CN112616662A

CN112616662A - Method for separating and purifying polyploid from chimeric polyploid by using pear regeneration system

Info

Publication number: CN112616662A
Application number: CN202011491016.9A
Authority: CN
Inventors: 徐凌飞; 向方昕; 王志刚; 翟锐
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2020-12-17
Filing date: 2020-12-17
Publication date: 2021-04-09

Abstract

本发明涉及一种利用梨再生体系从嵌合多倍体中分离纯化多倍体的方法，本发明包括以下步骤：1)外植体的建立；1.1)外植体的获得：以梨花器官作为外植体来源；1.2)外植体的消毒；1.3)外植体的预培养：将梨子房接种在PIM培养基上预培养；2)体细胞胚诱导成苗：将预培养的子房在无菌条件下转移至IM培养基上培养，诱导体细胞胚分化成苗；3)幼苗的倍性鉴定：选择健康叶片，利用解离液解离细胞后，用细胞流式仪鉴定倍性；4)幼苗的扩繁。本发明可以简单、短时、高效的从梨嵌合多倍体中分离、纯化出多倍体植株。The present invention relates to a method for separating and purifying polyploids from chimeric polyploids by utilizing a pear regeneration system. The present invention comprises the following steps: 1) establishment of explants; 1.1) acquisition of explants: using pear flower organs as Source of explants; 1.2) Sterilization of explants; 1.3) Pre-culture of explants: inoculate pear ovaries on PIM medium for pre-culture; 2) Induce somatic embryos into seedlings: place pre-cultured ovaries in Transfer to IM medium for culture under sterile conditions to induce somatic embryos to differentiate into seedlings; 3) Ploidy identification of seedlings: select healthy leaves, dissociate cells with dissociation solution, and identify ploidy with flow cytometry; 4) Propagation of seedlings. The invention can separate and purify polyploid plants from pear chimeric polyploid simply, in a short time and with high efficiency.

Description

Method for separating and purifying polyploid from chimeric polyploid by using pear regeneration system

Technical Field

The invention relates to the technical field of biology, in particular to a method for separating and purifying polyploids from chimeric polyploids by using a pear regeneration system.

Background

Polyploid breeding is one of the important means for germplasm innovation of horticultural plants. For pear varieties, the fruits of polyploid varieties are generally larger than those of diploids, and the fruits have excellent quality and strong stress resistance. The spontaneous bud mutation of fruit trees is an important source of the existing polyploid variety, but the bud mutation breeding still has a plurality of defects. For example, when spontaneous bud mutation occurs, the mutant cells and wild cells are associated with each other, so that chimera is easily formed and the chimeric type is difficult to control, thereby causing unstable mutant characters and easy loss of mutant characters. The instability not only restricts the development of bud mutation and seed selection, but also causes great loss of excellent mutation traits and special germplasm resources.

Disclosure of Invention

The invention provides a method for separating and purifying polyploidy from chimeric polyploidy by using a pear regeneration system, which can separate and purify polyploidy plants from the chimeric polyploidy simply, in a short time and efficiently.

The technical solution of the invention is as follows: the invention relates to a method for separating and purifying polyploids from chimeric polyploids by using a pear regeneration system, which is characterized by comprising the following steps: the method comprises the following steps:

1) establishing an explant;

1.1) obtaining of explants: taking pear flower organs as explant sources;

1.2) disinfection of explants;

1.3) preculture of explants: inoculating the pear ovaries on a PIM culture medium for pre-culture;

2) inducing somatic embryos into seedlings: transferring the pre-cultured ovaries to an IM (instant Messaging) culture medium under the aseptic condition for culture, and inducing somatic embryos to differentiate into seedlings;

3) ploidy identification of seedlings: selecting healthy leaves, dissociating cells by using a dissociation solution, and identifying ploidy by using a cell flow instrument;

4) and (5) propagation of seedlings.

Preferably, the chimeric polyploid pear 9712 is selected as a material in the step 1.1); selecting the pear 7 days before the full-bloom stage (the specific date depends on the climatic conditions of the current year, and using a non-flowering bud and an ovary as materials for establishing the explant.

Preferably, the specific steps of step 1.2) are as follows: washing the picked pear ovaries at 25 ℃ for 6 hours by clear water; sterilizing with 70% alcohol for 30 s; rinsing with sterile water for 30 seconds, and repeating for 4 times; sterilizing with 15% sodium hypochlorite for 20 min, and shaking for 4 times; finally, rinsing with sterile water for 30 seconds, and repeating for 4 times.

Preferably, the specific formula of the PIM medium in step 1.3) is: basic elements: 1/2QL medium (current commercial medium) supplemented with 24mg/LEDTA chelated iron, 250mg/L potassium nitrate, 97mg/L magnesium sulfate heptahydrate, 407mg/L ammonium nitrate; hormones: 0.2 mg/LIBA; carbon source: 30g/L of sucrose.

Preferably, the pre-culture conditions in step 1.3) are specifically: dark culture was carried out at 25 ℃ for 4 days.

Preferably, the inoculation mode in the step 1.3) is specifically as follows: cutting the sterile bud and the ovary at the lower part into two parts along the radial direction in a sterile super clean bench, exposing anther and ovule, cutting off redundant pedicel, and sticking the cut surface downwards on a culture medium.

Preferably, the specific formula of the IM medium in the step 2) is as follows: basic elements: 1/2QL medium (current commercial medium) supplemented with 24mg/LEDTA chelated iron, 250mg/L potassium nitrate, 97mg/L magnesium sulfate heptahydrate, 407mg/L ammonium nitrate, 2.2 mg/LTDZ; hormones: 0.2 mg/LIBA; carbon source: 30g/L of sucrose.

Preferably, the culture conditions for inducing the somatic embryos to differentiate into seedlings in the step 2) are as follows: culturing at 25 deg.C in dark for 60 days, and changing to new IM medium after culturing for 30 days.

Preferably, the specific steps of step 4) are as follows: after 4 weeks of seedling culture, selecting a tissue culture seedling with strong growth vigor, cutting off callus and redundant leaves on the lower part of the plant, keeping about 2-3 cm of the upper half part of the plant, transferring the plant into an MS culture medium for subculture and culture, wherein the culture temperature is 25 +/-2 ℃, the illumination culture is carried out, the light source is a fluorescent lamp, the illumination intensity is 2000lx, and the light cycle is 16 h.

The invention has the following advantages:

1. adopts a brand new induction formula: the invention reselects the element proportion and hormone concentration combination of the culture medium, adjusts the dark culture time, and leads the immature ovule and anther which are not pollinated to directly develop into a new plant.

2. Separating and purifying polyploid by adopting a brand new explant: the invention utilizes the cell totipotency and the gene type source unicity of the plant ovule and the anther to directly utilize the ovule and the anther to obtain a regeneration plant with a single gene type. The regenerated plants are all diploid or tetraploid through detection of a cell flow instrument, and no chimera appears.

Detailed Description

The method of the embodiment of the invention comprises the following steps:

1) establishing an explant;

1.1) obtaining of explants: taking pear flower organs as explant sources; selecting 9712 as a material for embedding polyploid pears; selecting the bud and ovary which are not bloomed as the materials for establishing the explant 7 days before the full-bloom period of the pear (the specific date depends on the climatic conditions of the current year).

1.2) disinfection of explants: washing the picked pear ovaries at 25 ℃ for 6 hours by clear water; sterilizing with 70% alcohol for 30 s; rinsing with sterile water for 30 seconds, and repeating for 4 times; sterilizing with 15% sodium hypochlorite for 20 min, and shaking for 4 times; finally, rinsing with sterile water for 30 seconds, and repeating for 4 times.

1.3) preculture of explants: inoculating the pear ovaries on a PIM culture medium for pre-culture; the specific formula of the PIM culture medium is as follows: basic elements: 1/2QL medium (current commercial medium) supplemented with 24mg/LEDTA chelated iron, 250mg/L potassium nitrate, 97mg/L magnesium sulfate heptahydrate, 407mg/L ammonium nitrate; hormones: 0.2 mg/LIBA; carbon source: 30g/L sucrose; the pre-culture conditions are specifically as follows: culturing at 25 deg.C in dark for 4 days; the inoculation mode is specifically as follows: cutting the sterile bud and the ovary at the lower part into two parts along the radial direction in a sterile super clean bench, exposing anther and ovule, cutting off redundant pedicel, and sticking the cut surface downwards on a culture medium.

2) Inducing somatic embryos into seedlings; transferring the pre-cultured ovaries to an IM (instant Messaging) culture medium under the aseptic condition for culture, and inducing somatic embryos to differentiate into seedlings; the specific formula of the IM culture medium is as follows: basic elements: 1/2QL medium (current commercial medium) supplemented with 24mg/LEDTA chelated iron, 250mg/L potassium nitrate, 97mg/L magnesium sulfate heptahydrate, 407mg/L ammonium nitrate, 2.2 mg/LTDZ; hormones: 0.2 mg/LIBA; carbon source: 30g/L sucrose; the culture conditions for inducing somatic embryo differentiation into seedlings are as follows: culturing at 25 deg.C in dark for 60 days, and changing to new IM medium after culturing for 30 days.

3) Ploidy identification of seedlings: healthy leaves are selected, and after cells are dissociated by using a dissociation solution, the ploidy is identified by using a cell flow meter.

4) And (3) propagation of seedlings: after 4 weeks of seedling culture, selecting a tissue culture seedling with strong growth vigor, cutting off callus and redundant leaves on the lower part of the plant, keeping about 2-3 cm of the upper half part of the plant, transferring the plant into an MS culture medium for subculture and culture, wherein the culture temperature is 25 +/-2 ℃, the illumination culture is carried out, the light source is a fluorescent lamp, the illumination intensity is 2000lx, and the light cycle is 16 h.

The regenerated plants obtained by the method for separating and purifying the polyploids from the chimeric polyploids by utilizing the pear regeneration system are detected by a cell flow instrument, and the regenerated plants are all diploid or tetraploid without chimeras.

The present invention and the technical contents not specifically described in the above embodiments are the same as the prior art.

The present invention is not limited to the above-described embodiments, and the present invention can be implemented with the above-described advantageous effects.

The above embodiments are only specific embodiments disclosed in the present invention, but the scope of the present invention is not limited thereto, and the scope of the present invention disclosed in the present invention should be subject to the scope of the claims.

Claims

1. a method utilizing pear regeneration system to separate and purify polyploid from chimeric polyploid is characterized in that: the method comprises the following steps:

1) Establishment of explants;

1.1) Acquisition of explants: use pear flower organs as the source of explants;

1.2) Sterilization of explants;

1.3) Pre-culture of explants: inoculate pear ovary on PIM medium for pre-culture;

2) Induction of somatic embryos into seedlings: the pre-cultured ovary is transferred to IM medium under aseptic conditions for cultivation, and the somatic embryos are induced to differentiate into seedlings;

3) Ploidy identification of seedlings: select healthy leaves, use dissociation solution to dissociate cells, and identify ploidy with cytometer;

4) Propagation of seedlings.

2. the method for separating and purifying polyploid from chimeric polyploid by utilizing pear regeneration system according to claim 1, is characterized in that: in described step 1.1), selecting chimeric polyploid pear 9712 as material; selecting The unbloomed flower buds and ovary were used as the material for establishing explants 7 days before the full flowering period of pears.

3. The method for separating and purifying polyploid from chimeric polyploid by utilizing pear regeneration system according to claim 2, is characterized in that: the concrete steps of described step 1.2) are as follows: the picked pear ovary is heated at 25°C , rinse with clean water for 6 hours; shake and disinfect with 70% alcohol for 30 seconds; rinse with sterile water for 30 seconds, repeat 4 times; disinfect with 15% sodium hypochlorite for 20 minutes, shake 4 times during the period; finally rinse with sterile water for 30 seconds, Repeat 4 times.

4. the method that utilizes pear regeneration system to separate and purify polyploid from chimeric polyploid according to claim 3, is characterized in that: the specific formula of PIM medium in described step 1.3) is: basic element: 1 Macro and trace elements in /2QL medium, supplemented with 24mg/L EDTA chelated iron, 250mg/L potassium nitrate, 97mg/L magnesium sulfate heptahydrate, 407mg/L ammonium nitrate; hormones: 0.2mg/L IBA; carbon source: 30g/L sucrose.

5. the method for separating and purifying polyploid from chimeric polyploid by utilizing pear regeneration system according to claim 4, is characterized in that: in described step 1.3), the pre-cultivation condition is specifically: 25 degrees Celsius, dark culture 4 sky.

6. the method that utilizes pear regeneration system to separate and purify polyploid from chimeric polyploid according to claim 5, is characterized in that: in described step 1.3), inoculation mode is specially: in aseptic ultra-clean bench Divide the sterile flower bud and the lower ovary into two radially to expose the anther and ovule, cut off the excess pedicel, and stick the cut side down on the medium.

7. the method that utilizes pear regeneration system to separate and purify polyploid from chimeric polyploid according to claim 6, is characterized in that: the concrete formula of IM medium in described step 2) is: basic element: 1 Macro and trace elements in /2QL medium, supplemented with 24mg/L EDTA chelated iron, 250mg/L potassium nitrate, 97mg/L magnesium sulfate heptahydrate, 407mg/L ammonium nitrate, 2.2mg/L TDZ; hormones: 0.2mg/L L IBA; carbon source: 30 g/L sucrose.

8. The method for separating and purifying polyploids from chimeric polyploids utilizing pear regeneration system according to claim 7, characterized in that: in the step 2), the culturing conditions for inducing somatic embryo differentiation into seedlings are specifically: : 25 degrees Celsius, dark culture for 60 days, and replace with new IM medium after 30 days of culture.

9. The method for separating and purifying polyploid from chimeric polyploid by utilizing pear regeneration system according to claim 8, is characterized in that: the concrete steps of described step 4) are as follows: after culturing the seedling for 4 weeks, select For tissue culture seedlings with robust growth vigor, excise the lower callus and redundant leaves of the plant, keep the upper half of the plant about 2-3 cm, and transfer it into MS medium for sub-generation and culture. The light source is fluorescent lamp, the light intensity is 2000lx, and the photoperiod is 16h.