CN105850714B - Dendrobium candidum breeding method and corresponding preparation method based on development of floral organs - Google Patents
Dendrobium candidum breeding method and corresponding preparation method based on development of floral organs Download PDFInfo
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- 241000026010 Dendrobium candidum Species 0.000 title claims abstract description 50
- 238000011161 development Methods 0.000 title claims abstract description 28
- 238000009395 breeding Methods 0.000 title claims abstract description 25
- 210000000056 organ Anatomy 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims description 8
- 239000000575 pesticide Substances 0.000 claims abstract description 81
- 230000010152 pollination Effects 0.000 claims abstract description 62
- 230000018109 developmental process Effects 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 27
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 claims abstract description 12
- GCKALTUGDMFFNE-UHFFFAOYSA-N 4-(4-aminophenyl)aniline;hydrogen peroxide Chemical compound OO.C1=CC(N)=CC=C1C1=CC=C(N)C=C1 GCKALTUGDMFFNE-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000010186 staining Methods 0.000 claims abstract description 9
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- 238000004043 dyeing Methods 0.000 claims description 13
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 239000011521 glass Substances 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000010586 diagram Methods 0.000 claims description 5
- 229960002163 hydrogen peroxide Drugs 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 3
- DMVOXQPQNTYEKQ-UHFFFAOYSA-N biphenyl-4-amine Chemical compound C1=CC(N)=CC=C1C1=CC=CC=C1 DMVOXQPQNTYEKQ-UHFFFAOYSA-N 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N diphenyl Chemical compound C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 claims 2
- 235000010290 biphenyl Nutrition 0.000 claims 1
- 239000004305 biphenyl Substances 0.000 claims 1
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- 239000003814 drug Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 244000025254 Cannabis sativa Species 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 241000254023 Locusta Species 0.000 description 2
- 206010044565 Tremor Diseases 0.000 description 2
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- CPWCKGMTKBLVHZ-UHFFFAOYSA-M 1,2,5-triphenyltetrazol-1-ium;chloride Chemical compound [Cl-].C1=CC=CC=C1C1=[N+](C=2C=CC=CC=2)N(C=2C=CC=CC=2)N=N1 CPWCKGMTKBLVHZ-UHFFFAOYSA-M 0.000 description 1
- LCJINPXTMHVZGV-UHFFFAOYSA-N 2,3,5-triphenyl-1h-tetrazol-4-ium;chloride Chemical class [Cl-].[NH2+]1N(C=2C=CC=CC=2)N(C=2C=CC=CC=2)N=C1C1=CC=CC=C1 LCJINPXTMHVZGV-UHFFFAOYSA-N 0.000 description 1
- 241000282452 Ailuropoda melanoleuca Species 0.000 description 1
- 206010010144 Completed suicide Diseases 0.000 description 1
- 241001523681 Dendrobium Species 0.000 description 1
- 241001076416 Dendrobium tosaense Species 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
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- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of dendrobium candidum breeding method based on development of floral organs：Take away and spend the flower pesticide of 8~14d to pollinate onto the column cap for blooming 8~21d.Present invention also offers a kind of method for obtaining above-mentioned dendrobium candidum breeding method, include the following steps：1), when dendrobium candidum branch begins to show petal, it is listed；2) little Hua of whole florescence different flowering number of days in same raceme, is won；3), using the whole florescence different flowering number of days of TTC Determination Stainings little Hua flower pesticide vigor；4) Enumerable infinite set of the stylet head of whole florescence different flowering number of days, is measured using benzidine hydrogen peroxide method；5), in same raceme, the energetic flower pesticide pollination of flower pesticide is selected to the strong column cap of Enumerable infinite set.According to the above method, 150 days or so, the capsule of consistent, the high fruit-setting rate of genetic background can be obtained.
Description
Technical field
The present invention relates to a kind of dendrobium candidum producing method for seed based on development of floral organs.
Background technology
Dendrobium candidum (Dendrobium officinale Kimura et Migo) is China's tradition rare traditional Chinese medicine, tool
There is the effect of reinforcing stomach reg fluid, nourishing Yin and clearing heat etc. are unique.Early in Qin Han dynasty,《Sheng Nong's herbal classic》With regard to recording dendrobium candidum " main wound
In, eliminating impediment, lower gas, the thin thin, Qiang Yin of tonifying five zang organs consumptive disease, long term usage thickness stomach "；Before more than 1000 years《Taoist Scriptures》Dendrobium candidum is classified as
First of " Chinese nine big celestial grass "；Li Shizhen (1518-1593 A.D.) exists《Compendium of Materia Medica》Middle evaluation dendrobium candidum " strong the moon strengthening the essence, thick stomach, mend in never
Foot, flat stomach gas, long muscle, intelligence development is except frightened, macrobiosis of making light of one's life by commiting suicide "；Among the people to be called help celestial grass, international medicinal plant circle is known as " medicine
Boundary giant panda ".Modern pharmacological research proves that dendrobium candidum has strengthen immunity, suppresses the effect such as cancer.For this reason, 2010 editions
《Pharmacopoeia of People's Republic of China》Spy individually records dendrobium candidum.Before the 1990s, dendrobium candidum relies primarily on open country
Production-goods source, since seed sprouts that difficult, growth and development is slow under natural conditions, plus people for a long time to progresss destructiveness
Excavation and the destruction of living environment, make its wild resource be in endangered, international community is included in《Wild plant state in imminent danger
Border trade pact CITES》, dendrobium candidum is also classified as national second class protection endangered plants by China.
In recent years, as the growth of " blowout " occur in plantation, the breakthrough of processing technology, dendrobium candidum cultivated area,
2015, national medicinal dendrobium cultivated area was up to more than 50000 mus, and cultivated area continues to increase, and needed 80,000 plants of seedling per acre
Left and right, the demand of seedling is big, and the seedling breeding of dendrobium candidum is most at present uses artificial pollination, dependent on tissue cultures
Seedling breeding production technology of growing directly from seeds.But breeding research theoretical foundation of the current China to orchids such as dendrobium candidums is weak,
The production of hybrid seeds (the different storeroom of genetic background carries out the pollination production of hybrid seeds) lack of standardization causes variet complexity, kind property shakiness, and quality differs,
Medicinal ingredient difference in these former plant varieties ultimately results in medicinal material drug effect shakiness, seriously hinders the further of dendrobium candidum
Utilization, become the bottleneck of industry development.And the racemose speciality of dendrobium candidum, i.e., every small Huadu have an anthocaulus with
Floral axis is regularly connected, there is the flower of different development degrees on whole floral axis, the effective guarantee consistent parent of genetic background
This material.Carry out the production of hybrid seeds pattern based on dendrobium candidum development of floral organs for this, the consistent capsule of a large amount of genetic backgrounds can be obtained
Fruit, effectively solves the Genetic stability problems of introduces a collection, it will help offer genetic background is consistent, medicinal ingredient is identical, drug effect is steady
Fixed high quality seedling.
Breeding research theoretical foundation of the current China to dendrobium candidum is weak, and the production of hybrid seeds is lack of standardization.Many enterprises are not directly
The pollination production of hybrid seeds is carried out between the different dendrobium candidum of the genetic backgrounds such as same kind, strain and source, causes variet complexity, kind property not
Connect with the heart steady, quality differs.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of dendrobium candidum producing method for seed based on development of floral organs, uses
This method can obtain the consistent capsule of a large amount of genetic backgrounds, effectively solve the Genetic stability problems of introduces a collection, it will help provide
Genetic background is consistent, medicinal ingredient is identical, the high quality seedling of efficacy stability.
In order to solve the above technical problem, the present invention provides a kind of dendrobium candidum breeding method based on development of floral organs：Take
Bloom 8~14d flower pesticide pollination on the column cap for blooming 8~21d.
The present invention also provides the method for obtaining above-mentioned dendrobium candidum breeding method at the same time, includes the following steps：
1), when dendrobium candidum branch begins to show petal, it is listed (that is, marking)；
2) little Hua of whole florescence different flowering number of days in same raceme, is won；
3), using TTC (2,3,5- triphenyltetrazolium chlorides, 2,3,5, Triphenyl-2H-Tetrazolium
Chloride) the vigor of the little Hua flower pesticide of the whole florescence different flowering number of days of Determination Staining；
4) Enumerable infinite set of the stylet head of whole florescence different flowering number of days, is measured using benzidine-hydrogen peroxide method；
5), in same raceme, the energetic flower pesticide pollination of flower pesticide is selected to the strong column cap of Enumerable infinite set.
According to the above method, 150 days or so, the capsule of consistent, the high fruit-setting rate of genetic background can be obtained.
Remarks explanation：In the present invention, according to coloration result, the development time table of flower pesticide and column cap is established, takes flower pesticide to live
The strong flower pesticide of power is pollinated onto the strong column cap of Enumerable infinite set.
Improvement as the method for the acquisition dendrobium candidum breeding method of the present invention：
After step 1),
The raceme chosen on a branch proceeds as follows：Record in the raceme, each little Hua is from flower
Bud forms the whole florescence started to flower at the end of withering；
Meanwhile choose the raceme on another branch and carry out above-mentioned steps 2)~step 5).
Further improvements in methods as the acquisition dendrobium candidum breeding method of the present invention：
TTC Determination Stainings in the step 3) are：The flower pesticide in the small flower pesticide cap of dendrobium candidum is taken out, is placed in centrifuge tube
It is interior, after adding TTC dye liquors not have flower pesticide, 37 DEG C of lucifuges to be incubated 15~30min, dye liquor is abandoned, with rinsed with sterile water flower pesticide, blotting paper is inhaled
Dry surface moisture；Flower pesticide after dyeing is placed in observation under stereoscopic zoom microscope to take pictures；Flower pesticide dyes red and more deeply feels bright work
Power is stronger.
Further improvements in methods as the acquisition dendrobium candidum breeding method of the present invention：
The preparation method of the TTC dye liquors is：By the phosphate buffer (PBS) of the TTC of 0.5g 0.1mol/L pH 7.5
Dissolve and be settled to 100ml, so as to be configured to the TTC dyeing liquors that concentration is 0.5%.
Remarks explanation：TTC dye liquors need to be kept in dark place, and cannot be used again if rubescent.
Further improvements in methods as the acquisition dendrobium candidum breeding method of the present invention：
Benzidine-hydrogen peroxide method of the step 4) is determined as：Small flower is connected anthocaulus to adopt down, removes all petals
(removal flower pesticide)；Gynostemium is cut along membrane with scalpel, removes stamen and exposes column cap nest；Column cap nest is placed on glass slide,
2~3 drop benzidine-hydroperoxidation liquid are added dropwise on column cap nest；Glass slide is immediately placed under stereoscopic zoom microscope and is seen
Examine and take pictures；The reaction solution of column cap surrounding present blueness is deeper, bubbles volume show column cap Enumerable infinite set it is stronger.
Further improvements in methods as the acquisition dendrobium candidum breeding method of the present invention：
The preparation method of benzidine-hydroperoxidation liquid is：It is 1% biphenyl amine aqueous solution by mass concentration, volumetric concentration
It is 3% hydrogenperoxide steam generator, distilled water according to 4:11:22 volume ratio is prepared and obtained.
Further improvements in methods as the acquisition dendrobium candidum breeding method of the present invention：In the step 5), Mei Gezong
Locusta 3~5 flowers of pollination.That is, each branch does 3-5 colored pollination.
Further improvements in methods as the acquisition dendrobium candidum breeding method of the present invention：Being listed for the step 1) be：
Label is hung up in the branch base portion for beginning to show petal.
Further improvements in methods as the acquisition dendrobium candidum breeding method of the present invention：By listed branch, clap daily
According to each little Hua in tracking or the diagram whole raceme of writing record at the end of being withered petal is formed to flower
The whole florescence.
The dendrobium candidum producing method for seed based on development of floral organs of the present invention, is according to the racemose spy of dendrobium candidum
Matter, i.e., every small Huadu have an anthocaulus to be regularly connected with floral axis, there is the flower of different development degrees on whole floral axis,
This is flower pesticide in the same little Hua of dendrobium candidum and stigma development is asynchronous provides effective material base, effective guarantee heredity
The consistent parent material of background.Carry out the production of hybrid seeds pattern based on dendrobium candidum development of floral organs for this, a large amount of heredity can be obtained
The consistent capsule of background, effectively solves the Genetic stability problems of introduces a collection, it will help offer genetic background is consistent, medicinal ingredient
Identical, efficacy stability high quality seedling.
The technical advantages of the present invention are that：
1st, the present invention is established total using TTC dyeing flower pesticide vigor and benzidine-hydrogen peroxide method measure Pistil recipient phase
Each little Hua is deployed into the flower pesticide at the whole florescence that flower withers and the development time table of column cap from petal in locusta, so
We can select energetic flower pesticide and the good column cap of Enumerable infinite set carry out pollination group according to column cap and the Developmental maturity of flower pesticide
The proportioning of conjunction, substantially increases the percentage of fertile fruit of capsule after pollination, efficiently solves in actual production that pollination amount is big and percentage of fertile fruit is low
Production bottleneck.
2nd, the present invention is proposed carries out the pollination production of hybrid seeds in same raceme, so can be with effective guarantee parent material
Genetic background it is consistent, obtain that substantial amounts of capsule genetic background is consistent, effectively solve the Genetic stability problems of introduces a collection, will help
In providing, genetic background is consistent, medicinal ingredient is identical, the high quality seedling of efficacy stability.
The vigor for the TTC Determination Staining dendrobium candidum flower pesticide that the present invention uses, has the following advantages：1) TTC dyeing is direct
Have drawn from flesh tissue, convenient and easy；2) overall process about 15~30min of dyeing, saves the time；3) need not in dyeing course
Special experiment equipment, required instrument and equipment are simple；4) within 3 day time, the TTC dye liquors of 10mL are 2-3 times reusable, greatly
18 sections can about be contaminated.
The present invention uses the flower of the TTC decoration methods dendrobium candidum consistent with benzidine-hydrogen peroxide method measure genetic background
Medicine vigor and Pistil recipient phase, disclose the asynchronous development phenomenon of flower pesticide and column cap.
Brief description of the drawings
The embodiment of the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is TTC Determination Staining flower pesticide vigor, i.e. the flower pesticide schematic diagram of the different female bloom dates after TTC dyeing；1d~30d
The flower pesticide bloomed 1-30 days is represented respectively.
Fig. 2 is benzidine-hydrogen peroxide Determination Staining Pistil recipient phase；I.e., after benzidine-hydrogen peroxide dyeing not
With the column cap schematic diagram at florescence；1d~30d represents the column cap bloomed 1-30 days respectively.
Fig. 3 is the percentage of fertile fruit of each pollination combination；
Flower pesticide is divided into 3 groups according to flower pesticide vigor and Pistil recipient phase：Bloom 1-7d (A), 8-14d of blooming (B), 14d of blooming
After (C)；Column cap is also classified into 3 groups：Bloom 1-7d (a), after 8-21d (b) of blooming, 21d of blooming (c).
Aa is represented：Bloom the flower pesticide of 1-7d (A) and the column cap of 1-7d (a) of blooming is combined pollination；
Ab is represented：Bloom the flower pesticide of 1-7d (A) and the column cap of 8-21d (b) of blooming is combined pollination；
Ac is represented：Bloom the flower pesticide of 1-7d (A) and the column cap of (c) after 21d of blooming is combined pollination；
Ba is represented：Bloom the flower pesticide of 8-14d (B) and the column cap of 1-7d (a) of blooming is combined pollination；
Bb is represented：Bloom the flower pesticide of 8-14d (B) and the column cap of 8-21d (b) of blooming is combined pollination；
Bc is represented：Bloom the flower pesticide of 8-14d (B) and the column cap of (c) after 21d of blooming is combined pollination；
Ca is represented：The column cap of the flower pesticide of (C) and 1-7d (a) of blooming after 14d of blooming are combined pollination；
Cb is represented：The column cap of the flower pesticide of (C) and 8-21d (b) of blooming after 14d of blooming are combined pollination；
Cc is represented：Blooming after 14d, the column cap of (c) is combined pollination after the flower pesticide of (C) and 21d of blooming；
Note：P≤0.05.
Embodiment
Embodiment 1, a kind of method for obtaining the dendrobium candidum breeding method based on development of floral organs, carry out following step successively
Suddenly：
1), label (that is, marking) is hung up in the base portion for the branch for beginning to show petal；
2 listed branches are proceeded as follows respectively, i.e. one of branch carries out following step 2), meanwhile, separately
One branch carries out following step 3)；
2), the branch takes pictures in tracking or the diagram whole raceme of writing record each little Hua from petal shape daily
Into the change for starting to flower the whole florescence at the end of withering, and flowering dates is indicated on label；
3), the branch is won in same raceme, the little Hua of whole florescence different flowering number of days；It is careful to take out iron sheet stone
2 pieces of massulas in the small flower pesticide cap of dry measure used in former times, are placed in centrifuge tube, add 0.3ml TTC dye liquors not have flower pesticide, and 37 DEG C of lucifuge water-baths are incubated
After educating 15min, dye liquor is abandoned, with rinsed with sterile water flower pesticide, blotting paper blots surface moisture；Flower pesticide after dyeing is placed in stereoscopic change
Observation is taken pictures under focusing microscope；
The specific preparation method of TTC dye liquors is as follows：
The TTC powder of 0.5g is weighed, is dissolved and is settled to the phosphate buffer (PBS) of 0.1mol/L pH 7.5
100ml, is configured to the TTC dyeing liquors that concentration is 0.5%.Solution is kept in dark place, and cannot be used again if rubescent.
By the above-mentioned little Hua for having taken out flower pesticide, it is close to membrane lower edge with scalpel and cuts gynostemium, cut away stamen and expose column
Head nest；Column cap nest is placed on glass slide, 2~3 drops (about 0.1ml) are added dropwise and drip benzidine-hydroperoxidation liquid in column cap nest
On；Glass slide is placed in observation under stereoscopic zoom microscope rapidly to take pictures；
The preparation method of benzidine-hydroperoxidation liquid is specific as follows：1% biphenyl amine aqueous solution, 3% hydrogen peroxide are molten
Liquid, distilled water are according to volume ratio 4:11:22 prepare.
In same raceme, the most strong flower pesticide pollination of flower pesticide vigor is selected to the most strong column cap of Enumerable infinite set.Flower pesticide passes through
TTC is dyed, flower pesticide dye it is red more to deeply feel bright vigor stronger, and non-staining flower pesticide is not vigourous；Benzidine-hydrogen peroxide dye
Color column cap, the reaction solution of column cap surrounding present blueness is deeper, bubbles volume show column cap Enumerable infinite set it is stronger；It is otherwise poorer.
According to coloration result, the development time table of flower pesticide and column cap is established, takes the most strong flower pesticide of flower pesticide vigor to pollinate most strong to Enumerable infinite set
Column cap on.
Remarks illustrate that corresponding content is in the development time table of flower pesticide：The flower pesticide of different flowering number of days, after TTC is dyed
The change of red weight, represents the change of flower pesticide vigor in the whole florescence；Corresponding content is in the development time table of column cap：No
With the column cap for number of days of blooming, the change of blue weight and bubbles volume after the dyeing of benzidine hydrogen peroxide, represents whole flower
The change of the Enumerable infinite set of interim column cap.
Acquired results are specific as follows：According to coloration result, the development time table of flower pesticide and column cap is established.According to flower pesticide vigor
Flower pesticide is divided into 3 groups with Pistil recipient phase：Bloom 1-7d (A), after 8-14d of blooming (B), 14d of blooming (C)；Column cap is also classified into 3
Group：Bloom 1-7d (a), after 8-21d (b) of blooming, 21d of blooming (c).
It is observed that：In above-mentioned 3 groups of flower pesticide, the flower pesticide red for 8~14d of blooming is most deep, i.e. the vigor of this group of flower pesticide is most
By force.In above-mentioned 3 groups of column caps, blueness that the reaction solution of the column cap surrounding for 8~21d of blooming is presented is most deep, bubbles volume is most, i.e.
The Enumerable infinite set of this group of column cap is stronger.
Experiment 1, take away and spend the flower pesticide of 10d to pollinate onto the column cap for blooming 15d.16 branches of pollination, each branch
Bar does 5 colored pollinations, pollinates 80, is repeated 3 times altogether.It turns out that the percentage of fertile fruit of pollination combination is 96.67%, such as
Described in Fig. 3.
According to the above method, 150 days or so, the capsule of consistent, the high fruit-setting rate of genetic background can be obtained.
Remarks explanation：Take away and spend the pollination of 8~14d flower pesticide of any day to blooming on the 8-21d column caps of any day,
Above-mentioned experiment 1 is repeated, percentage of fertile fruit is about 96~97%.
Comparative example 1, take away the flower pesticide pollination of colored 4d on the column cap for blooming 4d.16 branches of pollination, each branch do 5
Piece colored pollination, pollinates 80, is repeated 3 times altogether.It turns out that the percentage of fertile fruit of pollination combination (Aa) is 1.67%, such as Fig. 3
It is described.
Comparative example 2, take away the flower pesticide pollination of colored 4d on the column cap for blooming 15d.16 branches of pollination, each branch are done
5 colored pollinations, pollinate 80, are repeated 3 times altogether.It turns out that the percentage of fertile fruit of pollination combination (Ab) is 5.42%, such as scheme
Described in 3.
Comparative example 3, take away the flower pesticide pollination of colored 4d on the column cap for blooming 25d.16 branches of pollination, each branch are done
5 colored pollinations, pollinate 80, are repeated 3 times altogether.It turns out that the percentage of fertile fruit of pollination combination (Ac) is 1.67%, such as scheme
Described in 3.
Comparative example 4, take away the flower pesticide pollination of colored 10d on the column cap for blooming 4d.16 branches of pollination, each branch are done
5 colored pollinations, pollinate 80, are repeated 3 times altogether.It turns out that the percentage of fertile fruit of pollination combination (Ba) is 2.92%, such as scheme
Described in 3.
Comparative example 5, take away the flower pesticide pollination of colored 10d on the column cap for blooming 25d.16 branches of pollination, each branch
5 colored pollinations are done, pollinates 80, is repeated 3 times altogether.It turns out that the percentage of fertile fruit of pollination combination (Bc) is 1.25%, such as
Described in Fig. 3.
Comparative example 6, take away the flower pesticide pollination of colored 20d on the column cap for blooming 4d.16 branches of pollination, each branch are done
5 colored pollinations, pollinate 80, are repeated 3 times altogether.It turns out that the percentage of fertile fruit of pollination combination (Ca) is 2.08%, such as scheme
Described in 3.
Comparative example 7, take away the flower pesticide pollination of colored 20d on the column cap for blooming 15d.16 branches of pollination, each branch
5 colored pollinations are done, pollinates 80, is repeated 3 times altogether.It turns out that the percentage of fertile fruit of pollination combination (Cb) is 2.92%, such as
Described in Fig. 3.
Comparative example 8, take away the flower pesticide pollination of colored 20d on the column cap for blooming 25d.16 branches of pollination, each branch
5 colored pollinations are done, pollinates 80, is repeated 3 times altogether.It turns out that the percentage of fertile fruit of pollination combination (Cc) is 2.92%, such as
Described in Fig. 3.
From the figure 3, it may be seen that the pollination combination (Bb) of the column cap of the flower pesticide for 8-14d of blooming and 8-21d of blooming combination, percentage of fertile fruit reaches
To 96.67% or so, it is significantly higher than other combinations, experiment shows to obtain high percentage of fertile fruit, the flower pesticide of strong vigor and strong Enumerable infinite set
Column cap it is indispensable.
Shown in the comparing result table 1 specific as follows of above-mentioned case：
Table 1
| Scheme | Percentage of fertile fruit |
| Embodiment 1 | 96.67% |
| Comparative example 1 | 1.67% |
| Comparative example 2 | 5.42% |
| Comparative example 3 | 1.67% |
| Comparative example 4 | 2.92% |
| Comparative example 5 | 1.25% |
| Comparative example 6 | 2.08% |
| Comparative example 7 | 2.92% |
| Comparative example 8 | 2.92% |
Finally, it should also be noted that it is listed above be only the present invention several specific embodiments.Obviously, this hair
It is bright to be not limited to above example, there can also be many deformations.Those of ordinary skill in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (6)
1. the dendrobium candidum breeding method based on development of floral organs, it is characterized in that including the following steps：：
1), when dendrobium candidum branch begins to show petal, it is listed；
2) little Hua of whole florescence different flowering number of days in same raceme, is won；
3), using the whole florescence different flowering number of days of TTC Determination Stainings little Hua flower pesticide vigor；
4) Enumerable infinite set of the stylet head of whole florescence different flowering number of days, is measured using benzidine-hydrogen peroxide method；
5), in same raceme, take away and spend the flower pesticide pollination of 8~14d to bloom 8~21d's to same raceme
On column cap；
TTC Determination Stainings in the step 3) are：The flower pesticide in the small flower pesticide cap of dendrobium candidum is taken out, is placed in centrifuge tube,
After adding TTC dye liquors not have flower pesticide, 37 DEG C of lucifuges to be incubated 15~30min, dye liquor is abandoned, with rinsed with sterile water flower pesticide, blotting paper blots
Surface moisture；Flower pesticide after dyeing is placed in observation under stereoscopic zoom microscope to take pictures；Flower pesticide dyes red and more deeply feels bright vigor
It is stronger；
The preparation method of the TTC dye liquors is：The TTC of 0.5g is dissolved and determined with the phosphate buffer of 0.1mol/L pH 7.5
Hold to 100ml, so as to be configured to the TTC dyeing liquors that concentration is 0.5%；
Benzidine-hydrogen peroxide method of the step 4) is determined as：Small flower is connected anthocaulus to adopt down, removes all petals；With solution
Cut open cutter cuts gynostemium along membrane, removes stamen and exposes column cap nest；Column cap nest is placed on glass slide, 2~3 drop biphenyl are added dropwise
Amine-hydroperoxidation liquid is on column cap nest；Glass slide is immediately placed in observation under stereoscopic zoom microscope to take pictures；Column cap surrounding
Reaction solution present the deeper, bubbles volume of blueness show column cap Enumerable infinite set it is stronger.
2. the dendrobium candidum breeding method according to claim 1 based on development of floral organs, it is characterized in that：
After step 1),
The raceme chosen on a branch proceeds as follows：Record in the raceme, each little Hua is from petal shape
Into the whole florescence started to flower at the end of withering；
Meanwhile choose the raceme on another branch and carry out above-mentioned steps 2)~step 5).
3. the dendrobium candidum breeding method according to claim 2 based on development of floral organs, it is characterized in that：
The preparation method of benzidine-hydroperoxidation liquid is：It is 1% biphenyl amine aqueous solution, volumetric concentration 3% by mass concentration
Hydrogenperoxide steam generator, distilled water are according to 4:11:22 volume ratio is prepared and obtained.
4. the dendrobium candidum breeding method according to claim 3 based on development of floral organs, it is characterized in that：The step 5)
In, 3~5 flowers of each raceme pollination.
5. the dendrobium candidum breeding method according to claim 4 based on development of floral organs, it is characterized in that：The step 1)
It is listed and is：Label is hung up in the branch base portion for beginning to show petal.
6. the dendrobium candidum breeding method according to claim 5 based on development of floral organs, it is characterized in that：The step 1)
In, branch that will be listed, each little Hua is from petal shape in take pictures daily tracking or the diagram whole raceme of writing record
Into the whole florescence started to flower at the end of withering.
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