CN101878734B - Method for constructing collard cytoplasmic male sterility near-isogenic lines - Google Patents
Method for constructing collard cytoplasmic male sterility near-isogenic lines Download PDFInfo
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- CN101878734B CN101878734B CN2010102119695A CN201010211969A CN101878734B CN 101878734 B CN101878734 B CN 101878734B CN 2010102119695 A CN2010102119695 A CN 2010102119695A CN 201010211969 A CN201010211969 A CN 201010211969A CN 101878734 B CN101878734 B CN 101878734B
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Abstract
The invention discloses a method for constructing collard cytoplasmic male sterility near-isogenic lines, which is characterized by comprising the following steps of: A, selecting the variety of collard, performing selfing to the ninth generation, and using the ninth generation as a receptor; B, selecting a cytoplasmic male sterile Chinese cabbage selfed line as an CMS donor; C, using the donor as a female parent, using the receptor as a male parent, performing interspecific hybridization to obtain interspecific hybrid, and successfully transforming the CMS genes to collard; D, performing authenticity identification on the interspecific hybrid; E, using the interspecific hybrid in the interspecific hybridization as a female parent, using a receptor collard selfed line as a recurrent parent, and performing back crossing to the sixth generation to breed a collard cytoplasmic male sterility line which forms a pair of near-isogenic lines with the recurrent male parent; and F, analyzing the near-isogenic property between the back-crossed sixth generation and the recurrent parent. The invention provides a method for constructing collard cytoplasmic male sterility near-isogenic lines, obtains the collard cytoplasmic male sterility near-isogenic lines, and provides a parent material for producing advantageous hybrid of the collard.
Description
Technical field
The invention belongs to technical field of flower breeding, be specifically related to a kind of construction method of collard cytoplasmic male sterility near-isogenic lines.
Background technology
Kale (Brassica oleracea var.acephala L) belongs to flowers for the Cruciferae rape, and it is littoral to originate in Mediterranean.Kale rosette stage center vane color is gorgeous, and leaf gauffer is colourful, and cold resistance is strong, has important economy and ornamental value.
Kale is a cross pollinated plant, and hybrid vigour is obvious.When tying up to the kale hybrid seeding, kale male sterile can exempt artificial emasculation, and not only time saving and energy saving, can also guarantee the purity of crossbreed, significant for the heterosis utilization of kale.Utilizing the male sterile line production of hybrid seeds is one of main path of utilizing of the vegetables oil plant crop heterosis that belongs to together.
(cytoplasmic male sterility CMS) can not produce normal pollen to cytoplasmic male sterile line, but can be used as the hybridization female parent, gives the pollination of male sterile plant gynoecium with educating strain pollen, can be normally solid.The economic organ of kale is nutrition organs (leaf in rosette stage), but not seed, so in its crossbreed produces, need not to recover system.Therefore, the cultivation of collard cytoplasmic male sterility system and homotype maintenance line thereof can make up complete kale crossbreed production system, and this has great importance on simplification kale heterosis breeding.
(near isogenic line NIL) is meant except only a few proterties or gene NIL, two genetic stockss that other proterties or gene are all identical.Collard cytoplasmic male sterility system promptly can be a pair of NIL with its homotype maintenance line.Collard cytoplasmic male sterility near-isogenic lines is except that male fertile, two parts of genetic stockss that other proterties are all identical.Have only to obtain except that the male fertile proterties, a pair of genetic stocks that other proterties are all consistent just deserves to be called a pair of male sterility near-isogenic lines.
To sum up visible, making up collard cytoplasmic male sterility near-isogenic lines is the key that realizes kale two-line male sterile method heterosis utilization.The structure of collard cytoplasmic male sterility near-isogenic lines can provide parent material for kale advantage crossbreed produces.Do not see at present the method that makes up collard cytoplasmic male sterility near-isogenic lines as yet.
Summary of the invention
The construction method that the purpose of this invention is to provide a kind of collard cytoplasmic male sterility near-isogenic lines makes and can construct collard cytoplasmic male sterility near-isogenic lines.
Technical scheme provided by the invention is following:
A. receptor parent is cultivated: choose the kale range of goods, carry out the selfing purifying, to selfing the 9th generation (F
9), breed the kale inbred line, as receptor parent.
B. donor parents is prepared: choose and belong to vegetable crop Chinese cabbage cytoplasmic male sterility (CMS) inbred line together as the CMS donor parents, this donor parents is carried cytoplasmic male sterile gene.
C. interspecific cross, with cytoplasmic male sterile gene to the kale transformation: as female parent, as male parent, carry out interspecific cross with donor parents Chinese cabbage inbred line with receptor parent kale inbred line.
For overcoming interspecific cross compatibility obstacle, employing begins repeatedly to repeat pollination from the flower bud phase and solves, so that effectively obtain CMS species hybrid F
1, will be positioned at the CMS donor--the CMS gene success transformation on the Chinese cabbage is to the receptor parent kale.
D. species hybrid F
1Authenticity identification: because in the interspecific hybridization offspring; Pseudostationary possibly appear; Be necessary the interspecific cross offspring is identified, confirm the authenticity of interspecific hybrid, so that next step carries out saturated backcrossing with true hybrid between planting and recurrent parent (receptor parent).Species hybrid F
1Authenticity identification choose typical proterties as appraisal basis.
E. directed saturated backcrossing: with the cytoplasmic male sterility species hybrid F that obtains in the interspecific cross
1Be female parent, as the samsara male parent, backcross, the kale economic characters are pursued for transformation to CMS species hybrid, be returned to the 6th generation (BC with receptor parent kale inbred line
6), breed collard cytoplasmic male sterility system, this male sterile line and samsara male parent (kale inbred line) constitute a pair of NIL.
F. the property analysis such as near of NIL: choose 13 phenotypic characters, at the 6th generation (BC that backcrosses
6) and receptor parent between carry out property analyses such as near.
Good effect of the present invention is: set up a kind of method that makes up collard cytoplasmic male sterility near-isogenic lines, and obtained collard cytoplasmic male sterility near-isogenic lines, for kale advantage crossbreed produces parent material is provided.
Description of drawings
Fig. 1 is the cultivating process sketch map of receptor parent k13.
Fig. 2 is that collard cytoplasmic male sterility near-isogenic lines k13 and CMSk13 make up flow chart.
Fig. 3 is the cultivating process sketch map of receptor parent k10.
Fig. 4 is that collard cytoplasmic male sterility near-isogenic lines k10 and CMSk10 make up flow chart.
Fig. 5 is the cultivating process sketch map of receptor parent k04.
Fig. 6 is that collard cytoplasmic male sterility near-isogenic lines k04 and CMSk04 make up flow chart.
Embodiment
Embodiment 1: successfully made up kale male sterility near-isogenic lines k13 and CMSk13.
Concrete grammar is following:
A. receptor parent is cultivated: see Fig. 1, choose kale range of goods 0113, carry out the selfing purifying, to selfing the 9th generation (F
9), breed kale inbred line k13, highly neat between inbred line k13 strain system.The typical proterties of inbred line k13 is seen table 1.With inbred line k13 as receptor parent.
Table 1 receptor parent k13, donor parents A02 and species hybrid F thereof
1 (A02-k13)The comparison of 11 typical proterties
B. donor parents is prepared: choose cytoplasmic male sterility Chinese cabbage inbred line A02 as the CMS donor parents.The typical proterties of inbred line A02 is seen table 1.
C. interspecific cross: see Fig. 2, as female parent, as male parent, carry out interspecific cross with receptor parent k13 with donor parents A02.
Because parents are edge relation far away, exist cross compatibility obstacle to a certain degree, conventional florescence pollination can not be solid.
For solving this cross compatibility obstacle, carried out following solution:,, column cap is exposed with tweezers strip off bud in little the flowers are in blossom preceding 2 days of putting of maternal A02; The mature pollen of male parent k13 is applied on the column cap of maternal A02; After this, every morning is smeared pollination 1 time, pollinates continuously 3-4 days.This method has obtained fully-developed interspecific cross seed.
D. species hybrid F
1 (A02-k13)Authenticity identification: see table 1, with 11 typical proterties as identification of indicator.Through identifying species hybrid F
1 (A02-k13)The morphological feature (table 1) that has possessed parents.In 11 typical proterties, leaf table wax powder, the plant type in rosette stage, 3 proterties of lobus cardiacus color show as paternal form; Kernel seed coat colour, hypocotyl color, vein color, siphonal lobe color, leaf, 6 proterties of chromosome number of somatic show as parents' intermediate form; Staminody whether and 2 proterties of fertility show as maternal A02 type.Can assert that thus the interspecific cross offspring who obtains in the interspecific cross is the real hybrid between parents A02 and k13.The CMS gene success transformation that so far, will be positioned on the donor Chinese cabbage A02 has been arrived on the receptor parent kale inbred line k13.
In the evaluation of above-mentioned 11 typical proterties, the 1-10 proterties is a phenotypic character, adopts ocular estimate to identify.
The 11st proterties is a chromosome number of somatic, adopts the tip of a root pressed disc method of in-vitro inducing root to identify.The breeding method step of in-vitro inducing root is following:
(1) giving birth to tender stem with the armpit of hybrid seedling is explant; After 0.1% mercuric chloride sterilization 5 minutes; Being seeded in minimal medium is to induce newborn indefinite bud on MS, the medium that adds 2mg/L 6-benzyl aminoadenine and 0.2mg/L methyl; Condition of culture is 23-25 ℃, and intensity of illumination is 1000lx, illumination every day 16h.
(2) induce one month after, the newborn indefinite bud that induces is transferred on the MS minimal medium of additional 1mg/L 6-benzyl aminoadenine and 0.1mg/L methyl, carry out inducing of root system.
When (3) treating root length, cut the tip of a root for 0.5-0.8cm.At ambient temperature; With the tip of a root with saturated right-dichloro-benzenes aqueous solution preliminary treatment 2-5h; Flushing back is with the fixing 10-15h of Kano fixer (volume ratio according to 95% ethanol and glacial acetic acid is preparation in 3: 1), use dissociation solution (preparing) 10-15min that dissociates again according to 37.5% concentrated hydrochloric acid and equal-volume 95% ethanol after, after dyeing 1 minute with carbolfuchsin dyeing liquor (preparing) according to conventional method; Free-hand compressing tablet is examined under a microscope the counting chromosome number.
E. directed saturated backcrossing: see Fig. 2, with the cytoplasmic male sterility species hybrid F that obtains in the interspecific cross
1Being female parent, is the samsara male parent with k13, backcrosses, and the kale economic characters are pursued for transformation to cytoplasmic male sterility species hybrid, is returned to for the 6th generation, obtains BC
6 (k13)
F. the property analysis such as near of NIL: see table 2, table 3, choose 13 phenotypic characters, to BC
6 (k13)Property analyses such as near have been carried out with k13.
Investigation and measuring method: in the analysis of 13 phenotypic characters, kernel seed coat colour, hypocotyl color, vein color, siphonal lobe color, 5 nonumeric proterties of lobus cardiacus color adopt the colorimetric card (the 5th edition) compare (table 2) of The Royal Horticultural Society (RHS); Leaf table wax powder, 2 nonumeric proterties of plant type adopt ocular estimate; Plant height, siphonal lobe degree of development, 3 numerical value proterties of lobus cardiacus degree of development adopt tape measure; The outer number of sheets, lobus cardiacus number, several 3 the numerical value proterties direct count of leaf margin fold (table 3).Each proterties is at BC
6 (k13)The picked at random individual plant is investigated and is measured in strain system and the k13 strain system.6 numerical value proterties are chosen 15 individual plants at every turn, average, and repeat 3 times, carry out significance of difference analysis.
Table 2 BC
6 (k13)And the comparison of 7 nonumeric phenotypic characters between the k13
Table 3 BC
6 (k13)And the comparison of 6 numerical value phenotypic characters between the k13
The result shows, in 13 phenotypic characters, and BC
6 (k13)7 nonumeric proterties identical (seeing table 2) with k13; 6 numerical value proterties difference is not significantly (seeing table 3) all.
In addition, except that above-mentioned 13 phenotypic characters, BC
6 (k13)Identical with the stamen proterties of donor parents A02, i.e. staminody, male sterile rate and sterile degree are 100%.
This shows BC
6 (k13)Phenotype and recurrent parent k13 basic identical, with BC
6 (k13)Name and be that CMSk13, CMSk13 are a new kale male sterile line, k13 is the homotype maintenance line of CMSk13.So far, k13 and CMSk13 have constituted a pair of NIL.
Embodiment 2: successfully made up kale male sterility near-isogenic lines k10 and CMSk10.
Concrete grammar is following:
A. receptor parent is cultivated: see Fig. 3, choose kale range of goods 0113, carry out the selfing purifying,, breed kale inbred line k10 to the 9th generation of selfing, and highly neat between inbred line k10 strain system.The representative configuration characteristic of inbred line k10 is seen table 4.With inbred line k10 as receptor parent.
Table 4 receptor parent k10, donor parents A02 and species hybrid F thereof
1 (A02-k10)The comparison of 11 typical proterties
B. donor parents is prepared: choose cytoplasmic male sterility Chinese cabbage inbred line A02 as the CMS donor parents.The representative configuration characteristic of inbred line A02 is seen table 4.
C. interspecific cross: see Fig. 4, as female parent, as male parent, carry out interspecific cross with receptor parent k10 with donor parents A02.Hybridization pollination method between A02 and k10 is with the C item in the instance 1.Obtained the interspecific cross seed between fully-developed A02 and k10 in this example.
D. species hybrid F
1 (A02-k10)Authenticity identification: see table 4, through the evaluation of 11 typical proterties, species hybrid F
1 (A02-k10)The representative configuration characteristic that has possessed parents A02 and k10.The authentication method of 11 typical proterties of species hybrid offspring is with instance 1.Through identifying, the interspecific cross offspring who obtains in this routine C item is real A02 and the hybrid between k10.The CMS gene success transformation that so far, will be positioned on the donor Chinese cabbage A02 has been arrived on the receptor parent kale inbred line k10.
E. directed saturated backcrossing: see Fig. 4, with the cytoplasmic male sterility species hybrid F that obtains among this routine C
1 (A02-k10)Being female parent, is the samsara male parent with k10, backcrosses, and the kale economic characters are pursued for transformation to cytoplasmic male sterility species hybrid, is returned to for the 6th generation, obtains BC
6 (k10)
F. the property analysis such as near of NIL: see table 5, BC
6 (k10)And 7 nonumeric phenotypic characters between the k10 are identical; See table 6, BC
6 (k10)And 6 numerical value proterties differences between k10 are all not remarkable.The investigation of property such as 13 phenotypic characters are near and measuring method are with instance 1.
Table 5 BC
6 (k10)And the comparison of 7 nonumeric phenotypic characters between the k10
Table 6 BC
6 (k10)And the comparison of 6 numerical value phenotypic characters between the k10
In addition, except that above-mentioned 13 phenotypic characters, BC
6 (k10)Identical with the stamen proterties of donor parents A02.
This shows BC
6 (k10)Phenotype and recurrent parent k10 basic identical, with BC
6 (k10)Name and be that CMSk10, CMSk10 are a new kale male sterile line, k10 is the homotype maintenance line of CMSk10.So far, k10 and CMSk10 have constituted a pair of NIL.
Embodiment 3: successfully made up kale male sterility near-isogenic lines k04 and CMSk04.
Concrete grammar is following:
A. receptor parent is cultivated: see Fig. 5, choose kale range of goods 0104, carry out the selfing purifying,, breed kale inbred line k04 to the 9th generation of selfing, and highly neat between inbred line k04 strain system.The representative configuration characteristic of inbred line k04 is seen table 7.With inbred line k04 as receptor parent.
B. donor parents is prepared: choose cytoplasmic male sterility Chinese cabbage inbred line A02 as the CMS donor parents.The representative configuration characteristic of inbred line A02 is seen table 7.
C. interspecific cross: see Fig. 6, as female parent, as male parent, carry out interspecific cross with receptor parent k04 with donor parents A02.Hybridization pollination method between A02 and k04 is with the C item in the instance 1.
D. species hybrid F
1 (A02-k04)Authenticity identification: see table 7, through the evaluation of 11 typical proterties, species hybrid F
1 (A02-k04)The representative configuration characteristic that has possessed parents A02 and k04.The authentication method of 11 typical proterties of species hybrid offspring is with instance 1.Through identifying, the interspecific cross offspring who obtains in this routine C item is real A02 and the hybrid between k04.Wherein, proterties 1,3,4,5,7 shows as paternal form, and proterties 6,8,11 shows as type between parents' kind, and proterties 9,10 shows as maternal form, proterties 2 indifference between parents.The CMS gene success transformation that so far, will be positioned on the donor Chinese cabbage A02 has been arrived on the receptor parent kale inbred line k04.
Table 7 receptor parent k04, donor parents A02 and species hybrid F thereof
1 (A02-k04)The comparison of 11 typical proterties
E. directed saturated backcrossing: see Fig. 6, with the cytoplasmic male sterility species hybrid F that obtains among this routine C
1 (A02-k04)Be female parent, backcross for the samsara male parent, the kale economic characters are pursued for transformation to cytoplasmic male sterility species hybrid, be returned to the 6th generation (BC with k04
6).
F. the property analysis such as near of NIL: see table 8, BC
6And 7 nonumeric phenotypic characters are identical between k04; See table 9, BC
6 (k04)And 6 numerical value proterties differences between k04 are all not remarkable.The investigation of property such as 13 phenotypic characters are near and measuring method are with instance 1.
Table 8 BC
6 (k04)And the comparison of 7 nonumeric phenotypic characters between the k04
Table 9 BC
6 (k04)And the comparison of 6 numerical value phenotypic characters between the k04
In addition, except that above-mentioned 13 phenotypic characters, BC
6 (k04)Identical with the stamen proterties of donor parents A02.
This shows BC
6 (k04)Phenotype and recurrent parent k04 basic identical, with BC
6 (k04)Name and be that CMSk04, CMSk04 are a new kale male sterile line, k04 is the homotype maintenance line of CMSk04.So far, k04 and CMSk04 have constituted a pair of NIL.
Claims (1)
1. the construction method of a collard cytoplasmic male sterility near-isogenic lines is characterized in that:
A, receptor parent cultivate: choose the kale range of goods, carry out the selfing purifying, to the 9th generation of selfing, breed the kale inbred line, as receptor parent;
B, donor parents are prepared: choose cytoplasmic male sterility Chinese cabbage inbred line as the CMS donor parents;
C, interspecific cross: as female parent, as male parent, carry out interspecific cross with donor parents, adopt to begin repeatedly to repeat pollinating method from the flower bud phase and obtain species hybrid with receptor parent, with the CMS gene to kale success transformation;
D, species hybrid F
1Authenticity identification: be the pseudostationary that rejecting in orientation is backcrossed possibly occur, choose 11 typical proterties and carry out species hybrid F
1Authenticity identification; 11 typical proterties are kernel seed coat colour, hypocotyl color, vein color, leaf table wax powder, rosette stage plant type, rosette stage siphonal lobe color, rosette stage lobus cardiacus color, the rosette stage is leaf, whether stamen degenerates, stamen fertility, chromosome number of somatic;
E, directed saturated backcrossing: with the species hybrid F that obtains in the interspecific cross
1Be female parent, as recurrent parent, be returned to for the 6th generation with receptor parent kale inbred line, breed collard cytoplasmic male sterility system, this male sterile line and samsara male parent constitute a pair of NIL;
The property analysis such as near of F, NIL: choose 13 phenotypic characters as the Analysis and Identification index, analyze BC
6And the property such as near between recurrent parent; 13 phenotypic characters comprise 7 nonumeric phenotypic characters; Be kernel seed coat colour, cotyledon period hypocotyl color, rosette stage vein color, rosette stage siphonal lobe color, rosette stage lobus cardiacus color, leaf table wax powder, rosette stage plant type and comprise 6 numerical value phenotypic characters, be i.e. plant height, siphonal lobe degree of development, lobus cardiacus degree of development, the outer number of sheets, lobus cardiacus number, leaf margin fold number.
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| CN103461103B (en) * | 2013-09-11 | 2015-09-16 | 镇江瑞繁农艺有限公司 | A kind of wave leaf views and admires the breeding method of kale |
| CN108362649A (en) * | 2017-12-18 | 2018-08-03 | 温州科技职业学院 | A method of the detection purity of hybrid of the color based on Cauliflower hypocotyl |
| CN109662025A (en) * | 2018-12-29 | 2019-04-23 | 北京市农林科学院 | A kind of breeding method of colour cabbage new germ plasm |
| CN116649207A (en) * | 2023-06-20 | 2023-08-29 | 魏保权 | Dynamic polymerization hybrid crop breeding method |
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