CN114532222B

CN114532222B - Efficient induction method for polyploidy of fagoya grandis

Info

Publication number: CN114532222B
Application number: CN202210203557.XA
Authority: CN
Inventors: 杨虎彪; 冷青云; 李晓霞; 倪成凤; 张瑜; 虞道耿
Original assignee: Tropical Crops Genetic Resources Institute CATAS
Current assignee: Tropical Crops Genetic Resources Institute CATAS
Priority date: 2022-03-03
Filing date: 2022-03-03
Publication date: 2023-01-17
Anticipated expiration: 2042-03-03
Also published as: CN114532222A

Abstract

The invention provides a polyploid high-efficiency induction method of fava-bean, which comprises the steps of soaking white fava-bean seeds obtained by dark culture in colchicine solution added with dimethyl sulfoxide for assisting dissolution for shaking culture, wherein the concentration of colchicine in the colchicine solution is 0-200 mg/L and is not 0, and the culture time is 12-48 h; after the treatment is finished, washing the seeds for 3 to 5 times by using distilled water, and sowing the seeds in the pot; and selecting polyploidy from the obtained seedlings, and inducing to obtain the polyploidy pig dung bean. The invention adopts the germinated pig fagopyrum seed as the polyploid inducer, optimizes the colchicine concentration and the induction time, realizes the good mutation rate and the seedling rate of the pig fagopyrum seeds, has the polyploid induction rate of 13.33 percent at most, improves the seedling quality, has simple operation, low cost and high stability, is convenient for popularization and application, and has good application prospect.

Description

Efficient induction method for polyploidy of faecium guineense

Technical Field

The invention belongs to the technical field of plant polyploid seedling raising, and particularly relates to a method for efficiently inducing polyploid of fava bean.

Background

Canine thistle (Crotalaria pallida) is a plant of the genus Canine thistle of the subfamily Papiloideae of the family Leguminosae, and is an annual shrub-like herb. Widely distributed in africa, america, oceania, tropical and subtropical asia. The plant of the genus Canarium has wide adaptability in southern areas of China, and 19 provinces in the south of Qinling mountains are distributed. The fava has medicinal value and can be used for green manure, and has the feeding functional characteristics of fast growth, high yield, high crude protein content and the like. In a word, the faecium crenata is a plant which has great development and utilization potential as an agricultural resource.

The fava is a typical butterfly flower plant, has two stamens and is self-compatible, has no apomixis phenomenon, and is mainly bred by actively selfing to produce seeds. At present, the breeding of new variety of the Chinese yam fava is mainly based on systematic seed source selection, and artificial interspecific hybridization has fertilization obstacle, so that hybrid seeds cannot be obtained, which limits the breeding process of the new variety to a certain extent.

Polyploid plants often exhibit hypertrophic leaf, enlarged flower, enhanced resistance, etc. The economic value is 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, the utilization of polyploid material as parent is a key technology for overcoming the distant hybridization obstacle between the pig dung and the bean, and the key is how to obtain polyploid germplasm efficiently.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, and provides the efficient induction method for the polyploidy of the fagophora guineensis, which is simple to operate, high in stability, high in induction rate, high in identification efficiency and short in breeding period.

The invention provides a method for efficiently inducing polyploidy of fagophora guineensis, which comprises the following steps:

(1) Seed germination: cleaning and disinfecting the pig dung bean seeds, performing skin breaking treatment, and performing dark culture until the seeds are white; (2) polyploid induction: soaking the white seeds obtained by dark culture in colchicine solution added with dimethyl sulfoxide for assisting dissolution for shaking culture, washing the seeds with distilled water for 3 to 5 times after the treatment is finished, and sowing the seeds in a pot; (3) polyploid identification: and selecting polyploidy from the obtained seedlings, and inducing to obtain the polyploidy pig dung beans.

Wherein the concentration of the colchicine in the colchicine solution in the step (2) is 0-200 mg/L and is not 0, such as 10mg/L, 20mg/L, 30mg/L, 40mg/L, 50mg/L, 60mg/L, 70mg/L, 80mg/L, 100mg/L, 120mg/L, 150mg/L, 180mg/L, 200mg/L and the like.

Wherein the culture time in the step (2) is 12-48 h, such as 12h, 14h, 16h, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 36h, 38h, 40h, 44h, 48h and the like.

Preferably, in the step (2), the concentration of colchicine in the colchicine solution is 50-200 mg/L, and the culture time is 12h; or the concentration of colchicine in colchicine solution is 50mg/L, and the culture time is 12-48 h.

More preferably, in the step (2), the concentration of colchicine in the colchicine solution is 100-200 mg/L, the culture time is 12h, or the concentration of colchicine in the colchicine solution is 50mg/L, the culture time is 24h,

further preferably, in the step (2), the concentration of colchicine in the colchicine solution is 100mg/L, and the culture time is 12h.

Preferably, in the step (2), the concentration of colchicine in the colchicine solution is 50-100 mg/L, the culture time is 12-24 h, or the concentration of colchicine in the colchicine solution is 100-200 mg/L, the culture time is 24h, or the concentration of colchicine in the colchicine solution is 200mg/L, the culture time is 24-48 h, or the concentration of colchicine in the colchicine solution is 50-200 mg/L, and the culture time is 24h.

More preferably, the concentration of colchicine in the colchicine solution is 100mg/L and the cultivation time is 24h.

Preferably, step (1) is specifically: washing seed of the Chinese yam faecium with a detergent, soaking and disinfecting with benzalkonium bromide, washing with sterile water under high temperature sterilization, cutting seed coats with a knife, spreading the seed in a culture box which is fully wetted with sterile water and fully covered with paper towels at certain intervals, and carrying out dark culture in a constant-temperature artificial climate box at 25 ℃ for 24 hours in a dark culture box until the seeds are exposed to the white.

Preferably, the fagoya seeds are collected in the field in 3 months per year and stored on the branches, and the seeds with consistent size and full seeds are selected after being dried in the shade.

Preferably, during the polyploid identification in the step (3), the polyploid is determined as a variant seedling according to the external morphological change of the plant, and the young leaf grown from the variant seedling is taken for polyploid identification.

The second aspect of the invention provides the application of the colchicine solution in the high-efficiency induction of the polyploidy and/or the polyploidy of the fagoya.

Preferably, the application is performed according to the method of the first aspect of the invention.

Compared with the prior art, the invention has the following beneficial effects:

1. the polyploidy induction method of the Chinese red bean is simple and easy to implement, short in treatment time, simple to operate, low in cost, high in stability, convenient to popularize and apply and good in application prospect, and only needs mutation on germinated Chinese red bean seeds without tissue culture, sterile operation and the like.

2. The invention adopts the germinated pig fagoides seeds as the polyploidy inducer, optimizes the colchicine concentration and the induction time, realizes the good mutation rate and seedling rate of the pig fagoides, and improves the seedling quality, wherein the polyploidy induction rate can reach 13.33 percent to the maximum extent.

Drawings

FIG. 1 shows the shapes of the seedlings of the Canine Polianthes Canine treated by the present invention. The following steps: expanding hypocotyls; the method comprises the following steps: thickening the embryonic axis; the following: and (6) comparison.

FIG. 2 is a diagram of flow-based ploidy determination according to the present invention. B1: diploid; b2: tetraploid; b3: diploid/tetraploid hybrids. As can be seen from the DNA content profile of the leaf cells in FIG. 2, the intensity of fluorescence alone shows that a single peak at 6300 appears diploid (FIG. 2B 1); a single peak at 12600 as a tetraploid (fig. 2B 2); at the same time, a single peak of 2 x/4 x polyploid appeared at 6300 and 12600 (fig. 2B 3).

FIG. 3 shows the plant morphology of the processed fagophora suis. Left: diploid; and (3) right: and (4) tetraploid.

FIG. 4 is a drawing showing the identification of chromosomes of faecium Candidum treated by the present invention. D1: diploid; d2: tetraploid.

FIG. 5 shows the form of the leaves of the processed Canine soy beans of the present invention. The method comprises the following steps: diploid; the following: and (4) tetraploid.

FIG. 6 shows the seed morphology of the processed Canton procumbens after seed setting. The following steps: diploid; the following: and (4) tetraploid.

Detailed Description

The invention will be better understood by reference to the following description of specific embodiments with reference to the accompanying drawings. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

A, collecting and treating pig dung bean seeds: collecting the fava seeds lodged on the branches in the field in 3 months each year, and selecting the seeds with consistent size and full seeds for later use after drying in the shade.

B, seed germination: and B, cleaning the seeds selected in the step A by using a detergent, soaking and disinfecting by using benzalkonium bromide, washing by using high-temperature sterilized sterile water, cutting off the seed coats by using a knife, paving the seeds in a culture box which is fully covered with paper towels and fully moistened by using sterile water at certain intervals, and carrying out dark culture in a dark culture box in a constant-temperature artificial climate box at 25 ℃ for 24 hours until the seeds are exposed to the white.

C polyploid induction: dissolving colchicine in 2% dimethyl sulfoxide to obtain colchicine solution with concentration of 50mg/L, 100mg/L, and 200 mg/L. And B, respectively soaking the exposed white seeds obtained by dark culture in the step B in prepared colchicine solutions with different concentrations for shaking culture for different times (12 h, 24h and 48 h), wherein the temperature of a shaking table is 25 ℃, and the rotating speed is 150r/min. Each treatment was 3 replicates, 25 seeds per replicate. After the treatment is finished, washing the seeds for 3 to 5 times by using distilled water, and sowing the seeds in the pot.

D, variant seedling statistics: and D, after the seedlings treated in the step C grow for 14d, according to the external morphological change of the plants: the hypocotyl is expanded, and the whole hypocotyl is thickened, and the plant is short and small, etc. to be determined as a variant seedling.

E, polyploid identification: and (3) cracking young leaves grown from the variant seedlings obtained in the step D after 60D of culture by using a WPB (WPB) buffer solution, then carrying out flow cytometry ploidy detection, and further carrying out chromosome slide observation and determination on the suspected polyploid plants detected by the flow cytometry (figure 2).

(1) Influence on the radicle and hypocotyl of herba Cancriniae Collinae

The influence of different colchicine concentrations and treatment time on the length of the radicle of the faecium creosotum is larger. As shown in Table 1, when the treatment time is the same, the length of the radicle of the faecium guineense is gradually shortened along with the increase of the colchicine concentration, and the treatment concentrations are all obviously different (P is less than 0.05) except that no obvious difference is generated between 12h,50mg/L and a control (P is more than 0.05), namely the radicle growth is inhibited to different degrees along with the increase of the colchicine concentration; when the treatment concentration is the same, the radicle length of the faecium creosotum is also in a tendency of shortening along with the extension of the treatment time, the difference between the treatment times is obvious when the concentration is 50mg/L and 100mg/L (P is less than 0.05), the radicle length is the shortest when the concentration is 200mg/L, and the radicle length is obviously less than 12h (P is less than 0.05) when the concentration is 24h and 48h. The length of the radicle is subjected to variance analysis (table 2), the colchicine concentration has obvious influence on the growth of the radicle (P is less than 0.01), the treatment time has obvious influence on the growth of the radicle (P is less than 0.01), and the interaction of the two factors has obvious influence on the radicle (P is less than 0.01).

Colchicine concentration has an effect on the length of the hypocotyls, as shown in Table 1, the length of the hypocotyls in the colchicine-treated group was significantly lower than that in the control group (P < 0.05). When the treatment concentrations are the same, the embryonic axis lengths of the treatment groups of 24h and 48h are obviously lower than 12h (P < 0.05) when the treatment concentrations are 50 and 200mg/L, and the embryonic axis lengths of the treatment groups of 48h are obviously lower than 12h (P < 0.05) when the treatment concentrations are 100 mg/L. Analysis of variance was performed on the length of the hypocotyl (Table 3), the effect of colchicine concentration on the length of the hypocotyl field was very significant (P < 0.01), the effect of treatment time on the length of the radicle was significant (P < 0.05), and the effect of interaction of the two factors was not significant (P > 0.05).

Colchicine concentration has certain influence on the diameter of the hypocotyl, as shown in Table 1, when the time is the same, the diameter of the hypocotyl of each treatment concentration is obviously lower than that of a control group (P is less than 0.05), and the difference between each treatment group is not obvious (P is more than 0.05).

When the treatment concentrations are the same, the hypocotyl diameter is significantly lower than 48h (P < 0.05) at each treatment concentration at 12h and 24h, and the difference between the treatment concentrations at 12h and 48h is not significant (P > 0.05). Variance analysis is carried out on the diameter of the hypocotyl (Table 4), the effect of colchicine concentration on the diameter of the hypocotyl is very obvious (P is less than 0.01), the effect of treatment time on the diameter of the hypocotyl is very obvious (P is less than 0.01), and the effect of the interaction of colchicine concentration and time on the diameter of the hypocotyl is not obvious (P is more than 0.01).

TABLE 1 Effect of different concentrations of colchicine and treatment time on radicle and hypocotyl

Note: different capital letters in the same row indicate significant differences between colchicine concentrations, and different lower case letters in the same column indicate significant differences between treatment times.

TABLE 2 two-way ANOVA of the Effect of different concentrations of colchicine and treatment time on radicle Length

Sources of variation	Sum of squares	Degree of freedom	Mean square	F value	Significance P value
						Colchicine concentration	5870.191	3	1956.73	10716.03 ^**	＜0.01
Time of treatment	370.894	2	185.447	1015.601 ^**	＜0.01
						Concentration x time	549.032	6	91.505	501.129 ^**	＜0.01

Note: * Indicates a very significant difference P <0.01.

TABLE 3 two-way ANOVA of the Effect of different concentrations of colchicine and treatment time on the length of the embryonic Axis

Sources of variation	Sum of squares	Degree of freedom	Mean square	F value	Significance P value
						Colchicine concentration	10991.88	3	3663.96	5868.789 ^**	＜0.01
Time of treatment	4.475	2	2.238	3.584 ^*	0.043
						Concentration x time	4.764	6	0.794	1.272	0.307

Note: * Indicates a significant difference P < 0.05, indicates a very significant difference P <0.01.

TABLE 4 two-way ANOVA of the Effect of different concentrations of colchicine and treatment time on the diameter of the hypocotyl

Source of variation	Sum of squares	Degree of freedom	Mean square	F value	Significance ofP value
						Colchicine concentration	5.541	3	1.847	547.674 ^**	＜0.01
Time of treatment	0.111	2	0.055	16.386 ^**	＜0.01
						Concentration x time	0.034	6	0.006	1.681	0.169

Note: * Indicates that the difference is very significant P <0.01.

(2) Mutagenesis effect on pig faecium seed

The mutagenic effect of colchicine on the fagophora guianensis seeds at different concentrations and treatment times is shown in table 5. Treating the seeds for 12 hours by using a colchicine solution with the concentration of 100mg/L, wherein the tetraploid induction rate is 13.33 percent; seed is treated by colchicine solution with concentration of 50mg/L for 24h, and tetraploid inductivity is 8%. The two colchicine concentrations and time are combined to treat the white-spotted crotalaria seed to obtain higher inductivity, the combined inductivity is highest by treating 100mg/L +12h, and the effect is optimal.

TABLE 5 mutagenesis Effect of colchicine at different concentrations and treatment times on the seed of Canine

The seedling rate = seedling number/treated seed number × 100%;

tetraploid induction rate = tetraploid seedling number/treated seed number × 100%;

the induction rate of the polyploid =2 ×/4 × number of polyploid seedlings/number of treated seeds × 100%;

(3) The characteristic of the processed Chinese yam

The characteristic of the seedling of the Chinese limnoperna herb after being treated by the method is shown in figure 1, and the root system and the hypocotyl of the variation seedling of the Chinese limnoperna herb are obviously changed after being treated by colchicine. The method is divided into two types according to different variation characteristics. Type 1: compared with the control, the same point is that the plant height has no obvious difference and true leaves emerge, and the difference is that the hypocotyl of the variant is expanded and the radicle is shorter. Type 2: the plant growth is obviously inhibited, the whole embryonic axis is shortened and thickened, the growth is slow, no true leaf comes out, and the radicle is obviously short. Statistically, treatment combinations 12h,50mg/L and 12h,100mg/L belong to type 1, and the remaining treatment combinations are represented as type 2.

The plant morphology of the processed faecium swingle is shown in figure 3, and compared with a diploid plant, the tetraploid plant is short and strong, short and thick in internode and short and round in leaf and thick. The chromosome number of the root tip cell of the diploid plant was 2n =2 =16 and the chromosome number of the tetraploid root tip cell was 2n =4 =32 as observed by the chromosome preparation of the root tip (fig. 4).

The form of the leaves of the processed Chinese feverfew is shown in figure 5. The tetraploid and diploid plants have obvious difference in leaf morphology, the tetraploid leaves are short and round and are thick (figure 5), the leaf morphology characteristics are shown in a table 6 result, and the length, width, length-width ratio, petiole length and petiole width contrast difference of the diploid faecium pinnatum and the tetraploid central lobular leaves is obvious. The length, width and length-width ratio of the tetraploid central lobular leaf and the length and width of the three-complex petiole are respectively 0.94 times, 1.10 times, 0.84 times, 0.91 times and 1.2 times of that of the diploid.

TABLE 6 comparison of morphological characteristics of diploid and tetraploid horsebean leaves

The shapes of the seeds formed by the processed Chinese crinis bilineata seeds are shown in figure 6, and compared with diploid Chinese crinis bilineata seeds (figure 6, table 7), the area, the grain length and the grain width of the tetraploid Chinese crinis bilineata seeds are obviously increased. The tetraploid has 1.372g of hundred miles weight, which is 0.429g more than that of the diploid seeds.

TABLE 7 comparison of phenotypic characteristics of diploid and tetraploid limnoperna seed

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions for the present invention are within the scope of the present invention for those skilled in the art. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

1. The efficient induction method for the polyploidy of the fava bean is characterized by comprising the following steps:

(1) Seed germination: washing and disinfecting the faecium seed, then carrying out skin breaking treatment, and carrying out dark culture until the seed is white;

(2) Polyploid induction: soaking the white exposed seeds obtained by dark culture in colchicine solution added with dimethyl sulfoxide for assisting dissolution for shaking culture, wherein the concentration of colchicine in the colchicine solution is 100mg/L for 12h; or the concentration of colchicine in colchicine solution is 50mg/L, and the culture time is 24h; after the treatment is finished, washing the seeds for 3 to 5 times by using distilled water, and sowing the seeds in a pot;

(3) Polyploid identification: and selecting polyploidy from the obtained seedlings, and inducing to obtain the polyploidy pig dung beans.

2. The efficient induction method of the polyploidy of faecium guineense according to claim 1, wherein the step (1) is specifically as follows: washing seed pig dung beans with a detergent, soaking and disinfecting with benzalkonium bromide, washing with sterile water under high temperature sterilization, cutting off seed coats with a small knife, spreading the seed dung in a culture box which is fully wetted with sterile water and is fully covered with paper towels at certain intervals, and carrying out dark culture in a dark culture box in a constant-temperature artificial climate box at the temperature of 25 ℃ for 24 hours until the seeds are exposed to be white.

3. The efficient induction method of polyploidy of Canine thara herb, as claimed in claim 1, wherein Canine herb seeds are selected from the Canine herb seeds which are collected in the field and lodged on the branches at 3 months per year, and the seeds with consistent size and plump seeds are selected after drying in the shade.

4. The efficient induction method of polyploidy of fava guianensis according to claim 1, wherein in the step (3) of polyploidy identification, a variant seedling is determined according to the external morphological change of a plant, and young leaves grown from the variant seedling are taken for polyploidy identification.

5. Use of colchicine solution for the efficient induction of polyploids and/or polyploids of fagoya according to the method of any one of claims 1 to 4.