CN103718957A - Method for culturing male sterile tobacco haplont via unfertilized ovule - Google Patents
Method for culturing male sterile tobacco haplont via unfertilized ovule Download PDFInfo
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Abstract
The invention provides a method for culturing a male sterile tobacco haplont via an unfertilized ovule. According to the method for culturing the male sterile tobacco haplont, an ovule of which the development of an unpollinated male sterile tobacco bud is between a megasporocyte stage and a binucleate stage is used as a culture medium, after sterilization treatment, the ovule is sequentially subjected to embryoid induced culture, regeneration bud rooting culture and regenerated plant culture in a 28-DEG C illumination culture chamber, so that the male sterile tobacco haplont is finally cultured, and during the period of the culture, the illumination continuously lasts for 10 hours every day under a 1,500 Lux illumination condition. The method for culturing the male sterile tobacco haplont via the unfertilized ovule is adopted to culture male sterile tobaccos, so that the frequency of tobacco ovule embryoid induction and plant regeneration are remarkably improved.
Description
Technical field
The invention belongs to tobacco breeding technical field, be specifically related to cultivate by Unfertilized Ovules the haploid method of male sterile tobacco hybridization kind that obtains.
Background technology
Monoploid refers to the sporophyte with gametic chromosome composition.Haploid induction technology is as the chief component of modern high-efficient breeding system, there is following remarkable advantage: after crop monoploid material doubles, be pure lines (double haploid), excellently middlely select the excellent gene loci of Cross Combinations that can isozygoty fast after excellent, thereby make the breeding time limit shorten 3～4 years compared with conventional method; Secondly, because genotype and the phenotype of double haploid are in full accord, when screening breeding material, can greatly reduce and falsely drop frequency, obviously improve efficiency of selection, particularly with the obvious advantage especially when the merit of selecting by recessive gene control.In addition, utilize monoploid to cultivate and can obtain fast doubled haploid (DH) colony, can be the ideal material of carrying out crop Main Agronomic Characters molecular labeling, and utilize molecular labeling carry out the chief component that assisted Selection is modern high-efficient breeding system (positive .2007. crops haploid breeding research overview and thinking. Shandong agricultural science, NO.5.122-125).
Tobacco is important in the world economic crops.Conventionally, can pass through flower pesticide or the microspore Haploid production of culture in vitro tobacco, 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 the bad change of yield and quality proterties is serious after doubling to find this class monoploid, the value that breeding utilizes is limited, adding this class, haploid to add overtones band very low, conventionally only has 1% left and right, current this method is seldom used (Ren Xueliang in tobacco breeding, Li Jixin, Li Minghai .2007. American Tobacco Advances in Breeding overview. Chinese tobacco journal, NO.6.57-64).And there is obvious advantage from Unpollinated Ovaries of Nicotiana tabacum L induction haplobiont, 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 have just started Unpollinated Ovaries of Nicotiana tabacum L training as far back as 1979, and successfully obtained haplobiont (Burk LG, Gerstel DU, Wernsman EA.1979.Maternal Haploids of Nicotiana tabacum L.from Seed.Science., NO.2.585).They compare the double haploid after tobacco Genogenetic haploid and male doubling monoploids and their source parent, find that female double haploid is better than male double haploid, female double haploid is more similar to source parent, can not cause bad change (the Wernsman EA of yield and quality, Matzinger DF, Rebeca C Rufty.1989.Androgenetic vs.Gynogenetic Doubled Haploids of Tobacco.Crop Sci, NO.29.1151-1155).At present, they this technology has been 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 to utilize ovary or ovule to cultivate 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 in the death of cotyledon stage, the seedling of survival is the parent monoploid that is easy to resolution, utilizing the haploid petiole culture in vitro of this class to be easy to realize haploidly doubles naturally, and frequency is very high, conventionally reach more than 80% (Ren Xueliang, Li Jixin, Li Minghai .2007. American Tobacco Advances in Breeding overview. Chinese tobacco journal, NO.6.57-64).
Domestic scholars is wished Zhong Chun, the people such as Wu Haishan have carried out Unpollinated ovary culture (Zhu Zhongchun to common tobacco bred copus yeusuheku No.4, wheat and tobacco ovary that Wu sea coral .1979. never pollinates are turned out haplobiont. Acta Genetica Sinica, NO.2.181-183), then to common tobacco bred NC2326, great Ye tobacco and sterile tobacco pure line cultivar MS NC2326 have carried out Unpollinated ovary culture (Zhu Zhongchun, Wu sea coral .1981. turns out again haplobiont from the In Haploids In Nicotiana Tabacum L ovary of not pollinating. Acta Genetica Sinica, NO.1.63-65), and common tobacco bred innovation has been carried out to Unpollinated ovary culture (Zhu Zhongchun No. five, 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 are take ovary as explant, directly ovary is inoculated in to upper cultivation of H1 medium (appositional growth element IAA0.5mg/L and basic element of cell division KT2mg/L), but rarely seen copus yeusuheku No.4 cultivates the report that obtains haplobiont by ovary.Wu Baiji and Zheng state Chang have carried out Unpollinated ovary culture to common tobacco bred 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 to upper cultivation of H2 medium (appositional growth element IAA0.5mg/L and basic element of cell division 6-BA2mg/L), also obtained haplobiont (Wu Baiji, Zheng state Chang .1982. never pollinate cytology and the embryology research of tobacco ovary induction haplobiont. 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 appositional growth element IAA0.5mg/L of people's reports such as Wu uncle thoroughbred horse and the H2 medium of basic element of cell division 6-BA2mg/L, ovary is directly inoculated, changed 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 increased the risk of character variation.And, the people such as Zhu Zhongchun and Wu Baiji etc. per capita using Pollen stage as ovary cultivation period according to be not suitable for male organs degenerate male sterile tobacco.The people such as Zhu Zhongchun adopt N6 medium to carry out cultivating (Zhu Zhongchun to the great Ye tobacco ovary of not pollinating, Wu Haishan, Anqing female .1984. great Ye tobacco do not pollinate ovary cultivate and cytology research. Acta Genetica Sinica, NO.4.281-287), ovary by pollen in monokaryotic stage is directly inoculated in upper cultivation of N6 medium (appositional growth element IAA0.5mg/L, basic element of cell division KT6mg/L, inositol 100mg/L and sucrose 8g/L), obtained regeneration plant, but frequency is very low, only accounts for 4%; The consumption that improves growth hormone in medium, can induce vigorous callus, then differentiates a large amount of seedlings, but has produced various distortion of chromosome number, and haplobiont frequency is low.Applicant adopts same medium, take ovule as explant, male sterile tobacco is cultivated, result be in megasporocyte ovary in period ovule other explant produce that to expand the ratio of ovule minimum.The people such as Pan Li adopt H1 medium to carry out cultivating (Pan Li to the common tobacco bred 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, on the H1 medium of 4-D and 6-BA, cultivate, 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 μ mol/L, ovary can directly produce embryoid, and then forms seedling; When 6-BA concentration is 2.22 μ mol/L and 4.44 μ mol/L, ovary first produces callus, then is divided into embryoid, and then forms seedling, but haploid inductivity is low, forms the required time of plantlet long.They then cultivate common tobacco bred MC944, K326 × C319, G80, Coker319, Shanxi too No. 6, No. 1, distant cigarette and the sterile pure line cultivar MS NC89 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 common tobacco (Speight G-28 × Burley21) Fl, (Speight G28 × Ky56) Fl and Hongda.The ovule of selecting corolla approximately to grow than calyx in the bud of 1 centimetre is inoculated on the B5 and two kinds of minimal mediums of H of additional KT2mg/L, IAA0.5mg/L, activated carbon 10g/L, obtained regeneration plant, but do not identify ploidy, result is indefinite, and regeneration plant inductivity is extremely low.Applicant adopts the ovule of same medium, identical developmental stage to cultivate, even there are no the ovule obviously expanding.Although forefathers related to male sterile tobacco do not pollinate ovary cultivation (Zhu Zhongchun, Wu sea coral .1981. turns out again haplobiont from the 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 only limit to pure line cultivar, there are no sterile hybrid do not pollinate ovary cultivate report, especially by Unfertilized Ovules, cultivate the research that obtains male sterile In Haploids In Nicotiana Tabacum L, so far there are no openly reports.
Summary of the invention
The invention provides a kind of method that obtains male sterile tobacco hybridization kind haplobiont of cultivating by Unfertilized Ovules, adopt the method can obviously improve the induction frequency of embryoid and plant.
Technical scheme provided by the invention: described one is cultivated the haploid method of male sterile tobacco by Unfertilized Ovules, is characterized in that concrete steps are as follows:
(1) prepare medium
Adopt H1 medium or H2 medium as minimal medium, then adding growth hormone IAA, basic element of cell division KT(is kinetin KT), sucrose and agar is 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 respectively 0.5mg/L and 2mg/L; Described sucrose and the agar final concentration in embryoid induction medium is respectively 2% and 1%;
Adopt H1 medium as minimal medium, then add sucrose and agar to be prepared into the not solid shape regeneration bud inducing culture containing hormone, wherein, described sucrose and the agar final concentration in regeneration bud inducing culture is 2% and 1%;
Adopt H1 medium and N6 medium as minimal medium, then add growth hormone IAA, sucrose and agar to 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 agar final concentration in regeneration bud root media is respectively 8% and 0.8%;
(2) collection of culture materials, during male sterile Tobacco Flowering, adopt Ovule Development between megasporocyte period to the not male sterile tobacco bud of pollination between two core phases;
(3) disinfect and inoculate, it is 0.1% the mercuric chloride aqueous solution 7-9min that sterilizes that the male sterile tobacco bud gathering in step (2) is put into concentration, then use aseptic water washing 3-4 time, under aseptic condition, the base portion of bud is cut away, extrude ovary, and remove ovary wall or directly peel off after ovule, ovule is put into the embryoid induction medium of step (1) preparation;
(4) induction of embryoid, has the embryoid induction medium of ovule to be placed in 28 ℃ of illumination cultivation chambers inoculation in step (3), and every day, continuous illumination was cultivated 10 hours under 1500Lux illumination, and induction ovule produces embryoid;
(5) induction of regeneration bud, the embryoid obtaining in step (4) is transferred under aseptic condition on the regeneration bud inducing culture of preparation in step (1), continue to be placed in 28 ℃ of illumination cultivation chambers, every day, continuous illumination was cultivated 10 hours under 1500Lux illumination, cultivate 2-3 week, inducing embryoid body produces regeneration bud;
(6) cultivation of regeneration plant, the regeneration bud obtaining in step (5) is transferred on regeneration bud root media under aseptic condition, continue to be placed in 28 ℃ of illumination cultivation chambers, every day, continuous illumination was cultivated 10 hours under 1500Lux illumination, cultivate 2-3 week, make its formation plantlet of taking root, afterwards, to sprouting the regeneration plant of root, transfer on the regeneration bud root media of new preparation and under same condition, continue to cultivate, form male sterile tobacco monoploid regeneration plant.
The male sterile tobacco monoploid regeneration plant of cultivating in step (6) grows to that 2-3cm is high or while having 3-4 sheet blade, the leaf sample of getting gained regeneration plant carries out ploidy by flow cytometry analysis and determines.By flow cytometry analysis ploidy, be 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.The preparation method of described leaf sample: get the regeneration plant 1.3-1.6cm cultivating according to the method described above
2spire, (Tris-Hcl buffer in the cell pyrolysis liquid of 0.5ml, ph=7.2) with blade, shred, sample and solution mixture are passed through to aperture 30 μ m micro-pore-film filtrations in testing tube, add 20ul DAPI dyeing liquor [137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4,1.47mM KH2PO4,0.4 μ g propidium iodide (PI) (sigma), 0.1%tritonX-100, and4 μ g RNAse A] dye after 15min, sample is splined on to flow cytometer.
The bud outward appearance performance period gathering in step 2 between corolla be about to expose calyx period to corolla than calyx the not bud in stage between loose powder period of long a times and flower pesticide.
Described in step of the present invention (1), H1 medium, H2 medium and N6 medium are all take water as solvent, and add following formula material formulated according to every premium on currency, and the concrete material formula of every 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 is take ovule as explant, ovary removed to ovary wall or from bud, directly peel off ovule to be inoculated on embryoid induction medium, significantly improved the ratio that ovule expands in cultivation process;
(2) with Ovule Development period and corresponding bud outward appearance performance period thereof, the foundation of cultivating as collection ovary, the not pollination ovary or the ovule that are suitable for the various tobacco breds including male organs degeneration kind are cultivated, experimental results show that: get to grow in megasporocyte period and cultivate the best to ovule in the ovary in stage between two core phases, that is to say and get corolla to be about to expose calyx more best than the ovule inoculation situation of the bud of long one times of this one-phase of calyx to corolla, at us, draw materials in scope, grow early stage ovule and be more conducive to the induction of embryoid, grow more late induction result poorer, especially growing at the ovule in megasporocyte period is that the ovule that corolla is about to expose the bud in calyx period is all put up the best performance on H1 and H2 medium, being close to all explants has ovule to expand,
(3) the present invention is adjusted into 1% by the agar final concentration in embryoid induction medium, has not only effectively suppressed the generation of glass seedling, also helps the growth of embryoid;
(4) in the present invention, the embryoid also ovule being produced on embryoid induction medium is transferred on regeneration bud inducing culture and is continued to cultivate, effectively overcome the browning of embryoid on embryoid induction medium, obviously promoted growing thickly of regeneration bud.
The present invention utilize grow early stage ovule pass through different culture media and the hormone combinations induction Ovule Development haplobiont that becomes to regenerate, obviously improved 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 present invention's regeneration haplobiont of cultivating;
Fig. 3 is the flow cytometer Ploidy Identification contrast schematic diagram of normal ovary donor plant;
Fig. 4 be adopt five kinds of different embryoid induction medium carry out embryoid induction while cultivating ovule expand the ratio Line Chart of explant.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is further illustrated.
Describedly a kind ofly by Unfertilized Ovules, cultivate being prepared as follows of various medium in the haploid method of 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 respectively 0.5mg/L and 2mg/L; Described sucrose and the agar final concentration in embryoid induction medium is respectively 2% and 1%;
Regeneration bud inducing culture is using H1 medium as minimal medium, then adds sucrose and agar to be prepared into the not solid shape medium containing hormone, and wherein, described sucrose and the agar final concentration in regeneration bud inducing culture is 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 to 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 agar final concentration in regeneration bud root media is respectively 8% and 0.8%;
The collection of its material is during Tobacco Flowering, gathers the flue-cured tobacco sterile hybrid that tobacco bred KRK26(introduces from Zimbabwe) outward appearance shows as the not pollination bud in following five kinds of periods: K1: corolla is about to expose calyx; K2: corolla has grown 1 centimetre of calyx less than; K3: corolla is longer one times than calyx; K4: corolla is longer two times than calyx; K5: bud is about to open.The bud in five kinds of periods of taking is stripped to ovary, be made into according to a conventional method paraffin section, section is placed under microscope (DM2500, Leica, Germany), observe the developmental state of the interior ovule of ovary of different outward appearance performance buds 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 mono-: the ovary of five of above-mentioned K1, K2, K3, K4, K5 bud in period is removed to the embryoid induction medium of putting into respectively the embryoid induction medium of the preparation take H1 medium as minimal medium and the preparation take H2 medium as minimal medium after ovary wall and carry out embryoid induction test.
The specific operation process that the embryoid induction medium of wherein preparing take H1 medium as minimal medium carries out embryoid induction cultivation is as follows:
(1) material disinfecting and inoculating, the bud in above-mentioned five periods gathering is put into 0.1% the mercuric chloride solution 7-9min that sterilizes, then use aseptic water washing 3-4 time, then under aseptic condition, the bud base portion in five periods after sterilization is cut away, with tweezers, extrude ovary respectively, and after removing ovary wall, the ovule in five periods is put into respectively to five culture dishes that the embryoid induction medium of preparation take H1 medium as minimal medium is housed;
(2) five inoculations in step (1) are had to the culture dish of different times ovule, be placed in 28 ℃ of illumination cultivation chambers, under 1500Lux illumination, cultivate, 10 hours every days, induction ovule produces embryoid;
Cultivate the ovule that a Zhou Houneng observes five periods and have increase, the explant ratio that ovule in different development stage ovary expands in cultivation process is: K1 is that 99.8%, K2 is that 95.8%, K3 period is 53.5% period period, K4 is that 17.7%, K5 period is 0.0% period.
According to aforesaid operations process, the embryoid induction medium of the preparation take H1 medium as minimal medium is replaced with take H2 medium as minimal medium to the embryoid induction medium of preparation, respectively the ovule in five periods of K1, K2, K3, K4, K5 is carried out to embryoid induction cultivation.
Cultivate the ovule that a Zhou Houneng observes five periods and have increase, the explant ratio that ovule in different development stage ovary expands in cultivation process is: K1 is that 90.6%, K2 is that 75.2%, K3 period is 65.6% period period, K4 is that 43.9%, K5 period is 13.4% period.
Embodiment bis-: the ovary to five of K1, K2, K3, K4, K5 bud in period is directly peeled off ovule, and the embryoid induction medium of putting into respectively the embryoid induction medium of the preparation take H1 medium as minimal medium and the preparation take H2 medium as minimal medium carries out embryoid induction test, specific operation process is as embodiment mono-, and the result of the test obtaining is identical with embodiment mono-.
Embodiment tri-: to above-mentioned K1, K2, K3, K4, the embryoid induction medium that the ovary of five of K5 bud in period carries out putting into respectively after ring cutting the embryoid induction medium of the preparation take H1 medium as minimal medium and the preparation take H2 medium as minimal medium carries out embryoid induction test, ring cutting ovary inoculated and cultured can be observed the ovule exposing and obviously expands after one week, with the explant ovary wall of ovary wall, turing green thickens, do not observe ovary wall fracture phenomena, inner ovule does not expand sign substantially, now ovary wall is removed to still brownization death of ovule in cultivation process.
In above-mentioned three embodiment, the ovary in five periods of K1 to K5, removing ovary wall or directly peeling off ovule and cultivate, its ovule has the phenomenon of expanding, but can find out that with enforcement two K1 to K5 ovary in the period explant rate that ovule expands in dissimilar medium is all on a declining curve by implementing one, 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 expanding is the highest, so, in tested scope, Embryo Sac Development is more ripe, the proliferation-inducing of ovule is instead poorer, should gather ovule in developmental stage ovary early is relatively conducive to cultivate, get to grow in megasporocyte period and cultivate the best to ovule in the ovary in stage between two core phases, that is to say, in K1～K3 ovary in period, ovule inoculation situation is best.
Applicant continues K1～K3 ovule in period in embodiment mono-cultivation and obtains embryoid after one month in the embryoid induction medium take H1 medium as minimal medium, statistics shows, 60 ovules in megasporocyte period have just produced 516 embryoids, and its induction frequency is 860%; 40 ovules in monokaryotic stage period have produced 220 embryoids, and its induction frequency is 550%; 40 ovules in two periods core phase have produced 112 embryoids, and its induction frequency is 280%.
The embryoid that the induction of implementing in is cultivated is out transferred to not containing on the regeneration bud inducing culture of hormone under aseptic condition, continues to be placed in 28 ℃ of illumination cultivation chambers, under 1500Lux illumination, cultivates, and 10 hours every days, inducing embryoid body produces regeneration bud; Cultivate after two weeks, after the embryoid of cultivating in embodiment mono-, start to extend, obviously form the two poles of the earth; Continue to cultivate, most of polarity embryo differentiation, to similar cotyledonary embryos shape, grows palpus shape leaf subsequently, and most of embryoid that ovule induces in K1, K2, K3 ovary in period develops into thin and weak seedling gradually.After cultivation completes, statistical result showed, in 516 embryoids that produce, has 402 embryoids to develop into seedling at the ovule in megasporocyte period, and 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 that the ovule in two periods core phase produces, have 88 embryoids to develop into seedling, induction frequency is 78.5%.
By K1, K2, tri-regeneration buds that obtain period of K3 in above-mentioned steps, under aseptic condition, transfer on regeneration bud root media, continue to cultivate, after two weeks, regeneration bud starts to take root, grow fine, after three weeks, visible complete regeneration plant carries out subculture cultivation to the regeneration plant of sprouting root afterwards under this conditions and environment.
In embodiment mono-, to grow to 2-3cm high or while having 3-4 sheet blade for K1, K2, tri-regeneration plants of cultivating out period of K3, gets the blade of gained regeneration plant, utilizes the BD FACSCalibur flow cytometry analysis of U.S. company BD production to carry out ploidy and determine.Concrete grammar is: get the about 1.5cm of gained regeneration plant
2spire, (Tris-Hcl buffer in the cell pyrolysis liquid of 0.5ml, ph=7.2) with blade, shred, sample and solution mixture are passed through to aperture 30 μ m micro-pore-film filtrations in testing tube, add 20ul DAPI dyeing liquor [137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4,1.47mM KH2PO4,0.4 μ g propidium iodide (PI) (sigma), 0.1%tritonX-100, and4 μ g RNAse A] dye after 15min, sample is splined on to 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, adopt regeneration plant testing result that method of the present invention cultivates out as shown in Figure 2, and the result that normal ovary donor plant detects as shown in Figure 3.Contrast by Fig. 2 and Fig. 3 can be found out, adopt that of the present invention by Unfertilized Ovules, to cultivate most of plant that the haploid method of male sterile tobacco cultivates be out haplobiont, the seedling growth that is 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 represents by the curve in Fig. 4, the embryoid induction medium of H1 representative preparation take H1 medium as minimal medium in five curves in Fig. 4; The embryoid induction medium of H2 representative preparation take H2 medium as minimal medium; H3 representative is not added growth hormone KT in the embryoid induction medium of the preparation take H1 medium as minimal medium, and adds activated carbon, and its final concentration is 10g/L; The embryoid induction medium of N6 representative preparation take N6 medium as minimal medium; HB representative, in the embryoid induction medium of the preparation take H2 medium as minimal medium, replaces with basic element of cell division 6-BA by basic element of cell division KT.
Experiment one: the embryoid induction medium in embodiment mono-is replaced with to the induction of 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 and cultivate, as shown in Figure 4, on N6 embryoid induction medium, in K1 ovary in period, ovule, other explant produces that to expand the ratio of ovule minimum to result of the test.
Experiment two: the embryoid induction medium in embodiment mono-is not added to growth hormone KT, and interpolation activated carbon, its final concentration is 10g/L, by this medium called after H3 medium, as shown in Figure 4, then the induction of the ovary ovule in tetra-periods of K1, K2, K3, K4 being carried out according to the method described in the present invention to embryoid is cultivated, and experiment finds that the ovule in four periods of cultivating on this medium is there are no the ovule obviously expanding.
Experiment three: the 6-BA that is 2mg/L with final concentration by the embryoid induction medium in embodiment bis-replaces the KT that final concentration is 2mg/L, by this medium called after HB medium, find that ovule does not directly produce embryoid, but first produce callus, be divided into again embryoid, extended 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 cultivation process, and some embryoids there will be vitrifying.Applicant is also successively in embodiment one, increase sucrose content (2% → 4%), pH(5.5 → 5.8) and agar concentration (0.8% → 1%), the generation that the experiments such as mitogen content (2mg/L → 1.5mg/L) suppress glass seedling reduced.Found that, this several method can suppress the generation of glass seedling, but except the medium of 1% agar concentration affects embryoid growth nothing, other medium is all unfavorable to embryoid growth.
Claims (5)
1. by Unfertilized Ovules, cultivate the haploid method of male sterile tobacco, it is characterized in that concrete steps are as follows:
(1) prepare medium
Adopt H1 medium or H2 medium as minimal medium, then add growth hormone IAA, basic element of cell division KT, sucrose and agar to 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 respectively 0.5mg/L and 2mg/L; Described sucrose and the agar final concentration in embryoid induction medium is respectively 2% and 1%;
Adopt H1 medium as minimal medium, then add sucrose and agar to be prepared into the not solid shape regeneration bud inducing culture containing hormone, wherein, described sucrose and the agar final concentration in regeneration bud inducing culture is 2% and 1%;
Adopt H1 medium and N6 medium as minimal medium, then add growth hormone IAA, sucrose and agar to 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 agar final concentration in regeneration bud root media is respectively 8% and 0.8%;
(2) collection of culture materials, during male sterile Tobacco Flowering, adopt Ovule Development between megasporocyte period to the not male sterile tobacco bud of pollination between two core phases;
(3) disinfect and inoculate, it is 0.1% the mercuric chloride aqueous solution 7-9min that sterilizes that the male sterile tobacco bud gathering in step (2) is put into concentration, then use aseptic water washing 3-4 time, under aseptic condition, the base portion of bud is cut away, extrude ovary, and remove ovary wall or directly peel off after ovule, ovule is put into the embryoid induction medium of step (1) preparation;
(4) induction of embryoid, has the embryoid induction medium of ovule to be placed in 28 ℃ of illumination cultivation chambers inoculation in step (3), and every day, continuous illumination was cultivated 10 hours under 1500Lux illumination, and induction ovule produces embryoid;
(5) induction of regeneration bud, the embryoid obtaining in step (4) is transferred under aseptic condition on the regeneration bud inducing culture of preparation in step (1), continue to be placed in 28 ℃ of illumination cultivation chambers, every day, continuous illumination was cultivated 10 hours under 1500Lux illumination, cultivate 2-3 week, inducing embryoid body produces regeneration bud;
(6) cultivation of regeneration plant, the regeneration bud obtaining in step (5) is transferred on regeneration bud root media under aseptic condition, continue to be placed in 28 ℃ of illumination cultivation chambers, every day, continuous illumination was cultivated 10 hours under 1500Lux illumination, cultivate 2-3 week, make its formation plantlet of taking root, afterwards, to sprouting the regeneration plant of root, transfer on the regeneration bud root media of new preparation and under same condition, continue to cultivate, form male sterile tobacco monoploid regeneration plant.
2. one according to claim 1 is cultivated 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 of cultivating grows to that 2-3cm is high or while having 3-4 sheet blade, the leaf sample of getting gained regeneration plant carries out ploidy by flow cytometry analysis and determines in step (6).
3. one according to claim 1 is cultivated the haploid method of male sterile tobacco by Unfertilized Ovules, it is characterized in that: the bud outward appearance performance period gathering in step 2 between corolla be about to expose calyx period to corolla than calyx the not bud in stage between loose powder period of long a times and flower pesticide.
4. one according to claim 1 is cultivated the haploid method of male sterile tobacco by Unfertilized Ovules, it is characterized in that: in step (7), by flow cytometry analysis ploidy, be 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.
5. one according to claim 2 is cultivated the haploid method of male sterile tobacco by Unfertilized Ovules, it is characterized in that: the described leaf sample that carries out ploidy analysis by flow cytometer is to take regeneration plant 1.3cm
2-1.6cm
2spire, shreds with blade at the cell pyrolysis liquid of 0.5ml, and sample and solution mixture are passed through to aperture 30 μ m micro-pore-film filtrations in testing tube, adds after 20ul DAPI dyeing liquor dyeing 15min, and sample is splined on to flow cytometer.
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