CN101971774A - Method for transforming hot-pepper nucleo-cytoplasmic interactive male-sterile line, homo-maintainer line and homo-restorer line - Google Patents
Method for transforming hot-pepper nucleo-cytoplasmic interactive male-sterile line, homo-maintainer line and homo-restorer line Download PDFInfo
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Abstract
The invention discloses a method for transforming a hot-pepper nucleo-cytoplasmic interactive male-sterile line, a homo-maintainer line and a homo-restorer line, which comprises the following steps of: hybridizing by using a hot-pepper selfing line as a male parent and the hot-pepper nucleo-cytoplasmic interactive male-sterile line as a female parent, simultaneously selfing a male parent plant, selecting sterile plants or other inflorescences in 4-50 corresponding male-parent filial single plants and hot-pepper filial generations for paired test crossing when in filial-generation fertility separation, selfing each male-parent single plant, and respectively processing according to the group fertility expression of test crossing generations to select the male-sterile line, the homo-maintainer line and the homo-restorer line. When in filial-generation group fertility separation, the invention not only can be used for transforming the hot-pepper nucleo-cytoplasmic interactive male-sterile line and the homo-maintainer line from the hot-pepper selfing line, but also can be used for breeding the homo-restorer line, expands the selecting space of the restored male parents of other homologous sterile lines and can be used for screening more excellent three-line combined varieties. A hot-pepper hybrid is produced by adopting a male-sterile three-line technology, all the complex pollination procedures are simplified, and the economic benefit is quite remarkable.
Description
Technical field
The present invention relates to the agricultural planting production method, particularly a kind of method that the transformation of capsicum inbred line is become nucleo-cytoplasmic interaction male sterile line, homotype maintenance line and homotype recovery system.
Background technology
Capsicum is the favourite a kind of solanaceous vegetables of Chinese, and along with the popularization of capsicum crossbreed and the raising of peasant household's planting benefit, area under cultivation is increasing.All the time, most capsicum crossbreed all are to pollinate by artificial hybridization to carry out seed production, and carry out seed production with common artificial hybridization pollinating method, not only want before the pollination will pollinate in the clear fruit of blowing, pollinating process mark, pollination also needs blowing clearly really to avoid the loss of nutrient after finishing, receive when planting and also will recognize the pollination mark, waste time and energy, and always be difficult to avoid the appearance of pseudostationary with manpower, this will influence the prestige of seed manufacturing enterprise, cause to a certain degree loss also can for planting base peasant household.Along with the raising day by day of cost of human resources, breeding cost also will restrict further developing of capsicum industrialization.Adopt capsicum nucleo-cytoplasmic interreaction male sterility system, maintenance line and recovery system (being called for short three is) to produce the capsicum crossbreed, under isolation condition preferably, only need directly to give open flower pollination to get final product.Because the three line method production of hybrid seeds simplified numerous and diverse pollination program, save the seed production and pollination labour cost, reduce the seed production cost, improve first generation of hybrid purity greatly and be subjected to the favor of domestic and international breeding man.
Seed selection capsicum nucleo-cytoplasmic interreaction male sterility system, maintenance line and recovery system, normally the key inbred line transformation of existing capsicum is become male sterile line and homotype maintenance line after, utilize the male sterile line screening to recover system again.Key inbred line of capsicum (making male parent) and the hybridization of nucleo-cytoplasmic interreaction male sterility system, male parent strain selfing simultaneously, 3 kinds of situations can appear in filial generation colony fertility usually: offspring Quan Keyu, offspring's complete sterility or fertility are separated.The processing method that forefathers' monograph and the paper of publishing have mainly been introduced the filial generation complete sterility or can have been educated entirely, seed selection male sterile line and maintenance line did not but have effective way when the fertility separation was appearred in filial generation.The inventor finds that in correlative study the probability that the fertility separation appears in filial generation reaches 83.33% or higher, in this case, finds a kind of method seed selection male sterile line of strong operability and maintenance line just to have bigger practical significance.Previous finding is thought: a kind is if having hold facility or recovery capability, hold facility and recovery capability no significant difference between its same kind or its strain individual plant.Therefore, the inventor handles according to conventional method: select 1-2 individual plant offspring of corresponding male parent and the sterile strain pairing backcrossing of filial generation, all as a result backcross progeny fertility continue to separate (through investigating sterile plant rate or rising, reduce or fearing), the offspring who repeatedly backcrosses is still like this, the situation of backcross progeny fertility complete sterility is difficult to occur, even the backcross progeny fertility is recovered entirely, cause and utilize the research work of key inbred line transformation male sterile line of capsicum and homotype maintenance line to enter the dead angle.This is a disaster to good key inbred line material transformation male sterile line and homotype maintenance line, can cause a lot of good key inbred lines can not select male sterile line and homotype maintenance line.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of transformation capsicum nucleo-cytoplasmic interreaction male sterility system, homotype maintenance line and homotype to recover the method for system, it can select capsicum nucleo-cytoplasmic interreaction male sterility system, homotype maintenance line and homotype simultaneously and recover system when the capsicum inbred line is separated with the filial generation fertility of nucleo-cytoplasmic interreaction male sterility system, to overcome the deficiencies in the prior art.
The present invention is achieved in that the capsicum inbred line is made male parent and capsicum nucleo-cytoplasmic interreaction male sterility system hybridizes as female parent, male parent strain selfing simultaneously, when the filial generation fertility is separated, select 4～50 male parent filial generation individual plants to make sterile strain or the paired test cross of its inflorescence in male parent and the capsicum filial generation, each male parent individual plant selfing, do to handle respectively according to three kinds of situations that test cross progeny population fertility will occur:
(1) test cross offspring complete sterility, then corresponding male parent is the maintenance line individual plant, select 1～2 individual plant of this male parent self-bred progeny in pairs continuous multi-generation is saturated with it and backcross, this male parent individual plant selfing simultaneously, final transformation goes out the nucleo-cytoplasmic interreaction male sterility system and the homotype maintenance line of this capsicum inbred line;
(2) test cross offspring Quan Keyu, then corresponding male parent is that the homotype recovery is an individual plant, the male parent selfing is reserved seed for planting and is homotype recovery system;
(3) test cross offspring fertility is separated, and screening or superseded continues to backcross.
Art designs of the present invention is:
Capsicum inbred line that will transformation is done male parent and capsicum nucleo-cytoplasmic interreaction male sterility system (doing maternal) hybridization, (the fertility separation appears in male parent self progeny in male parent strain selfing simultaneously, eliminate this combination and male parent thereof), make to handle respectively (offspring's fertility evaluation employing flower bud phase pollen microscopic examination * 160 times, microscopy adopts the IKI decoration method) according to three kinds of situations that filial generation colony fertility may occur:
1 filial generation colony complete sterility, then Dui Ying male parent is the maintenance line individual plant, select this male parent self progeny typical case individual plant continuous multi-generation is saturated in pairs with it and backcross, this male parent individual plant selfing simultaneously finally selects the nucleo-cytoplasmic interreaction male sterility system and the homotype maintenance line of this capsicum inbred line.
2 filial generation colonies can educate entirely, and then corresponding male parent is an individual plant for recovering, and the male parent selfing is reserved seed for planting to be and recovered system.
(this is a modal situation in the sterile line breeding process to the fertility separation of 3 filial generation colonies, can select capsicum nucleo-cytoplasmic interreaction male sterility system, homotype maintenance line and homotype simultaneously in order to following method and recover system), with the sterile strain in the filial generation or its inflorescence and the paired test cross of a corresponding male parent self progeny 4-50 individual plant (making male parent), each male parent individual plant selfing, do to handle respectively according to three kinds of situations that test cross progeny population fertility will occur:
⑴ test cross offspring complete sterility, then corresponding male parent is the maintenance line individual plant, select this male parent self progeny typical case individual plant continuous multi-generation is saturated in pairs with it and backcross, this male parent individual plant selfing simultaneously finally selects the nucleo-cytoplasmic interreaction male sterility system and the homotype maintenance line of this capsicum inbred line.
⑵ test cross offspring Quan Keyu, then corresponding male parent is that the homotype recovery is an individual plant, the male parent selfing is reserved seed for planting and is homotype recovery system.
⑶ test cross offspring fertility is separated, and screening or superseded continues to backcross.
For further checking the present invention, therefore carried out following test:
One, material and method
The male sterile line chilli seedling 2007A that introduces is little peppery type long red pepper, growth potential is strong, wide leaf, the vertical footpath of fruit 1O～12 ㎝, transverse diameter 2.0 cm, peel smooth, the fruit color depth is green, and open male sterile line corolla is roomy, the thick flower pesticide of also being longer than slightly of column cap, flower pesticide is wide flat, abortion is thorough, and flower bud phase microscopy WUHUAFEN grain belongs to the complete sterile type of typical WUHUAFEN.Kind (M8～M55 with 48 various types of inbred lines and introduction, comprise goat's horn green pepper, long red pepper, pimento and capsicum annum fasciculatum, all through above individual plant selection of four generations purifying) be male parent, with sterile strain hybridization, show according to the fertility of filial generation colony and to screen the maintenance line individual plant and recovery is an individual plant.It is pointed out that each hybrid combination plants the 3O strain at least, observe and identify whether decision needs further to backcross again so that make up colony's fertility.100 % are sterile for filial generation, select corresponding male parent self progeny's individual plant pairing backcrossing with it, and the combination plantation (more than 50 strains) as far as possible in a large number of backcrossing is to improve the accuracy of colony's sterile plant rate investigation, so that breeding of new sterile line launched further checking.Fertility is observed and is identified all employings range estimation back flower bud phase microscopy (bud bleaches) earlier, and microscopy adopts the IKI decoration method.
Two, seed selection process
Project research is shuttled back and forth in Kweiyang and Sanya and is carried out, to quicken breeding process.(other 6 show as 100% fertility restorer for M8A～M55A), carry out fertility at spring and summer in 2008 and identify, M11A complete sterility wherein, and all the other all show as fertility and separate in 48 hybrid combinations of winter in 2007 preparation.6 combinations that fertility is recovered entirely, its male parent then is that the recovery of this male sterile line is an individual plant, selects its offspring typical case individual plant selfing to reserve seed for planting to be and recovers system, so that new male sterile line carries out the screening that good recovery is after breeding; To the combination that complete sterility separates with fertility, choosing 2 sterile strains wherein are carried out to returning (survey) friendship, in the hope of screening and the dissimilar male sterile line of transformation with two fine individual plants of corresponding male parent self progeny.Sowed back (survey) in winter in 2008 and hand over 29 of generation combinations in Hainan, only two of M1l combinations of backcrossing show as 100% sterile, through saturated male sterile line and the homotype maintenance line of backcrossing and obtaining M11 in winter in 2009 of 4 generations, to name and be Y92A and Y92B, this male sterile line (maintenance line) is the goat's horn green pepper, little peppery, plant type is tall and big, growth potential is strong, and the field comprehensive disease resistance is good, and Y92A sterile plant rate and sterile degree all reach 100%, the Y92B pollen amount is big, and self-fruitfulness is good.Spring and summer in 2009 to the male sterile line Y92A of new seed selection carry out combining ability test (with 6 that select and remain recover system's preparation and recover combination), the winter in 2009 filtered out Y92A in the Sanya 2 good recoveries are SY09-3, X-1 and recover combination S Y09-3A and X-1A accordingly.
Because showing as fertility all the time with unmarried desirable A, separates the combination small fruit type capsicum annum fasciculatum PBC521A that repeatedly backcrosses, spring and summer in 2009, proceed pairing backcrossing with improved transfer method, it is PBC521C and to recover towards the unmarried desirable A of the unmarried desirable male sterile line of sky taper green pepper, the unmarried desirable B of homotype maintenance line, homotype be unmarried desirable C that the winter in the same year goes out in the Sanya transformation that male sterile line PBC521A, homotype maintenance line PBC521B towards lamb angle, sky green pepper PBC521, homotype recover.
Winter in 2009, recovering with the homotype of new seed selection was PBC521C and the unmarried desirable A of male sterile line, unmarried desirable C and the PBC521A crisscrossing newly bred in the Sanya, and spring and summer in 2010 is observed and identified that two combinations all show as fertility and recover entirely.
Three, the male sterile line of seed selection and homotype maintenance line morphological feature and pollen grain specific activity are
With Y92A and Y92B is example, in fertility in earlier stage, the male sterile line of seed selection and homotype maintenance line plant, except that the fertility difference, the equal indifference of other economical characters; Enter late growth stage, the male sterile line plant is than homotype maintenance line height, and the later stage highly differs and can reach more than one times, and the parthenocarpy phenomenon is arranged.Think, enter after the fruiting period, compare that a little less than the male sterile line reproductive growth, it is stronger to nourish and grow, just cause obviously to surpass maintenance line on late growth stage male sterile line plant height, degree of development, the branch amount with maintenance line.In addition, observe according to winter in 2009 and 2010 two generations of spring and summer and to find, Y92A viral diseases ability than Y92B a little less than, this is consistent with former research results.
The male sterile line floral organ of seed selection is compared with maintenance line, and flower pesticide shrinks, and reducing runs dry, the column cap elongation that attenuates, and anther dehiscence range estimation WUHUAFEN, this morphosis all is unfavorable for self-fertility, but the gynoecium fertility is normal, and the pollination energy is solid; The short and thick plumpness of the flower pesticide of maintenance line, anther dehiscence range estimation pollen amount is big.Iodination reaction shows: maintenance line pollen grain circle, and inclusion is enriched, and iodination reaction is navy blue, and self-fruitfulness is good, and male sterile line can not form pollen grain, and inclusion is thin, and iodination reaction is not painted, belongs to the complete sterile type of WUHUAFEN.
Four, the genetic mechanism of the male sterile line Y92A of seed selection
The hybridization of the male sterile line of seed selection and its maintenance line, it is 100% sterile that the offspring keeps, and according to the Sears Heredity theory, proves that the nuclear gene of male sterile line and maintenance line has identical recessive sterile gene; Maintenance line self progeny 100% can educate, and the cytoplasm that proves maintenance line has can educate gene, to sum up analyzes, and proves that the sterile of seed selection is the sterile type of nucleo-cytoplasmic interaction.Winter in 2008 in the Sanya to educating the F of combination entirely
2Carry out fertility for colony and separate statistics,, can educate the law of segregation that strain and sterile strain meet 3:1 through test.Illustrate that the male sterile line of introducing is the nucleo-cytoplasmic interreaction male sterility system (CMS) that is subjected to single recessive gene control.
Five, the stability of the male sterile line Y92A of seed selection
Male sterile line Y92A mixes microscopy through field observation and florescence in the transformation process of shuttling back and forth of Kweiyang and Sanya, sterile rate in isolation implant field and sterile degree all reach 100%; Late growth stage in the Sanya has the parthenocarpy phenomenon, but does not have seed mostly or seed does not have germination rate.Winter in 2008, male sterile line met with under the situation of continuous low temperature (14～20 ℃) flowering stage in the Sanya, and it is 100% sterile that microscopy all can reach, and illustrated that the male sterile line sterility of introducing has good stability.
Six, the homotype maintenance line of seed selection and homotype are recovered the economical character contrast of system
Economical character height unanimity, self-fruitfulness are all good.
Utilize the dissimilar male sterile line of capsicum inbred line and capsicum nucleo-cytoplasmic interreaction male sterility system hybridization transformation, when the filial generation fertility was separated, male sterile line, homotype maintenance line and the homotype of utilizing the present invention can select this capsicum inbred line were recovered system.When utilizing the hybrid vigour cross-breeding, the inbred line of an excellence often can be bred different hybrid combination as male parent with maternal the utilization simultaneously.If this inbred line can only transformation become male sterile line and homotype maintenance line, just can only be when the triple crossing breed breeding as the female parent utilization, it has just been lost as the value of male parent so.Under the prerequisite that the filial generation fertility is separated, the present invention not only can become male sterile line and homotype maintenance line with the capsicum inbred line transformation of excellence, also it transformation can be become homotype to recover system, this capsicum inbred line also can be used as and recovers male parent and continue to play a role like this, enlarge the selection space that other homology male sterile lines recover male parent, can breed how good triple crossing kind.Adopt the male sterility three line technology to produce the capsicum crossbreed, under isolation condition preferably, only need directly to give open flower pollination to get final product, simplified all numerous and diverse pollination programs.Utilize this method production of hybrid seeds, first generation of hybrid purity can reach 100%, saves pollination labour cost 80.77%, and whole seed production cost reduces by 33.07%, and economic benefit is very remarkable, has good industrial prospect.After realizing three series mating; because only needing male sterile line (doing maternal) and recovering is that (making male parent) carries out the production production of hybrid seeds; breeder parent (maintenance line) will obtain safekeeping, and breeding units can be protected the intellectual property of oneself effectively, also can further promote capsicum production of hybrid seeds commercialization.
Embodiment
Embodiments of the invention 1: transformation capsicum nucleo-cytoplasmic interreaction male sterility system, homotype maintenance line and homotype are recovered the method for system, winter in 2007 is in the Sanya, the capsicum nucleo-cytoplasmic interreaction male sterility system that does male parent and introduction towards lamb angle, sky green pepper PBC521 hybridizes, and male parent strain selfing is received respectively and planted; Spring and summer in 2008 is observed filial generation colony fertility and is separated in Kweiyang, select the sterile strain in 2 individual plants of male parent filial generation and the filial generation to carry out pairing backcrossing, and male parent strain selfing is received respectively and planted; Winter in 2008, two backcross progenies showed as colony's fertility separation once more; Spring and summer in 2009 selects the sterile strain in 20 male parent filial generation individual plants and the backcross progeny to carry out pairing backcrossing, and male parent strain selfing is received respectively and planted; Winter in 2009 observable backcross progeny the fertility situation: 3 backcross progeny complete sterilities are male sterile line PBC521A, and then corresponding male parent is homotype maintenance line PBC521B; 5 test cross offspring Quan Keyu, then corresponding male parent is that homotype is recovered system; 12 test cross offspring fertility are separated, and eliminate.
Embodiments of the invention 2: transformation capsicum nucleo-cytoplasmic interreaction male sterility system, homotype maintenance line and homotype are recovered the method for system, winter in 2007 is in the Sanya, the unmarried ideal of capsicum annum fasciculatum is done the capsicum nucleo-cytoplasmic interreaction male sterility system hybridization of male parent and introduction, and male parent strain selfing is received respectively and planted; Spring and summer in 2008 is observed filial generation colony in Kweiyang be that fertility is separated, and selects the sterile strain in 2 individual plants of male parent filial generation and the filial generation to carry out pairing backcrossing, and male parent strain selfing is received respectively and planted; Winter in 2008, two backcross progenies showed as colony's fertility separation once more; Spring and summer in 2009 selects the sterile strain in 4 male parent filial generation individual plants and the backcross progeny to carry out pairing backcrossing, and male parent strain selfing is received respectively and planted; Winter in 2009 observable backcross progeny the fertility situation: 1 backcross progeny complete sterility is the unmarried desirable A of male sterile line, and then corresponding male parent is the unmarried desirable B of homotype maintenance line; 2 test cross offspring Quan Keyu, then corresponding male parent is that homotype is recovered system; 1 test cross offspring fertility is separated, and eliminates.
Embodiments of the invention 3: transformation capsicum nucleo-cytoplasmic interreaction male sterility system, homotype maintenance line and homotype are recovered the method for system, and spring and summer in 2009 is done male parent and male sterile line Y92A hybridization with line green pepper inbred line X-1, and male parent strain selfing is received respectively and planted; Observing filial generation colony in winter in 2009 is that fertility is separated, and selects the inflorescence of 50 individual plants of male parent filial generation and the sterile strain of filial generation to carry out pairing backcrossing, and male parent strain selfing is received respectively and planted; The fertility situation of the observable backcross progeny of spring and summer in 2010: 5 backcross progeny complete sterilities, then corresponding male parent is the maintenance line individual plant, choosing wherein 1 strain in pairs continuous multi-generation is saturated with it backcrosses, male parent individual plant selfing simultaneously finally can select male sterile line X-1A and the homotype maintenance line X-1B of X-1; 39 test cross offspring Quan Keyu, then corresponding male parent is an individual plant for recovering, and selects wherein 1 strain selfing to reserve seed for planting and is homotype recovery system; 6 test cross offspring fertility are separated, and eliminate.
Claims (1)
1. transformation capsicum nucleo-cytoplasmic interreaction male sterility system, homotype maintenance line and homotype are recovered the method for system, it is characterized in that: the capsicum inbred line makes male parent and capsicum nucleo-cytoplasmic interreaction male sterility system hybridizes as female parent, male parent strain selfing simultaneously, when the filial generation fertility is separated, select 4～50 male parent filial generation individual plants to make sterile strain or the paired test cross of its inflorescence in male parent and the capsicum filial generation, each male parent individual plant selfing, do to handle respectively according to three kinds of situations that test cross progeny population fertility will occur:
(1) test cross offspring complete sterility, then corresponding male parent is the maintenance line individual plant, select 1～2 individual plant of this male parent self-bred progeny in pairs continuous multi-generation is saturated with it and backcross, this male parent individual plant selfing simultaneously, final transformation goes out the nucleo-cytoplasmic interreaction male sterility system and the homotype maintenance line of this capsicum inbred line;
(2) test cross offspring Quan Keyu, then corresponding male parent is that the homotype recovery is an individual plant, the male parent selfing is reserved seed for planting and is homotype recovery system;
(3) test cross offspring fertility is separated, and screening or superseded continues to backcross.
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| CN105075855A (en) * | 2015-09-22 | 2015-11-25 | 镇江瑞繁农艺有限公司 | Method for breeding male sterility restoring line of yellow sprout pepper cytoplasm |
| CN107372096A (en) * | 2017-08-14 | 2017-11-24 | 贵州省蚕业研究所 | A kind of method of quick breeding nucleo-cytoplasmic interreaction sterile line |
| CN107535352A (en) * | 2017-10-20 | 2018-01-05 | 青岛市农业科学研究院 | A kind of selection towards day type male sterile line chilli seedling |
| CN107787827A (en) * | 2016-08-31 | 2018-03-13 | 辽宁东亚农业发展有限公司 | A kind of spicy green pepper new variety selective breeding technology of precocious lower temperature resistance |
| CN108849483A (en) * | 2018-08-24 | 2018-11-23 | 贵州省蚕业研究所 | A kind of quick breeding method of capsicum CMS line |
| CN109156336A (en) * | 2018-08-20 | 2019-01-08 | 青岛农业大学 | A kind of selection of High color values and slow sweet tea pigment green pepper restorer of fading |
| CN111406644A (en) * | 2020-04-20 | 2020-07-14 | 山西省农业科学院蔬菜研究所 | Breeding method of nucleus-cytoplasm interactive male sterile line of cluster pod pepper and application of three-line matching system |
| CN111758564A (en) * | 2020-07-23 | 2020-10-13 | 贵州省蚕业研究所(贵州省辣椒研究所) | Cluster breeding method for pepper sterile line |
| CN114885831A (en) * | 2022-06-08 | 2022-08-12 | 三明市农业科学研究院 | Reverse pepper breeding method |
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| CN105075855A (en) * | 2015-09-22 | 2015-11-25 | 镇江瑞繁农艺有限公司 | Method for breeding male sterility restoring line of yellow sprout pepper cytoplasm |
| CN107787827A (en) * | 2016-08-31 | 2018-03-13 | 辽宁东亚农业发展有限公司 | A kind of spicy green pepper new variety selective breeding technology of precocious lower temperature resistance |
| CN107372096A (en) * | 2017-08-14 | 2017-11-24 | 贵州省蚕业研究所 | A kind of method of quick breeding nucleo-cytoplasmic interreaction sterile line |
| CN107535352A (en) * | 2017-10-20 | 2018-01-05 | 青岛市农业科学研究院 | A kind of selection towards day type male sterile line chilli seedling |
| CN107535352B (en) * | 2017-10-20 | 2019-12-31 | 青岛市农业科学研究院 | Breeding method of upturned chili male sterile line |
| CN109156336A (en) * | 2018-08-20 | 2019-01-08 | 青岛农业大学 | A kind of selection of High color values and slow sweet tea pigment green pepper restorer of fading |
| CN108849483A (en) * | 2018-08-24 | 2018-11-23 | 贵州省蚕业研究所 | A kind of quick breeding method of capsicum CMS line |
| CN111406644A (en) * | 2020-04-20 | 2020-07-14 | 山西省农业科学院蔬菜研究所 | Breeding method of nucleus-cytoplasm interactive male sterile line of cluster pod pepper and application of three-line matching system |
| CN111758564A (en) * | 2020-07-23 | 2020-10-13 | 贵州省蚕业研究所(贵州省辣椒研究所) | Cluster breeding method for pepper sterile line |
| CN114885831A (en) * | 2022-06-08 | 2022-08-12 | 三明市农业科学研究院 | Reverse pepper breeding method |
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Application publication date: 20110216 |