阅读说明：本技术 一种基于萱草花粉管通道导入ems诱变育种的方法 (Method for introducing EMS mutation breeding based on day lily pollen tube channel ) 是由 谭雨馨 倪迪安 张铮 董书琦 于 2021-10-11 设计创作，主要内容包括：本发明公开了一种基于萱草花粉管通道导入EMS诱变育种的方法,包括如下步骤：(1)于田间选取萱草花朵自交进行授粉；(2)将授粉的花剥离雄蕊和花瓣,截取并保留2mm长的花柱,用微量注射器吸取10μl诱变剂,沿花柱柱头纵轴注射,然后用湿棉花包住子房；诱变剂为浓度0.00001-0.00005％的EMS溶液,以磷酸缓冲液作为溶剂；(3)35d后收获种子,将种子在水中浸泡12h后剥去外种皮,在育苗盒中于植物培养间25℃条件下培养,本发明通过花粉管通道注射诱变剂的方法,授粉后使EMS溶液能够沿着花粉管通道形成的途径渗透,经过珠心进入胚囊,最终诱变尚不具备正常细胞壁的合子或早期胚胎细胞,相比于传统的诱变育种方法,该方法操作简单,容易掌握,省时省力。(The invention discloses a method for introducing EMS mutation breeding based on a hemerocallis fulva pollen tube channel, which comprises the following steps: (1) selecting flowers of hemerocallis fulva in the field for selfing and pollinating; (2) stripping stamens and petals of pollinated flowers, cutting and reserving a style with the length of 2mm, sucking 10 mu l of mutagen by using a micro-injector, injecting along the longitudinal axis of the style stigma, and then wrapping ovaries by using wet cotton; the mutagen is EMS solution with concentration of 0.00001-0.00005%, and phosphate buffer is used as solvent; (3) and harvesting seeds after 35d, soaking the seeds in water for 12h, peeling off the episperms, and culturing in a seedling box at the temperature of 25 ℃ in a plant culture room.)

1. The method for introducing EMS mutation breeding based on day lily pollen tube passage is characterized by comprising the following steps:

(1) selecting flowers of hemerocallis fulva in the field for selfing and pollinating;

(2) stripping stamens and petals of pollinated flowers, cutting and reserving a style with the length of 2mm by using a blade, sucking 10 mu l of mutagen by using a micro-injector, injecting along the longitudinal axis of the style stigma, and then wrapping ovaries by using wet cotton soaked in distilled water; the mutagen is EMS solution with the concentration of 0.00001-0.00005%, and phosphate buffer is used as solvent;

(3) after 35d, harvesting seeds, soaking the seeds in water for 12h, peeling off the episperms, and culturing in a seedling box at 25 ℃ in a plant culture room.

2. The method for introducing EMS mutation breeding based on day lily pollen tube channel as claimed in claim 1, wherein in step (1), the time of self-pollination is 7-8 a.m.

3. The method for introducing EMS mutation breeding based on day lily pollen tube channel as claimed in claim 1, wherein in step (2), the time for injecting mutagen is 24h after self-pollination in step (1).

4. The method for introducing EMS mutation breeding based on day lily pollen tube channel as claimed in claim 1, wherein in the step (2), the method for injecting mutagen is: the first injection of 5. mu.l of mutagen into the ovary 2/3, gentle withdrawal of the microsyringe into the ovary 1/3, and another injection of 5. mu.l mutagen.

5. The method for introducing EMS mutation breeding based on the hemerocallis fulva pollen tube channel as claimed in claim 1, wherein in the step (2), the EMS solution has a concentration of 0.00001%, 0.00002% or 0.00005%.

6. The method for introducing EMS mutation breeding based on day lily pollen tube channel as claimed in claim 1, wherein the concentration of phosphate buffer solution in step (2) is 0.1% mol/L, pH is 7, and the mixture is used after autoclaving.

7. The method of claim 1, wherein in step (2), the phosphate buffer is disposed by introducing EMS mutation breeding through a hemerocallis fulva pollen tube channel: 3.852g of disodium hydrogenphosphate dodecahydrate was dissolved and the volume was fixed to 100mL, 1.561g of sodium dihydrogenphosphate dihydrate was dissolved and the volume was fixed to 100mL, 61mL of the disodium hydrogenphosphate dodecahydrate solution was taken as solution 1 out of 100mL, 39mL of the sodium dihydrogenphosphate dihydrate solution was taken as solution 1 out of 100mL, and solution 1 and solution 2 were mixed to prepare 100mL of a phosphate buffer.

Technical Field

The invention relates to the technical field of plant breeding, in particular to a method for introducing EMS mutation breeding based on a hemerocallis fulva pollen tube channel.

Background

Hemerocallis fulva is a perennial root herbaceous plant of Hemerocallis of Liliaceae, native to southern China, southern Europe and Japan, and is widely cultivated in the southern China and northern China. The variety of hemerocallis fulva is more than ten thousand, and the hemerocallis fulva becomes an important ornamental and cut flower and is also the most variety of liliaceae flowers. Hemerocallis fulva flower is also called as 'mother flower' and 'forgetfulness grass', is rich in germplasm resources in China and has a long cultivation history. Since ancient times, hemerocallis are popular flowers and have been commonly used as singing topics by historical culture personnel. However, hemerocallis have many defects in mutation breeding, such as: complex operation, time and labor waste and the like. Therefore, there is an urgent need to develop a breeding method based on introducing EMS into the hemerocallis fulva pollen tube channel to induce mutation so as to solve the above technical problems.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a method for introducing EMS mutation breeding based on a day lily pollen tube channel, which injects a mutagen through the pollen tube channel, so that EMS solution can permeate along a path formed by the pollen tube channel after pollination, enters embryo sacs through a nucellus, and finally mutates zygotes or early embryo cells without normal cell walls. Compared with the traditional mutation breeding method, the method is simple to operate, easy to master, time-saving and labor-saving, has important significance for researching double breeding of the hemerocallis fulva, has wide application prospect, and is beneficial to popularization and application.

In order to achieve the above object, the method for introducing EMS mutation breeding based on the hemerocallis fulva pollen tube channel provided by the invention comprises the following steps:

(1) selecting flowers of hemerocallis fulva in the field for selfing and pollinating;

(2) stripping stamens and petals of pollinated flowers, cutting and reserving a style with the length of 2mm by using a blade, sucking 10 mu l of mutagen by using a micro-injector, injecting along the longitudinal axis of the style stigma, and then wrapping ovaries by using wet cotton soaked in distilled water; the mutagen is EMS solution with the concentration of 0.00001-0.00005%, and phosphate buffer is used as solvent;

(3) after 35d, harvesting seeds, soaking the seeds in water for 12h, peeling off the episperms, and culturing in a seedling box at 25 ℃ in a plant culture room.

Preferably, in the step (1), the time of self-pollination is 7-8 hours in the morning.

Preferably, in the step (2), the time for injecting the mutagen is 24 hours after the self-pollination in the step (1).

Preferably, in the step (2), the method for injecting the mutagen is as follows: the first injection of 5. mu.l of mutagen into the ovary 2/3, gentle withdrawal of the microsyringe into the ovary 1/3, and another injection of 5. mu.l mutagen.

Preferably, in the step (2), the concentration of the EMS solution is 0.00001%, 0.00002%, or 0.00005%.

Preferably, in the step (2), the phosphate buffer solution has a concentration of 0.1% mol/L and a pH of 7, and is used after autoclaving.

Preferably, in the step (2), the preparation method of the phosphate buffer solution comprises: 3.852g of disodium hydrogenphosphate dodecahydrate was dissolved and the volume was fixed to 100mL, 1.561g of sodium dihydrogenphosphate dihydrate was dissolved and the volume was fixed to 100mL, 61mL of the disodium hydrogenphosphate dodecahydrate solution was taken as solution 1 out of 100mL, 39mL of the sodium dihydrogenphosphate dihydrate solution was taken as solution 1 out of 100mL, and solution 1 and solution 2 were mixed to prepare 100mL of a phosphate buffer.

The method for introducing EMS mutation breeding based on day lily pollen tube channel has the following beneficial effects.

According to the method, through the method of injecting the mutagen into the pollen tube channel, after pollination, the EMS solution can permeate along the path formed by the pollen tube channel, enters the embryo sac through the nucellus, and finally mutates zygotes or early embryo cells without normal cell walls. Compared with the traditional mutation breeding method, the method is simple to operate, easy to master, time-saving and labor-saving, and has important significance for researching double breeding of hemerocallis fulva.

Detailed Description

The present invention will be further described with reference to specific examples to assist understanding of the invention.

The invention provides a method for introducing EMS mutation breeding based on a day lily pollen tube channel, which comprises the following steps:

(1) selecting hemerocallis fulva flowers in the field for selfing for pollination, wherein the self-pollination time is 7-8 hours in the morning;

(2) stripping stamens and petals of pollinated flowers, cutting and reserving a style with the length of 2mm by using a blade, sucking 10 mu l of mutagen by using a micro-injector, injecting along the longitudinal axis of the style stigma, and then wrapping ovaries by using wet cotton soaked in distilled water;

the method for injecting the mutagen comprises the following steps: the first injection of 5. mu.l of mutagen into the ovary 2/3, gentle withdrawal of the microsyringe into the ovary 1/3, and another injection of 5. mu.l mutagen. The time for injecting the mutagen is 24h after self-pollination in the step (1).

The mutagen is EMS solution with the concentration of 0.00001-0.00005, and phosphate buffer is used as a solvent. Preferably, the EMS solution has a concentration of 0.00001%, 0.00002%, or 0.00005%.

The concentration of the phosphate buffer solution is 0.1% mol/L, and the preparation method comprises the following steps: 3.852g of disodium hydrogenphosphate dodecahydrate was dissolved and the volume was fixed to 100mL, 1.561g of sodium dihydrogenphosphate dihydrate was dissolved and the volume was fixed to 100mL, 61mL of the disodium hydrogenphosphate dodecahydrate solution was taken as solution 1 out of 100mL, 39mL of the sodium dihydrogenphosphate dihydrate solution was taken as solution 1 out of 100mL, and solution 1 and solution 2 were mixed to prepare 100mL of a phosphate buffer. The pH was 7, and the mixture was autoclaved and used.

(3) After 35d, harvesting seeds, soaking the seeds in water for 12h, peeling off the episperms, and culturing in a seedling box at 25 ℃ in a plant culture room.

Example 1

A method for introducing EMS mutation breeding based on day lily pollen tube channel comprises the following steps:

(1) selecting 50 flowers of hemerocallis middendorf Sterla variety in the field for selfing for pollination at 7-8 am;

(2) after self-pollination is carried out for 24 hours, the pollinated flower is stripped of stamens and petals, a 2mm long style is cut by a blade and reserved, 10 mu l of mutagen is absorbed by a micro-injector and injected along the longitudinal axis of the style stigma, and then the ovary is wrapped by wet cotton soaked in distilled water;

the method for injecting the mutagen comprises the following steps: the first injection of 5. mu.l of mutagen into the ovary 2/3, gentle withdrawal of the microsyringe into the ovary 1/3, and another injection of 5. mu.l mutagen.

The mutagen is EMS solution with the concentration of 0.00001%, and phosphate buffer is used as a solvent. The concentration of the phosphate buffer solution is 0.1% mol/L, and the preparation method comprises the following steps: 3.852g of disodium hydrogenphosphate dodecahydrate was dissolved and the volume was fixed to 100mL, 1.561g of sodium dihydrogenphosphate dihydrate was dissolved and the volume was fixed to 100mL, 61mL of the disodium hydrogenphosphate dodecahydrate solution 100mL was used as solution 1, 39mL of the sodium dihydrogenphosphate dihydrate 100mL was used as solution 2, and solution 1 and solution 2 were mixed to prepare 100mL of a phosphate buffer solution having a pH of 7, which was used after autoclaving.

(3) After 35d, harvesting seeds, and recording the number of harvested seeds, the maturing rate and the germination rate. Soaking the seeds in water for 12h, peeling off the testa, and culturing in a seedling box at 25 deg.C.

Example 2

A method for introducing EMS mutation breeding based on day lily pollen tube channel comprises the following steps:

(1) selecting 50 flowers of hemerocallis middendorf Sterla variety in the field for selfing for pollination at 7-8 am;

(2) after self-pollination is carried out for 24 hours, the pollinated flower is stripped of stamens and petals, a 2mm long style is cut by a blade and reserved, 10 mu l of mutagen is absorbed by a micro-injector and injected along the longitudinal axis of the style stigma, and then the ovary is wrapped by wet cotton soaked in distilled water;

the method for injecting the mutagen comprises the following steps: the first injection of 5. mu.l of mutagen into the ovary 2/3, gentle withdrawal of the microsyringe into the ovary 1/3, and another injection of 5. mu.l mutagen.

The mutagen is EMS solution with the concentration of 0.00002%, and phosphate buffer is used as a solvent. The concentration of the phosphate buffer solution is 0.1% mol/L, and the preparation method comprises the following steps: 3.852g of disodium hydrogenphosphate dodecahydrate was dissolved and the volume was fixed to 100mL, 1.561g of sodium dihydrogenphosphate dihydrate was dissolved and the volume was fixed to 100mL, 61mL of the disodium hydrogenphosphate dodecahydrate solution 100mL was used as solution 1, 39mL of the sodium dihydrogenphosphate dihydrate 100mL was used as solution 2, and solution 1 and solution 2 were mixed to prepare 100mL of a phosphate buffer solution having a pH of 7, which was used after autoclaving.

(3) After 35d, harvesting seeds, and recording the number of harvested seeds, the maturing rate and the germination rate. Soaking the seeds in water for 12h, peeling off the testa, and culturing in a seedling box at 25 deg.C.

Example 3

A method for introducing EMS mutation breeding based on day lily pollen tube channel comprises the following steps:

(1) selecting 50 flowers of hemerocallis middendorf Sterla variety in the field for selfing for pollination at 7-8 am;

(2) after self-pollination is carried out for 24 hours, the pollinated flower is stripped of stamens and petals, a 2mm long style is cut by a blade and reserved, 10 mu l of mutagen is absorbed by a micro-injector and injected along the longitudinal axis of the style stigma, and then the ovary is wrapped by wet cotton soaked in distilled water;

the method for injecting the mutagen comprises the following steps: the first injection of 5. mu.l of mutagen into the ovary 2/3, gentle withdrawal of the microsyringe into the ovary 1/3, and another injection of 5. mu.l mutagen.

The mutagen is EMS solution with the concentration of 0.00005%, and phosphate buffer is used as a solvent. The concentration of the phosphate buffer solution is 0.1% mol/L, and the preparation method comprises the following steps: 3.852g of disodium hydrogenphosphate dodecahydrate was dissolved and the volume was fixed to 100mL, 1.561g of sodium dihydrogenphosphate dihydrate was dissolved and the volume was fixed to 100mL, 61mL of the disodium hydrogenphosphate dodecahydrate solution 100mL was used as solution 1, 39mL of the sodium dihydrogenphosphate dihydrate 100mL was used as solution 2, and solution 1 and solution 2 were mixed to prepare 100mL of a phosphate buffer solution having a pH of 7, which was used after autoclaving.

(3) After 35d, harvesting seeds, and recording the number of harvested seeds, the maturing rate and the germination rate. Soaking the seeds in water for 12h, peeling off the testa, and culturing in a seedling box at 25 deg.C.

Comparative example 1

A method for introducing EMS mutation breeding based on day lily pollen tube channel comprises the following steps:

(1) selecting 50 flowers of hemerocallis middendorf Sterla variety in the field for selfing for pollination at 7-8 am;

(2) after self-pollination is carried out for 24 hours, the pollinated flower is stripped of stamens and petals, a 2mm long style is cut by a blade and reserved, 10 mu l of mutagen is absorbed by a micro-injector and injected along the longitudinal axis of the style stigma, and then the ovary is wrapped by wet cotton soaked in distilled water;

the method for injecting the mutagen comprises the following steps: the first injection of 5. mu.l of mutagen into the ovary 2/3, gentle withdrawal of the microsyringe into the ovary 1/3, and another injection of 5. mu.l mutagen.

The mutagen is 0.0001% EMS solution, and phosphate buffer is used as solvent. The concentration of the phosphate buffer solution is 0.1% mol/L, and the preparation method comprises the following steps: 3.852g of disodium hydrogenphosphate dodecahydrate was dissolved and the volume was fixed to 100mL, 1.561g of sodium dihydrogenphosphate dihydrate was dissolved and the volume was fixed to 100mL, 61mL of the disodium hydrogenphosphate dodecahydrate solution 100mL was used as solution 1, 39mL of the sodium dihydrogenphosphate dihydrate 100mL was used as solution 2, and solution 1 and solution 2 were mixed to prepare 100mL of a phosphate buffer solution having a pH of 7, which was used after autoclaving.

(3) After 35d, harvesting seeds, and recording the number of harvested seeds, the maturing rate and the germination rate. Soaking the seeds in water for 12h, peeling off the testa, and culturing in a seedling box at 25 deg.C.

Comparative example 2

A method for introducing EMS mutation breeding based on day lily pollen tube channel comprises the following steps:

(1) selecting 50 flowers of hemerocallis middendorf Sterla variety in the field for selfing for pollination at 7-8 am;

(2) after self-pollination is carried out for 24 hours, the pollinated flower is stripped of stamens and petals, a 2mm long style is cut by a blade and reserved, 10 mu l of mutagen is absorbed by a micro-injector and injected along the longitudinal axis of the style stigma, and then the ovary is wrapped by wet cotton soaked in distilled water;

the method for injecting the mutagen comprises the following steps: the first injection of 5. mu.l of mutagen into the ovary 2/3, gentle withdrawal of the microsyringe into the ovary 1/3, and another injection of 5. mu.l mutagen.

The mutagen is 0.0002% EMS solution, and phosphate buffer is used as a solvent. The concentration of the phosphate buffer solution is 0.1% mol/L, and the preparation method comprises the following steps: 3.852g of disodium hydrogenphosphate dodecahydrate was dissolved and the volume was fixed to 100mL, 1.561g of sodium dihydrogenphosphate dihydrate was dissolved and the volume was fixed to 100mL, 61mL of the disodium hydrogenphosphate dodecahydrate solution 100mL was used as solution 1, 39mL of the sodium dihydrogenphosphate dihydrate 100mL was used as solution 2, and solution 1 and solution 2 were mixed to prepare 100mL of a phosphate buffer solution having a pH of 7, which was used after autoclaving.

(3) After 35d, harvesting seeds, and recording the number of harvested seeds, the maturing rate and the germination rate. Soaking the seeds in water for 12h, peeling off the testa, and culturing in a seedling box at 25 deg.C.

Comparative example 3

A method for introducing EMS mutation breeding based on day lily pollen tube channel comprises the following steps:

(1) selecting 50 flowers of hemerocallis middendorf Sterla variety in the field for selfing for pollination at 7-8 am;

(2) after self-pollination is carried out for 24 hours, the pollinated flower is stripped of stamens and petals, a 2mm long style is cut by a blade and reserved, 10 mu l of mutagen is absorbed by a micro-injector and injected along the longitudinal axis of the style stigma, and then the ovary is wrapped by wet cotton soaked in distilled water;

the method for injecting the mutagen comprises the following steps: the first injection of 5. mu.l of mutagen into the ovary 2/3, gentle withdrawal of the microsyringe into the ovary 1/3, and another injection of 5. mu.l mutagen.

The mutagen is EMS solution with the concentration of 0.0005 percent, and phosphate buffer is used as a solvent. The concentration of the phosphate buffer solution is 0.1% mol/L, and the preparation method comprises the following steps: 3.852g of disodium hydrogenphosphate dodecahydrate was dissolved and the volume was fixed to 100mL, 1.561g of sodium dihydrogenphosphate dihydrate was dissolved and the volume was fixed to 100mL, 61mL of the disodium hydrogenphosphate dodecahydrate solution 100mL was used as solution 1, 39mL of the sodium dihydrogenphosphate dihydrate 100mL was used as solution 2, and solution 1 and solution 2 were mixed to prepare 100mL of a phosphate buffer solution having a pH of 7, which was used after autoclaving.

(3) After 35d, harvesting seeds, and recording the number of harvested seeds, the maturing rate and the germination rate. Soaking the seeds in water for 12h, peeling off the testa, and culturing in a seedling box at 25 deg.C.

The statistics of the number of harvested seeds, the maturing rate and the germination rate of the above examples 1 to 3 and comparative examples 1 to 3 are shown in Table 1.

TABLE 1 Effect of different concentrations of mutagen on the number of seeds harvested from Hemerocallis fulva, the seed set and the germination percentage

From the above test results, it can be seen that: with the increase of EMS concentration, the number of seeds harvested, the seed setting rate and the germination rate of the hemerocallis gradually decrease, and the number of seeds harvested, the seed setting rate and the germination rate of the hemerocallis in the comparative examples 1 to 3 are significantly lower than those in the examples 1 to 3 of the present invention, i.e., the EMS concentration selected by the present invention makes the hemerocallis still have higher seed setting rate and germination rate. Therefore, it could be demonstrated that the method of the present invention for mutagenesis breeding by introducing EMS through the pollen tube pathway is completely feasible.

According to the method, through the method of injecting the mutagen into the pollen tube channel, after pollination, the EMS solution can permeate along the path formed by the pollen tube channel, enters the embryo sac through the nucellus, and finally mutates zygotes or early embryo cells without normal cell walls. Compared with the traditional mutation breeding method, the method is simple to operate, easy to master, time-saving and labor-saving, and has important significance for researching double breeding of hemerocallis fulva.

The inventive concept is explained in detail herein using specific examples, which are given only to aid in understanding the core concepts of the invention. It should be understood that any obvious modifications, equivalents and other improvements made by those skilled in the art without departing from the spirit of the present invention are included in the scope of the present invention.