CN103718957B - The haploid method of male sterile tobacco is cultivated by Unfertilized Ovules - Google Patents
The haploid method of male sterile tobacco is cultivated by Unfertilized Ovules Download PDFInfo
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Abstract
The invention provides a kind of by the haploid method of Unfertilized Ovules cultivation male sterile tobacco.The haploid method of described cultivation male sterile tobacco is the male sterile tobacco bud of the not pollinating ovule of growing between megasporocyte period to two core phases is culture materials, after disinfecting in 28 DEG C of illumination cultivation rooms, cultivate through embryoid induction successively, cultivate after the cultivation of regeneration bud culture of rootage and regeneration plant and form, cross the every day of continuous illumination ten hours under 1500Lux illumination condition in incubation.Adopt method of the present invention to cultivate male sterile tobacco, significantly improve the induction of tobacco ovule embryoid and the frequency of plant regeneration.
Description
Technical field
The invention belongs to tobacco breeding technical field, be specifically related to be cultivated by Unfertilized Ovules obtain the haploid method of male sterile tobacco hybridization kind.
Background technology
Monoploid refers to the sporophyte with gametic chromosome composition.Haploid induction technology is as the chief component of modern efficient breeding system, there is following remarkable advantage: after crop monoploid material doubles, be pure lines (double haploid), excellent middle select excellent after can to isozygoty fast the favorable genes site of Cross Combinations, thus make the breeding time limit comparatively conventional method shorten 3 ~ 4 years; Secondly, due to the genotype of double haploid and phenotype completely the same, greatly can reduce when screening breeding material and falsely drop frequency, significantly improve efficiency of selection, particularly with the obvious advantage especially when selecting the merit controlled by recessive gene.In addition, utilize monoploid to cultivate and can obtain doubled haploid (DH) colony fast, can be the ideal material carrying out crop Main Agronomic Characters molecular labeling, and utilize molecular labeling to carry out chief component that assisted Selection is modern efficient breeding system (positive .2007. crops haploid breeding research overview and thinking. Shandong agricultural science, NO.5.122-125).
Tobacco is economic crops important in the world.Usually, can by the flower pesticide of culture in vitro tobacco or microspore Haploid production, i.e. the monoploid of flower pesticide origin.The nineties in 20th century, some tobacco scientific research institutions of the U.S., particularly large quantity research has done in Tobacco Genetic Breeding project team of Bei Ka state university, often yield and quality characters deterioration is serious after doubling to find this kind of monoploid, the value of breeding utilization is limited, add this kind of haploid double frequency very low, usually about 1% is only had, current this method seldom uses (Ren Xueliang in tobacco breeding, Li Jixin, Li Minghai .2007. American Tobacco Advances in Breeding overview. Chinese tobacco journal, NO.6.57-64).And from Unpollinated Ovaries of Nicotiana tabacum L induction haplobiont, there is obvious advantage, as ploidy and character variation little, offspring is more stable, closer to (Pan Li such as original parents, Yang Tie encourages the research of .2000. Unpollinated Ovaries of Nicotiana tabacum L embryoid induction. northwest Botany Gazette, NO.1.59-63).
The people such as foreign scholar Wernsman just started Unpollinated Ovaries of Nicotiana tabacum L training as far back as 1979, and have successfully been obtained haplobiont (BurkLG, GerstelDU, WernsmanEA.1979.MaternalHaploidsofNicotianatabacumL.from Seed.Science., NO.2.585).Double haploid after tobacco Genogenetic haploid and male doubling monoploids and their source parent compare by they, find that female double haploid is better than male double haploid, female double haploid is more similar to source parent, deterioration (the WernsmanEA of yield and quality can not be caused, MatzingerDF, RebecaCRufty.1989.Androgeneticvs.GynogeneticDoubledHaplo idsofTobacco.CropSci, NO.29.1151-1155).At present, they this technology to be successfully applied in tobacco breeding (Ren Xueliang, Li Jixin, Li Minghai .2007. American Tobacco Advances in Breeding overview. Chinese tobacco journal, NO.6.57-64).But this method is not directly utilize ovary or Ovule development to obtain monoploid, but by Nicotiana gossei N.africana as parent sire of hybrid pigs, obtain a large amount of high germination power seeds, filial generation seedling great majority are dead at cotyledon stage, the seedling of survival is the parent monoploid being easy to differentiate, this kind of haploid petiole culture in vitro is utilized to be easy to realize haploid Natural double, and frequency is very high, usually more than 80% (Ren Xueliang is reached, Li Jixin, Li Minghai .2007. American Tobacco Advances in Breeding overview. Chinese tobacco journal, NO.6.57-64).
Domestic scholars wishes Zhong Chun, the people such as Wu Haishan have carried out Unpollinated ovary culture (Zhu Zhongchun to Nicotiana tabacum kind copusyeusuhekuNo.4, the wheat that Wu sea coral .1979. never pollinates and tobacco Ovary culture go out haplobiont. Acta Genetica Sinica, NO.2.181-183), then to Nicotiana tabacum kind NC2326, great Ye tobacco and sterile tobacco pure line cultivar MSNC2326 have carried out Unpollinated ovary culture (Zhu Zhongchun, Wu sea coral .1981. turns out haplobiont again from In Haploids In Nicotiana Tabacum L ovary of not pollinating. Acta Genetica Sinica, NO.1.63-65), and No. five are reformed to Nicotiana tabacum kind carried out Unpollinated ovary culture (Zhu Zhongchun, Liu Zhenyue, the embryoid of the .1981. Unpollinated Ovary of Nicotiana Tabacum Cultivated In Vitros such as Wu Haishan is grown. Botany Gazette, NO.6.499-501), they take ovary as explant, directly ovary is inoculated in H1 medium (appositional growth element IAA0.5mg/L and basic element of cell division KT2mg/L) upper cultivation, but rarely seen copusyeusuhekuNo.4 obtains the report of haplobiont by Ovary culture.Wu Baiji and Zheng state Chang has carried out Unpollinated ovary culture to Nicotiana tabacum kind long leaf tobacco, Venus cigarette, yellow seedling elm and Aztec tobacco kind Gansu Nicotiniana rustica, also be using ovary as explant, directly ovary is inoculated in H2 medium (appositional growth element IAA0.5mg/L and basic element of cell division 6-BA2mg/L) upper cultivation, also obtain haplobiont (Wu Baiji, Zheng state Chang .1982. never pollinates the tobacco ovary induction cytology of haplobiont and embryology research. Botany Gazette, but concrete kind is not quite clear NO.2.125-129).And applicant adopts the medium identical with the people such as the people such as Zhu Zhongchun and Wu Baiji, ovary mutually of the same period, identical inoculation method to cultivate male sterile Unpollinated Ovaries of Nicotiana tabacum L, do not observe that ovule expands, ovary wall is pushed to top, and then differentiates the phenomenon of regeneration plant.Wherein, adopt the H2 medium of the appositional growth element IAA0.5mg/L and basic element of cell division 6-BA2mg/L of people's reports such as Wu uncle thoroughbred horse, ovary directly inoculated, change into and remove ovary wall and strip ovule inoculation, ovule is directly embryoid formation shape body not, and then growth seedling, but first form callus, then form embryoid by Calli Differentiation, finally grow up to plant, but haplobiont frequency is lower, and add the risk of character variation.And, people and the Wu Baiji etc. such as Zhu Zhongchun per capita using Pollen stage as Ovary culture period according to the male sterile tobacco being not suitable for male organs and degenerating.The people such as Zhu Zhongchun adopt N6 medium to carry out cultivating (Zhu Zhongchun to great Ye tobacco ovary of not pollinating, Wu Haishan, Anqing female .1984. great Ye tobacco is not pollinated Ovary culture and cytology research thereof. Acta Genetica Sinica, NO.4.281-287), ovary pollen being in monokaryotic stage is directly inoculated in N6 medium (appositional growth element IAA0.5mg/L, basic element of cell division KT6mg/L, inositol 100mg/L and sucrose 8g/L) upper cultivation, obtain regeneration plant, but frequency is very low, only account for 4%; Improve the consumption of growth hormone in medium, can induce vigorous callus, then differentiate a large amount of seedlings, but create various distortion of chromosome number, haplobiont frequency is low.Applicant adopts same medium, is that explant is cultivated male sterile tobacco with ovule, and result is that other explant produces that to expand the ratio of ovule minimum except ovule in megasporocyte ovary in period.The people such as Pan Li adopt H1 medium to carry out cultivating (Pan Li to Nicotiana tabacum kind NC89 ovary of not pollinating, Liu Zongcai, like the effect of Unpollinated Ovaries of Nicotiana tabacum L being cultivated into peace .1998. exogenous hormone. Agricultural University Of He'nan's journal, NO.2.167-170), using ovary as explant, directly be inoculated in additional 2, the H1 medium of 4-D and 6-BA is cultivated, and result shows, 2,4-D can give birth to callus by elicitor house property, is then divided into embryoid; When 6-BA concentration is 8.89 μm of ol/L, ovary directly can produce embryoid, and then forms seedling; When 6-BA concentration is 2.22 μm of ol/L and 4.44 μm of ol/L, ovary then first produces callus, then is divided into embryoid, and then forms seedling, but haploid inductivity is low, and the time formed needed for plantlet is long.They then cultivate Nicotiana tabacum kind MC944, K326 × C319, G80, Coker319, Shanxi too No. 6, No. 1, distant cigarette and sterile pure line cultivar MSNC89 ovary of not pollinating, and inquired into the influence factor (Pan Li of embryoid induction, Yang Tie encourages the research of .2000. Unpollinated Ovaries of Nicotiana tabacum L embryoid induction. northwest Botany Gazette, but finally have no and obtain the report of regeneration plant NO.1.59-63).Ran Bangding has carried out Unpollinated ovary culture to Nicotiana tabacum (SpeightG-28 × Burley21) Fl, (SpeightG28 × Ky56) Fl and Hongda.Corolla is selected to be inoculated on B5 and H two kinds of minimal mediums of additional KT2mg/L, IAA0.5mg/L, activated carbon 10g/L than the ovule in the bud of about long 1 centimetre of calyx, obtain regeneration plant, but do not identify ploidy, result is indefinite, and regeneration plant inductivity is extremely low.Applicant adopts same medium, the ovule of identical developmental stage is cultivated, even there are no the ovule obviously expanded.Although forefathers relate to male sterile tobacco do not pollinate ovary cultivation (Zhu Zhongchun, Wu sea coral .1981. turn out haplobiont again from In Haploids In Nicotiana Tabacum L ovary of not pollinating. Acta Genetica Sinica, NO.1.63-65; Pan Li, Yang Tie encourages the research of .2000. Unpollinated Ovaries of Nicotiana tabacum L embryoid induction. northwest Botany Gazette, NO.1.59-63), but be only limitted to pure line cultivar, not pollinate there are no sterile hybrid the report of Ovary culture, cultivate the research obtaining male sterile In Haploids In Nicotiana Tabacum L especially by Unfertilized Ovules, so far there are no openly reports.
Summary of the invention
The invention provides a kind of method of being cultivated acquisition male sterile tobacco hybridization kind haplobiont by Unfertilized Ovules, adopt the method can significantly improve the induction frequency of embryoid and plant.
Technical scheme provided by the invention: described one cultivates the haploid method of male sterile tobacco by Unfertilized Ovules, it is characterized in that concrete steps are as follows:
(1) medium is prepared
Adopt H1 medium or H2 medium as minimal medium, then add growth hormone IAA, basic element of cell division KT (i.e. kinetin KT), sucrose and agar and be prepared into embryoid induction medium, wherein growth hormone IAA and the final concentration of basic element of cell division KT in embryoid induction medium are 0.5mg/L and 2mg/L respectively; Described sucrose and the final concentration of agar in embryoid induction medium are 2% and 1% respectively;
Adopt H1 medium as minimal medium, then add sucrose and agar is prepared into not containing the solid shape regeneration bud inducing culture of hormone, wherein, described sucrose and the final concentration of agar in regeneration bud inducing culture are 2% and 1%;
Adopt H1 medium and N6 medium as minimal medium, then add growth hormone IAA, sucrose and agar and be prepared into solid shape regeneration bud root media, wherein the final concentration of growth hormone IAA in regeneration bud root media is 10mg/L, and sucrose and the final concentration of agar in regeneration bud root media are 8% and 0.8% respectively;
(2) collection of culture materials, during male sterile Tobacco Flowering, adopts the male sterile tobacco bud of not pollinating of Ovule Development between megasporocyte period to two core phases;
(3) disinfect and inoculate, the male sterile tobacco bud gathered in step (2) is put into concentration be 0.1% the mercuric chloride aqueous solution to sterilize 7-9min, then aseptic water washing 3-4 time is used, aseptically the base portion of bud is cut away, extrude ovary, and remove ovary wall or after directly peeling off ovule, ovule is put into the embryoid induction medium of step (1) preparation;
(4) induction of embryoid, have the embryoid induction medium of ovule to be placed in 28 DEG C of illumination cultivation rooms inoculation in step (3), every day, continuous illumination cultivated 10 hours under 1500Lux illumination, and induction ovule produces embryoid;
(5) induction of regeneration bud, the embryoid obtained in step (4) is aseptically transferred on the regeneration bud inducing culture of preparation in step (1), continue to be placed in 28 DEG C of illumination cultivation rooms, every day, continuous illumination cultivated 10 hours under 1500Lux illumination, cultivate 2-3 week, inducing embryoid body produces regeneration bud;
(6) cultivation of regeneration plant, the regeneration bud obtained in step (5) is aseptically transferred on regeneration bud root media, continue to be placed in 28 DEG C of illumination cultivation rooms, every day, continuous illumination cultivated 10 hours under 1500Lux illumination, cultivate 2-3 week, make its formation plantlet of taking root, afterwards, the regeneration plant sprouting root is transferred on the new regeneration bud root media prepared and continues under identical condition to cultivate, form male sterile tobacco monoploid regeneration plant.
The male sterile tobacco monoploid regeneration plant cultivated in the step (6) grow to 2-3cm high or have a 3-4 sheet blade time, the leaf sample getting gained regeneration plant carries out ploidy by flow cytometry analysis and determines.That be adjusted to 40channels, 10000 cells are at least analyzed at each curve peak, and repeat 4 times with normal ovary donor plant DNA content standard in contrast by flow cytometry analysis ploidy.The preparation method of described leaf sample: get the regeneration plant 1.3-1.6cm cultivated according to the method described above
2spire, (Tris-Hclbuffer in the cell pyrolysis liquid of 0.5ml, ph=7.2) shred with blade, sample and solution mixture are passed through 30 μm, aperture micro-pore-film filtration in testing tube, add 20ulDAPI dyeing liquor [137mMNaCl, 2.7mMKCl, 4.3mMNa2HPO4,1.47mMKH2PO4,0.4 μ gpropidiumiodide (PI) (sigma), 0.1%tritonX-100, and4 μ gRNAseA] after the 15min that dyes, sample is splined on flow cytometer.
The bud appearance period gathered in step 2 between corolla be about to expose calyx period to corolla than calyx long one times and the bud in stage between flower pesticide non-loose powder period.
H1 medium, H2 medium and N6 medium described in step of the present invention (1) are all take water as solvent, and it is formulated to add following formula material according to every premium on currency, and the concrete material formula of often kind of medium is as shown in table 1.
Table 1H1 culture fluid, H2 culture fluid and N6 culture fluid formula
Beneficial effect of the present invention:
(1) the present invention take ovule as explant, ovary is removed ovary wall or from bud, directly peel off ovule to be inoculated on embryoid induction medium, significantly improves the ratio that ovule expands in incubation;
(2) with the bud appearance period of Ovule Development period and correspondence thereof, the foundation of cultivating is carried out as collection ovary, be suitable for do not pollinate ovary or the Ovule development of the various tobacco breds comprising male organs degeneration kind, experiment proves: get grow the stage between megasporocyte period to two core phases ovary in ovule carry out cultivations the best, that is getting corolla, to be about to expose calyx best to the ovule inoculation situation of the bud of corolla one times of this one-phase longer than calyx, at us draw materials in scope, grow the induction that early stage ovule is more conducive to embryoid, grow more late induction result poorer, especially grow the ovule being about to expose the bud in calyx period at the ovule in megasporocyte period and corolla all to put up the best performance on H1 and H2 medium, being close to all explants has ovule to expand,
(3) the agar final concentration in embryoid induction medium is adjusted to 1% by the present invention, not only restrained effectively the generation of vitreous shoot, also helps the growth of embryoid;
(4) in the present invention, also the embryoid that ovule produces on embryoid induction medium is transferred on regeneration bud inducing culture and continue to cultivate, effectively overcome the browning of embryoid on embryoid induction medium, obviously facilitate growing thickly of regeneration bud.
The present invention utilize grow early stage ovule become regeneration haplobiont by different culture media and hormone combinations induction Ovule Development, significantly improve the induction of tobacco ovule embryoid and the frequency of plant regeneration.
Accompanying drawing explanation
Fig. 1 is the developmental state of blastular corresponding to the ovary of five kinds of different development stages;
Fig. 2 is the flow cytometer ploidy identification schematic diagram of the regeneration haplobiont that the present invention cultivates;
Fig. 3 is the flow cytometer ploidy identification contrast schematic diagram of normal ovary donor plant;
Fig. 4 is that the different embryoid induction medium of employing five kinds carries out ovule when embryoid induction is cultivated and expands the ratio Line Chart of explant.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is further illustrated.
Described a kind of being prepared as follows by various medium in the haploid method of Unfertilized Ovules cultivation male sterile tobacco:
Embryoid induction medium is using H1 medium or H2 medium as minimal medium, then add growth hormone IAA, basic element of cell division KT, sucrose and agar to make, wherein growth hormone IAA and the final concentration of basic element of cell division KT in embryoid induction medium are 0.5mg/L and 2mg/L respectively; Described sucrose and the final concentration of agar in embryoid induction medium are 2% and 1% respectively;
Regeneration bud inducing culture is using H1 medium as minimal medium, and then add sucrose and agar and be prepared into not containing the solid shape medium of hormone, wherein, described sucrose and the final concentration of agar in regeneration bud inducing culture are 2% and 1%;
Regeneration bud root media is using H1 medium and N6 medium as minimal medium, then add growth hormone IAA, sucrose and agar and be prepared into solid shape, wherein the final concentration of growth hormone IAA in regeneration bud root media is 10mg/L, and sucrose and the final concentration of agar in regeneration bud root media are 8% and 0.8% respectively;
The collection of its material is during Tobacco Flowering, gathers the bud of not pollinating that tobacco bred KRK26 (the flue-cured tobacco sterile hybrid from Zimbabwe introduces) appearance is following five kinds of periods: K1: corolla is about to expose calyx; K2: corolla has grown calyx less than 1 centimetre; K3: corolla is longer than calyx one times; K4: corolla is longer than calyx two times; K5: bud is about to open.The bud in take five kinds of periods is stripped ovary, is made into paraffin section according to a conventional method, under section being placed in microscope (DM2500, Leica, Germany), the developmental state of ovule in the ovary of observing different appearance bud in period; Ovary observed result: as shown in Figure 1, wherein, K1 is megasporocyte period to Ovule Development situation corresponding to five kinds of developmental stage ovarys; K2 is monokaryotic stage; K3 was two core phases; K4 is four cores and eight core phases; K5 is mature embryo sac.
Embodiment one: put into the embryoid induction medium prepared for minimal medium with H1 medium after ovary wall is removed to the ovary of above-mentioned K1, K2, K3, K4, K5 five bud in period respectively and carry out embryoid induction test with the embryoid induction medium that H2 medium is prepared for minimal medium.
The specific operation process of wherein carrying out embryoid induction cultivation for embryoid induction medium that minimal medium is prepared with H1 medium is as follows:
(1) the disinfecting and inoculating of material, the mercuric chloride solution bud in above-mentioned five periods gathered being put into 0.1% is sterilized 7-9min, then aseptic water washing 3-4 time is used, then aseptically the bud base portion in five periods after sterilization is cut away, extrude ovary with tweezers respectively, and after removing ovary wall, the ovule in five periods is put into respectively the culture dish that five are equipped with the embryoid induction medium prepared for minimal medium with H1 medium;
(2) five inoculations in step (1) are had the culture dish of different times ovule, be placed in 28 DEG C of illumination cultivation rooms, cultivate under 1500Lux illumination, 10 hours every days, induction ovule produces embryoid;
Cultivate a Zhou Houneng to observe the ovule in five periods and have increase, the explant ratio that ovule in different development stage ovary expands in incubation is: K1 to be period 99.8%, K2 be period 95.8%, K3 period is 53.5%, K4 is 17.7%, K5 period period is 0.0%.
According to aforesaid operations process, by with H1 medium for the embryoid induction medium that minimal medium is prepared replaces with the embryoid induction medium prepared for minimal medium with H2 medium, respectively embryoid induction cultivation is carried out to the ovule in five periods of K1, K2, K3, K4, K5.
Cultivate a Zhou Houneng to observe the ovule in five periods and have increase, the explant ratio that ovule in different development stage ovary expands in incubation is: K1 to be period 90.6%, K2 be period 75.2%, K3 period is 65.6%, K4 is 43.9%, K5 period period is 13.4%.
Embodiment two: directly ovule is peeled off to the ovary of K1, K2, K3, K4, K5 five bud in period, and put into the embryoid induction medium prepared for minimal medium with H1 medium respectively and carry out embryoid induction test with the embryoid induction medium that H2 medium is prepared for minimal medium, specific operation process is as embodiment one, and the result of the test obtained is identical with embodiment one.
Embodiment three: to above-mentioned K1, K2, K3, K4, put into the embryoid induction medium prepared for minimal medium with H1 medium after the ovary of K5 five bud in period carries out ring cutting respectively and carry out embryoid induction test with the embryoid induction medium that H2 medium is prepared for minimal medium, the ovule that ring cutting ovary inoculated and cultured can be observed to expose after one week obviously expands, explant ovary wall with ovary wall turns green and thickens, do not observe ovary wall fracture phenomena, inner ovule does not expand sign substantially, still brownization is dead now ovary wall to be removed ovule in incubation.
In above-mentioned three embodiments, the ovary in five periods of K1 to K5, cultivate at removal ovary wall or direct ovule of peeling off, its ovule has and expands phenomenon, but can find out that the explant rate that K1 to K5 ovary in period ovule in dissimilar medium expands is all on a declining curve by enforcement one and enforcement two, in K1 ovary in period, ovule is all put up the best performance on different culture media, being close to all explants has ovule to expand, and the ovule ratio expanded is the highest, so, in tested scope, Embryo Sac Development is more ripe, the proliferation-inducing of ovule is instead poorer, ovule in developmental stage ovary comparatively early should be gathered compare and be conducive to cultivating, get grow the stage between megasporocyte period to two core phases ovary in ovule carry out cultivations the best, that is, in K1 ~ K3 ovary in period, ovule inoculation situation is best.
K1 ~ K3 ovule in period in embodiment one is continued cultivation and obtains embryoid after one month by applicant in the embryoid induction medium being minimal medium with H1 medium, statistics shows, 60 ovules being in megasporocyte period just create 516 embryoids, and its induction frequency is 860%; 40 ovules being in monokaryotic stage period create 220 embryoids, and its induction frequency is 550%; 40 ovules being in for two periods core phase create 112 embryoids, and its induction frequency is 280%.
By the embryoid out of the Fiber differentiation in enforcement one, aseptically transfer to not containing on the regeneration bud inducing culture of hormone, continue to be placed in 28 DEG C of illumination cultivation rooms, cultivate under 1500Lux illumination, 10 hours every days, inducing embryoid body produces regeneration bud; After cultivating two weeks, start after the embryoid cultivated in embodiment one to extend, obviously form the two poles of the earth; Continue to cultivate, most of polarity embryo differentiation is to similar cotyledonary embryos shape, and grow palpus shape leaf subsequently, major part is in the embryoid that in K1, K2, K3 ovary in period, ovule induces and develops into thin and weak seedling gradually.After cultivation completes, statistical result showed, in 516 embryoids that the ovule in megasporocyte period produces, have 402 embryoids to develop into seedling, induction frequency is 77.9%; In 220 embryoids that the ovule in monokaryotic stage period produces, have 157 embryoids to develop into seedling, induction frequency is 71.4%; In 112 embryoids of the ovule generation in two periods core phase, have 88 embryoids to develop into seedling, induction frequency is 78.5%.
By tri-regeneration buds obtained period of K1, K2, K3 in above-mentioned steps, aseptically transfer on regeneration bud root media, continue to cultivate, after two weeks, regeneration bud starts to take root, grow fine, regeneration plant complete as seen after three weeks, carries out squamous subculture to the regeneration plant sprouting root afterwards under this conditions and environment.
In embodiment one K1, K2, K3 tri-regeneration plants cultivating out period grow to 2-3cm high or have a 3-4 sheet blade time, get the blade of gained regeneration plant, utilize the BDFACSCalibur flow cytometry analysis of U.S. company BD production to carry out ploidy and determine.Concrete grammar is: get gained regeneration plant and be about 1.5cm
2spire, (Tris-Hclbuffer in the cell pyrolysis liquid of 0.5ml, ph=7.2) shred with blade, sample and solution mixture are passed through 30 μm, aperture micro-pore-film filtration in testing tube, add 20ulDAPI dyeing liquor [137mMNaCl, 2.7mMKCl, 4.3mMNa2HPO4,1.47mMKH2PO4,0.4 μ gpropidiumiodide (PI) (sigma), 0.1%tritonX-100, and4 μ gRNAseA] after the 15min that dyes, sample is splined on flow cytometer.And with normal ovary donor plant DNA content standard in contrast, be adjusted to 40channels.10000 cells are at least analyzed at each curve peak, and repeat 4 times, and as shown in Figure 2, the result that normal ovary donor plant detects as shown in Figure 3 for the regeneration plant testing result adopting method of the present invention to cultivate out.Can be found out by the contrast of Fig. 2 and Fig. 3, the most of plant cultivating out by the haploid method of Unfertilized Ovules cultivation male sterile tobacco of the present invention is adopted to be haplobiont, the seedling growth being wherein haplobiont is slow, and leaf and plant height can not show a candle to parental plant.
Applicant has also been following contrast experiment, and result of the test is represented by the curve in Fig. 4, and in five curves in Fig. 4, H1 represents the embryoid induction medium prepared for minimal medium with H1 medium; H2 represents the embryoid induction medium prepared for minimal medium with H2 medium; H3 representative does not add growth hormone KT in the embryoid induction medium prepared for minimal medium with H1 medium, and adds activated carbon, and its final concentration is 10g/L; N6 represents the embryoid induction medium prepared for minimal medium with N6 medium; Basic element of cell division KT, in the embryoid induction medium prepared for minimal medium with H2 medium, is replaced with basic element of cell division 6-BA by HB representative.
Experiment one: the embryoid induction medium in embodiment one is replaced with the Fiber differentiation carrying out embryoid in the embryoid induction medium (adding growth hormone IAA0.5mg/L, basic element of cell division KT6mg/L, inositol 100mg/L, agar 1%, sucrose 8% in N6 basic culture solution) of N6 medium preparation, as shown in Figure 4, on N6 embryoid induction medium, except ovule in K1 ovary in period, other explant produces that to expand the ratio of ovule minimum to result of the test.
Experiment two: the embryoid induction medium in embodiment one is not added growth hormone KT, and add activated carbon, its final concentration is 10g/L, by this medium called after H3 medium, as shown in Figure 4, then the ovary ovule in tetra-periods of K1, K2, K3, K4 is carried out according to the method described in the present invention the Fiber differentiation of embryoid, experiment finds that the ovule in four periods of cultivating on this medium is there are no the ovule obviously expanded.
Experiment three: by the embryoid induction medium final concentration in embodiment two be 2mg/L 6-BA replace final concentration be the KT of 2mg/L, by this medium called after HB medium, find that ovule does not directly produce embryoid, but first produce callus, be divided into embryoid again, extend the time of embryoid induction, and embryoid induction frequency obviously reduces, the concrete ovule in five periods expands rate as shown in Figure 4.
Tobacco is in embryoid induction incubation, and some embryoids there will be vitrifying.Applicant is also a kind of in embodiment successively, increase sucrose content (2% → 4%), pH (5.5 → 5.8) and agar concentration (0.8% → 1%), reduce the generation that the experiments such as mitogen content (2mg/L → 1.5mg/L) suppress vitreous shoot.Found that, this several method can suppress the generation of vitreous shoot, but except the medium of 1% agar concentration is on except the impact of embryoid growth nothing, other medium is all unfavorable to embryoid growth.
Claims (4)
1. cultivate the haploid method of male sterile tobacco by Unfertilized Ovules, it is characterized in that concrete steps are as follows:
(1) medium is prepared
Adopt H1 medium or H2 medium as minimal medium, then add growth hormone IAA, basic element of cell division KT, sucrose and agar and be prepared into embryoid induction medium, wherein growth hormone IAA and the final concentration of basic element of cell division KT in embryoid induction medium are 0.5mg/L and 2mg/L respectively; Described sucrose and the final concentration of agar in embryoid induction medium are 2% and 1% respectively;
Adopt H1 medium as minimal medium, then add sucrose and agar is prepared into not containing the solid shape regeneration bud inducing culture of hormone, wherein, described sucrose and the final concentration of agar in regeneration bud inducing culture are 2% and 1%;
Adopt H1 medium and N6 medium as minimal medium, then add growth hormone IAA, sucrose and agar and be prepared into solid shape regeneration bud root media, wherein the final concentration of growth hormone IAA in regeneration bud root media is 10mg/L, and sucrose and the final concentration of agar in regeneration bud root media are 8% and 0.8% respectively;
In step (1), the concrete formula of H1 medium, H2 medium and N6 medium is as follows:
(2) collection of culture materials, during male sterile Tobacco Flowering, adopt the male sterile tobacco bud of not pollinating of Ovule Development between megasporocyte period to two core phases, bud appearance period in this stage between corolla be about to expose calyx period to corolla than calyx long one times and the bud in stage between flower pesticide non-loose powder period;
(3) disinfect and inoculate, the male sterile tobacco bud gathered in step (2) is put into concentration be 0.1% the mercuric chloride aqueous solution to sterilize 7-9min, then aseptic water washing 3-4 time is used, aseptically the base portion of bud is cut away, extrude ovary, and remove ovary wall or after directly peeling off ovule, ovule is put into the embryoid induction medium of step (1) preparation;
(4) induction of embryoid, have the embryoid induction medium of ovule to be placed in 28 DEG C of illumination cultivation rooms inoculation in step (3), every day, continuous illumination cultivated 10 hours under 1500Lux illumination, and induction ovule produces embryoid;
(5) induction of regeneration bud, the embryoid obtained in step (4) is aseptically transferred on the regeneration bud inducing culture of preparation in step (1), continue to be placed in 28 DEG C of illumination cultivation rooms, every day, continuous illumination cultivated 10 hours under 1500Lux illumination, cultivate 2-3 week, inducing embryoid body produces regeneration bud;
(6) cultivation of regeneration plant, the regeneration bud obtained in step (5) is aseptically transferred on regeneration bud root media, continue to be placed in 28 DEG C of illumination cultivation rooms, every day, continuous illumination cultivated 10 hours under 1500Lux illumination, cultivate 2-3 week, make its formation plantlet of taking root, afterwards, the regeneration plant sprouting root is transferred on the new regeneration bud root media prepared and continues under identical condition to cultivate, form male sterile tobacco monoploid regeneration plant.
2. one according to claim 1 cultivates the haploid method of male sterile tobacco by Unfertilized Ovules, it is characterized in that the method is further comprising the steps of: the male sterile tobacco monoploid regeneration plant cultivated in the step (6) grow to 2-3cm high or have a 3-4 sheet blade time, the leaf sample getting gained regeneration plant carries out ploidy by flow cytometry analysis and determines.
3. one according to claim 2 cultivates the haploid method of male sterile tobacco by Unfertilized Ovules, it is characterized in that: be with normal ovary donor plant DNA content standard in contrast by flow cytometry analysis ploidy after step (6), be adjusted to 40channels, 10000 cells are at least analyzed at each curve peak, and repeat 4 times.
4. one according to claim 2 cultivates male sterile tobacco haploid method by Unfertilized Ovules, it is characterized in that: the described leaf sample carrying out ploidy analysis by flow cytometer takes regeneration plant 1.3cm
2-1.6cm
2spire, shreds at the cell pyrolysis liquid blade of 0.5ml, and sample and solution mixture are passed through 30 μm, aperture micro-pore-film filtration in testing tube, after adding 20ulDAPI dyeing liquor dyeing 15min, sample is splined on flow cytometer.
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| CN106106555A (en) * | 2016-06-29 | 2016-11-16 | 固镇县华原家庭农场 | A kind of germinator being effectively improved Caulis et Folium Lactucae sativae percentage of seedgermination |
| CN107439370B (en) * | 2017-07-10 | 2019-06-28 | 湖北大学 | A method of diploid rice strain is created using tetraploid Unfertilized Ovaries culture |
| CN107873514A (en) * | 2017-11-09 | 2018-04-06 | 西北农林科技大学 | A kind of efficient tobacco anther arrhenokaryon direct development abductive approach |
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