CN106561461A - Rapid cultivation method for octoploid homozygous tobacco plant - Google Patents
Rapid cultivation method for octoploid homozygous tobacco plant Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a rapid cultivation method for an octoploid homozygous tobacco plant. The method comprises the following steps: subjecting a tobacco seedling or a stem tip of a tobacco plant to chromosome doubling under in-vivo induction of colchicine; carrying out tissue culture on a variant plant or variant leaves; and detecting a regenerated plant so as to obtain the octoploid homozygous tobacco plant. The method provided by the invention guarantees the success rate of induction and is easy to operate and short in cultivation time.
Description
Technical field
The invention belongs to tobacco breeding technical field, it is related to a fast culture process for growing tobacco octoploid homozygous plants.
Background technology
Ploidy breeding is one of important directions of current plant breeding.Compared to common diploid material, polyploid is outside
See form, active constituent content, the adaptability aspect to adverse circumstance and all there is certain advantage.Segmental polypoid is due to single
The solid ability of property and be widely used in agricultural production.
Nicotiana tabacum L. for China some areas pillar industry, be also that part rural area is rely the chief crop got rich, to national Jing
The impact of Ji is huge.Tobacco breeding is mainly using the hybridization between different cultivars and many generation backcrossings or selfing.Due to breeding mode
Restriction and resource scarcity so that tobacco breeding faces huge bottleneck.The application of new breeding method has one side
Help the expansion of tobacco germplasm, on the other hand put into practice providing important reference to tobacco breeding.
The main kind for adopting is tetraploid tobacco bred in tobacco leaf production, and this is primarily due to Nicotiana tabacum L. for heterologous four times
Body species, i.e. chromosome set become 2n=4x=SSTT=48.Have no other materials for production report, in breeding research
Also it is less to refer to.To expand breeding resources, it is extremely necessary to formulate other ploidy materials.The acquisition of other ploidy materials can lead to
Cross Anther Culture, microspores culture and obtain monoploid, chromosome doubling obtains polyploid, and Different Ploidy intermolecular hybrid obtains polyploid
And aneuploid.Visible stain body doubles to occupy very important position in the acquisition of other ploidy materials.
Currently acquired plant chromosome doubles the method for plant various, such as：K cryogenic treatment, high-temperature process, chemical reagent
Process, Irradiation etc., wherein most commonly seen with chemical reagent process.It is the widest with Colchicine application in chemical reagent process
It is general.Carrying out processing method using Colchicine also has various, such as：Seed is processed, stem apex is processed, is processed tissue cultured seedling etc., first two
Although method is simple, mostly chimera is obtained, though later approach can obtain homozygosis polyploid, nothing need to be pre-build
Bacterium cultivating system, it is more time-consuming.
The content of the invention
It is an object of the invention to provide a fast culture process for growing tobacco octoploid homozygous plants, solves prior art
Present in problem, operation is simple, and the time is shorter.
The technical solution adopted in the present invention is：One fast culture process for growing tobacco octoploid homozygous plants, by Nicotiana tabacum L.
Seedling or tobacco plant stem apex Jing Colchicine live body induced chromosomes are doubled, and taking variation plant or variation blade carries out tissue training
Support, regeneration plant is detected, obtain octoploid plant.
Another technical scheme of the present invention is：One fast culture process for growing tobacco octoploid homozygous plants, tool
Body is followed the steps below：
Seed treatment：
Step 1, it is some to take tetraploid tobacco seed, pulls out after soaked overnight 8-10h in 30 DEG C of warm water, in 25 DEG C of moisturizings
Dark accelerating germination, to improve seed germination rate and sprout regularity；
Step 2, after seed shows money or valuables one carries unintentionally, is induced with 0.2% colchicine solution immersion 48h, makes chromosome doubling；This
When embryo be in fast growing period, be conducive to octoploid to induce；
Step 3, removes colchicine solution, and the seed that shows money or valuables one carries unintentionally is rinsed 3 times in distilled water, and is soaked 2 times, each 2h, with
Remove the Colchicine of residual in seed；
Step 4, the seed after cleaning cultivates 3-5d under 25 DEG C of illumination, treats that cotyledon is opened, and grows seedling；
Above 1-4 step is chromosome doubling induction.
Step 5, after cotyledon is fully opened, chooses and grows slow, short strong variation plant for material；
Step 6, the plant chosen is removed after kind of shell, in being wrapped in warp layer cloth, is pricked at gauze opening with fine rule, in
0.1% HgCl2Carry out surface sterilizing in solution, after sterile water wash 3 times proliferative induction culture medium MS+1.0mg/ is inoculated in
On L6-BA+0.2mg/L NAA；
Step 7, after 40d, treats that adventitious bud grows to 3-5cm, cuts adventitious bud in root media 1/2MS+2.0mg/L
In IBA, can take root after 7-10d；
Above 5-7 step is tissue culture.
Step 8, Ploidy Identification and transplanting：Cutting tip of a root flow cytometer carries out Ploidy Identification, and employing goes wall hypotonic
Flame seasoning carries out being transplanted after chromosome sectioning is confirmed, you can screening obtains octoploid plant.
Another technical scheme of the present invention is：One fast culture process for growing tobacco octoploid homozygous plants, tool
Body is followed the steps below：
Stem apex process：
Step 1, the blade for launching near the tobacco plant stem apex of fast-growth and will launch is plucked, it is ensured that Colchicum autumnale
Plain solution can immerse stem apex fast-growth part；0.2% Colchicine parcel stem apex 5d, plastic bag parcel Cotton Gossypii are dipped with Cotton Gossypii
Moisturizing；
Step 2, to process and remove Cotton Gossypii after 5d, treats stem apex free growth, after 7d, stem apex mounted blade；
Above 1-2 step is chromosome doubling induction.
Step 3, induction of learning from else's experience processes the blade that the plants stems tip position of simultaneously continued growth occurs metamorphosis, carries out surface
Sterilizing；Blade is wrapped in warp layer cloth, is pricked at gauze opening, in 0.1% HgCl with fine rule2Carry out surface in solution to go out
It is inoculated on proliferative induction culture medium MS+1.0mg/L 6-BA+0.2mg/L NAA after bacterium, sterile water wash 3 times；
Step 4, after 40d, treats that adventitious bud grows to 3-5cm, cuts adventitious bud in root media 1/2MS+2.0mg/L
In IBA, can take root after 7-10d；
Above 3-4 step is tissue culture.
Step 5, Ploidy Identification and transplanting：Cutting tip of a root flow cytometer carries out Ploidy Identification, and employing goes wall hypotonic
Flame seasoning carries out being transplanted after chromosome sectioning is confirmed, you can screening obtains octoploid plant.
The invention has the beneficial effects as follows after by the seed Germination And Seedling after process, the seedling that form slection state morphs carries out group
Knit culture, it is ensured that the success rate of induction；After stem apex growth a period of time after process, the tissue for taking morphological variation is organized
Culture, equally ensure that the success rate of induction；Tissue culture is carried out due to material process and after morphing, in tissue culture's bar
Under part, regeneration plant mostly is unicellular origin.Therefore, regeneration plant is the plant of homozygosis.
Description of the drawings
Fig. 1 is K326 octoploids (2n=8x=96) plant chromosome (DAPI dyeing) figure.
Fig. 2 a：K326 tetraploid flow cytomery results；Fig. 2 b：K326 octoploid flow cytomery results；
Fig. 2 c：K326 tetraploids and octoploid biased sample flow cytomery result.
Fig. 3 is octoploid (2n=8x=96) the plant chromosome of cloud and mist 87 (DAPI dyeing) figure.
Fig. 4 a：The tetraploid flow cytomery result of cloud and mist 87；Fig. 4 b：The octoploid flow cytomery of cloud and mist 87 is tied
Really；Fig. 4 c：The tetraploid of cloud and mist 87 and octoploid biased sample flow cytomery result.
Specific embodiment
With reference to the accompanying drawings and detailed description the present invention is described in detail.
One fast culture process for growing tobacco octoploid homozygous plants, first by tobacco seedling or tobacco plant stem apex Jing autumns
Narcissus element live body induced chromosome is doubled, and taking variation plant or variation blade carries out tissue culture, and regeneration plant is detected,
Obtain homozygosis octoploid plant.Specifically, including：The big step of chromosome doubling induction, tissue culture, Ploidy Identification and transplanting three
Suddenly.
Herein, the method for inducing again tissue culture using first chromosome doubling, has on the one hand ensured what material was hurt
Degree improves survival rate.On the other hand reduce and pre-build tissue culture system, for the method induced tissue cultured seedling with tradition,
The time is saved.
This method is had the advantage that compared with traditional method：
With tradition immersion tissue cultured seedling method compared with, this method take induced and occurred morphological variation young plant and
The blade of the field plant morphed Jing after induction.This has carried out primary election equivalent to the material after induction, improves and doubles
The success rate of plant screening.
With tradition immersion tissue cultured seedling method compared with, this method without pre-building tissue culture quick breeding system, in a short time
(2 months) can obtain octoploid plant.
Compared with the stem apex infusion method of field, this method carries out tissue training by the effective plant to morphological variation and blade
Support, because the regeneration plant under conditions of tissue culture mostly is unicellular origin, the Chromosome doubling plant for being obtained mostly is homozygosis
Body plant.
The present invention emphasis show money or valuables one carries unintentionally for seed after Jing colchicine-induced Hou Zai Jing tissue cultures seedling.
Embodiment 1
K326 octoploids are induced
Seed treatment：
(1) (germination rate is higher, and regularity is also higher) the K326 seed tobacco seeds for taking then are some, in warm water (30 DEG C)
Pull out after middle soaked overnight (8-10h), in 25 DEG C of moisturizing dark accelerating germination, to improve seed germination rate and sprout regularity.
(2) after seed shows money or valuables one carries unintentionally, induced with 0.2% colchicine solution immersion 48h, made chromosome doubling；Now
Embryo is in fast growing period, is conducive to octoploid to induce.
(3) colchicine solution is removed, the seed that shows money or valuables one carries unintentionally is rinsed 3 times in distilled water, and is soaked 2 times, each 2h, to remove
The Colchicine of residual in seed；
(4) seed after cleaning cultivates 3-5d under 25 DEG C of illumination, treats that cotyledon is opened, and grows seedling；
(5) after cotyledon is fully opened, choose and grow slow, short strong variation plant for material；
(6) plant chosen is removed after kind of shell, in being wrapped in warp layer cloth, is pricked at gauze opening, in 0.1% with fine rule
HgCl2Carry out surface sterilizing in solution, after sterile water wash 3 times proliferative induction culture medium (MS+1.0mg/L6-BA+ is inoculated in
0.2mg/L NAA) on；
(7) after 40d, treat that adventitious bud grows to 3-5cm, cut adventitious bud in root media (1/2MS+2.0mg/L
IBA in), can take root after 7-10d；
Above 5-7 step is tissue culture.
(8) Ploidy Identification and transplanting：Cutting tip of a root flow cytometer carries out Ploidy Identification, and the hypotonic flame of wall is removed in employing
Seasoning carries out being transplanted after chromosome sectioning is confirmed, you can screening obtains octoploid plant.
Embodiment 2
Stem apex process：
(1) blade for launching near the tobacco plant stem apex of fast-growth and will launch is plucked, it is ensured that Colchicine
Solution can immerse stem apex fast-growth part；0.2% Colchicine parcel stem apex 5d is dipped with Cotton Gossypii, parcel Cotton Gossypii protects plastic bag
It is wet；
(2) process after 5d and remove Cotton Gossypii, treat stem apex free growth, after 7d, stem apex mounted blade；
(3) induction of learning from else's experience processes the blade that the plants stems tip position of simultaneously continued growth occurs metamorphosis, carries out surface and goes out
Bacterium；Blade is wrapped in warp layer cloth, is pricked at gauze opening with fine rule, in 0.1% mercuric chloride aqueous solution surface sterilizing is carried out,
It is inoculated in proliferative induction culture medium (MS+1.0mg/L 6-BA+0.2mg/L NAA) after sterile water wash 3 times；
(4) after 40d, treat that adventitious bud grows to 3-5cm, cut adventitious bud in root media (1/2MS+2.0mg/L
IBA in), can take root after 7-10d；
(5) Ploidy Identification and transplanting：Cutting tip of a root flow cytometer carries out Ploidy Identification, and the hypotonic flame of wall is removed in employing
Seasoning carries out being transplanted after chromosome sectioning is confirmed, you can screening obtains octoploid plant.In this example, obtained
Plant octoploid ratio is up to 86% (detecting 43 plants) in 50 plants, and all homozygote plant of Jing flow cytomeries.
Fig. 1 K326 octoploids (2n=8x=96) plant chromosomes (DAPI dyeing).
Flow cytomery result (Fig. 2 a of Fig. 2 K326 octoploid plant：K326 tetraploid flow cytomeries
As a result；Fig. 2 b：K326 octoploid flow cytomery results；Fig. 2 c：K326 tetraploids and octoploid biased sample streaming are thin
Born of the same parents' instrument testing result.) result shows：The octoploid for being obtained is homozygous plants.
Embodiment 3
The octoploid of cloud and mist 87 is induced
Seed treatment：
(1) the 87 seed tobacco seeds of (germination rate is higher, and regularity is also higher) cloud and mist for taking then are some, in warm water (30
DEG C) in pull out after soaked overnight (8-10h), in 25 DEG C of moisturizings dark accelerating germination, to improve seed germination rate and sprout regularity.
(2) after seed shows money or valuables one carries unintentionally, induced with 0.2% colchicine solution immersion 48h, made chromosome doubling；Now
Embryo is in fast growing period, is conducive to octoploid to induce.
(3) colchicine solution is removed, the seed that shows money or valuables one carries unintentionally is rinsed 3 times in distilled water, and is soaked 2 times, each 2h, to remove
The Colchicine of residual in seed；
(4) seed after cleaning cultivates 3-5d under 25 DEG C of illumination, treats that cotyledon is opened, and grows seedling；
(5) after cotyledon is fully opened, choose and grow slow, short strong variation plant for material；
(6) plant chosen is removed after kind of shell, in being wrapped in warp layer cloth, is pricked at gauze opening, in 0.1% with fine rule
HgCl2Carry out surface sterilizing in solution, after sterile water wash 3 times proliferative induction culture medium (MS+1.0mg/L6-BA+ is inoculated in
0.2mg/L NAA) on；
(7) after 40d, treat that adventitious bud grows to 3-5cm, cut adventitious bud in root media (1/2MS+2.0mg/L
IBA in), can take root after 7-10d；
(8) Ploidy Identification and transplanting：Cutting tip of a root flow cytometer carries out Ploidy Identification, and the hypotonic flame of wall is removed in employing
Seasoning carries out being transplanted after chromosome sectioning is confirmed, you can screening obtains octoploid plant.
Embodiment 4
Stem apex process：
(1) blade for launching near the tobacco plant stem apex of fast-growth and will launch is plucked, it is ensured that Colchicine
Solution can immerse stem apex fast-growth part；0.2% Colchicine parcel stem apex 5d is dipped with Cotton Gossypii, parcel Cotton Gossypii protects plastic bag
It is wet；
(2) process after 5d and remove Cotton Gossypii, treat stem apex free growth, after 7d, stem apex mounted blade；
(3) induction of learning from else's experience processes the blade that the plants stems tip position of simultaneously continued growth occurs metamorphosis, carries out surface and goes out
Bacterium；Blade is wrapped in warp layer cloth, is pricked at gauze opening with fine rule, in 0.1% mercuric chloride aqueous solution surface sterilizing is carried out,
It is inoculated in proliferative induction culture medium (MS+1.0mg/L 6-BA+0.2mg/L NAA) after sterile water wash 3 times；
(4) after 40d, treat that adventitious bud grows to 3-5cm, cut adventitious bud in root media (1/2MS+2.0mg/L
IBA in), can take root after 7-10d；
(5) Ploidy Identification and transplanting：Cutting tip of a root flow cytometer carries out Ploidy Identification, and the hypotonic flame of wall is removed in employing
Seasoning carries out being transplanted after chromosome sectioning is confirmed, you can screening obtains octoploid plant.In this example, obtained
Plant octoploid ratio is up to 86% (detecting 43 plants) in 50 plants, and all homozygote plant of Jing flow cytomeries.
Octoploid (2n=8x=96) the plant chromosome of Fig. 3 clouds and mists 87 (DAPI dyeing).
Flow cytomery result (Fig. 4 a of the octoploid plant of Fig. 4 clouds and mists 87：The tetraploid flow cytometer of cloud and mist 87 is examined
Survey result；Fig. 4 b：The octoploid flow cytomery result of cloud and mist 87；Fig. 4 c：The tetraploid of cloud and mist 87 and octoploid biased sample
Flow cytomery result.) result shows：The octoploid for being obtained is homozygous plants.
Claims (3)
1. a fast culture process for growing tobacco octoploid homozygous plants, it is characterised in that by tobacco seedling or tobacco plant stem
Sharp Jing Colchicines live body induced chromosome is doubled, and taking variation plant or variation blade carries out tissue culture, and regeneration plant is entered
Row detection, obtains octoploid plant.
2. a fast culture process for growing tobacco octoploid homozygous plants, it is characterised in that specifically follow the steps below：
Seed treatment：
Step 1, it is some to take tetraploid tobacco seed, pulls out after soaked overnight 8-10h in 30 DEG C of warm water, in 25 DEG C of moisturizing dark
Accelerating germination, to improve seed germination rate and sprout regularity；
Step 2, after seed shows money or valuables one carries unintentionally, is induced with 0.2% colchicine solution immersion 48h, makes chromosome doubling；Now embryo
In fast growing period, octoploid is conducive to induce；
Step 3, removes colchicine solution, and the seed that shows money or valuables one carries unintentionally is rinsed 3 times in distilled water, and is soaked 2 times, each 2h, to remove
The Colchicine of residual in seed；
Step 4, the seed after cleaning cultivates 3-5d under 25 DEG C of illumination, treats that cotyledon is opened, and grows seedling；
Step 5, after cotyledon is fully opened, chooses and grows slow, short strong variation plant for material；
Step 6, the plant chosen is removed after kind of shell, in being wrapped in warp layer cloth, is pricked at gauze opening, in 0.1% with fine rule
HgCl2Carry out surface sterilizing in solution, after sterile water wash 3 times proliferative induction culture medium MS+1.0mg/L 6-BA+ is inoculated in
On 0.2mg/L NAA；
Step 7, after 40d, treats that adventitious bud grows to 3-5cm, cuts adventitious bud in root media 1/2MS+2.0mg/L IBA
In, can take root after 7-10d；
Step 8, Ploidy Identification and transplanting：Cutting tip of a root flow cytometer carries out Ploidy Identification, and the hypotonic flame of wall is removed in employing
Seasoning carries out being transplanted after chromosome sectioning is confirmed, you can screening obtains octoploid plant.
3. a fast culture process for growing tobacco octoploid homozygous plants, it is characterised in that specifically follow the steps below：
Stem apex process：
Step 1, the blade for launching near the tobacco plant stem apex of fast-growth and will launch is plucked, it is ensured that Colchicine is molten
Liquid energy immersion stem apex fast-growth part；0.2% Colchicine parcel stem apex 5d is dipped with Cotton Gossypii, parcel Cotton Gossypii protects plastic bag
It is wet；
Step 2, to process and remove Cotton Gossypii after 5d, treats stem apex free growth, after 7d, stem apex mounted blade；
Step 3, induction of learning from else's experience processes the blade that the plants stems tip position of simultaneously continued growth occurs metamorphosis, carries out surface and goes out
Bacterium；Blade is wrapped in warp layer cloth, is pricked at gauze opening, in 0.1% HgCl with fine rule2Surface sterilizing is carried out in solution,
It is inoculated on proliferative induction culture medium MS+1.0mg/L 6-BA+0.2mg/L NAA after sterile water wash 3 times；
Step 4, after 40d, treats that adventitious bud grows to 3-5cm, cuts adventitious bud in root media 1/2MS+2.0mg/L IBA
In, can take root after 7-10d；
Step 5, Ploidy Identification and transplanting：Cutting tip of a root flow cytometer carries out Ploidy Identification, and the hypotonic flame of wall is removed in employing
Seasoning carries out being transplanted after chromosome sectioning is confirmed, you can screening obtains octoploid plant.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107750514A (en) * | 2017-11-06 | 2018-03-06 | 河南省农业科学院烟草研究所 | The one quick method for doubling for growing tobacco polyploid |
| CN116941529A (en) * | 2023-07-26 | 2023-10-27 | 湖北省烟草科学研究院 | Tobacco haploid plant doubling and rapid propagation integrated method |
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| US7102056B1 (en) * | 1997-04-29 | 2006-09-05 | The Regents Of The University Of California | Compositions and methods for plant transformation and regeneration |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107750514A (en) * | 2017-11-06 | 2018-03-06 | 河南省农业科学院烟草研究所 | The one quick method for doubling for growing tobacco polyploid |
| CN116941529A (en) * | 2023-07-26 | 2023-10-27 | 湖北省烟草科学研究院 | Tobacco haploid plant doubling and rapid propagation integrated method |
| CN116941529B (en) * | 2023-07-26 | 2024-05-17 | 湖北省烟草科学研究院 | Tobacco haploid plant doubling and rapid propagation integrated method |
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