnoy's fixative for 12-24 hr, transferring into 70% ethanol, and storing at 4 deg.C;

washing anther taken out from the preserved flower bud with distilled water, dyeing with modified phenol fuchsin solution for 10-15min, lightly tabletting, rapidly passing through flame for 6-7 times, observing with OLYMPUS DP73 microscope, taking picture,

thereby recording the number of the dichotomies, the trichromatics and the tetrads,

wherein the Carnot's stationary liquid is composed of absolute ethyl alcohol and glacial acetic acid with the volume ratio of 3:1.

In some embodiments of the various aspects of the invention, wherein step (b) comprises: the diameter of the flower buds on the stalks to be treated is measured and recorded, and then the cotton wool soaked with colchicine solution is wrapped on the flower buds for treatment for, for example, 3 days (for example, 6 to 7 stalks per concentration treatment, not less than 4 flower buds per stalk).

In some embodiments of the various aspects of the invention, a flower bud is photographed (e.g., 10 a.m.:00 a.) and the flower bud diameter is measured using Image J Image processing software.

In some embodiments of the various aspects of the invention, step (b) comprises: 2n pollen induction (e.g. treatment of flower buds with colchicine solution for 3 days) is performed when the pollen is in a meiotic stage selected from: microsporocyte stage, fine line stage-even line stage, pachytene stage-terminal change stage, metaphase I-telase I or prophase II-telase II. In some embodiments, the meiotic period is judged according to measured bud diameter. In some embodiments, said step (b) comprises treating the flower bud with colchicine solution for 3 days (i.e., performing 2n pollen induction) when the pollen is in the fine-even phase, the pachytene-terminal phase, or the metaphase-telogen phase.

In some embodiments of various aspects of the invention, the natural 2n pollen rate is about 0.68-1.78%. In some embodiments of various aspects of the invention, the post-induction 2n pollen rate is at least 6.5%, such as at least 10%, such as 10.04%.

In some embodiments of the various aspects of the invention, the Phalaenopsis is a species of Phalaenopsis, such as Phalaenopsis miniata (Phalaenopsis equiquesis), phalaenopsis aenopsis blue-white variety (Phalaenopsis equiquesis var. Coerulea), 'ann' Phalaenopsis (Phalaenopsis Anna-Larati sokardi), 'fennel Purple' Phalaenopsis (Phalaenopsis Tzu Chiang Sapphire.), 'matamantanus' Phalaenopsis (Phalaenopsis Queen Beer 'Red Sky'), and/or 'amethyst' Phalaenopsis (Phalaenopsis pure Crystal).

In some embodiments of the various aspects of the present invention, the variety of phalaenopsis is phalaenopsis miniata, and the corresponding relationship between the microsporocyte phase, the leptosporium phase-the idogenous phase, the pachytene phase-the terminal transition phase, the metaphase phase minus the terminal phase I, the prophase phase minus the terminal phase II and the bud diameter is 0-3.30, 3.30-4.72, 4.72-5.40, 5.40-5.62, 5.62-6.20mm, such as 0-less than 3.30, greater than 3.30-less than 4.72, greater than 4.72-less than 5.40, greater than 5.40-less than 5.62, greater than 5.62-less than 6.20mm, whereby the meiosis phase can be judged on the basis of the measured bud diameter.

In some embodiments of the various aspects of the invention, the butterfly orchid variety is the blue-white variety of butterfly orchid of small orchid, and the correspondence is 0-3.07, 3.07-4.37, 4.37-5.64, 5.64-5.83, 5.83-6.46mm. For example 0 to less than 3.07, greater than 3.07 to less than 4.37, greater than 4.37 to less than 5.64, greater than 5.64 to less than 5.83, greater than 5.83 to less than 6.46mm.

In some embodiments of the various aspects of the invention, the species of phalaenopsis is 'anna' phalaenopsis, and the correspondence is 0-4.06, 4.06-5.16, 5.16-5.54, 5.54-5.90, 5.90-6.57 mm. For example, from 0 to less than 4.06, from greater than 4.06 to less than 5.16, from greater than 5.16 to less than 5.54, from greater than 5.54 to less than 5.90, from greater than 5.90 to less than 6.57mm.

In some embodiments of the various aspects of the invention, the variety of phalaenopsis is phalaenopsis of the species 'fennel purple' and the correspondence is 0-4.21, 4.21-6.53, 6.53-7.36, 7.36-7.60, 7.60-7.89 mm. For example 0 to less than 4.21, more than 4.21 to less than 6.53, more than 6.53 to less than 7.36, more than 7.36 to less than 7.60, more than 7.60 to less than 7.89mm.

In some embodiments of the various aspects of the invention, the variety of phalaenopsis is 'amanita red' phalaenopsis and the correspondence is 0-4.37, 4.37-7.96, 7.96-10.76, 10.76-11.18, 11.18-12.74mm. For example, 0 to less than 4.37, greater than 4.37 to less than 7.96, greater than 7.96 to less than 10.76, greater than 10.76 to less than 11.18, greater than 11.18 to less than 12.74mm.

In some embodiments of various aspects of the invention, the variety of phalaenopsis is 'amethyst' phalaenopsis, and the correspondence is 0-8.24, 8.24-10.20, 10.20-12.00, 12.00-12.84, 12.84-13.55mm. For example from 0 to less than 8.24, from greater than 8.24 to less than 10.20, from greater than 10.20 to less than 12.00, from greater than 12.00 to less than 12.84, from greater than 12.84 to less than 13.55mm. In some embodiments of the various aspects of the invention, the bud diameter is the transverse bud diameter.

In some embodiments of the various aspects of the present invention, when referring to "judging the meiosis period based on the measured bud diameter" or the like, it means that the meiosis period is judged by the diameter of the bud based on the correspondence of the bud diameter to the meiosis period as described herein. In some embodiments, the "meiotic stage" of the present invention refers to a pollen meiotic stage, which is part of the microsporocyte development stage.

In some embodiments of various aspects of the invention, step (a) or (c) comprises:

(a/c 1) randomly taking 3-5 pollen blocks of each butterfly orchid variety, placing the pollen blocks on a glass slide, grinding the pollen blocks by using forceps, randomly counting three visual fields, and repeating for 3 times;

(a/c 2) putting the flower buds into Carnoy's stationary liquid (absolute ethyl alcohol: glacial acetic acid = 3:1) for fixing for 12-24h, then transferring into 70% ethanol, and storing in a refrigerator at 4 ℃ for later use; washing with distilled water for 2-3 times, placing anther on glass slide, mashing with tweezers, removing residue, adding 1 drop of improved phenol fuchsin solution, dyeing for 10-15min, slightly tabletting, rapidly passing through flame for 6-7 times, tapping with pencil with rubber to make chromosome distribution uniform, and observing and taking picture with OLYMPUS DP73 microscope; and

(a/c 3) calculating the 2n pollen rate using the following formula: 2n pollen rate = (2Dy + Tr)/(2Dy +3Tr +4 Te), wherein Dy is the number of dyads, tr is the number of tritads, and Te is the number of tetrads.

In some embodiments of the various aspects of the invention, step (b) comprises:

(b1) Dividing the absorbent cotton into small pieces with equal size and side length of about 2cm, and immersing the small pieces in prepared colchicine solution with concentration of 0.01%, 0.05% or 0.1%;

(b2) Taking a picture at 10:00 a.m. and measuring the diameter of a flower bud on a flower stalk to be treated, wherein the diameter of the flower bud is measured by adopting Image J Image processing software;

(b3) Wrapping the flower buds with absorbent cotton soaked in colchicine solution, and treating for 3 days, wherein each concentration treats 6-7 flower stalks, and each flower stalk is not less than 4 flower buds.

The invention utilizes colchicine to induce 2n pollen of 6 phalaenopsis varieties (species and varieties) such as phalaenopsis miniata, phalaenopsis miniata blue-white variety, 'anna' phalaenopsis, 'pinus purple' phalaenopsis, 'all-weather red' phalaenopsis and 'amethyst' phalaenopsis, and finds that the concentration and the treatment period of the colchicine have great influence on the induction of the 2n pollen of the phalaenopsis miniata variety, particularly the phalaenopsis miniata. And (3) counting the yield of the natural 2n pollen of the 6 phalaenopsis varieties, wherein the 2n pollen rate is about 0.68-1.78%. When the colchicine with the concentration of 0.01 percent and 0.05 percent is used for inducing buds in the I reduction period, the 2n pollen rate which is not lower than 6.5 percent can be obtained; the highest induction rate can reach 10.04 percent by treating the strain with 0.05 percent colchicine for 3 days in a thin line period-even line period.

When the colchicine is used for inducing the 2n pollen of the phalaenopsis, the prior art lacks the relevant discussion on the proper concentration and the treatment period of the colchicine. This is mainly because the colchicine treatment period and concentration are not well controlled, buds wither and fall when the treatment period is too early or the treatment concentration is too high, and the 2n pollen induction rate is low when the treatment time is not suitable or the treatment concentration is low. In the research, by treating the flower buds of the phalaenopsis varieties (such as the phalaenopsis miniata) in different meiosis stages with colchicine in different concentrations, colchicine-induced proper concentration and meiosis stage which are suitable for treatment and can generate more 2n pollen are found, and the 2n pollen induction rate reaches over 10 percent. Therefore, the method provides support for obtaining more butterfly orchid 2n pollen and cultivating new butterfly orchid germplasm with large and bright flowers and high ornamental value, and has wide application prospect in cultivation of new butterfly orchid polyploid varieties.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.

Fig. 1 is a flow chart of a method for inducing moth orchid to produce 2n pollen using colchicine according to an embodiment of the invention.

Fig. 2 is a photograph showing a butterfly orchid flower morphology of small orchid (fig. 2A), a butterfly orchid blue-white variety flower morphology of small orchid (fig. 2B), 'ann' butterfly orchid flower morphology (fig. 2C), 'fennel violet' butterfly orchid flower morphology (fig. 2D), 'sky red' butterfly orchid native flower morphology (fig. 2E), and 'amethyst' butterfly orchid flower morphology (fig. 2F), according to an embodiment of the present invention.

Fig. 3 is a photomicrograph showing natural unreduced male gametophyte development of moth orchid of the small orchid (fig. 3A), natural unreduced male gametophyte development of the bluish-white variety of moth orchid of the small orchid (fig. 3B), 'ann' moth orchid natural unreduced male gametophyte development (fig. 3C), 'fennel purple' moth orchid natural unreduced male gametophyte development (fig. 3D), 'dahurian red' butterfly orchid natural unreduced male gametophyte development (fig. 3E), and 'purple' moth orchid natural unreduced male gametophyte development (fig. 3F), according to an embodiment of the invention.

Fig. 4 is a photomicrograph showing unreduced male gametogenesis of phalaenopsis miniorchid after colchicine treatment (fig. 4A) and before colchicine treatment (fig. 4B), according to embodiments of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Aiming at the problems in the prior art, the invention provides a method for inducing phalaenopsis to generate 2n pollen by colchicine.

Referring to fig. 1, in methods according to some embodiments of the invention, the method of inducing butterfly orchid to produce 2n pollen using colchicine of the invention comprises the following steps:

s101, counting the natural 2n pollen rate of the phalaenopsis: randomly placing 3-5 pollen blocks in a glass slide for each butterfly orchid variety, grinding the pollen blocks by using forceps, randomly counting three visual fields, and repeating the steps for 3 times;

s102, dividing the absorbent cotton into small blocks with the same size and the side length of about 2cm, and immersing the small blocks into a prepared colchicine solution for later use, wherein the concentrations are 0.01%, 0.05% and 0.1% respectively;

s103, colchicine treatment: taking a picture at 10 a.m.:00 a.to record the diameter of the flower buds on the flower stalks to be treated, then wrapping absorbent cotton with colchicine solutions with different concentrations on the flower buds for treatment for 3 days, wherein each concentration treats 6-7 flower stalks, and each flower stalk is not lower than 4 flower buds;

s104, counting the 2n pollen rate after colchicine induction: when the pollen is mature, collecting flower buds, counting the numbers of dichotomies, trichromatics and tetrads, and calculating the 2n pollen rate.

S105, data statistics: excel2016 is adopted for data recording and data analysis, SPSS2.0 is adopted for variance analysis, and an LSD multiple comparison method is adopted, so that data variation range values are standard deviations of all processed data values.

The invention uses colchicine with different concentrations to induce the flower buds of the phalaenopsis at different meiosis stages, thereby obviously improving the 2n pollen rate of the phalaenopsis. The invention discusses the proper concentration and treatment period of the colchicine treated buds and lays a foundation for polyploidy induced cultivation of a new variety of phalaenopsis with large and bright flowers and high ornamental value. The invention is further described with reference to the following examples.

Examples

1. Test materials

Taking the phalaenopsis miniata, the blue-white variety of phalaenopsis miniata, ' Anna ' phalaenopsis, ' fennel purple ' phalaenopsis, ' all-weather red phalaenopsis and ' amethyst ' phalaenopsis as materials, counting the natural 2n pollen rate of the phalaenopsis, and carrying out colchicin induction on 2n pollen by taking the phalaenopsis miniata as a material. The experiments were carried out at the vegetable and flower institute of Chinese academy of agricultural sciences, with all materials planted in the greenhouse. The flower morphology is shown in fig. 2.

2. Data statistics

Excel2016 is adopted for data analysis, SPSS2.0 is adopted for variance analysis, an LSD multiple comparison method is adopted, data variation range values (+/-) in a table are mean values +/-standard deviations, and when multiple comparisons are carried out on percentages, the percentages are subjected to arcsine conversion and then are analyzed.

Example 1: natural 2n pollen yield statistics

The mature butterfly orchid pollen exists in a tetrad mode, the number of bipartites, tritons and tetrads in the mature butterfly orchid pollen is observed and recorded, the natural 2n pollen rate is calculated, 3-5 pollen blocks of each butterfly orchid variety are randomly placed on a glass slide, are crushed by using forceps, randomly count three visual fields and are repeated for 3 times. The 2n pollen proportion = (2Dy + Tr)/(2Dy +3Tr + 4Te), wherein Dy is the number of dyads, tr is the number of triads, and Te is the number of tetrads.

As a result, it was found that, in all five species of phalaenopsis, the presence of dyads and triploids was observed (fig. 3A to D, fig. 3F), except for amanita red (fig. 3E) in which normal pollen could not be produced due to synaptogenesis. The natural 2n pollen rates of five phalaenopsis are counted, and the results are shown in table 1, and the natural 2n pollen rates are found to be remarkably different, and the 2n pollen rates of the five phalaenopsis are different from 0.68% to 1.78%. The natural 2n pollen production rate of 4 diploid varieties of the phalaenopsis miniata, the phalaenopsis miniata variety, the phalaenopsis exigua and the phalaenopsis amethyst is not more than 1%, and the natural 2n pollen rate of the diploid phalaenopsis anna is 1.78%.

Table 1: incidence frequency of unreduced male gametes of butterfly orchid

Note: in the table, "-" indicates data missing, and different lower case letters in the same column indicate significant difference (P < 0.05).

Example 2: inducing 2n pollen with colchicine

The absorbent cotton is divided into small pieces with equal size and side length of about 2cm, and immersed in prepared colchicine solution for use, the concentration is 0.01%, 0.05% and 0.1% respectively. Taking pictures at 10 a.m.:00 a.m., recording the diameter of the flower bud on the flower stalk to be treated, then wrapping absorbent cotton with colchicine solutions with different concentrations on the flower bud for 3 days, treating 6-7 flower stalks with each concentration, wherein each flower stalk is not lower than 4 flower buds, and mainly investigating the influence of meiosis period and colchicine concentration on the 2n pollen induction rate of the phalaenopsis. When the flower buds reached the state of mature pollen, the flower buds were collected, the numbers of dichotomies, trichromates and tetrads were counted in the same manner as in example 1, and the 2n pollen rate was calculated.

And (3) carrying out colchicine treatment on the flower buds of the phalaenopsis miniata in different reduction periods (judged according to the flower bud diameters), collecting pollen when the flower buds grow to a split period, wherein the 2n pollen statistical results are shown in table 2. The proportion of bipartite and tripartite bodies after colchicine treatment (fig. 4A) was significantly increased compared to the untreated (fig. 4B). Significance analysis of 2n pollen induction rate showed that both factors, i.e. meiosis period, colchicine treatment concentration and interaction effect between them, had very significant effect on the 2n pollen induction rate of phalaenopsis (table 3). As can be seen from Table 2, not all combinations of treatments were able to harvest pollen, and some treatments were likely to cause premature bud fall, dry and death due to high concentrations or too tender buds, and no pollen was collected. The 2n pollen rate obtained by the treatment in the microsporocyte period is lower, and higher induction rate can be obtained by the treatment in the earlier stage of I reduction, wherein the 2n pollen rate obtained by the treatment of 0.05 percent colchicine in the thin line period to the even line period for 3 days is the highest and can reach 10.04 percent; when the treatment concentration is different, the 2n pollen induction rate is changed, wherein 0.05 percent of colchicine has better induction effect and can reach 10.04 percent, and when the concentration is 0.1 percent, the 2n pollen induction rate is reduced to some extent and has stronger toxicity to small buds.

TABLE 2 pollen Rate inducing chromosome doubling of butterfly orchid pollen under different treatment conditions

Note: in the table, "-" indicates data missing, and different lower case letters in the same column indicate significant difference (P < 0.05); and the diameters of the flower buds corresponding to the microsporocyte period, the thin line period-even line period, the thick line period-final change period, the I reduction middle period-I reduction end period and the II reduction early period-II reduction end period are respectively 0-3.30, 3.30-4.72, 4.72-5.40, 5.40-5.62 and 5.62-6.20mm.

Table 3 analysis of variance of pollen induction rate of phalaenopsis miniata 2n in different treatment combinations

Therefore, when the 2n pollen is obtained by wrapping flower buds with colchicine absorbent cotton and inducing, factors such as colchicine concentration, meiosis period of microspore mother cells and the like are crucial to whether the 2n pollen is successfully induced and the ideal 2n pollen rate is obtained.

In 2n pollen induction studies of spring sword (Fan Bin, 2012) and dendrobium (Wang Aihua et al, 2017), 2n pollen rates of 2.65% and 6.22% at the highest were obtained respectively by observing the meiotic stage of microsporocytes and carrying out colchicine injection treatment on flower buds at the early stage of I reduction. Reports have shown that the optimal induction period for chromosome doubling of eucalyptus pollen is the two-line phase-terminal transition phase, the mid-phase of subtraction-the terminal phase of subtraction (Yang et al, 2016); the optimal induction period for chromosome doubling of populus alba pollen is the terminal fine line period-pachytene period (Li et al, 2014); the optimal induction period for 2n pollen of Populus sieboldii is in pachytene (Zhou Qing et al, 2020). In this example, the fine-line phase, the adventitious phase, in which meiosis of the microspore mother cells of phalaenopsis miniata occurs, is the most effective phase.

In addition to the meiotic phase, the concentration of colchicine is also important for induction. Reports showed that 1000mg/l (0.1%) of colchicine induced the best effect on young flower buds of Phalaenopsis amabilis (Azmi et al, 2016 a). However, the inventors found that in this example, 0.05% colchicine was used to wrap the flower buds in the filament-couple phase to induce the best effect, and the 2n pollen induction rate was 10.04% at the maximum.

In addition, the injection method is mainly adopted in pollen chromosome doubling induction studies of dendrobium nobile (Wang Aihua and the like, 2017), chun jian (Fan Bin, 2012), poplar (Zhou Qing and the like, 2020). The invention also tries to induce the phalaenopsis by adopting an injection method, but the flower buds basically can not grow normally and wither and fall after 2 to 3 days. The reason may be that the bud of phalaenopsis is tender and not opened, and the cell tissue is injured during injection to cause death.

Example 3: establishing the corresponding relation between the bud diameter of the butterfly orchid and the meiosis period of the pollen

At the end of 5-8 months in 2020, randomly selecting three smaller flower buds for observing the growth change of the flower buds at 10 am: about 00, taking a picture to record the diameter of the flower bud, recording once a day, and continuously recording until the flower bud blooms.

Meanwhile, 6 butterfly orchid buds with different sizes are picked in a sunny day of 10 to 14 percent (10 to 20 buds with different sizes are picked each and repeated for 3 times), the diameters of the buds are recorded by photographing, the buds are fixed in Carnoy fixing solution (absolute ethyl alcohol: glacial acetic acid = 3:1) for 12 to 24 hours and then transferred into 70 percent ethanol to be stored in a refrigerator at the temperature of 4 ℃ for later use. Washing with distilled water for 2-3 times, placing the anther on a glass slide, mashing with tweezers, removing residue, adding 1 drop of modified phenol fuchsin solution, dyeing for 10-15min, gently tabletting, rapidly passing through flame for 6-7 times, tapping with pencil with rubber to make chromosome distribution uniform, and observing and taking picture with OLYMPUS DP73 microscope.

Cytological observations of butterfly flower buds at different microspore development stages revealed a significant correlation between meiotic progression and bud diameter variation (table 4).

TABLE 4 correspondence between developmental stages of microsporocytes of Phalaenopsis and morphological changes outside flower buds

Note: 1: butterfly orchid of small orchid, 2: phalaenopsis glauca white variety, 3: 'Anna' moth orchid, 4: 'fennel purple' moth orchid, 5: 'Mantianhong' moth orchid, 6: phalaenopsis amabilis' amethyst

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.

Reference to the literature

Fan Bin artificial induction of 2n pollen in Chun Jian [ M ]. University of Sichuan agriculture, master academic paper 2012.

Li, guo Qian, wang Jun, tian Jun, kang Xiangyang colchicine induced poplar pollen chromosome doubling and its cytological effect study [ J ]. Nuclear agro-Proc.2014, 28.

Wang Aihua, wu Qingqing, yang Lan, chen Zhilin. Research on the cellular mechanisms of dendrobium officinale 2n pollen induction and its formation [ J ]. North-west plant Proc. 2017,37 (2): 0273-0278.

zhuang Donggong, qu Ying, xu Daxiong, li Jun, chen Zhiling, 2007. Analysis of chromosome number and morphology of several varieties (lines) of moth orchid. Horticulture proceedings, (05): 1257-1262.

zhu Jiao, liu Yiyang, zeng Ruizhen, li Yanghui, guo and Rong, xie Li, yisheng, zhang Zhisheng. 2014 Generation of unreduced male gametes in different ploidy butterfly orchids and their cytological mechanisms were explored. 2132-2138. Zhou Qing, wu Jian, sang Yaru, zhao Zhengyang, liu Meiqin, zhang Pingdong colchicine treatment induced 2n pollen of populus alba and hybrid triploid creation [ J ]. University of Beijing, 2020, 42.

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Claims (4)

1. A method for inducing phalaenopsis to produce 2n pollen by using colchicine comprises the following steps: selecting one of meiotic stages according to the bud diameter based on the correspondence of the bud diameter to the pollen meiotic stage, treating the bud with a colchicine solution to induce 2n pollen production, wherein the meiotic stage comprises a thin line stage-even line stage and a thick line stage-final phase stage, the concentration of the colchicine solution is 0.01% -0.05%, wherein:

the butterfly orchid variety is the butterfly orchid of small orchid ((A), (B), (C) and (C))Phalaenopsis equestris) The corresponding relation between the fine line period, the even line period, the pachytene period, the final change period and the bud diameter is 3.30-4.72 mm and 4.72-5.40mm;

the phalaenopsis variety is the blue-white variety of phalaenopsis miniata (Phalaenopsis equestris var. coerulea) The corresponding relation is 3.07-4.37 mm, 4.37-5.64 mm;

the butterfly orchid variety is 'Anna' butterfly orchidPhalaenopsis Anna-Larati soekardi) The corresponding relation is 4.06-5.16 mm and 5.16-5.54 mm;

the butterfly orchid variety is 'fennel purple' butterfly orchid (A)Phalaenopsis Tzu Chiang Sapphire) The corresponding relation is 4.21-6.53 mm, 6.53-7.36 mm;

the phalaenopsis variety is 'Mantianhong' phalaenopsis (A)Phalaenopsis Queen Beer ‘Red Sky’) The corresponding relation is 4.37-7.96 mm, 7.96-10.76 mm; or alternatively

The phalaenopsis variety is 'amethyst' phalaenopsis (Phalaenopsis Purple Crystal) The corresponding relation is 8.24-10.20 mm, 10.20-12.00 mm.

2. The method of claim 1, wherein treating the flower buds with a colchicine solution comprises: immersing absorbent cotton in a colchicine solution, and then wrapping the absorbent cotton soaked in the colchicine solution on a flower bud for treatment, wherein the concentration of the colchicine solution is 0.03-0.05%.

3. The method of claim 1, wherein the correspondence of bud diameter to meiosis stage is established by:

(i) Measuring the bud diameter: measuring and recording the bud diameter;

(ii) Microsporocyte development period observation: (ii) collecting the flower buds of step (i), and performing chromosome tabletting analysis on the pollen therein to determine the development period of microsporocytes; and

(iii) (iii) performing statistics and analysis on the data obtained in steps (i) and (ii) to establish a correspondence between meiosis time periods and bud diameters.

4. The method of claim 1, wherein the concentration of the colchicine solution is 0.05%.

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