CN114868653B

CN114868653B - Method for efficiently inducing citrus single embryo variety to generate tetraploid

Info

Publication number: CN114868653B
Application number: CN202210674420.2A
Authority: CN
Inventors: 郭文武; 任杰; 解凯东; 段耀园
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2022-06-15
Filing date: 2022-06-15
Publication date: 2023-04-25
Anticipated expiration: 2042-06-15
Also published as: CN114868653A

Abstract

The invention discloses a method for efficiently inducing a single embryo variety of citrus to generate tetraploid, belonging to the technical field of biological breeding of citrus. The method of the invention comprises the following steps: when the single embryo seed grows to 5-7cm high, cutting off the seedling below the first true leaf of the seedling with a blade to keep the length of the epicotyl 2-3cm; inducing incision under dark condition to form callus; after the callus is formed, the callus is treated by colchicine solution with the concentration of 0.0125-0.1 percent, and the callus is induced to regenerate buds under the illumination condition; when the buds germinate and grow 2-4 true leaves, screening the single embryogenic tetraploid by combining the flow cytometry ploidy identification with the citrus single multi-embryo specific molecular marker. The invention has wide applicability, low homozygosity and chimeric rate of the obtained tetraploid by utilizing the characteristic of single cell regeneration of the callus, high induction efficiency, simple and easy operation, no need of tissue culture and great improvement of the obtaining efficiency of the tetraploid of the citrus single embryo variety.

Description

Method for efficiently inducing citrus single embryo variety to generate tetraploid

Technical Field

The invention belongs to the technical field of biological breeding of citrus, and particularly relates to a method for efficiently inducing a single embryo variety of citrus to generate tetraploid.

Background

Citrus is the most important fruit tree in the south of China, and the citrus industry in China rapidly develops in recent years, so that the citrus industry becomes a large prop industry of rural economy in the main production area in the south of China. Seedless citrus is favored because of its convenience in eating and excellent quality, and therefore seedless citrus is an important breeding goal. The triploid is a natural seedless type because of abnormal meiosis and male and female gametes are sterile, and the cultivation of the triploid is an important way for obtaining the seedless germplasm of citrus, and has important breeding application value. The natural occurrence frequency of triploid in citrus is low, and the most effective breeding route for obtaining triploid is hybridization of tetraploid with diploid. The diploid is used as female parent and tetraploid is used as male parent for ploidy hybridization, but the method is difficult to obtain triploid seeds due to endosperm abortion during hybridization, and the embryo rescue technology is needed to obtain hybrid seedlings, so that time and labor are wasted. The single embryo tetraploid is used as female parent to hybridize with diploid ploidy, so that the seed is normal in development, the embryo rescue process can be omitted, and the breeding efficiency is greatly improved. However, most of the existing tetraploid resources of citrus are multi-embryogenic and single-embryogenic resources are seriously lacking. The multi-embryo tetraploid is used as a female parent to be hybridized with diploid ploidy, and the female parent bead core embryo can compete with the sexual embryo for nutrition space, so that the sexual embryo of the triploid is in post-development, and the embryo is also needed to be saved to improve the triploid obtaining rate, so that the breeding efficiency is low. Therefore, the creation of the single embryogenic tetraploid germplasm is a necessary condition for breaking through the bottleneck of limiting the improvement of the efficiency of three times of sports of citrus.

Among citrus, the most widely used method for obtaining single embryogenic tetraploids is colchicine induction. The traditional method generally selects seeds, adult young shoots or young buds as materials for induction, and generally has the problems of low induction efficiency, difficult bud formation, high chimera ratio and the like, and is difficult to popularize and utilize on a large scale.

Disclosure of Invention

Aiming at the defects of low induction efficiency, difficult sprouting, high chimera proportion and the like in the prior art for inducing the citrus varieties to generate tetraploids, the invention aims to provide a method for efficiently inducing the citrus single embryogenic varieties to generate tetraploids, which comprises the following steps: namely, single embryo seed is grown into seedling to grow to 5-7cm in height, the seedling is cut off below the first true leaf of the seedling by a blade, and the length of the epicotyl is kept to 2-3cm; inducing incision under dark condition to form callus; after the callus is formed, colchicine solution with the mass fraction ratio of 0.0125% -0.1% is used for treating the callus, and the callus is induced to regenerate buds under the illumination condition; when the buds germinate and grow 2-4 true leaves, screening the single embryogenic tetraploid by combining the flow cytometry ploidy identification with the citrus single multi-embryo specific molecular marker. The method utilizes the characteristic of callus single cell regeneration, the obtained tetraploid is homozygous, the chimeric rate is low, the induction efficiency is high, the method is simple and convenient and easy to operate, tissue culture is not needed, and the obtaining efficiency of the citrus single embryo tetraploid is greatly improved.

In order to achieve the above purpose, the specific technical scheme of the invention is as follows:

a method for efficiently inducing a citrus single embryo variety to produce tetraploid, comprising the following steps:

(1) Taking out seeds from mature fruits of single embryo type citrus varieties, removing pectin, and peeling off exocarp; accelerating germination in an incubator, and transplanting the germinated seed radicle into a nutrition pot for culture.

(2) The citrus seedling grows to 5-7cm in height, is cut off below the first true leaf of the seedling by a blade, and meanwhile lateral buds are removed, the length of the epicotyl is kept to 2-3cm, and the incision is kept moist to promote callus formation under the dark condition. Wherein the incision is kept moist, preferably by reversing the suction head over the incision.

(3) After the incision callus is formed, soaking the incision callus in colchicine solution, wrapping the top of the nutrition pot with a preservative film to keep the incision moist, and placing the incision callus under the illumination condition to promote the callus to sprout.

(4) Screening the single embryogenic tetraploid after the callus is regenerated and germinated and 2-4 true leaves are grown.

Further, the method for efficiently inducing the citrus single embryo variety to generate tetraploid comprises the following steps:

(1) Picking fruits and peeling seeds after the citrus single embryo variety fruits are ripe, and soaking the citrus single embryo variety fruits in NaOH solution to remove pectin; cleaning with clear water, peeling off exocarp, placing in a culture dish paved with wet filter paper, and placing in an incubator for germination acceleration; after the seed radicle germinates, the seed radicle is sown in a nutrition pot and is placed in a growth chamber for culture, and the top of the nutrition pot is wrapped with a preservative film for moisture preservation.

Among them, the pectin removal conditions are preferably: soaking in 1mol/L NaOH solution for 10min. The temperature of the incubator for germination is preferably 28 ℃. The nutrition pot is preferably a nutrition pot with the length of 20cm multiplied by 20 cm. The conditions for growth chamber culture are preferably: the temperature is 25+/-1 ℃; light is applied for 16h and dark is applied for 8h.

(2) 10-15d after sowing, and when the seedling grows to 5-7cm in height. Cutting off the seedling at 2-3cm above the ground (without leaving leaves), and retaining moisture by reversely buckling the seedling at the incision with a suction head; the black cloth is used for wrapping the top of the nutrition pot to avoid light, so that the formation of callus is promoted.

(3) After 6-12d, the incision is formed into callus, the callus is soaked in colchicine water solution, the top of the nutrition pot is wrapped with preservative film for preserving moisture, and the callus is placed under the illumination condition to promote the germination of the callus. Among them, soaking of the callus with the colchicine aqueous solution is preferably achieved by reversely buckling a tip filled with the colchicine aqueous solution to the incised callus.

(4) And (3) regenerating a large number of buds at the incision part after 10-20d, and screening the single embryogenic tetraploid when 2-4 true leaves grow out from the buds.

In the above method, the colchicine aqueous solution preferably contains colchicine 0.0125% -0.1% (mass fraction ratio) and dimethyl sulfoxide 1-2% (volume fraction ratio). The time for soaking the callus in colchicine aqueous solution is preferably 1-2 hours.

Among the above methods, the method for screening for a single embryogenic tetraploid is preferably: and (3) carrying out preliminary screening according to morphological characteristics of polyploid leaves, carrying out ploidy identification on suspected polyploid regeneration buds by using a flow cytometry, and carrying out single-multiple embryo marking identification on tetraploids with definite ploidy by using a citrus specific single-multiple embryo molecular marking.

The invention has high tetraploid induction rate and low chimeric rate, and provides a high-efficiency novel method and novel germplasm for citrus polyploid breeding. Compared with the prior art, the invention has the following beneficial effects:

(1) In the invention, colchicine treatment is carried out by taking the regenerated callus of the citrus epicotyl as a material, and compared with the treatment of adult buds by directly utilizing colchicine, the method has stronger differentiation capability and higher regeneration efficiency; by utilizing the characteristics of callus single cells, the induced tetraploid is more homozygous, and the occurrence rate of chimerism is low; as the callus is tender compared with the mature buds, the callus needs to be treated by colchicine with low concentration, and the colchicine has little toxic effect on materials and higher induction efficiency.

(2) The invention utilizes the callus regenerated by the embryogenic axis of the citrus seedling to induce citrus polyploidy, can be used for manually pollinating and selecting proper parents, then carries out colchicine induction doubling on the callus regenerated by the embryogenic axis of the offspring seedling, combines single-embryo molecular marker identification and screening of single-embryo tetraploid, and creates a new tetraploid germplasm of single-embryo citrus variety with excellent characters.

(3) The method does not need aseptic operation of tissue culture in the whole process, is simple and convenient, is easy to operate, only needs 35-45 days from the treatment by colchicine solution to the acquisition of polyploid, and has high breeding efficiency.

(4) The invention has wide applicability, and at present, 13 tetraploids of single embryo varieties of citrus are obtained by using the invention. The citrus fruit type comprises cultivated citrus fruit such as lemon, pomelo, wide-peel orange, sweet orange and lemon.

Drawings

FIG. 1 is a flow chart of the present invention; a: mature single embryo variety fruit; b: accelerating germination of seeds; c: the state of seedlings before transverse cutting; d: a state after transverse cutting of seedlings; e: inducing callus by black cloth in dark place; f: the white suction head is filled with colchicine solution to induce incision callus; g: wrapping the preservative film for moisturizing; h: regenerating buds of the callus; i: flow cytometer ploidy identification; j: the induced tetraploid is compared with diploid morphology.

FIG. 2 shows the regenerated callus of dark-induced epicotyl (a: 0d cut callus state; b: 9d cut callus state) and regenerated bud of colchicine-treated callus (c) of citrus monoterminal variety Qiu Hui orange of example 1 induced by the present invention;

FIG. 3 shows the morphology difference between tetraploid plants and diploid plants induced by the present invention for the citrus mono-embryogenic variety Qiu Hui orange of example 1 (left) and the morphology difference between leaves of the Qiu Hui orange (right);

FIG. 4 shows the tetraploid induced by the present invention of citrus monoterminal variety Qiu Hui orange of example 1, identified by flow cytometry (a: diploid; b: tetraploid) and root tip chromosome tablet (c: diploid; d: tetraploid);

FIG. 5 shows the identification of the tetraploid, single multi-embryo molecular marker induced by the present invention for the citrus single embryo variety Qiu Hui orange of example 1;

FIG. 6 shows the morphological differences between tetraploid plants and diploid plants induced by the present invention in the citrus single embryo variety HB pomelo of example 2;

FIG. 7 shows the morphological differences between tetraploid plants and diploid plants induced by colchicine in citrus monotropus variety of example 3;

FIG. 8 is a morphological difference of tetraploid plants and diploid plants induced by colchicine from the single embryogenic variety amber sweet orange of example 4;

FIG. 9 shows three different developmental stages of the single embryo type HB shaddock of example 14 spring tips (a: non-lignified spring tips; b: semi-lignified spring tips; c: lignified spring tips) from 5-10cm from the top of the shoots, transecting the induced callus, colchicine post-induction state (d: non-lignified spring tips dark condition forming callus state; e: semi-lignified spring tips dark condition forming callus state; f: lignified spring tips dark condition forming callus state; g: non-lignified spring tips colchicine post-induction, light condition callus state; h: semi-lignified spring tips colchicine post-induction, light condition callus state; i: lignified spring tips colchicine post-induction, light condition callus state).

Detailed Description

For a clear and complete description of the objects, technical solutions and advantages of the present invention, the details of the present invention are further described below with reference to the accompanying drawings and examples. The described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

This example relates to the creation of a new germplasm of citrus mono-embryo variety Qiu Hui citrus tetraploid, the mature seed of citrus mono-embryo variety Qiu Hui citrus is harvested from the Huazhong university citrus germplasm resource nursery. The method specifically comprises the following steps:

(1) Picking up and peeling seeds after the mature of Qiu Hui orange fruits, soaking the fruits for 10min by using a 1mol/L NaOH solution to remove pectin, cleaning the fruits by using clear water, peeling off the exocarp, placing the seeds in a culture dish paved with wet filter paper, accelerating germination in a 28 ℃ incubator, sowing the seeds into a 20cm multiplied by 20cm nutrition pot after the radicle of the seeds germinates, placing the seeds in a growth chamber for culture (the growth chamber condition is that the temperature is 25+/-1 ℃, the illumination is 16h and the darkness is 8 h), and sowing 40-50 seeds in one pot. Wrapping the top of the nutrition pot with a preservative film to keep the soil in the nutrition pot moist.

(2) When the seedling grows to about 5cm in height (15 d after sowing). Cutting off the seedling at 2-3cm above ground (without leaving leaves), and retaining moisture in the incision with suction head (white suction head with volume of 20 μl commonly used in laboratory pipettes); the black cloth is used for wrapping the top of the nutrition pot to avoid light, so that the formation of callus is promoted. The growth chamber temperature was maintained during callus formation as follows: the temperature is 25+/-1 ℃ and the whole process is dark and light-proof.

(3) After 9d, callus is formed at the incision; sucking the colchicine water solution (colchicine mass fraction ratio is 0.05% and 1% dimethyl sulfoxide is added according to volume ratio) prepared in advance by a syringe, and adding the colchicine water solution into a white suction head; the white suction head filled with colchicine solution is reversely buckled on the cut callus (in order to prevent the liquid from leaking downwards, the tip of the suction head is sealed by flame roasting), so that the colchicine solution is soaked in the callus for 2 hours; and then the suction head is taken down, the top of the nutrition pot is wrapped by a preservative film for preserving moisture, and the nutrition pot is placed under the conditions of 25+/-1 ℃ and 16 hours of illumination and 8 hours of darkness to promote the germination of the callus.

(4) About 10-20d, a large number of buds can be regenerated at the incision; screening suspected polyploids according to morphological characteristics (such as thickening, width, darkening, large oil cells, low density and the like of the polyploid leaves when buds germinate and grow into 2-4 true leaves, and carrying out ploidy identification on the suspected polyploids by using a flow cytometer; and (3) carrying out single-embryo and multi-embryo marking identification on the tetraploid with definite ploidy by using citrus specific single-embryo molecular marking primers (P/M-F, P/M-R), and screening the single-embryo tetraploid. And simultaneously taking the tetraploid branches generated by induction, cutting for rooting, taking root tips for chromosome tabletting, and carrying out chromosome counting statistics. The morphology difference map of the Qiu Hui orange tetraploid plant and the diploid plant and the morphology difference of the Qiu Hui orange different ploidy leaves are shown in figure 3. Flow cytometer ploidy identification and root tip chromosome tabletting are shown in figure 4.

(5) The induced tetraploid uses primers P/M-F: TCTCTGGTTCATTGAGAATCC and P/M-R: CTGAGCACCAGGCAACAACTAC the identification of single multi-embryo molecular markers is carried out.

Single multi-embryo molecular identification is carried out on the Qiu Hui orange tetraploid induced by 70 strains, only 31 strains are found, two bands are multi-embryos, and the rest are single embryos. The identification result of the single multi-embryo molecular marker is shown in fig. 5.

Example 2

The same procedure as in example 1 was followed, and citrus single embryo variety HB pomelo seeds were harvested from citrus germplasm resource nursery at Huazhong university, and the morphology differences between the induced tetraploid plants and diploid plants were shown in FIG. 6.

Example 3

The method is the same as in example 1, citrus single embryo variety citric acid seeds are obtained from Yunnan De Hongzhang, and the morphology difference between tetraploid plants and diploid plants is induced and is shown in figure 7.

Example 4

The same method as in example 1, the citrus single embryo variety amber sweet orange seeds were harvested from citrus germplasm resource nursery at the university of Huazhong agriculture, and the morphology differences between the induced tetraploid plants and diploid plants are shown in FIG. 8.

Examples 5 to 13

The method is the same as in example 1, and the varieties are Hua Nonggong pomelo, thunder god vine, chrysanthemum-core pomelo, high-spot pomelo, huang Lingmiao orange, shatian pomelo, qing Xponkan, kleman Ding Ju Xponkan and bergamot.

Examples 1-13 induced tetraploid induction and chimerism occurrence for different citrus monotonous varieties are shown in Table 1 below.

TABLE 1 tetraploid Induction Rate and chimeric Generation Rate of different monoterminous varieties compared

Example 14

The method is similar to example 1, except that a citrus single embryo type HB pomelo, a non-lignified spring tip, a semi-lignified spring tip and a lignified spring tip are selected, and a colchicine induction part is transversely cut at a position 5-10cm away from the top end of a branch. The results show that the shoots at three different development periods can form callus in the dark, but after colchicine induction, the callus can not regenerate into buds under the illumination condition. The callus formed by the epicotyl of the HB in example 2 can normally regenerate buds after colchicine induction and tetraploid plants are obtained, which shows that the epicotyl of citrus is the best site for colchicine induced polyploidy due to the strong regeneration capacity. The spring tip induction results for three different developmental stages are shown in FIG. 9.

The above embodiments are intended to facilitate understanding by those skilled in the art that the embodiments of the present invention are not limited to the above embodiments, and any modifications, equivalent substitutions, combinations, improvements, etc. made within the spirit and principle of the present invention are included in the scope of protection of the present invention.

Claims

1. A method for efficiently inducing a citrus single embryo variety to generate tetraploid is characterized in that: the method comprises the following steps:

(1) Taking out seeds from mature fruits of single embryo type citrus varieties, removing pectin, and peeling off exocarp; accelerating germination in an incubator, and transplanting the germinated seed radicle to a nutrition pot for culture;

(2) The citrus seedling grows to 5-7cm in height, the citrus seedling is cut off below the first true leaf of the seedling by a blade, the length of the epicotyl is kept to be 2-3cm, the incision is kept moist, and the formation of callus is promoted under the condition of light shielding;

(3) After the incision callus is formed, soaking the incision callus in colchicine solution, wrapping the top of the nutrition pot with a preservative film to keep the incision moist, and placing the incision callus under the illumination condition to promote the callus to sprout;

the colchicine water solution contains 0.0125% -0.1% of colchicine and 1-2% of dimethyl sulfoxide; soaking the callus in colchicine water solution for 1-2h;

2. The method for efficiently inducing the production of tetraploids from citrus monoterminous varieties according to claim 1, which is characterized in that: in step (2), the incision is kept moist by reversing the tip over the incision.

3. The method for efficiently inducing the production of tetraploids from citrus monoterminous varieties according to claim 1, which is characterized in that: the method comprises the following steps:

(1) Picking fruits and peeling seeds after the citrus single embryo variety fruits are ripe, and soaking the citrus single embryo variety fruits in NaOH solution to remove pectin; cleaning with clear water, peeling off exocarp, placing in a culture dish paved with wet filter paper, and placing in an incubator for germination acceleration; after the seed radicle germinates, sowing the seed radicle in a nutrition pot, placing the nutrition pot in a growth chamber for culture, and wrapping the top of the nutrition pot with a preservative film for moisture preservation;

(2) When the seedling grows to 5-7cm in height; cutting off the seedling at 2-3cm above ground with a blade, and inversely fastening the seedling at the incision with a suction head for moisturizing; the top of the nutrition pot is wrapped by black cloth to avoid light, so that callus formation is promoted;

(3) After the incision callus is formed, soaking the callus by colchicine water solution, wrapping the top of the nutrition pot by using a preservative film for preserving moisture, and placing the nutrition pot under the illumination condition to promote the callus to sprout;

(4) After the buds are regenerated at the incision, screening the single embryogenic tetraploid when the buds germinate and grow 2-4 true leaves.

4. A method of efficiently inducing the production of tetraploids from citrus monoterminous varieties according to claim 3, characterized in that: in the step (1), pectin is removed under the following conditions: soaking in 1mol/L NaOH solution for 10min; the germination accelerating temperature of the incubator is 28 ℃; the conditions of the growth chamber culture are as follows: the temperature is 25+/-1 ℃; light is applied for 16h and dark is applied for 8h.

5. A method of efficiently inducing the production of tetraploids from citrus monoterminous varieties according to claim 3, characterized in that: in step (3), soaking the calli with the colchicine aqueous solution is achieved by reversing the tip filled with colchicine aqueous solution on the cut calli.

6. A method for efficiently inducing the production of tetraploids from citrus monoterminous varieties according to claim 1 or 3, characterized in that: the method for screening the single embryogenic tetraploid comprises the following steps: and (3) carrying out preliminary screening according to morphological characteristics of polyploid leaves, carrying out ploidy identification on suspected polyploid regeneration buds by using a flow cytometry, and carrying out single-multiple embryo marking identification on tetraploids with definite ploidy by using a citrus specific single-multiple embryo molecular marking.