CN103323441A - Fluorescence microscopy method for rapidly and efficiently observing camellia plant pollen tube - Google Patents
Fluorescence microscopy method for rapidly and efficiently observing camellia plant pollen tube Download PDFInfo
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- CN103323441A CN103323441A CN2013102366662A CN201310236666A CN103323441A CN 103323441 A CN103323441 A CN 103323441A CN 2013102366662 A CN2013102366662 A CN 2013102366662A CN 201310236666 A CN201310236666 A CN 201310236666A CN 103323441 A CN103323441 A CN 103323441A
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Abstract
The invention relates to a fluorescence microscopy method for rapidly and efficiently observing a camellia plant pollen tube. The method comprises the following steps: (1) dissecting and separating a pistil material of a camellia plant to be observed into an operation unit; and (2) sequentially soaking the separated material in NaClO solution and NaOH solution until the material is transparent and gets softened, dyeing through a water-soluble aniline blue dye solution, flaking by employing a double-glass slide attachment both-sided observation method, and finally performing pollen tube fluorescence microscopy. The method is easy, convenient and feasible to operate, the experiment efficiency is greatly improved, and the observation effect is optimized.
Description
Technical field
The present invention relates to plant microtechnic field, particularly a kind of fluorescence microscopy method of fast, efficiently observing Camellia plant flowers tube cell.
Background technology
Pollen tube fluorescence microscopy observation is to utilize the low concentration water-soluble aniline blue dyestuff (containing fluorchrome in the azure dye) under the alkali condition to contaminate vegetable material, make the fluorescent dye that is adsorbed in vegetable cell through the ultraviolet excitation of fluorescent microscope, exciting light is absorbed by material, just produce and discharge fluorescence, show the method for pollen tube growth track intuitively.
Traditional pollen tube fluorescence microscopy, softening to vegetable material (as style or ovary) earlier, use the azure dye dip dyeing treatment again, by carrying out Fluirescence observation behind " cover glass+material+microslide " combination compressing tablet.In the classic method, vegetable material only is softened, and opaque, and pollen tube is to grow in transmitting tissue, observes sight line or is blocked.In addition, the light and heavy degree of compressing tablet is difficult to be affectedly bashful, and compressing tablet kicks the beam then overlapping between the tissue, influences observing effect; Compressing tablet is overweight, material fragmentation, can't true reappearance growth track and growth course, and classic method apply to more the tiny plant of gynoecium and only single face observe, but then effect is not good enough for the bigger plants such as Camellia of gynoecium.
Summary of the invention
At the problems referred to above, the purpose of this invention is to provide a kind of fluorescence microscopy method of fast, efficiently observing Camellia plant flowers tube cell.The biology event of described method in can clear and accurate research Camellia plant zoogamy process optimized classic method simultaneously, obtains better observing effect.
The fluorescence microscopy method of observation Camellia plant flowers tube cell of the present invention comprises the steps:
(1) the gynoecium material of anatomical isolation Camellia plant to be seen is to operating unit;
(2) material after will separating is immersed in NaClO and the NaOH solution successively, and is transparent and softening up to material, again through the dyeing of water-soluble aniline blue dye liquor, adopt " two microslide applyings two sides observation " film-making after, carry out the pollen tube fluorescence microscopy and observe.
Wherein, step (1) is structure and the object of observation according to Camellia plant gynoecium, and the anatomical isolation of gynoecium material is divided into style anatomical isolation and ovary anatomical isolation.
If described gynoecium material is style, operating unit is an independently style; If described gynoecium material is ovary, operating unit be row or one give birth to ovule on placenta.
During the style anatomical isolation, neatly excise ovary from the style base portion, obtain an independently style; The anatomical isolation of the style of the gynoecium that described method is applicable to the gynoecium of firm collection, preserve with alcohol.
During the ovary anatomical isolation, neatly excise style, carpopodium from the style base portion, stay ovary, scrape off ovary wall, downcut each ventricle from axis, the orientation of giving birth to according to ovule in each ventricle again and continue cutting, give birth to if the ovule left-right symmetric, then cut from middle symmetry, guaranteeing that ovule is as much as possible is retained on the placenta, if ovule irregular give birth to, then separate in the mode of single ovule, also be retained on the placenta as much as possible.The anatomical isolation of the ovary of the gynoecium that described method is used in the gynoecium of firm collection equally, preserve with alcohol.
Wherein, the available chlorine content of NaClO solution is 8000-10000mg/L in the step (2), and the NaOH solution concentration is 7-9mol/L.
Preferably, the available chlorine content of NaClO solution is 9000mg/L in the step (2), and the NaOH solution concentration is 8mol/L.
Wherein, water-soluble aniline blue dye liquor mass concentration is 0.2-0.3% in the step (2), is that 8 phosphate buffer is formulated with pH.
Preferably, water-soluble aniline blue dye liquor mass concentration is 0.2% in the step (2), is that 8 phosphate buffer is formulated with pH.
Wherein, in transparent, softening in the step (2), when dyeing, used the airtight container splendid attire material that covers, and preferably, described container is measuring cup or penicillin bottle.
Slide thickness is 0.8-1.5mm.Preferably, slide thickness is 1.0mm.
Wherein, material soaks 2-3h earlier in the step (2) in NaClO solution, soaks 2-3h again in NaOH solution after the washing, the 6-8h that in the water-soluble aniline blue dye liquor, dyes after the washing, and film-making is observed and Taking Pictures recording.
Concrete, step (2) is: the material after will separating is through alcohol rehydrations at different levels (50% alcohol → 30% alcohol → distilled water), in NaClO solution, soak 2h, distillation is soaked 2h after washing 3 times in NaOH solution, distillation washing 3 times and flowing water are washed the 6h that dyes behind the 30min in the water-soluble aniline blue dye liquor, film-making is observed.
Wherein, the method that " two microslide applyings two sides observation " described in the step (2) carries out the film-making observation is: get a microslide A, material after the dyeing of water-soluble aniline blue dye liquor is placed on it, drip the water-soluble aniline blue dye liquor around material, get the identical microslide B of another specification, froth is on material, make two microslides be in fit-state, adopt the integrated mode of " microslide A+ material+microslide B ", without compressing tablet, can observe.
Want the bubble in the dye liquor between two microslides of emptying during film-making, make dye liquor and material be in the state of fitting tightly.During observation, can be earlier with the A microslide up, the B microslide deposits observation, and observation subsequently can be inverted.Traditional pressed disc method is " cover glass+material+microslide " combination, and the single face observation only of this method can not be inverted and carry out the two sides observation, and the dynamics of compressing tablet is difficult to grasp.
Preferably, described Camellia plant is oil tea.
The method of the invention is applicable to Camellia plant gynoecium material anatomical isolation and the observation of pollen tube fluorescence microscopy of field " immobile liquid is fixed-the ethanolic solution preservation " too.
The fluorescence microscopy method of fast, efficiently observing Camellia plant flowers tube cell of the present invention has following effect:
(1) the method for the invention initiative " two microslide applyings two sides observation " is carried out the observation of pollen tube fluorescence microscopy, has greatly improved conventional efficient, has optimized observing effect and easy to operation.
(2) the method for the invention has been utilized the transparent effect of NaClO, and it as clarifier, be need not material is dewatered when handling, and has not only avoided material to shrink but also shortened clearing time.
(3) the method for the invention is used clarifier (NaClO) simultaneously and is handled material with softening agent (NaOH), has optimized observing effect.
(4) the method for the invention can directly apply to the Camellia plant gynoecium material of field " immobile liquid is fixed-the ethanolic solution preservation ", and observing effect is good.
Description of drawings
Fig. 1-1 is excision style and carpopodium and polar view (style end) picture (* 15) of oil tea ' Asus ' when not pollinating that scrapes off ovary wall, and each ventricle exposes ovule.
Fig. 1-2 is excision style and carpopodium and equatorial view (upper end the be style end) picture (* 15) of oil tea ' Asus ' when not pollinating that scrapes off ovary wall, and each ventricle exposes ovule.
Fig. 2 is the observation picture (* 15) of the ovule material after the ovary anatomical isolation of oil tea ' Asus ' when not pollinating, and illustrates a row ovule and living on axile placenta.
Fig. 3-1 is traditional " cover glass+material+microslide " film-making, compressing tablet observation simplified schematic diagram.
Fig. 3-2 is " microslide A+ material+microslide B " of the present invention film-making, " two microslide applyings two sides observation " simplified schematic diagram.
Ovule is not transparent, softening through NaClO and NaOH in order to pollinate for Fig. 4-1, after the aniline blue dyeing, " two microslide applyings two sides observation " film-making, the ovule picture (* 40) that arrives with fluorescence microscope.
Fig. 4-2 for do not pollinate only, aniline blue dyeing softening through NaOH of ovule, " two microslide applyings two sides observation " film-making after, the ovule picture (* 40) that arrives with fluorescence microscope.
Fig. 5-1 is to be female parent with oil tea ' Asus ', ' Hunan woods XLC15 ' is male parent, fluorescence microscopy picture (* 40) behind the cross-pollination 132h after the dyeing of ovule, aniline blue transparent, softening through NaClO and NaOH, " two microslide applyings two sides observation " film-making is the observations of " on the A microslide-B microslide under " combination.
Fig. 5-2 is same material shown in Fig. 5-1, is the observations (* 40) of " on the B microslide-A microslide under " combination.
Fig. 6-1 is to be female parent with oil tea ' Asus ', ' Hunan woods XLC15 ' is male parent, fluorescence microscopy picture (* 100) behind the cross-pollination 132h after only, aniline blue dyeing softening through NaOH of ovule, " two microslide applyings two sides observation " film-making is the observations of " on the A microslide-B microslide under " combination.
Fig. 6-2 is same material shown in Fig. 6-1, is the observations (* 100) of " on the B microslide-A microslide under " combination.
Fig. 7-1, Fig. 7-2 are for oil tea ' Asus ' ovule of not pollinating, with the fluorescence microscopy picture (* 40) after tradition " cover glass+material+microslide " pressed disc method film-making.
After Fig. 8-1 is oil tea ' Asus ' self-pollination 72h, style is transparent, softening through NaClO and NaOH, after the aniline blue dyeing, " two microslide applyings two sides observation " film-making, the fluorescence microscopy picture (* 40) of pollen tube, the style full figure that is spliced through Photoshop software is the observations of " on the A microslide-B microslide under " combination.
Fig. 8-2 is same material shown in Fig. 8-1, is the observations (* 40) of " on the B microslide-A microslide under " combination.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, used experiment material, reagent, instrument etc. all are commercially available in the embodiment of the invention, if specifically do not indicate, used technological means is conventional means well-known to those skilled in the art among the embodiment.
State S-SC-CO-011-2009), ' Hunan woods XLC15 ' (numbering: state S-SC-CO-015-2006) be state and examine the oil tea breeding, be commercially available oil tea ' Asus ' (numbering:.
Embodiment 1 material anatomical isolation
The anatomical isolation process of oil tea ' Asus ' gynoecium is divided into the anatomical isolation of ovary and the anatomical isolation of style, and corresponding anatomical isolation method is as follows:
The anatomical isolation of oil tea ' Asus ' ovary: the oil tea gynoecium after will not pollinating or pollinate takes off from the tree body, fixedly is placed in 70% alcohol with the Ka Nuoshi immobile liquid and preserves.When dissected, the gynoecium taking-up is placed in the double dish that is full of 70% alcohol, and place the anatomical lens visual field, at anatomical lens incision oil removing camellia post, carpopodium, scrape off ovary wall, each ventricle exposes ovule (shown in Fig. 1-1, Fig. 1-2), downcut each ventricle from axis, the orientation of giving birth to according to ovule in each ventricle again and continue cutting, give birth to if the ovule left-right symmetric, then cut from middle symmetry, guaranteeing that ovule is as much as possible is retained on the placenta, if ovule irregular give birth to, then separate in the mode of single ovule, also be retained on the placenta as much as possible.Isolated oil tea ovule illustrates a row ovule and living on axile placenta (* 15) as shown in Figure 2.
The anatomical isolation of oil tea ' Asus ' style: the oil tea gynoecium after will not pollinating or pollinate takes off from the tree body, fixedly is placed in 70% alcohol with the Ka Nuoshi immobile liquid and preserves.When dissected is placed on the gynoecium taking-up in the double dish that is full of 70% alcohol, and places the anatomical lens visual field, from style base portion excision oil tea ovary, stays an independently style under anatomical lens.
The anatomical isolation process of the gynoecium of the oil tea of other kinds and Camellia other plant is identical with said method.
The described method of present embodiment is equally applicable to the ovary of the firm gynoecium of gathering and the anatomical isolation of style.
Embodiment 2
In measuring cup or penicillin bottle, with the material to be seen (operating unit) that separates through alcohol rehydrations at different levels (50% alcohol → 30% alcohol → distilled water), in being the NaClO solution of 9000mg/L, available chlorine content soaks 2h, in being the NaOH solution of 8mol/L, concentration soaks 2h after the distillation washing 3 times, after distillation washing 3 times and flowing water are washed 30min, (be that 8 phosphate buffer formulated with pH) dyeing 6h in mass concentration is 0.2% water-soluble aniline blue dye liquor, film-making is observed.
The concrete operation method of " two microslide applyings two sides observation " is (shown in Fig. 3-2): getting a thickness is the microslide A of 1.0mm, material to be seen after the dyeing of water-soluble aniline blue dye liquor is placed on it, around material, drip the water-soluble aniline blue dye liquor, get the identical microslide B of another specification, froth makes two microslides be in fit-state on material, forms the integrated mode of " microslide A+ material+microslide B ", without compressing tablet, can observe.Want the bubble in the dye liquor between two microslides of emptying during film-making, make dye liquor and material be in the state of fitting tightly.
Embodiment 3
The ovule material (pollination) of pressing the oil tea ' Asus ' that embodiment 1 method separates is transparent through NaClO solution, NaOH solution softening, after the aniline blue dyeing, " two microslide applyings two sides observation " film-making, the fluorescence microscope effect is shown in Fig. 4-1.
After ovule material (pollination) NaOH solution in the conventional view method of pressing the oil tea ' Asus ' of embodiment 1 method separation softens, aniline blue dyes and handles, " two microslide applyings two sides observation " film-making is not (except having the transparent step of NaClO solution, other steps are identical with embodiment 2 described methods), the fluorescence microscope effect is shown in Fig. 4-2.
Comparison diagram 4-1 and Fig. 4-2 can find out, uses method of the present invention, and material is after NaClO solution is transparent, and observing effect has had significant improvement.Among Fig. 4-1 the ovule clear-cut as seen, integument, the hole of bead, blastular orientation and clear-cut as seen, and among Fig. 4-2 ovule to observe sight line dim, can not differentiate integument, the hole of bead, blastular orientation and profile.
Embodiment 4
Be female parent with oil tea ' Asus ', ' Hunan woods XLC15 ' is male parent, behind the cross-pollination 132h, presses embodiment 1 described method and obtains oil tea ovule material to be seen, adopt embodiment 2 described methods to material processed, film-making, observations is shown in Fig. 5-1, Fig. 5-2.
Wherein, Fig. 5-1 is that ovule is transparent, softening through NaClO and NaOH, the fluorescence microscopy picture (* 40) after the aniline blue dyeing, " two microslide applyings two sides observation " film-making, is the observations of " on the A microslide-B microslide under " combination.Can be found out that by Fig. 5-1 the pollinia pipe has entered ovary, pollen tube all enters blastular by the hole of bead of two ovules.Fig. 5-2 is the fluorescence microscopy picture (* 40) of be inverted after the above-mentioned same material film-making " on the B microslide-A microslide under ".Entered ovary by Fig. 5-2 also visible pollinia pipe, but that pollen tube enters the situation of two ovules is completely different, wherein the pollen tube situation that whether enters the upper position ovule can't be observed, and enters down the orientation ovule and see pollen tube only.This shows that use the advantage of " two microslide applyings two sides observation " film-making to be crucial biology event in the seizure zoogamy process of maximal efficiency, traditional compressing tablet observation can only be observed the poor efficiency that then seems by single face.
Fig. 6-1 for ovule by only, the aniline blue dyeing softening through NaOH of conventional fluorescent microscopy observation method, film-making (except not having the transparent step of NaClO solution, other steps are identical with embodiment 2 described methods) after fluorescence microscopy picture (* 100), be the observations of " on the A microslide-B microslide under " combination.Can be found out that by Fig. 6-1 pollen tube has passed ovary outside placenta arrives ovule, whether enters blastular through the hole of bead but can't observe, the track of pollen tube growth is very unclear yet.Fig. 6-2 is the fluorescence microscopy picture (* 100) of be inverted after the above-mentioned same material film-making " on the B microslide-A microslide under ".Also can be observed pollen tube by Fig. 6-2 and arrived outside the ovule, can't observe whether enter blastular through the hole of bead equally.This shows that behind traditional pollen tube fluorescence microscopy observation processing material, though adopt " two microslide applyings two sides observation " film-making, the result that the two sides is observed is all not satisfactory.So, in ovule is observed, use successively that NaClO solution is transparent, NaOH softening, after the aniline blue dyeing, in conjunction with " two microslide applyings two sides observation " film-making, observing effect just can reach optimum.
Can be found out that by Fig. 4-2, Fig. 6-1, Fig. 6-2 in the classic method, only with NaOH solution material softening is handled, this processing mode causes the material transparent effect relatively poor, the observation sight line is not good enough; And the present invention adopts the transparent back of NaClO solution, the softening method of NaOH solution to handle material, observing effect has had significant improvement, the ovule clear-cut as seen, integument, the hole of bead, blastular orientation and clear-cut are as seen, the pollen tube growth track is clear, and the situation that enters blastular is understood.
Embodiment 5
Fig. 7-1, Fig. 7-2 is the fluorescence microscopy picture (* 40) of the ovule of not pollinating with oil tea ' Asus ' after the film-making of tradition " cover glass+material+microslide " pressed disc method (as Fig. 3-1).As seen from the figure, the ovule structure is destroyed, loses the observation meaning.
Can be found out that by Fig. 7-1, Fig. 7-2 in the classic method, adopt " cover glass+material+microslide " pressed disc method to observe, dynamics is difficult to grasp, and easily makes material crushing, distortion, has destroyed real structure, and has observed from one side only; And the present invention adopts " microslide A+ material+microslide B " applying two sides observation (as Fig. 3-2), without compressing tablet, and turningly all can observe from the two sides, has guaranteed the authenticity of material structure, and the simple flow of film-making simultaneously is row and rapidly and efficiently easily.
Embodiment 6
With 72h after oil tea ' Asus ' self-pollination, press the style under the embodiment 1 described method acquisition oil tea gynoecium material cutting to be seen, pressing embodiment 2 described methods handles, fluorescence microscopy picture after " two microslide applyings two sides observation " film-making, observations is shown in Fig. 8-1, Fig. 8-2, Fig. 8-1 is the observations (* 40) of " on the A microslide-B microslide under " combination, and Fig. 8-2 be the observations (* 40) that " on the B microslide-A microslide under " makes up.
Can be found out by Fig. 8-1, Fig. 8-2, the observations on two sides is totally different, and the true growth track of the clear reproduction pollen tube of Fig. 8-1 in style can be determined the position that pollen tube arrives in corresponding growth time, Fig. 8-2 then only shows style exocuticle, invisible pollen tube.
By each embodiment as can be known, the method for the invention is easy to operation, has greatly improved conventional efficient, and has optimized observing effect.
The fluorescence microscopy method of observation Camellia plant flowers tube cell of the present invention is not only applicable to oil tea, and is applicable to the fluorescence microscopy observation of the pollen tube of other Camellia plants.
Though, above used general explanation, embodiment and test, the present invention is described in detail, on basis of the present invention, can make some modifications or improvements it, and this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a fluorescence microscopy method of observing Camellia plant flowers tube cell is characterized in that, said method comprising the steps of:
(1) the gynoecium material of anatomical isolation Camellia plant to be seen is to operating unit;
(2) material after will separating is immersed in NaClO and the NaOH solution successively, and is transparent and softening up to material, again through the dyeing of water-soluble aniline blue dye liquor, adopt " two microslide applyings two sides observation " film-making after, carry out the pollen tube fluorescence microscopy and observe.
2. method according to claim 1 is characterized in that, in the step (1), if described gynoecium material is style, operating unit is an independently style; If described gynoecium material is ovary, operating unit be row or one give birth to ovule on placenta.
3. method according to claim 2 is characterized in that, in the step (1), during the style anatomical isolation, neatly excises ovary from the style base portion, obtains an independently style; During the ovary anatomical isolation, neatly excise style, carpopodium from the style base portion, stay ovary, scrape off ovary wall, downcut each ventricle from axis, the orientation of giving birth to according to ovule in each ventricle again and continue cutting, give birth to if the ovule left-right symmetric, then cut from middle symmetry, make that ovule is as much as possible to be retained on the placenta, if ovule irregular give birth to, then separate in the mode of single ovule, ovule is retained on the placenta as much as possible.
4. method according to claim 1 is characterized in that, the available chlorine content of NaClO solution is 8000-10000mg/L in the step (2), and the NaOH solution concentration is 7-9mol/L.
5. method according to claim 1 is characterized in that, water-soluble aniline blue dye liquor mass concentration is 0.2-0.3% in the step (2).
6. method according to claim 1 is characterized in that, slide thickness is 0.8-1.5mm in the step (2).
7. method according to claim 1 is characterized in that, material soaks 2-3h earlier in the step (2) in NaClO solution, soaks 2-3h again in NaOH solution after the washing, and the washing back is at water-soluble aniline blue dye liquor dyeing 6-8h, and film-making is observed.
8. method according to claim 7, it is characterized in that, step (2) is: with the material after the anatomical isolation through alcohol rehydrations at different levels, in NaClO solution, soak 2h, distillation is soaked 2h after washing 3 times in NaOH solution, after distillation washing 3 times and flowing water are washed 30min, the 6h that in the water-soluble aniline blue dye liquor, dyes, film-making is observed.
9. method according to claim 1, it is characterized in that, the method that " two microslide applyings two sides observation " described in the step (2) carries out the film-making observation is: get a microslide A, material after the dyeing of water-soluble aniline blue dye liquor is placed on it, around material, drip the water-soluble aniline blue dye liquor, get the identical microslide B of another specification, froth is on material, make two microslides be in fit-state, adopt the integrated mode of " microslide A+ material+microslide B ", without compressing tablet, observe.
10. method according to claim 9 is characterized in that, wants the bubble in the dye liquor between two microslides of emptying during film-making, makes dye liquor and material be in the state of fitting tightly.
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| CN104359734A (en) * | 2014-11-12 | 2015-02-18 | 云南省农业科学院花卉研究所 | Production method for fluorescent microscopic slices of ovule of pollinated azalea |
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