CN105794647A - Method for culturing Brassica rapa microspores with sodium butyrate-NaB as histone deacetylase inhibitor - Google Patents

Method for culturing Brassica rapa microspores with sodium butyrate-NaB as histone deacetylase inhibitor Download PDF

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Publication number
CN105794647A
CN105794647A CN201610216009.5A CN201610216009A CN105794647A CN 105794647 A CN105794647 A CN 105794647A CN 201610216009 A CN201610216009 A CN 201610216009A CN 105794647 A CN105794647 A CN 105794647A
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nab
sporidiole
embryoid
chinese cabbage
culture
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冯辉
张露
章云
唐小燕
刘志勇
李承彧
冀瑞琴
王玉刚
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Shenyang Agricultural University
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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
    • 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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  • Life Sciences & Earth Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Environmental Sciences (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention provides a method for culturing Brassica rapa microspores with sodium butyrate-NaB as a histone deacetylase inhibitor. Compared with a conventional culturing method, the method is simple, and NaB is added to an inducing medium NLN, so that the embryogenesis rate and the direct seedling development rate of the Brassica rapa microspores are increased. The embryogenesis rate of the Brassica rapa microspores is increased by 1.54-3.09 times and the direct seedling development rate is increased by 1.22-1.60 times in the inducing medium NLN with addition of 0.5 mu M-8 mu M of NaB. With the adoption of the method, the problems of low embryogenesis rate, low direct seedling development rate and long culturing period of the microspores in the conventional microspore culturing technology can be solved.

Description

Antibiotic FR 901228 sodium butyrate-NaB is utilized to cultivate Chinese cabbage sporidiole Method
Technical field
The present invention relates to Chinese cabbage microspore-isolated culture method, belong to plant cell breeding technical field, particularly a kind of Promote raw and directly seedling the method for Chinese cabbage microspore embryoid fetal hair (Crinis Carbonisatus).
Background technology
Chinese cabbage (Brassica rapa ssp. chinensisL.) belongs to typical Cruciferae cross-pollinated plant, has aobvious The hybrid vigor write.The necessary first purification parent of advantage cross-breeding to be utilized, traditional time needed for continuous selfing mode is relatively Long, take a lot of work, arduously；Utilize the microspore-isolated culture method acquisition homozygotic time to have only to 1 year, i.e. save the time, again Alleviate workload, and the regeneration plant cultivated in this way is possible not only to formulate the breeding material made new advances, and Between the generation of plant, stability is strong, and hybrid vigor is the strongest.Recent decades some researcheres both at home and abroad are swum about brassicaceous vegetable Do a lot of work from microspores culture, be devoted to optimize sporidiole and become embryo and embryoid seedling system, but still have many at present Embryo's incidence rate of genotype sporidiole is low and embryoid planting percent is low, limits this technology in actual breeding work Range of application.
Sodium butyrate-NaB is a kind of Antibiotic FR 901228, and it is carried by the activity of suppression deacetylase The degree of acetylation of high histone, at present about the application mainly work on clinical antitumor agents or animal embryo of NaB With, it is relevant with cell differentiation and cell proliferation.
Summary of the invention
It is an object of the invention to provide a kind of culture medium prescription by improvement Chinese cabbage microspore-isolated culture and promote Chinese cabbage The embryo of sporidiole occurs and the method for direct seedling, to set up efficient Chinese cabbage microspore-isolated culture system, for breeding work Lay the foundation.
Antibiotic FR 901228 sodium butyrate-the NaB that the present invention provides new work in Chinese cabbage microspores culture With, comprise the steps:
1. choosing Chinese cabbage microspore development period at mid-late uninucleate stage to double-core alabastrum in early days, now the feature of alabastrum is petal Alabastrum, between 1/2～3/4, after rinsing alabastrum 2～3 times with flowing water, is put into sterilized by the ratio of length and anther length In 100mL beaker, with concentration 75% ethanol solution surface sterilizing 30s, 0.1% HgCl2Solution surface sterilizing 6～8min, sterilized water Wash 3 times, each 5min, add 10～15mL containing 130 g L-1The B5 fluid medium of sucrose, with aseptic pestle extruding flower Flower bud, makes sporidiole free out.Suspension containing sporidiole is first sieved through filter with 300 mesh cells, then sieves with the cell of 500 mesh Filtering, collection filtrate is in 50mL centrifuge tube, and 1000～1200 rpm are centrifuged 3 min.Abandon supernatant, add 10～15mL and contain 130 g·L-1The B5 fluid medium of sucrose, 1000～1200 rpm are centrifuged 3 min, and gained precipitate is pure little spore Son.
2. it is diluted cultivating by the NLN culture medium that the sporidiole precipitate dissociating pure is conventional.Make when dilution Cell density is maintained at 1 × 105～2 × 105Individual/mL (counts with blood counting chamber), every 5 mL microspore suspension is distributed into In being the sterile glass culture dish of 60 mm × 15 mm, every ware adds the agarose 0.5g L after 100 μ L sterilizings-1, activated carbon 10g·L-1Mixed liquor；Add with the variable concentrations NaB (0,0.5,1,2,4,8 μMs) after ultra-pure water dissolution filter sterilizing.Use stone After cere sealing, first Heat thermostability 1d in 33 DEG C of calorstats, then go to stand at 25 DEG C light culture, after 9～10 days, naked eyes can Seeing spheroidal embryo, transfer to light culture on 25 DEG C of shaking tables together with culture dish, shaking table revolution is 50rpm.
3. within the 17th day after inoculation sporidiole, transfer embryoid to adding 30 g L-1Sucrose, 7.5 g L-1Agar, 0.1 g·L-1In the MS culture medium of activated carbon, its pH is 5.8～6.0, high-temperature heat sterilization.Inoculum concentration is every 100mL wide-mouth triangular flask Containing 50mL culture medium inoculated 3～5 embryoids；In 25 ± 1 DEG C, cultivate under the conditions of 16h hour illumination/sky；Embryo after 15～20 days Shape body grows the seedling for root, stem, leaf substantially stalwartness.
4.B5 culture medium prescription: pH 5.8～5.84, solvent is that ultra-pure water, solute and final concentration thereof are respectively (NH4)2SO4 0.134g·L-1, KNO3 0.25g·L-1, CaCl2·2H2O 0.15g·L-1, MgSO4·7H2O 0.25g·L-1, NaH2PO4·H2O 0.15g·L-1, MnSO4·4H2O 0.01g·L-1, ZnSO4·7H2O 0.002g·L-1, H3BO3 0.003g·L-1, Na2MnO4·2H2O 0.00025g·L-1, KI 0.00075g L-1, CuSO4·5H2O 0.000025g· L-1, CoCl2·6H2O 0.000025g·L-1, VB1 0.01g·L-1, VB6 0.001g·L-1, nicotinic acid 0.001g L-1, inositol 0.1g·L-1, Na2EDTA 0.0373g·L-1, FeSO4·7H2O 0.0278g·L-1, sucrose 130g L-1, high temperature is damp and hot to go out Bacterium.
5.NLN culture medium prescription: pH 5.8～5.84, solvent is that ultra-pure water, solute and final concentration thereof are respectively Ca (NO3)2·4H2O 0.025g·L-1, MgSO4·7H2O 0.0625g·L-1, KNO3 0.0625g·L-1, KH2PO4 0.0625g·L-1, MnSO4·4H2O 0.0223g·L-1, ZnSO4·7H2O 0.0086g·L-1, H3BO3 0.0062g·L-1, KI 0.00083g·L-1, Na2MoO4·2H2O 0.00025g·L-1, CuSO4·5H2O 0.000025g·L-1, CoCl2· 6H2O 0.000025g·L-1, thiamine hydrochloride 0.005g L-1, pyridoxine hydrochloride 0.0005g L-1, inositol 0.1g L-1, Nicotinic acid 0.005g L-1, folic acid 0.0005g L-1, glycine 0.002g L-1, biotin 0.00005g L-1, Na2EDTA 0.0373g·L-1, FeSO4·7H2O 0.0278g·L-1, sucrose 130 g L-1, glutathion 0.03g L-1, L silk ammonia Acid 0.1g L-1, L glutamine 0.8g L-1, filtration sterilization.
6.MS culture medium prescription: pH 5.8, solvent is that distilled water, solute and final concentration thereof are respectively NH4NO3 1.65g·L-1, KH2PO4 0.17g·L-1, KNO3 1.9g·L-1, MgSO4·7H2O 0.37g·L-1, CaCl2 0.33g·L-1, MnSO4·4H2O 0.0169g·L-1, H3BO3 0.0062g·L-1, Na2MoO4·2H2O 0.00025g·L-1, CuSO4· 5H2O 0.000025g·L-1, CoCl2·6H2O 0.000025g·L-1, KI 0.00083g L-1, ZnSO4·7H2O 0.0086g·L-1, thiamine hydrochloride 0.0004g L-1, Pyridoxin hydrochloride 0.0005g L-1, nicotinic acid 0.0005g L-1, flesh Alcohol 0.1g L-1, glycine 0.002g L-1, Na2EDTA 0.0373g·L-1, FeSO4·7H2O 0.0278g·L-1, sucrose 30g·L-1, agar 5.5g L-1, activated carbon 0.1 g L-1, high-temperature heat sterilization.
The positive effect of the present invention:
1., by adding 0.5 μM ~ 8 μMs NaB in NLN inducing culture in the embryoid induction stage, promote the little of Chinese cabbage Spore embryo occurs, and microspore embryoid incidence rate is up to 34.53 embryos/flower bud, improves 1.54 ~ 3.09 times than comparison.
2. have only to about 35 days to obtaining regeneration plant from inoculation Chinese cabbage sporidiole, lure at the NLN adding 0.5 μM of NaB Leading in culture medium, the direct planting percent of Chinese cabbage is the highest, has reached 88.36%, in the NLN inducing culture adding 2 μMs of NaB, The direct planting percent of Chinese cabbage takes second place, and has reached 82.99%, has improve 1.22 ~ 1.60 times than comparison.
3. operation is simple, only need to add the Antibiotic FR 901228 fourth of certain concentration at induction period Acid sodium (NaB), both can occur to produce facilitation, embryoid seedling can be produced facilitation again the embryo of sporidiole.
Accompanying drawing explanation
Fig. 1 is to add the embryoid that 2 μMs of NaB produce in Chinese cabbage learned genotype NLN culture medium.
Fig. 2 is the embryoid being inoculated in MS culture medium.
Fig. 3 is the direct seedling of embryoid.
Detailed description of the invention
Described in following embodiment, method is if no special instructions, is conventional method.
Reagent described in following embodiment, the most commercially obtains.
Culture medium used in following embodiment is with the B5 in summary of the invention, NLN, MS culture medium.
Chinese cabbage microspores culture method main process of the present invention includes:
One, microspore-isolated culture process
Choosing Chinese cabbage microspore development period at mid-late uninucleate stage to double-core alabastrum in early days, now the feature of alabastrum is that petal is long Alabastrum, between 1/2～3/4, after rinsing alabastrum 2～3 times with flowing water, is put into sterilized by the ratio of degree and anther length In 100mL beaker, with 75% ethanol solution surface sterilizing 30s, 0.1% HgCl2Solution surface sterilizing 6～8min, sterilized water washs 3 times, each 5min, add 10～15mL containing 130 g L-1The B5 fluid medium of sucrose, extrudes alabastrum with aseptic pestle, Make sporidiole free out.Suspension containing sporidiole is first sieved through filter with 300 mesh cells, then is sieved through with the cell of 500 mesh Filter, collection filtrate is in 50mL centrifuge tube, and 1000～1200 rpm are centrifuged 3 min.Abandon supernatant, add 10～15mL containing 130 g·L-1The B5 fluid medium of sucrose, 1000～1200 rpm are centrifuged 3 min, and gained precipitate is pure sporidiole.
It is diluted cultivating by the NLN culture medium that the sporidiole precipitate dissociating pure is conventional.Make thin when dilution Born of the same parents' density is maintained at 1 × 105～2 × 105Individual/mL (counts with blood counting chamber), every 5 mL microspore suspension are distributed into into In the sterile glass culture dish of 60 mm × 15 mm, every ware adds the agarose 0.5g L after 100 μ L sterilizings-1, activated carbon 10g·L-1Mixed liquor.After sealing with Parafilm, first Heat thermostability 1d in 33 DEG C of calorstats, then go to stand secretly at 25 DEG C Cultivating, spheroidal embryo seen from naked eyes after 9～10 days, transfer to light culture on 25 DEG C of shaking tables together with culture dish, shaking table revolution is 50rpm。
After inoculation sporidiole, the 18th day switching embryoid is to adding 30 g L-1Sucrose, 7.5 g L-1Agar, 0.1 g·L-1In the MS culture medium of activated carbon, its pH is 5.8～6.0, high-temperature heat sterilization.Every 3d observes once, records each embryo Growth situation, direct seedling, Secondary embryos, the number of callus are added up simultaneously.
Two, the process of Antibiotic FR 901228 NaB
Adding the NaB of variable concentrations in NLN culture medium, concentration is 0,0.5,1,2,4,8 μMs.After sealing with Parafilm, first exist Heat thermostability 1d in 33 DEG C of calorstats, then go to stand at 25 DEG C light culture, to embryoid seen from naked eyes, it is then transferred to shaking table Upper (50rpm) continues light culture.Adding up embryoid number after 17d, microspore embryoid inductivity produces embryoid number with each alabastrum Represent.
The present invention is through experimental study, and its result is reported as follows:
1 material: for examination breeds of Chinese cabbage 2, including Wal 918 and learned.
2 test methods and culture medium
2.1 test methods are with (one, microspore-isolated culture process).
2.2 NLN culture medium: add 130 g L-1Sucrose, pH is 5.8.The histon deacetylase (HDAC) suppression added The process of agent sodium butyrate (NaB), and filtration sterilization: NaB process: concentration is 0,0.5,1,2,4,8 μMs.MS culture medium, adds Add 30g L-1Sucrose, 7.5g L-1Agar, 0.1 g L-1Activated carbon, pH is 5.8, high-temperature heat sterilization.
3 result of the tests
Impact that Chinese cabbage Wal 918 and learned genotype microspore embryoid are occurred by 3.1 NaB (see Fig. 1,2):
NaB has facilitation for the embryo of Chinese cabbage sporidiole, compared with matched group, for Chinese cabbage Wal 918, learned base Because type microspore embryoid incidence rate has been respectively increased 1.54,3.09 times (tables 1), NaB is for Chinese cabbage Wal 918 and learned genotype The optimum concentration of facilitation is 2 μMs, and microspore embryoid incidence rate can respectively reach 6.83 and 34.53 embryo flower buds-1。
The impact that table 1 NaB is raw on Chinese cabbage microspore embryoid fetal hair (Crinis Carbonisatus)
Table 1 Effect of NaB on formation of microspore-derived embryos in pakchoi
Note: different lower cases are that difference reaches significantly (α=0.05) level
Note: The mean values that are followed by the different letters are significant at the 5% level
3.2 NLN inducing cultures add NaB on Chinese cabbage Wal 918 and the impact of learned genotype sporidiole embryoid seedling (see figure 3)
In table 2 NLN inducing culture, NaB concentration is on Wal 918 and the impact of learned genotype embryoid seedling
Table 2 The effects of NaB on the plant regeneration in NLN medium
Note: different lower cases are that difference reaches significantly (α=0.05) level
Note: The mean values that are followed by the different letters are significant at the 5% level
In NLN inducing culture, add NaB, 2 kinds of genotype Chinese cabbage seedlings are had facilitation (table 2).To Chinese cabbage Wal 918 Genotype, when NaB concentration is 2 μMs, direct planting percent can reach 72.90%, improves 1.60 times than comparison；To Chinese cabbage Learned genotype, when the concentration of NaB is 0.5 μM, and direct planting percent is 88.36%, improves 1.22 times than comparison.

Claims (1)

1. utilizing the method that Antibiotic FR 901228 sodium butyrate-NaB cultivates Chinese cabbage sporidiole, its feature includes Following steps:
(1) NaB promotes that Chinese cabbage microspore embryoid fetal hair (Crinis Carbonisatus) is raw
Use the conventional Chinese cabbage isolated and purified sporidiole of microspore-isolated culture method, be 0.5 μM ~ 8 μMs by the concentration containing NaB NLN culture medium culturing sporidiole, sporidiole density is 1 × 105～2 × 105Individual/mL NLN culture medium；Every 5 mL sporidioles are hanged Supernatant liquid be distributed into be 60 mm × 15 mm sterile glass culture dish in, every ware adds the agarose 0.5g after 100 μ L sterilizings L-1, activated carbon 10g L-1Mixed liquor, after sealing with Parafilm, first Heat thermostability 1d in 33 DEG C of calorstats, then go to 25 Stand light culture at DEG C, to embryoid seen from naked eyes, be then transferred on the shaking table that revolution is 50rpm continue light culture；
(2) NaB promotes the Chinese cabbage direct seedling of sporidiole embryoid
After inoculation sporidiole, the 18th day switching embryoid is to adding 30 g L-1Sucrose, 7.5 g L-1Agar, 0.1 g L-1 In the MS culture medium of activated carbon, its pH is 5.8～6.0, high-temperature heat sterilization；Inoculum concentration is that every 100mL wide-mouth triangular flask contains 5 embryoids of 50mL culture medium inoculated；In 25 ± 1 DEG C, cultivate under the conditions of 16h hour illumination/sky；After 15～20 days, embryoid is sent out Educate the seedling for root, stem, leaf substantially stalwartness.
CN201610216009.5A 2016-04-10 2016-04-10 Method for culturing Brassica rapa microspores with sodium butyrate-NaB as histone deacetylase inhibitor Pending CN105794647A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111657146A (en) * 2020-06-30 2020-09-15 富阿丽 Induction culture medium and induction culture method for culture breeding of green Chinese onion flowers
CN111685038A (en) * 2020-05-29 2020-09-22 邹传海 Culture medium for improving cell division frequency of dendrobium officinale protoplast
CN117121809A (en) * 2022-05-19 2023-11-28 南京农业大学 Method for cultivating microspore plant of non-heading Chinese cabbage

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111685038A (en) * 2020-05-29 2020-09-22 邹传海 Culture medium for improving cell division frequency of dendrobium officinale protoplast
CN111657146A (en) * 2020-06-30 2020-09-15 富阿丽 Induction culture medium and induction culture method for culture breeding of green Chinese onion flowers
CN117121809A (en) * 2022-05-19 2023-11-28 南京农业大学 Method for cultivating microspore plant of non-heading Chinese cabbage

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