CN112640779B - Method for creating watermelon mutant by using EMS mutagenesis - Google Patents

Abstract

The invention belongs to the technical field of watermelon breeding, and particularly relates to a method for creating watermelon mutants by using EMS mutagenesis. The method comprises the steps of collecting fully opened male anthers, fully stirring the anthers and EMS-paraffin oil solution to obtain EMS-treated pollen, dipping the pollen to pollinate female flowers opened on the day, uniformly spraying EMS pollen to pollinate female flower ovaries with the Tigualing with proper concentration, harvesting and planting seeds of M0 generations, and obtaining EMS mutant individuals of watermelons with rich M1 generations of variation. The invention can obtain various mutants such as male sterility, dwarfing, tetraploid, leaf deformity and the like, and effectively solves the technical bottlenecks of narrow genetic background, less genetic variation, deficient excellent seeds, low germplasm creating efficiency and the like in the research on functional genes of watermelons and the breeding of varieties, thereby being used for constructing a watermelon mutant library, facilitating the research on the functional genes, greatly promoting the excavation and application of the excellent genes of the watermelons and providing material support for the breeding of excellent new varieties.

Description

Method for creating watermelon mutant by using EMS mutagenesis

Technical Field

The invention relates to the technical field of watermelon breeding, in particular to a method for generating watermelon mutants by using EMS mutagenesis, which can create the watermelon mutants with rich variation.

Background

Watermelon (Citrullus lanatus) belongs to the cucurbitaceae watermelon, is an important economic crop worldwide and accounts for about 7 percent of the vegetable production area in the world. According to the annual data of Chinese agricultural statistics, the seeding area of watermelons in China in 2016 reaches 189.08 ten thousand hectares, the yield is 7940 ten thousand tons, which accounts for about 70% of the world production of watermelons, and the production of watermelons plays an important role in horticultural crops.

The watermelon industry in China generally has the problem of variety homogenization, the phenomenon of homogeneous and heterogeneous is prominent, and the root cause is that the watermelon genetic background is narrow, the available effective variation is less, the research of watermelon functional genes and the selective breeding of innovative varieties are not in the future, so the induction of genetic variation is one of the important ways for developing the research of watermelon functional genes and creating new germplasm.

Ethyl Methane Sulfonate (EMS) is one of the most widely used chemical mutagens, and EMS mutagenesis has the advantages of convenient operation, high mutation frequency, little chromosome aberration and adverse variation and the like, is mostly dominant mutation, and is easy to screen. At present, EMS mutagenesis is proved to be an effective means for creating variation, is widely applied to crop breeding, and plays an important role in the construction of a crop mutant library, basic research of reverse genetics and the like.

At present, the mutant is created by adopting an EMS (energy management system) seed treatment method, and available mutants are successfully created on crops such as rice (CN105695477A), wheat (CN109729972A), peanut (CN111670806A), rape (CN110578015A) and the like, but the induction efficiency is generally low, and the mutant phenotype can be observed only in M2 generation, so that the cost for processing the seed creation mutant by using the EMS is high, and the mutant is particularly obvious for cucurbit crops with low planting density. Many researchers put a lot of manpower, material resources and financial resources on important cucurbits such as cucumber and watermelon, and use EMS to treat seeds to create mutants, such as 1.5% EMS to treat cucumber seeds by CHEN Chen, and a series of mutants of dwarfing, yellow leaves, pericarp yellowing and leaf shrinkage are obtained in M2 (CHEN Chen et al (2018) An EMS mutant for cumber, Journal of Integrated Agriculture,17(7): 1612-1619); 1.0% of EMS is utilized to treat watermelon seeds in Houyan, a mutant library of 40 phenotypic variations is constructed (the construction and the phenotypic analysis of the mutant library of the watermelon are induced by EMS in Houyan et al (2016), the study on northwest plant reports, 12, 2411-; mutant materials with important characters such as male sterility, pulp color, fruit type and the like are not obtained, so that no report of further gene mining is carried out by other people, and the obtained cucumber and watermelon mutant library is low in efficiency or useless.

The method for creating the mutant by using EMS to treat pollen is a method for applying the mutant on the corn to be mature. Many researchers have tried on other crops due to the ability to rapidly create dominant mutants, but there has been no case of successful application, on one hand, because most of the crop pollen is not easy to collect; on the other hand, the paraffin oil does not cause damage to the filaments of the female flowers of the corn and can fruit normally, and the paraffin oil can seriously damage the female flowers of crops such as watermelon, cucumber and the like, so that fruit set is not caused or the fruit set rate is extremely low. Therefore, a new EMS mutagenesis method is needed to be explored on watermelon crops, on one hand, a mutant germplasm resource with economic value is created, and on the other hand, a large number of mutants needed for researching functional genes of the watermelon crops are provided.

Disclosure of Invention

In order to solve the problems, the invention provides a method for producing watermelon mutants by using EMS mutagenesis, which comprises the following steps:

(1) collecting male flowers and picking up anthers by using tweezers after the male flowers are fully opened in the morning on the pollination day;

(2) putting the anther picked in the step 1 into 0.1% EMS-paraffin oil solution, filtering the anther by a screen to obtain EMS pollen mixed treatment liquid, and then stirring the EMS pollen mixed treatment liquid on a magnetic stirrer for 30-50 min;

(3) standing or centrifuging the EMS pollen mixed treatment solution obtained in the step 2 for 10-20min, pouring out the supernatant, and filtering the pollen precipitate solution by using filter paper to obtain the EMS treated pollen;

(4) dipping the pollen in step 3 into the female flowers which open on the day, pollinating by using an inoculation pen or a fine hair pen, wherein the using time of the pollen treated by EMS each time is not more than 30min, retreating fresh pollen after 30min, and marking the pollinated female flowers by using marks;

(5) uniformly spraying the pollinated female ovary in the step 4 with the "Tiaoguanling" (such as forchlorfenuron, forchlorfenuron and the like) 16 pm on the day;

(6) collecting M0 generation seeds after the fruits are ripe;

(7) and (4) sowing the seeds of the M0 generation harvested in the step 6, observing and investigating the phenotype of the M1 generation mutant in the whole growth period, and counting the proportion of each mutant phenotype in the population to obtain the watermelon EMS mutant population with rich variation.

Furthermore, the method can be implemented under the condition that the watermelon can normally bear fruit.

Further, the male flowers and the female flowers are both from the watermelon high-generation inbred line 19Wa 00032.

Further, the EMS-paraffin oil solution in the step 2 is preferably stirred for 40 min.

Further, the EMS pollen mixed treatment solution in the step 3 needs to be stood for 20min or centrifuged at 500rpm for 10min, supernatant is discarded, and the pollen precipitate is filtered again by using filter paper if necessary to obtain the EMS treated pollen;

further, in the step 4, pollen treated by EMS is dipped by an inoculation pen or a fine hair pen for pollination, the service time of the pollen treated by EMS is not more than 30min, and the pollen treated by EMS is required to be prepared again by fresh pollen after 30 min;

further, in step 5, the female ovary of the pollinated pollen treated by EMS is uniformly sprayed with 0.1 percent of forchlorfenuron 150-250 times liquid or other trichosanthes kirilowii (forchlorfenuron and the like) with proper concentration in the afternoon.

Further, the phenotype of the mutant in step 7 includes male sterility, dwarfing, tetraploidy, plant type variation, leaf greening, fruit shape variation, pericarp band loss or variation, leaf malformation, etc.

The invention achieves the following beneficial effects:

(1) by utilizing the method for EMS treatment of watermelon pollen provided by the invention, the defect that EMS damages female flowers of watermelon is overcome, the influence of EMS on fruit setting of watermelon is reduced, and the mutagenesis efficiency is improved;

(2) by utilizing the method for EMS treatment of watermelon pollen, a large number of dominant mutant individuals with consistent mutant genetic background can be obtained in M1 generations, the time is short, the efficiency is high, the inheritance can be stabilized, and the method can be used for rapidly creating dominant genetic mutation, genetic analysis of important characters, clone research of mutant genes and application in the field of genetic breeding;

(3) the method provided by the invention can be implemented under the condition that the watermelon can normally bear fruit, and is not influenced by seasons and facility conditions;

(4) the method provided by the invention can be used for obtaining a large number of mutants with research and application values, and the properties of the mutants include but are not limited to male sterility, dwarfing, tetraploid, plant type variation, green leaves, fruit shape variation, peel strip loss or variation, leaf deformity and other dominant properties, so that the large-scale watermelon germplasm innovation can be favorably carried out, the method can be used for constructing a watermelon mutant library, the creation cost is lower, and the method has better applicability.

Drawings

FIG. 1 is a comparison of "male flower abortion" male sterile mutants with wild type, where A is the plant comparison of wild type (left) and mutant (right), B is the leaf comparison of wild type (left) and mutant (right), and C is the melon male flower comparison of wild type (left) and mutant (right).

FIG. 2 is a comparison of "pollen-aborted" male sterile mutants with wild type, where A is the comparison of plants from wild type (left) to mutant (right) and B is the comparison of male flowers from wild type (left) to mutant (right).

FIG. 3 is a graph comparing plant dwarfing mutants with wild type, where A is the comparison of plants with wild type (left) and mutant (right) and B is the comparison of melon vine and internode between wild type (left) and mutant (right).

FIG. 4 is a graph comparing tetraploid mutants with wild type, where A is the plant comparison of wild type (left) and mutant (right), B is the leaf comparison of wild type (left) and mutant (right), C is the unopened male flower comparison of wild type (left) and mutant (right), and D is the opened male flower comparison of wild type (left) and mutant (right).

FIG. 5 is a comparison of leaf malformed mutants with wild type, wherein A is the comparison of plants with wild type (left) and mutant (right), B is the comparison of leaves with wild type (left) and mutant (right), and C is the comparison of fruit set with wild type (left) and mutant (right).

FIG. 6 is a plot comparing the post-leaf green mutant to wild type plants, where A is the comparison of wild type (left) to mutant (right) plants and B is the comparison of wild type (left) to mutant (right) leaves.

FIG. 7 is a graph comparing plant type mutants with wild type, wherein A is the plant comparison of wild type (left) and mutant (right), B is the leaf comparison of wild type (left) and mutant (right), and C is the local plant status of mutant.

FIG. 8 is a graph comparing the appearance of the mutant compared to the wild type, where the left is the fruit of the wild type and the right is the fruit of the mutant.

FIG. 9 is a graph comparing the leaf malformation mutants M1 and M2, wherein A is the wild type (left) and mutant (right) plants of generation M1 and B is the wild type (left) and mutant (right) plants of generation M2.

Detailed Description

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the present invention is described in detail below with reference to specific embodiments, but these embodiments are only exemplary and do not limit the scope of the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are intended to be within the scope of the invention.

The test methods in the following examples, which are not specified in specific conditions, are generally conducted under conventional conditions, and the steps thereof will not be described in detail since they do not relate to the invention.

In the following examples, the experimental material is watermelon inbred line 19Wa00032, the weight of a single watermelon of the inbred line is about 2.5kg, the bottom color of the peel is dark green, and is covered with dark green sawtooth strips, the peel has large red flesh, and the content of soluble solids in the center of the fruit is about 11%. EMS (Ethyl methanesulfonate) and paraffin oil were purchased from Sigma.

Example 1 creation of watermelon mutants by EMS mutagenesis

The invention provides a method for creating watermelon mutants by using EMS mutagenesis, which comprises the following steps:

1) preparation of EMS-paraffin oil mother liquor

Under the premise of taking protective measures (such as experimental clothes, goggles, masks, double-layer gloves and the like), 2ml of EMS stock solution is sucked in a fume hood and added into 198ml of paraffin oil to prepare 1% EMS mutagen mother solution, the EMS mutagen mother solution is stirred on a magnetic stirrer overnight, the mother solution is stored in a glass bottle with a plug, and aluminum foil paper is wrapped outside the glass bottle for standby. EMS-paraffin oil mother liquor is prepared in advance and can be stored for 1 to 2 years.

2) Preparation of female flowers

Normally managing until more than 30% of watermelon plants are about to open for the first 1 day (at the moment, the phenotype of the female flowers is full and bright yellow), selecting single plants with good growth vigor and normal development of the female flowers, and covering the female flowers with paper caps folded by newspaper or similar paper to prevent the pollination of insects; in the facilities (such as a greenhouse or a net room) for isolating insects, no paper caps are needed.

3) EMS-preparation of paraffin oil mother liquor

And (3) stirring the EMS-paraffin oil mother liquor prepared in advance in the step (1) in a fume hood for more than 1h by using a magnetic stirrer, wherein the early morning of pollination is about 6: 30.

4) Preparation and concentration of EMS treatment solution

On the premise of making protective measures (such as experimental clothes, goggles, masks, double-layer gloves and the like), in the morning of pollination, about 7:30, in a fume hood, the EMS-paraffin oil mother liquor fully stirred in the step 3 is absorbed and added into proper amount of paraffin oil to prepare EMS treatment liquid with the volume ratio of 0.1%, and each 20ml of EMS treatment liquid is subpackaged in conical bottles with plugs.

5) Anther collection

According to the weather condition, fully opened fresh male flowers are collected in the morning on the day of pollination so as to ensure enough strong pollen activity, and the anthers of the male flowers are picked up by tweezers and placed on absorbent paper to be kept dry.

6) Pollen EMS mutagenesis treatment

And (3) putting the anther in the step (5) into EMS treatment liquid with the volume ratio of 0.1% on the premise of taking protective measures (such as experimental clothes, goggles, masks, double-layer gloves and the like), and filtering the anther by using a screen after the pollen is fully stirred and eluted in the EMS treatment liquid, so as to obtain the EMS pollen mixed treatment liquid with the pollen fully dispersed in the EMS-paraffin oil solution to the maximum extent. The obtained EMS pollen mixed treatment liquid is placed on a magnetic stirrer to be stirred for 30-50min, so that the full effect of the pollen and the EMS is ensured.

7) Obtaining pollinated pollen

And (3) on the premise of taking protective measures (such as experiment clothes, goggles, masks, double-layer gloves and the like), standing the EMS pollen mixed treatment solution fully stirred in the step (6) for 20min or centrifuging the mixed treatment solution at 500rpm for 10min to precipitate the pollen, removing the supernatant, and filtering the pollen precipitation solution by using filter paper to remove paraffin oil to the maximum extent, and only keeping the pollen treated by the EMS to obtain the pollen treated by the EMS.

8) Pollination is carried out

On the premise of taking protective measures (such as experimental clothes, goggles, masks, double-layer gloves and the like), around 9 am, dipping the pollen in the step 7 by using an inoculation pen or a fine hair pen, pollinating female flowers which are open on the day, wherein the pollination time of the pollen does not exceed 30min each time, and retreating by using fresh pollen after 30 min; one group of 2 persons, 1 person responsible for pollination, and the other 1 person responsible for capping and marking with a marker (only marking under insect isolation conditions).

EMS treatment pollen pollination tests are carried out in spring and autumn, and the fruit setting rates in different seasons are shown in table 1. It can be seen from table 1 that the fruit setting rate in spring is significantly higher than that in autumn, while the fruit setting rates of the wild-type control material 19Wa00032 are all 100%. The fruit setting rate is influenced by various conditions such as weather, temperature, humidity and the like, the inventor respectively carries out experiments in spring and autumn, continuous treatment is carried out each time, statistics is carried out for 15 days, and the fruit setting rate in spring obtained after analysis is only 4.38% at the lowest, reaches 49.24% at the highest and is 9.74% on average. And the average fruit setting rate in autumn is lower and is only 5.38 percent.

TABLE 1 EMS treatment of pollen pollination fruit setting rate

Season of treatment	Pollination number (number)	Fruit set number (number)	Percentage of fertile fruit (%)
				Spring season	4180	407	9.74
Autumn	3623	195	5.38

9) Treatment of Tibet melon

Because the vitality of the pollen after EMS-paraffin oil treatment is reduced, and the paraffin oil damages female flowers, the fruit setting is not easy, and the fruit setting rate is extremely low, the fruit setting rate and the efficiency are improved by adopting the fruit setting agent. On the premise of taking protective measures (experimental clothes, goggles, masks, double-layer gloves and the like), a pollinated female flower ovary is uniformly sprayed with 0.1 percent of forchlorfenuron 150-fold liquid or other types of custard (forchlorfenuron and the like) with proper concentration after 16 pm of the day, and fruit setting is promoted.

The invention carries out the experiments of improving the fruit setting rate of the fruit setting by using the Guaping treatment in autumn, the detailed results are shown in the table 2, and the fruit setting rate of the fruit setting after using the Guaping treatment is obviously improved from the table 2, and the average fruit setting rate is up to 28.52%.

TABLE 2 treatment effect of Tibet Guaping

Treatment of	Number of pollinations(A)	Fruit set number (number)	Percentage of fertile fruit (%)
				Sizhiling spray for fruit of Cucurbita moschata	270	77	28.52
Control	254	44	17.32

10) Harvesting of fruits

And after the fruits are ripe, collecting M0 generation seeds.

11) M1 generation mutant phenotype survey

Sowing seeds of M0 generations harvested in the step 10, sowing 6600 plants and 6126 plants in total, observing and investigating phenotypes of M1 generation mutants by taking an original material 19Wa00032 as a control in the whole watermelon growth period, counting the proportion of each mutant phenotype in the population to obtain a watermelon EMS mutant population with abundant variation, and counting the mutant phenotypes, wherein the statistical result is shown in Table 3, and the mutation percentage is 1.18%. The obtained mutant has characteristics of male sterility, plant dwarfing, tetraploid, leaf malformation, premature senility, fruit shape lengthening, pericarp strip variation and the like, wherein the characteristic comparison graphs of typical mutants are shown in figures 1-8.

TABLE 3 mutant phenotype statistics (type, number of plants, percentage)

Phenotypic variationModel (III)	Number of plants	Percent (%)
			Male sterility	6	0.10
Dwarfing of plants	1	0.02
			Tetraploid	3	0.05
Deformity of the blade	4	0.07
			Variation of plant type	15	0.24
Plant premature senility	3	0.05
			Rear green of leaf	1	0.02
Variation of leaves	15	0.24
			The fruit shape is lengthened	3	0.05
Loss or greening of pericarp bands	10	0.16
			No growing point	5	0.08
The peel has green bottom color	4	0.07
			Variation of fruit peel	2	0.03
In total	72	1.18

12) Genetic analysis of leaf curl mutant M2 generation

Selfing the mutant single plants screened in the step 11, and respectively collecting seeds. A leaf curl mutant single strain Me52 was selected for genetic analysis of M2 generation to clearly obtain the genetic stability of the mutant. Randomly taking 72 seeds (M2 generation) of the Me52 mutant single plant selfing seeds, soaking the seeds in a 5% sodium hypochlorite solution for 10min, washing the seeds clean by running water, horizontally placing the seeds in a culture dish paved with two layers of absorbent paper, placing the culture dish in a constant-temperature incubator at 30-32 ℃ for accelerating germination, and sowing the seeds in a 72-hole tray after the seeds are exposed to white. The seedling culture medium is peat: vermiculite: perlite (3:1:1), growth chamber temperature is kept at 26 ℃/16 ℃ (day/night), humidity is 60% or so. The phenotype of the plant is observed in the 1-leaf stage of 5 leaves (FIG. 9), the phenotype and genetic analysis results are shown in Table 4, and the rolling is carried out in the M2 generation plant56 leaves, 16 normal leaves, chi-square test (X)²Less than 3.84) shows that the leaf curl character of the mutant individual strain Me52 can be stably inherited and conforms to a single dominant gene inheritance model.

TABLE 4 leaf malformation mutant M2 representative segregation ratio and genetic analysis

The method for performing EMS mutagenesis on pollen provided by the invention is also suitable for other cucurbit crops such as melons, cucumbers, pumpkins and the like, and can obtain a dominant mutant with rich variation in the M1 generation.

Claims (6)

1. A method for creating watermelon mutants by using EMS mutagenesis is characterized by comprising the following steps:

(1) collecting male flowers and picking up anthers by using tweezers after the male flowers are fully opened in the morning on the pollination day;

(2) putting the anther picked in the step 1 into EMS-paraffin oil solution for stirring, filtering to remove the anther, obtaining EMS pollen mixed treatment liquid, and then fully stirring the mixed treatment liquid, wherein the EMS-paraffin oil solution is EMS solution with the volume concentration of 0.1% prepared by using paraffin oil, and the stirring time is 30-50 minutes;

(3) standing or centrifuging the EMS pollen mixed treatment solution obtained in the step 2 for 10-20 minutes, pouring out the supernatant, and filtering the pollen precipitate to obtain the EMS treated pollen;

(4) dipping the female flowers which are pollinated by pollen in the step 3 and open on the same day, wherein the using time of the pollen treated by EMS each time is not more than 30 minutes, and after 30 minutes, the step 2-3 needs to be repeated;

(5) uniformly spraying the pollinated female ovary in the step 4 with the Tibet Guaping in the afternoon of the same day, wherein the effective component of the Tibet Guaping is forchlorfenuron, and the concentration of the forchlorfenuron is 150 times of forchlorfenuron solution with the concentration of 0.1 percent;

(6) after the fruits are ripe, harvesting M0 generation seeds in a mixing manner;

(7) and (4) sowing seeds of M0 generations collected in the step 6, and observing and investigating the phenotype of the M1 generation mutant in the whole growth period to obtain the watermelon EMS mutant population.

2. The method of claim 1, wherein the sufficient agitation of the mixed process fluid is by use of a magnetic stirrer.

3. The method according to claim 1, wherein the filtering to remove anthers in step 2 is performed using a mesh.

4. The method as claimed in claim 1, wherein the filtering method of the pollen precipitate in step 3 is to use filter paper.

5. The method of claim 1, wherein the dipping in step 4 is dipping using an inoculating pen.

6. The method of claim 5, wherein the inoculation pen comprises a fine writing brush.

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