CN107047317A - A kind of Orychophragmus violaceus embryoid and the cultural method of plant - Google Patents
A kind of Orychophragmus violaceus embryoid and the cultural method of plant Download PDFInfo
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- CN107047317A CN107047317A CN201710443184.2A CN201710443184A CN107047317A CN 107047317 A CN107047317 A CN 107047317A CN 201710443184 A CN201710443184 A CN 201710443184A CN 107047317 A CN107047317 A CN 107047317A
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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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a kind of Orychophragmus violaceus embryoid and the cultural method of plant, comprise the following steps：Orychophragmus violaceus is planted, microspores culture and plant regeneration, transplanting；In the microspores culture stage, heat shock incubation time and concentration of activated carbon can be impacted to embryoid yield, wherein, the addition of activated carbon is with the mass volume ratio (mg/mL) of the suspensions of microspore NLN 13：1:4~3:4；Heat shock condition of culture is specially：32~35 DEG C of light cultures 1~7 day.The present invention can quickly obtain the regeneration plant of a fairly large number of Orychophragmus violaceus microspore embryoid and inheritance stability, be conducive to accelerating the process of Orychophragmus violaceus rearing new variety.
Description
Technical field
The invention belongs to plant culture field, specifically, it is related to a kind of Orychophragmus violaceus embryoid and the cultural method of plant.
Background technology
Orychophragmus violaceus [Orychophragmus violaceus (L.) O.E.Schulz] is Cruciferae (Cruciferae)
Orychophragmus plant, is mainly distributed on China, has NATURAL DISTRIBUTION or artificial introducing and planting in many areas.Orychophragmus violaceus often by with
Make gardens, urban afforestation cover plant and parterre flower, also known as " two pale blue (orchid) ".Except itself has higher ornamental value
Outside, Orychophragmus violaceus is also possible to be the genetic donor excellent material for creating safflower sightseeing type rape variety.By rape kindred plant cabbage mustard
(Brassica alboglabra) hybridizes with Orychophragmus violaceus, and hybrid is out purplish red or pink flower (Yin Jiaming etc., 1998).In addition, all
The vegetables and feed germ plasm resource of Pueraria lobota dish or great DEVELOPMENT PROSPECT.
Orychophragmus violaceus is wide in China's distribution, and hereditary variation is big, and diversity is enriched, and this provides material base for the seed selection of Orychophragmus violaceus
Plinth.Monoploid and zygoid can be quickly obtained by flower pesticide or pollen cultures, is conducive to the seed selection of Orychophragmus violaceus new material
And research.Wu waits that (Wu is along friend, the Anther Culture gardening journals of Luo Peng .1996. Orychophragmus violaceus, 23 (4) along friend:404-406.) enter
Row Orychophragmus violaceus Anther Culture, has obtained a small amount of embryoid and regeneration plant, and gained plant is mixoplod, and the tissue of plant
Source is that Anther Somatic or microspore are not known.(Jia Yongjiong, Tang Lin, Lin Honghui, are displayed Jia Yongjiong etc., Wang Youping
.1999. the research Sichuan Universitys journal (natural science edition) of Plant Regeneration From Orychophragmus Violaceus Pollen, 36 (6):
Microspore 1106-1110.) is separated again from 4 DEG C of low-temperature treatments Orychophragmus violaceus flower pesticide of 4 days to be cultivated, and obtains callus
And regeneration plant, its Regeneration Ways is microspore callus, and regeneration plant easily morphs.However, on Zhuge
Dish microspore-isolated culture obtains embryoid and regeneration plant has not been reported.
The content of the invention
In view of this, there is provided a kind of Orychophragmus violaceus embryoid and the cultural method of plant for the problem of present invention is directed to above-mentioned.
In order to solve the above-mentioned technical problem, the invention discloses a kind of Orychophragmus violaceus embryoid and the cultural method of plant, bag
Include following steps：
1) mid or late September, by orychophiagmus violaceus seed in field seeding nursery, by soil in seedling replanting to glasshouse,
Per Wo Shi composite fertilizers 5g after surviving, water as needed；Greenhouse illumination is natural light, and temperature does not do Special controlling；Post flowering takes
Sample carries out microspores culture；
2) microspores culture：In at 8~9 points in morning florescence, the long buds of 4~5mm are taken from healthy growth plant, good fortune is replaced through 5% ammonia
Sterile water wash is used after 10~15min of people's solution disinfection；Sterilization bud is placed in culture dish, plus appropriate B5- 13 nutrient solutions
(pH5.8, containing 13% sucrose), pollen, 400 mesh stainless steel mesh filtering, by pollen suspension are squeezed out with the grinding of syringe inner cylinder
It is transferred in centrifuge tube and centrifuges, abandons supernatant, add B5- 13 nutrient solutions shake up centrifugation；After such repeated centrifugation 3 times, use
PH6.0 NLN-13 nutrient solutions press 1 flower bud pollen/mL density suspensions；Microspore NLN-13 suspensions are mixed, ф 6cm trainings are sub-packed in
Ware is supported, activated carbon storing liquid is added in every ware, heat shock culture is carried out after sealed membrane sealing, is then transferred at 25 DEG C and continues dark
Culture, is transferred in shaken cultivation case during the visible embryoid of naked eyes and is cultivated, obtain cotyledonary embryos；
3) plant regeneration, transplanting：Cotyledonary embryos after shaken cultivation are transferred on agar medium, trained under illumination condition
Support；After budding by bud cut succeeding preservation and expand it is numerous to each pollen plant clone have 3~5 buds；Transplant first 1 month, will
Bud, which is transferred on root media, takes root；Test tube seedling after taking root is transplanted to experimental field after 1 week through hardening, with white in initial 1 week
Color plastic foil covers.
Further, step 2) in activated carbon storing liquid prepared by following steps：Add in 100mL ultra-pure waters
Stored for future use after entering 1g activated carbons and 0.5g agaroses, 121 DEG C of high-temperature sterilizations.
Further, step 2) in centrifugal condition be：Rotating speed is 900-1500r/min, and the time is 1-5min.
Further, step 2) in be per the mass volume ratio (mg/mL) of activated carbon and microspore NLN-13 suspensions in ware：
1:4~3:4.
Further, step 2) in heat shock condition of culture be specially：32~35 DEG C of light cultures 1-7 days.
Further, step 2) in condition of culture in shaken cultivation case be：Rotating speed is 55-65r/min, the time is 14~
20d, temperature is 25 DEG C.
Further, step 3) in the agar medium that is transferred in agar medium of cotyledonary embryos be：1/2MS+3% sucrose
+ 7g/L agar, pH=5.8；Illumination condition is 20 DEG C, 1000Lux16h/d.
Further, step 3) in root media be：1/2MS+0.5mg/L IBA+3% sucrose+7g/L agar cultures
Base, pH=5.8.
Compared with prior art, the present invention can be obtained including following technique effect：
1) activated carbon and heat shock culture are required to embryoid induction.The buds of 4mL 1 flower is cultivated in ф 6cm culture dishes
During powder/mL microspore NLN-13 suspensions, added per ware 1mg activated carbons and 32 DEG C of heat shocks 3 days culture cotyledon shape embryoid and
Total embryoid yield highest, respectively 0.92 ± 0.18/bud and 1.32 ± 0.25/bud.
2) cotyledon shape embryoid germination rate on 1/2MS culture mediums is 27.73%.Natural doubling rate is in pollen plant
25%, it is 24 to double plant chromosome number, and Chromosomes of Haploid number is 12.Double plant strong compared with haplobiont growth
Strong, flower and bud are larger.
3) it is 58.35~76.03% to double plant fertile pollen rate, and selfing is per really setting seeds 0~0.65, and outcrossing is tied per fruit
Seed 10.20~16.40；Haplobiont fertile pollen rate is 0, it is impossible to set seeds.
4) present invention is using the microspore culture purified, by adding activated carbon and heat shock culture, embryoid yield
Height, or and regeneration plant to double plant, or be haplobiont, plant it is tissue-derived clear, be microspore.
5) present invention obtains embryo shape using the NLN-13 culture suspensions of microspore by adding activated carbon and heat shock culture
Body, Regeneration Ways are microspore embryoid, and variation is less likely to occur regeneration plant, and stable offspring is obtained beneficial to quick.
6) present invention can quickly obtain the regeneration plant of a fairly large number of Orychophragmus violaceus microspore embryoid and inheritance stability,
Be conducive to accelerating the process of Orychophragmus violaceus rearing new variety.
Certainly, any product for implementing the present invention it is not absolutely required to while reaching all the above technique effect.
Brief description of the drawings
Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes the part of the present invention, this hair
Bright schematic description and description is used to explain the present invention, does not constitute inappropriate limitation of the present invention.In the accompanying drawings：
Fig. 1 is Orychophragmus violaceus microspore embryogenesis of the present invention；Wherein, A：Microspore is expanded for part；B：Microspore is equal
Isotomy；C：Microspore asymmetric division；D：Many cells group；E：Has the proembryo of suspensor shape structure；F：Spherical proembryo；G：It is spherical
Embryo；H：Has the spheroidal embryoid of suspensor shape structure；I：Ovoid embryoid；J：Peach heart embryoid；K：Heart-shaped embryoid；
L：Embryoid after cultivating 28 days；
Fig. 2 is Orychophragmus violaceus microspore embryoid shape volume morphing of the present invention；Wherein, A：Dicotyledonous embryoid；B：Three cotyledon embryoids；
C：Has the cotyledon embryoid of 1 radicle shape structure three；D：Unifacial leaf embryoid；E：The double embryos of symphysis；F：Torpedo embryoid；G：Heart-shape embryo
Shape body；H：Spherical embryoid；I：Irregular shape embryoid；Scale=2mm；
Fig. 3 is regeneration, form, fertility and the ploidy of Orychophragmus violaceus microspore plant of the present invention；Wherein, A：Embryoid is sprouted；
B：The microspore plant doubled；C：Haplobiont；D：Double the flower and bud with haplobiont；E：Double plant pollen；
F：Haplobiont pollen；G：Double plant pollen mother cell chromosome (2n=24)；H：Haplobiont pollen mother cell contaminates
Colour solid (2n=12)；Scale=10 μm.
Embodiment
Describe embodiments of the present invention in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section can fully understand and implement according to this to solve technical problem and reach the implementation process of technology effect.
The invention discloses a kind of Orychophragmus violaceus embryoid and the cultural method of plant, comprise the following steps：
1) by orychophiagmus violaceus seed in field seeding nursery, per nest after in soil in seedling replanting to glasshouse, surviving
Composite fertilizer about 5g is applied, is watered as needed；Greenhouse illumination is natural light, and temperature does not do Special controlling；Post flowering sampling carries out small
Spore cultivation；
2) microspores culture：In at 8~9 points in morning florescence, the long buds of 4~5mm are taken from healthy growth plant, good fortune is replaced through 5% ammonia
Sterile water wash is used after 10~15min of people's solution disinfection；Sterilization bud is placed in culture dish, plus appropriate B5- 13 nutrient solutions
(pH5.8, containing 13% sucrose), pollen, 400 mesh stainless steel mesh filtering, by pollen suspension are squeezed out with the grinding of syringe inner cylinder
It is transferred in centrifuge tube, 900-1200r/min centrifugation 1-5min abandon supernatant, add B5- 13 nutrient solutions shake up centrifugation；Such as
After this repeated centrifugation 3 times, 1 flower bud pollen/mL density suspensions are pressed with NLN-13 nutrient solutions (pH6.0), microspore NLN-13 is obtained and hangs
Liquid；Microspore NLN-13 suspensions are mixed, culture dish is sub-packed in, activated carbon storing liquid, each culture dish are added in culture dish
In the mass volume ratio (mg/mL) of activated carbon and microspore NLN-13 suspensions be：1:4~3:4；After sealed membrane sealing 32~
35 DEG C of light cultures 1~7 day, are then transferred at 25 DEG C and continue light culture, and 25 DEG C of vibration training is transferred to during the visible embryoid of naked eyes
Support 55-65r/min in case and cultivate 14~20d, obtain cotyledonary embryos；
Wherein, activated carbon storing liquid is prepared by following steps：In 100mL ultra-pure waters add 1g activated carbons and
Stored for future use after 0.5g agaroses, 121 DEG C of high-temperature sterilizations；
3) plant regeneration, transplanting：Cotyledonary embryos after shaken cultivation are transferred to 1/2MS+3% sucrose+7g/L agar cultures
On base (pH5.8), cultivated under 20 DEG C, 1000Lux 16h/d illumination conditions；Bud is cut into succeeding preservation after budding and expands numerous arrive
Each pollen plant clone has 3~5 buds.Transplant first 1 month, by bud be transferred to 1/2MS+0.5mg/L IBA+3% sucrose+
Taken root on 7g/L agar mediums (pH5.8)；Test tube seedling after taking root is transplanted to experimental field after 1 week through hardening, is used in initial 1 week
White plastic film covers.
Embodiment 1
Orychophiagmus violaceus seed is collected in wild open pollination plant in Beibei, chongqing Southwestern University campus, by Chongqing City's rape work
Journey Technical Research Center breed conservation.September in 2010 20 days is in field seeding nursery, and November 7 is by seedling replanting to southwestern big
In school in glasshouse in soil, 100 plants are transplanted altogether, per Wo Shi composite fertilizers about 5g after surviving, are watered as needed.Greenhouse light
According to for natural light, temperature does not do Special controlling.Post flowering sampling in 2 months 2011 carries out microspores culture, plants microspore October
Strain is transplanted to experimental field, and next year Post flowering carries out morphologic observation and fecundity identification, and samples observation pollen fertility and chromosome
Number.
Wherein, microspores culture is specially：In at 8~9 points in morning florescence, the long buds of 4~5mm are taken from healthy growth plant, passed through
5% ammonia uses sterile water wash after replacing 10~15min of good fortune people's solution disinfection.Sterilization bud is placed in culture dish, plus appropriate B5- 13 trainings
Nutrient solution (pH5.8, containing 13% sucrose), pollen, 400 mesh stainless steel mesh filtering, by pollen are squeezed out with the grinding of syringe inner cylinder
Suspension is transferred in centrifuge tube, 1200r/min centrifugation 3min, is abandoned supernatant, is added B5- 13 nutrient solutions shake up centrifugation.So
After repeated centrifugation 3 times, 1 flower bud pollen/mL density suspensions are pressed with NLN-13 nutrient solutions (pH6.0).Microspore NLN-13 suspensions are mixed
It is even, ф 6cm culture dishes are sub-packed in, per ware 4mL, 1mg activated carbon are added in every ware, in 32 DEG C of light cultures after sealed membrane sealing
3 days, it is then transferred at 25 DEG C and continues light culture, 60r/min in 25 DEG C of shaken cultivation case is transferred to during the visible embryoid of naked eyes
Cultivate and embryoid yield is counted after 14~20d.Wherein, activated carbon storing liquid is prepared by following steps：It is ultrapure in 100mL
Stored for future use after adding 1g activated carbons and 0.5g agaroses, 121 DEG C of high-temperature sterilizations in water；
Microspore plant is transplanted to and is specially experimental field：14~20d of shaken cultivation cotyledonary embryos are transferred to 1/2MS+
On 3% sucrose+7g/L agar mediums (pH5.8), cultivated under 20 DEG C, 1000Lux 16h/d illumination conditions.By bud after budding
Cut succeeding preservation and expand it is numerous to each pollen plant clone have 3~5 buds.Transplant first 1 month, bud is transferred to 1/2MS+
Taken root on 0.5mg/L IBA+3% sucrose+7g/L agar mediums (pH5.8).Test tube seedling after taking root is transplanted after 1 week through hardening
To experimental field, covered in initial 1 week with white plastic film.
Embodiment 2
Orychophiagmus violaceus seed is collected in wild open pollination plant in Beibei, chongqing Southwestern University campus, by Chongqing City's rape work
Journey Technical Research Center breed conservation.September in 2010 20 days is in field seeding nursery, and November 7 is by seedling replanting to southwestern big
In school in glasshouse in soil, 100 plants are transplanted altogether, per Wo Shi composite fertilizers about 5g after surviving, are watered as needed.Greenhouse light
According to for natural light, temperature does not do Special controlling.Post flowering sampling in 2 months 2011 carries out microspores culture, plants microspore October
Strain is transplanted to experimental field, and next year Post flowering carries out morphologic observation and fecundity identification, and samples observation pollen fertility and chromosome
Number.
Wherein, microspores culture is specially：In at 8~9 points in morning florescence, the long buds of 4~5mm are taken from healthy growth plant, passed through
5% ammonia uses sterile water wash after replacing 10~15min of good fortune people's solution disinfection.Sterilization bud is placed in culture dish, plus appropriate B5- 13 trainings
Nutrient solution (pH5.8, containing 13% sucrose), pollen, 400 mesh stainless steel mesh filtering, by pollen are squeezed out with the grinding of syringe inner cylinder
Suspension is transferred in centrifuge tube, 900r/min centrifugation 5min, is abandoned supernatant, is added B5- 13 nutrient solutions shake up centrifugation.It is so heavy
After centrifuging 3 times again, 1 flower bud pollen/mL density suspensions are pressed with NLN-13 nutrient solutions (pH6.0).Microspore NLN-13 suspensions are mixed,
ф 6cm culture dishes are sub-packed in, per ware 4mL, 3mg activated carbon are added in every ware, in 35 DEG C of light cultures 1 after sealed membrane sealing
My god, it is then transferred at 25 DEG C and continues light culture, 55r/min in 25 DEG C of shaken cultivation case is transferred to during the visible embryoid of naked eyes and is trained
Support and embryoid yield is counted after 20d.Wherein, activated carbon storing liquid is prepared by following steps：Add in 100mL ultra-pure waters
Stored for future use after entering 1g activated carbons and 0.5g agaroses, 121 DEG C of high-temperature sterilizations；
Microspore plant is transplanted to and is specially experimental field：Shaken cultivation 20d cotyledonary embryos are transferred to 1/2MS+3% sugarcanes
On sugar+7g/L agar mediums (pH5.8), cultivated under 20 DEG C, 1000Lux16h/d illumination conditions.After budding by bud cut after
Generation preserve and expand it is numerous to each pollen plant clone have 3~5 buds.Transplant first 1 month, bud is transferred to 1/2MS+0.5mg/
Taken root on LIBA+3% sucrose+7g/L agar mediums (pH5.8).Test tube seedling after taking root is transplanted to experiment after 1 week through hardening
Ground, is covered in initial 1 week with white plastic film.
Embodiment 3
Orychophiagmus violaceus seed is collected in wild open pollination plant in Beibei, chongqing Southwestern University campus, by Chongqing City's rape work
Journey Technical Research Center breed conservation.September in 2010 20 days is in field seeding nursery, and November 7 is by seedling replanting to southwestern big
In school in glasshouse in soil, 100 plants are transplanted altogether, per Wo Shi composite fertilizers about 5g after surviving, are watered as needed.Greenhouse light
According to for natural light, temperature does not do Special controlling.Post flowering sampling in 2 months 2011 carries out microspores culture, plants microspore October
Strain is transplanted to experimental field, and next year Post flowering carries out morphologic observation and fecundity identification, and samples observation pollen fertility and chromosome
Number.
Wherein, microspores culture is specially：In at 8~9 points in morning florescence, the long buds of 4~5mm are taken from healthy growth plant, passed through
5% ammonia uses sterile water wash after replacing 10~15min of good fortune people's solution disinfection.Sterilization bud is placed in culture dish, plus appropriate B5- 13 trainings
Nutrient solution (pH5.8, containing 13% sucrose), pollen, 400 mesh stainless steel mesh filtering, by pollen are squeezed out with the grinding of syringe inner cylinder
Suspension is transferred in centrifuge tube, 1500r/min centrifugation 1min, is abandoned supernatant, is added B5- 13 nutrient solutions shake up centrifugation.So
After repeated centrifugation 3 times, 1 flower bud pollen/mL density suspensions are pressed with NLN-13 nutrient solutions (pH6.0).Microspore NLN-13 suspensions are mixed
It is even, ф 6cm culture dishes are sub-packed in, per ware 4mL, 2mg activated carbon are added in every ware, in 33 DEG C of light cultures after sealed membrane sealing
7 days, it is then transferred at 25 DEG C and continues light culture, 65r/min in 25 DEG C of shaken cultivation case is transferred to during the visible embryoid of naked eyes
Cultivate and embryoid yield is counted after 14d.Wherein, activated carbon storing liquid is prepared by following steps：In 100mL ultra-pure waters
Stored for future use after adding 1g activated carbons and 0.5g agaroses, 121 DEG C of high-temperature sterilizations；
Microspore plant is transplanted to and is specially experimental field：Shaken cultivation 14d cotyledonary embryos are transferred to 1/2MS+3% sugarcanes
On sugar+7g/L agar mediums (pH5.8), cultivated under 20 DEG C, 1000Lux16h/d illumination conditions.After budding by bud cut after
Generation preserve and expand it is numerous to each pollen plant clone have 3~5 buds.Transplant first 1 month, bud is transferred to 1/2MS+0.5mg/
Taken root on LIBA+3% sucrose+7g/L agar mediums (pH5.8).Test tube seedling after taking root is transplanted to experiment after 1 week through hardening
Ground, is covered in initial 1 week with white plastic film.
Illustrate the technique effect of the present invention with reference to specific experimentation：
1 materials and methods
1.1 material
Orychophiagmus violaceus seed is collected in wild open pollination plant in Beibei, chongqing Southwestern University campus, by Chongqing City's rape work
Journey Technical Research Center breed conservation.September in 2010 20 days is in field seeding nursery, and November 7 is by seedling replanting to southwestern big
In school in glasshouse in soil, 100 plants are transplanted altogether, per Wo Shi composite fertilizers about 5g after surviving, are watered as needed.Greenhouse light
According to for natural light, temperature does not do Special controlling.Post flowering sampling in 2 months 2011 carries out microspores culture, plants microspore October
Strain is transplanted to experimental field, and next year Post flowering carries out morphologic observation and fecundity identification, and samples observation pollen fertility and chromosome
Number.
1.2 method
1.2.1 microspores culture program
In at 8~9 points in morning florescence, the long buds of 4~5mm are taken from healthy growth plant, through 5% ammonia for good fortune people solution disinfection 10~
Sterile water wash is used after 15min.Sterilization bud is placed in culture dish, plus appropriate B5-13 nutrient solutions (pH5.8, containing 13% sucrose),
Pollen is squeezed out with the grinding of syringe inner cylinder, pollen suspension is transferred in centrifuge tube by the filtering of 400 mesh stainless steel mesh,
1200r/min centrifuges 3min, abandons supernatant, adds B5-13 nutrient solutions and shake up centrifugation.After such repeated centrifugation 3 times, NLN- is used
13 nutrient solutions (pH6.0) press 1 flower bud pollen/mL density suspensions.Microspore NLN-13 suspensions are mixed, ф 6cm culture dishes are sub-packed in,
Per ware 4mL, a certain amount of activated carbon is added in every ware, in 32 DEG C of light culture a couple of days after sealed membrane sealing, 25 are then transferred into
Continue light culture at DEG C, 60r/min in 25 DEG C of shaken cultivation case is transferred to during the visible embryoid of naked eyes and cultivates statistics after 14~20d
Embryoid yield.
1.2.2 the preparation of activated carbon storing liquid
Stored for future use after adding 1g activated carbons and 0.5g agaroses, 121 DEG C of high-temperature sterilizations in 100mL ultra-pure waters.
1.2.3 influence of the heat shock incubation time to embryoid yield
The addition activated carbon storing liquid (100uL) per ware, reaches concentration of activated carbon and heat shock culture is distinguished at 1mg/ wares, 32 DEG C
(i.e. 32 DEG C light cultures) 0,1,3,5 and 7d, influence of the research heat shock incubation time to embryoid yield.After first flower 5d in 1 week
Repeat materials 3 times, often handle and repeat 5 wares every time.
1.2.4 influence of the concentration of activated carbon to embryoid yield
Addition 0,1,2 and 3mg activated carbons per ware, heat shock culture 3 days at 32 DEG C, Study On The Activated Carbon concentration are produced to embryoid
The influence of amount.Repeat to draw materials 3 times in 1 week after first flower 5d to be tested, often handle and repeat 5 wares every time.
1.2.5 embryoid generating process and morphologic observation
Using every ware add 1mg activated carbons, 32 DEG C of heat shocks 3 days culture as object, cultivated through 1,3,5,7,10 and 14d,
Embryoid generating process is observed under inverted microscope respectively.After 14~20d of shaken cultivation, embryo shape is observed under Stereo microscope
Volume morphing.
1.2.6 plant regeneration, transplanting, fertility and Ploidy Identification
14~20d of shaken cultivation cotyledonary embryos are transferred on 1/2MS+3% sucrose+7g/L agar mediums (pH5.8),
Cultivated under 20 DEG C, 1000Lux 16h/d illumination conditions.Bud is cut into succeeding preservation after budding and expands numerous to each pollen plant
Clone has 3~5 buds.Transplant first 1 month, bud is transferred to 1/2MS+0.5mg/L IBA+3% sucrose+7g/L agar cultures
Taken root on base (pH5.8).Test tube seedling after taking root is transplanted to experimental field after 1 week through hardening, is hidden in initial 1 week with white plastic film
Cover.
At the florescence, morphologic observation is carried out to plant, flower will be opened by taking, take out flower pesticide and extrude 2% acetic acid of pollen
Observation counts pollen fertility under the microscope after the dyeing of fuchsin dye liquor.Take young tender bud Ka Nuoshi fixers (3 part of 95% second
Alcohol：1 part of glacial acetic acid) fix and 75% alcohol is transferred to after 24 hours is saved backup in 4 DEG C in refrigerator, dyed with improvement carbolfuchsin liquid
Observe pollen mother cellses later stage I or early stage II chromosome numbers.Artificial supplementary pollination is used certainly to the plant doubled
Hand over, to be known from handing over Setting percentage；Meanwhile, auxiliary is awarded with natural Orychophragmus violaceus pollen mixture, to understand outcrossing Setting percentage.
2 results and analysis
2.1 embryoid generating processes and form
After microspores culture 1 day, part microspore volume is significantly increased, rounded；Another part microspore volume is then
There is no significant change (Figure 1A).After culture 3 days, it was observed that the 1st cell division occurs for some microspores.Divisional mode has 2 kinds,
Mostly symmetrical fissions, form the cell (Figure 1B) that 2 sizes are closely waited；Another is non-symmetrical fissions, forms 2 sizes substantially not
Same cell (Fig. 1 C).After culture 5 days, many cells group (Fig. 1 D) and the proembryo (Fig. 1 E) with suspensor shape structure can be observed.
After culture 7 days, embryonic development to spherical proembryo stage (Fig. 1 F).After culture 10 days, embryonic development to globular embryo period (Fig. 1 G).Culture
14 days, the visible tiny white granular culture of naked eyes was viewed as spherical embryoid and heart-shape embryo shape under inverted microscope
Body, including spheroidal (Fig. 1 H) and ovoid (Fig. 1 I) embryoid and peach heart-shaped (Fig. 1 J) and heart-shaped embryoid (Fig. 1 K).
Continue shaken cultivation after 14 days, cotyledon period (Fig. 1 L) is arrived in embryoid development.
Embryoid form is observed after 14~20d of shaken cultivation under Stereo microscope.Embryoid can be divided into 5 types：Son
Leaf embryoid (Fig. 2A, 2B, 2C, 2D, 2E), torpedo embryoid (Fig. 2 F), heart-shaped embryoid (Fig. 2 G), spherical embryoid
(Fig. 2 H) and irregular shape embryoid (Fig. 2 I).Same type of embryoid has diversified form again.Such as cotyledon period embryo shape
Body just has dicotyledonous embryoid (Fig. 2A), three cotyledon embryoids (Fig. 2 B, 2C), unifacial leaf embryoid (Fig. 2 D) and 2 dicotyledonous
Various morphologic appearances such as embryoid symphysis (Fig. 2 E)；Spherical embryoid has rugby shape, elliposoidal, ovoid and spheroidal embryo
The morphological differences such as shape body (Fig. 2 H).
Influence of the 2.2 heat shock incubation times to embryoid yield
Result of the test (table 1) shows that, relative to 25 DEG C of cultures are continued, 32 DEG C of heat shock cultures are for embryoid induction and formation
It is required.The embryoid of any form of induced synthesis is unable to without heat shock culture, heat shock culture just can induce generation embryo shape for 1 day
Body.Test the total embryoid yield highest obtained with heat shock culture 3 days, cotyledon shape embryoid yield also highest.During heat shock culture
Between extend again, total embryoid yield and cotyledon shape embryoid yield have the trend of reduction.
Orychophragmus violaceus microspore embryoid yield (individual/flower bud) under the different heat shock periods of table 1
Note：Letter is different after same column of figure represents the significant difference in 0.05 level.
Influence of 2.3 concentration of activated carbon to embryoid yield
Result of the test (table 2) shows that addition activated carbon is required for the formation of embryoid, without the place of activated carbon
Reason does not obtain the embryoid of any form.Per ware add 1mg activated carbons when total embryoid yield and cotyledon shape embryoid yield most
Height, but with every ware add activated carbon 2mg when total embryoid yield and cotyledon shape embryoid yield compared be not statistically significant
On significant difference.Addition activated carbon crosses the formation for being at most unfavorable for embryoid, and the formation influence of the leaf embryoid of antithetical phrase is more
Greatly.
Orychophragmus violaceus microspore embryoid yield (individual/flower bud) under the different activities charcoal concentration of table 2
Note：Letter is different after same column of figure represents the significant difference in 0.05 level.
2.4 plant regenerations, form, fertility and ploidy
Cotyledon shape embryoid is transferred on 1/2MS solid mediums, gradually browning is dead for some embryoids；Some embryo shapes
Body embryo elongate axis but do not sprout；Also embryoid just starts to sprout and sprout quickly, its hypocotyl elongation grows the root of white,
Put out new shoots at cotyledonary node (Fig. 3 A).Cotyledonary embryos 119 are co-cultured, were cultivated by 30 days, there are 33 embryoids to sprout budding,
Germination rate is 27.73%.The root that embryoid sprouting is produced is slim and frahile and quantity is few, and bud is cut and is transferred to 1/2MS solid mediums
Succeeding preservation and expanding propagation have 3~5 buds to each clone.Transplant first 1 month, by 32 transfers of clone of succeeding preservation
Taken root on to 1/2MS+IBA0.5mg/L solid mediums, there are 22 clones to take root, rooting rate is 68.75%.After taking root
Test tube seedling is transplanted to experimental field after 1 week through hardening, and 22 clones are transplanted altogether, have 12 survivals to florescence in 2012, transplanting
Survival rate is 54.55%.
Florescence is observed, and according to plant forms particularly floral organ size, plant can be divided into 2 types, that is, the plant doubled
Strain (Fig. 3 B) and haplobiont (Fig. 3 C).Double plant strain growth healthy and strong, bud is more, and flower and bud are larger；And monoploid
Plant is more slim and frahile, and bud is less, and flower and bud are smaller (Fig. 3 B, 3C, 3D).In 12 pollen plant clones of survival
In, it is Natural double plant to have 3, and Natural doubling rate is 25%.Double plant flower pesticide full, include a large amount of fertile pollens (figure
3E).Pollen fertility is variant between difference doubles plant, 3 double pollen plant fertile pollen rate be respectively 58.35%,
62.68% and 76.03%.Haplobiont flower pesticide is shrivelled, and it is few to include pollen grain, and observation under the microscope is all small and can not contaminated
The abortive pollen (Fig. 3 F) of color.Chromosome quantitative observation shows, doubles plant chromosome for 24 (Fig. 3 G), haplobiont dye
Colour solid is 12 (Fig. 3 H).A small amount of seed can be tied by doubling plant selfing, and performance selfing height is not affine, and 3 double pollen plant
Average is respectively 0,0.25 and 0.65 per number of really setting seeds.3 plants are doubled with plant to aid in awarding then with natural Orychophragmus violaceus pollen mixture
More seed is tied, average is respectively 10.20,12.65 and 16.40 per number of really setting seeds.Haplobiont is open to pollinate and artificial
Auxiliary is awarded and can not set seeds with natural Orychophragmus violaceus pollen mixture.
3 discuss
Microspore embryoid fetal hair is given birth to and embryoid morphologic observation shows, the life of Orychophragmus violaceus microspore embryoid fetal hair experienced impartial or non-
In the periods such as equalcytokinesis, many cells group, spherical proembryo, globular embryo, heart-shape embryo, torpedo-shape embryo and cotyledonary embryos, this is with being all
The situation of the brassica plant of Cruciferae is similar.The present invention is it was additionally observed that the embryo with suspensor shape structure.Because tool suspensor is small
Spore embryo and zygotic embryo are more like, therefore tool suspensor microspore embryoid development pathway induction system is in terms of embryonic development mechanism is studied
There is important value and be taken seriously.In cabbage type rape, microspores culture temperature and time is that influence suspensor spline structure is formed
Key factor.The present invention observes that suspensor shape structure can continue until torpedo embryoid (Fig. 2 F), and on cotyledon shape embryoid
Suspensor shape structure is not observed.
Temperature stress is the induction embryogenetic key factor of microspore in crucifer microspores culture, is commonly used
Processing mode be that (25 DEG C) cultures of normal temperature are transferred to after 32~35 DEG C of Heat thermostability 1 to a couple of days.Result of the present invention shows, heat shock
Play an important roll to Orychophragmus violaceus microspore embryoid, for 25 DEG C of cultures are continued, 32 DEG C of heat shock cultures are for embryo
Shape body is induced and formation is required.Low temperature stress is similar with mature embryo shape volume morphing under traditional high temperature stress, and embryoid is sprouted
Hair planting percent and pollen plant chromosome spontaneous doubling rate are all not significantly different, and the lower embryoid of low temperature stress higher temperatures stress
Yield is much lower, and required incubation time is also longer.In addition, high temperature stress processing is obtained in crucifer microspores culture
To extensive use, and applicability of the microspore low temperature stress cultivating system on other Genotypes also needs further research.
Activated carbon has the ability for suppressing composition and toxic metabolic products adsorbed and reduced in culture medium, is frequently used for planting
Improve the growth and development of cell in thing tissue cultures.Present invention illustrates activated carbon to microspore embryoid fetal hair give birth to and plant again
Life shows facilitation, and as a result the importance in microspores culture show, addition activated carbon is to Orychophragmus violaceus microspore embryo
It is required.
Some preferred embodiments of invention have shown and described in described above, but as previously described, it should be understood that invention is not
Form disclosed herein is confined to, the exclusion to other embodiment is not to be taken as, and available for various other combinations, modification
And environment, and can be carried out in invention contemplated scope described herein by the technology or knowledge of above-mentioned teaching or association area
Change., then all should be in the appended power of invention and the change and change that those skilled in the art are carried out do not depart from the spirit and scope of invention
In the protection domain that profit is required.
Claims (8)
1. a kind of Orychophragmus violaceus embryoid and the cultural method of plant, it is characterised in that comprise the following steps：
1) mid or late September, by orychophiagmus violaceus seed in field seeding nursery, by soil in seedling replanting to glasshouse, survive
Afterwards per Wo Shi composite fertilizers 5g, water as needed；Greenhouse illumination is natural light, and temperature does not do Special controlling；Post flowering sample into
Row microspores culture；
2) microspores culture：In at 8~9 points in morning florescence, the long buds of 4~5mm are taken from healthy growth plant, it is molten for the good fortune people through 5% ammonia
Sterile water wash is used after 10~15min of liquid disinfectant；Sterilization bud is placed in culture dish, plus appropriate B5- 13 nutrient solutions, wherein, B5-
The pH5.8 of 13 nutrient solutions, containing 13% sucrose, squeezes out pollen, the filtering of 400 mesh stainless steel mesh will with the grinding of syringe inner cylinder
Pollen suspension is transferred in centrifuge tube and centrifuged, and abandons supernatant, adds B5- 13 nutrient solutions shake up centrifugation；Such repeated centrifugation 3 times
Afterwards, 1 flower bud pollen/mL density suspensions are pressed with pH6.0 NLN-13 nutrient solutions；Microspore NLN-13 suspensions are mixed, ф is sub-packed in
6cm culture dishes, activated carbon storing liquid is added in every ware, heat shock culture is carried out after sealed membrane sealing, is then transferred at 25 DEG C
Continue light culture, be transferred in shaken cultivation case and cultivated during the visible embryoid of naked eyes, obtain cotyledonary embryos；
3) plant regeneration, transplanting：Cotyledonary embryos after shaken cultivation are transferred on agar medium, cultivated under illumination condition；
After budding by bud cut succeeding preservation and expand it is numerous to each pollen plant clone have 3~5 buds；Transplant first 1 month, bud is turned
Move on on root media and take root；Test tube seedling after taking root is transplanted to experimental field after 1 week through hardening, with white modeling in initial 1 week
Expect film masking.
2. Orychophragmus violaceus embryoid according to claim 1 and the cultural method of plant, it is characterised in that the step 2) in
Activated carbon storing liquid prepared by following steps：1g activated carbons and 0.5g agaroses are added in 100mL ultra-pure waters,
Stored for future use after 121 DEG C of high-temperature sterilizations.
3. Orychophragmus violaceus embryoid according to claim 1 and the cultural method of plant, it is characterised in that the step 2) in
Centrifugal condition is：Rotating speed is 900-1500r/min, and the time is 1-5min.
4. Orychophragmus violaceus embryoid according to claim 1 and the cultural method of plant, it is characterised in that the step 2) in
The mass volume ratio (mg/mL) of activated carbon and microspore NLN-13 suspensions is in per ware：1:4~3:4.
5. Orychophragmus violaceus embryoid according to claim 1 and the cultural method of plant, it is characterised in that the step 2) in
Heat shock condition of culture is specially：32~35 DEG C of light cultures 1-7 days.
6. Orychophragmus violaceus embryoid according to claim 1 and the cultural method of plant, it is characterised in that the step 2) in
Condition of culture in shaken cultivation case is：Rotating speed is 55-65r/min, and the time is 14~20d, and temperature is 25 DEG C.
7. Orychophragmus violaceus embryoid according to claim 1 and the cultural method of plant, it is characterised in that the step 3) in
The agar medium that cotyledonary embryos are transferred in agar medium is：1/2MS+3% sucrose+7g/L agar, pH=5.8；Illumination bar
Part is 20 DEG C, 1000Lux16h/d.
8. Orychophragmus violaceus embryoid according to claim 1 and the cultural method of plant, it is characterised in that the step 3) in
Root media is：1/2MS+0.5mg/L IBA+3% sucrose+7g/L agar mediums, pH=5.8.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113545290A (en) * | 2021-08-11 | 2021-10-26 | 中国林业科学研究院亚热带林业研究所 | Olive nutrition somatic embryogenesis and plant regeneration method |
| CN114507361A (en) * | 2022-02-28 | 2022-05-17 | 新疆农业大学 | Agar activated carbon hydrogel for soilless culture seeds and preparation method thereof |
-
2017
- 2017-06-13 CN CN201710443184.2A patent/CN107047317A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| 吴沿友: "十字花科几种植物的胚培养研究", 《广西植物》 * |
| 吴沿友等: "诸葛菜的花药培养", 《园艺学报》 * |
| 牛永超等: "基因型和处理温度对芥菜型游离小孢子培养的影响", 《西南大学学报（自然科学版）》 * |
| 胡颂平等: "《植物组织培养技术》", 31 August 2014, 中国农业大学出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113545290A (en) * | 2021-08-11 | 2021-10-26 | 中国林业科学研究院亚热带林业研究所 | Olive nutrition somatic embryogenesis and plant regeneration method |
| CN114507361A (en) * | 2022-02-28 | 2022-05-17 | 新疆农业大学 | Agar activated carbon hydrogel for soilless culture seeds and preparation method thereof |
| CN114507361B (en) * | 2022-02-28 | 2024-01-19 | 新疆农业大学 | Agar activated carbon hydrogel for soilless culture seeds and preparation method thereof |
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Application publication date: 20170818 |