CN1442043A - Breeding method of humi grass using low energy heavy ion radiation mutagenesis - Google Patents
Breeding method of humi grass using low energy heavy ion radiation mutagenesis Download PDFInfo
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- CN1442043A CN1442043A CN03113207.3A CN03113207A CN1442043A CN 1442043 A CN1442043 A CN 1442043A CN 03113207 A CN03113207 A CN 03113207A CN 1442043 A CN1442043 A CN 1442043A
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- 238000009395 breeding Methods 0.000 title claims description 5
- 230000005855 radiation Effects 0.000 title abstract description 13
- 238000002703 mutagenesis Methods 0.000 title description 2
- 231100000350 mutagenesis Toxicity 0.000 title description 2
- 244000025254 Cannabis sativa Species 0.000 title 1
- 238000000034 method Methods 0.000 claims abstract description 11
- 230000035772 mutation Effects 0.000 claims abstract description 7
- 238000012216 screening Methods 0.000 claims abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims abstract 3
- 102000004169 proteins and genes Human genes 0.000 claims abstract 3
- 240000004928 Paspalum scrobiculatum Species 0.000 claims description 27
- 235000003675 Paspalum scrobiculatum Nutrition 0.000 claims description 27
- 206010020649 Hyperkeratosis Diseases 0.000 claims description 22
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- 230000001488 breeding effect Effects 0.000 claims description 4
- 230000004069 differentiation Effects 0.000 claims description 4
- 230000002068 genetic effect Effects 0.000 claims description 4
- 238000005286 illumination Methods 0.000 claims description 3
- 230000007226 seed germination Effects 0.000 claims description 2
- 238000012258 culturing Methods 0.000 abstract 1
- 231100000219 mutagenic Toxicity 0.000 abstract 1
- 230000003505 mutagenic effect Effects 0.000 abstract 1
- 238000009304 pastoral farming Methods 0.000 abstract 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 11
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- 239000006160 differential media Substances 0.000 description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 239000005556 hormone Substances 0.000 description 6
- 229940088597 hormone Drugs 0.000 description 6
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- 235000007164 Oryza sativa Nutrition 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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Abstract
A mutagenic method with low-energy heavy ion radiation for culturing the seeds of Humi herb used to build the grazing land in shoal and nunja includes such steps as low-energy heavy ion radiation to the embryonic calli of Humi herb to lead and mutation of genetic material, differentiating to generate regenerated seedlings, growing in artificial greenhouse, screening excellent line by testing its characteristics including protein content, and comparing DNA variation to obtain a new variety.
Description
One, technical field
The invention belongs to biological technical field, relate to a kind of method of utilizing low-energy heavy ion radioinduction seed selection fox rice grass high-quality new lines.
Two, background technology
Fox rice grass (Spartina patens) is a kind of high-quality salt tolerant herbage, also is simultaneously a kind of common tool kind that salt marsh vegetation is recovered.The Deposits in Eastern Coastal China area has a large amount of exposed beach, lacks the vegetation that is fit to.The introducing a fine variety and developing on such beach of fox rice grass high-quality new lines recovers all significant to China's solonchak agricultural development and beach wetland.
Fox rice grass is introduced a fine variety in China and is started from the eighties in last century, and domestic variety is single, and does not have the report of its genetic breeding aspect so far.The report of ion beam irradiation mutation breeding in the past all concentrates on crop seed and microorganism, and does not see the plant embryos callus.The halophytes biotechnology center of Delaware, USA university, carried out the research of fox rice grass tissue culture aspect first, and one piece of (the Xianggan Li et al. that publishes thesis, 1995.Plantregeneration from callus cultures of salt marsh hay, Spartina patens, and itscellular-based salt tolerance.Aquatic botany 51 103-113), but does not report its regeneration efficiency.This method inventor and partner also once with regard to fox rice grass tissue culture deliver two pieces of articles (Cai Xiaoning etc., the regeneration of 2000. fox rice grass isolated culture plant strain. Jiangsu agricultural journal 16 (3), 143-147; Zhou Lu et al., 2003.Effect of brassinolide on callus growth and regeneration in Spartina patens (Poaceae) .Plant cell, tissueand organ culture 73,87-89), and the technical patent (patent No. 00112526.5) of having applied for laboratory large-scale production fox rice grass regrowth.But all working does not all relate to the screening of fox rice grass genetic improvement and new quality product system, and used tissue culture technique complicated operation, reagent waste.
Three, summary of the invention
The objective of the invention is: utilize the low-energy heavy ion radiation to set up complete fox rice natural plant height and imitate breeding method, utilization this invention changes the single present situation of fox rice grass species genetic structure, for the breeding of fox rice grass provides raw material and technical support, and filter out the high-quality fox rice grass seeds matter resource that is suitable for China's coastal tidal pasture construction and revegetation.Technical scheme of the present invention is:
1. the preparation of fox rice grass embryo callus
With about the non-embryo tip cut-off of fox rice grass seed 1mm, seed germination rate is brought up to about 70% by 5%.Get the aseptic seedlings basal part of stem, be equipped with appropriate hormones, induce callus.
The key of this work is to obtain the embryo individuality from a large amount of callus.Under illumination condition, portion of tissue (about 5%) green particles occurs at early growth period, immediately itself and white is on every side organized careful separation and is cultivated separately, and in one month, one finds the white tissue, i.e. excision at once afterwards.After two months, other is not changeed green piece of tissue full scale clearance.
Green compact tissue is an embryo callus, and subculture under new hormone combination was changed a subculture in every month, and material is promptly numerous with 1: 8 the rapid expansion of ratio.
The basis of used callus induction and embryo material subculture medium is in this method:
MS basic element (Sigma finished product) 4.6g/L+ sucrose 30g/L+Phytagel agarose 2g/L
Hormone combination is respectively:
Inducing culture: 2,4-D2.0mg/L+IAA 1.0mg/L
Subculture medium: NAA 1.0mg/L+2,4-D0.5mg/L+BA 0.5mg/L
2. low-energy heavy ion radiation
Embryo callus is taken out from subculture medium, soak 45min in the DMSO aqueous solution of 2% sterilization, the surface water mark that dries in the air in the ultra-clean operating room is put into the ion beam implanter target chamber afterwards.With the energy is 20KeV, and dosage range is 2.6 * 10
15Ions cm
-2To 15.6 * 10
15Ions cm
-2N
+Radiative material.The every work 10s of implanter, a breath 10s.Target chamber must vacuumize before injecting.Material after the radiation is received again on the subculture medium and was recovered 3 months, and about 27.4% tissue can be survived.
3. tissue differentiation and growth of seedlings
The embryo callus that will be after the radiation recovers growth inserts the bud differential medium, treats that young shoot grows to the 4cm height and it downcut access root differential medium from piece of tissue when above.Remaining piece of tissue can continue to induce sprouting.The bud differential medium basis that adopts in this method is the same, and hormone is BA 3mg/L; Root differential medium basis changes 2.3g/L+ sucrose 15g/L+Phytagel agarose 2g/L into, does not add hormone in addition.
When root grows to 1cm when above, can with seedling replanting to weather can manually operated closed greenhouse in.Medium of seedling bed is based on wood sawdust and carbon slag (10: 1).Seedling plantation density is 15 strains/m
2Water once improved Hoagland nutrient solution or artificial seawater weekly with manual type, keep matrix moistening with the automatic dripping irrigation running water at ordinary times.
Summer in the greenhouse (6～August), day highest temperature was average 34.5 ℃, day average 583.9 μ mols of maximum light intensity
-1m
-2Winter (12～February), day lowest temperature was average 5.7 ℃, day average 217.5 μ mols of maximum light intensity
-1m
-2Mean annual humidity 72.7%.
Improved Hoagland nutrient solution and artificial seawater prescription are:
| The Hoagland nutrient solution | Artificial seawater | ||||
| Macroelement | Trace element | Macroelement | |||
| Salt | Content (g/L) | Salt | Content (mg/L) | Salt | Content (g/L) |
| ??KH 2PO 4 | ????0.14 | ??H 3BO 3 | ????2.86 | ????NaCl | ???22.40 |
| ??KNO 3 | ????0.51 | ??MnCl 2 | ????1.13 | ???CaSO 4 | ????0.76 |
| ??Ca(NO 3) 2 | ????0.82 | ??ZnSO 4 | ????0.12 | ?????KCl | ????0.80 |
| ??MgSO 4 | ????0.49 | ??CuSO 4 | ????0.05 | ???MgCl 2 | ????1.22 |
| Ironic citrate | ????0.02 | ?H 2MoO 4 | ????0.02 | ???MgSO 4 | ????0.93 |
Utilization this method, every anti-green embryo callus can differentiate 3～4 batches of seedling, amounts to about 30 strains.Seedling reaches more than 95% in survival rate on the seedbed.
4. comparison of plant quality trait and DNA detection
After growing 3 months on the seedbed, the plant height, internode of measuring every strain regrowth respectively apart from, leaf is long, leaf is wide, stem is thick, tiller number, ground biomass, underground biomass, on the ground/quality trait indexs such as the ratio of underground biomass, crude protein content, and contrast with one-way analysis of variance and without the original regrowth related data of radiation callus formation.To the sample labeling of forward variation takes place, extract DNA with the CTAB method, with Operon primer A
18, A
10, A
4, A
3, B
I0, C
2, C
11, D
7Each marker samples is carried out the RAPD amplification, comparison dna variation situation.
Through N in this method
+After the irradiation treatment, about 73.7% regeneration plant can detect the difference of the index of quality, and wherein the forward variation accounts for 1/3.In the sample of forward variation occurring, there is plant about 64.9% that the variation of DNA (account for sum 15.1%) takes place again.
5. the plantlet of transplant that quality obviously improves and DNA also morphs is gone into new seedbed, preserve as new germ plasm resource.Effect of the present invention is: 1) set up the complete fox rice grass mutation breeding program of a cover first.2) in the callus subculture, white non-embryonal connective tissue is thoroughly rejected.Existing experimental results show that occupies most white tissue differentiation regeneration capacity extreme differences, do not have value substantially.Only expand numerous green embryonal connective tissue and can save labour and reagent greatly, and, help simple to operateization because of there are not problems such as mucous modification, brownization in chlorenchyma.Culture medium prescription had saved coconut milk in subculture medium more in the past, had saved IAA in the bud differential medium, had saved whole hormones and active carbon in the root differential medium, and basic element and sucrose have also reduced half.Prescription after the change is to not influence of embryo callus growth, but can simplify the operation and save reagent.3) with the acceptor of plant embryos callus as the low-energy heavy ion radiation, this material division is vigorous, and is responsive more than the seed of dormancy to radiation, is easy to generate sudden change.4) regrowth is planted in the greenhouse of relative closure, but climatic factor Artificial Control such as illumination, temperature, humidity can reduce the influence of envirment factor to vine growth and development as far as possible.5) energy is 20KeV, and dosage range is 2.6 * 10
15Ions cm
-2To 15.6 * 10
15Ions cm
-2N
+Radiation is very high to fox rice grass embryo callus mutagenesis mutation rate, by each quality trait of regrowth is quantized comparison, finds subtle difference easily simultaneously, can prepare the mass mutation material for fox rice grass genetic breeding.
This invention can within a short period of time recovers to provide fine germplasm resources for the construction of coastal tidal pasture and wetland.
Four, embodiment example 1. is got 100 of fox rice grass seeds, sprouts after excising non-embryo end, obtains 61 strain seedling altogether.Get the aseptic seedlings basal part of stem and containing MS basic element 4.6g/L, sucrose 30g/L, 2,4-D2.0mg/L, induce callus on the inducing culture of IAA1.0mg/L and Phytagel agarose 2g/L, and therefrom isolate 4 parts of green embryonal connective tissues, put into and contain MS basic element 4.6g/L, sucrose 30g/L, NAA 1.0mg/L, 2, cultivate on the subculture medium of 4-D0.5mg/L, BA0.5mg/L and Phytagel agarose 2g/L.Expand numerously after some, therefrom select 20 block organizations and immerse 2%DMSO solution 45min, imposing energy behind the surface water mark that dries in the air is 20KeV, and dosage is 10.4 * 10
15Ions cm
-2N
+Radiation.Recover to cultivate after 3 months the survival of 6 block organizations.It is changed over to induce on the bud differential medium that contains MS basic element 4.6g/L, sucrose 30g/L, BA3mg/L and Phytagel agarose 2g/L sprout, again bud is downcut, forward on the root differential medium that contains MS basic element 2.3g/L, sucrose 15g/L and Phytagel agarose 2g/L and induce root.Amount to and obtain regrowth strain more than 150.Growth was carried out attributional analysis after 3 months in Nanjing University's intelligent greenhouse, found to have 92 strains one or many index variation to occur, wherein forward variation sample 29 strains, and have 19 samples the DNA variation to occur in the forward variation sample.Wherein have a strain plant height, internode apart from, leaf is long, leaf is wide, stem is thick, tiller number, ground biomass, underground biomass, on the ground/ratio and the careless equal no significant difference of original fox rice of underground biomass, but crude protein content is up to 6.5% (fresh weight ratio), be 1.8 times of original fox rice grass, the corresponding primer C of original fox rice grass
11The fragment of 250bp disappears in this strain in the amplified production, illustrates the variation on the genetics has taken place.This strain system is considered to the high-quality new lines that a suitable beach pasture is built because of nutritive value obviously improves, and is kept in the Nanjing University greenhouse.Example 2. is got 100 of fox rice grass seeds, sprouts after excising non-embryo end, obtains 78 strain seedling altogether.Get the aseptic seedlings basal part of stem and containing MS basic element 4.6g/L, sucrose 30g/L, 2,4-D2.0mg/L, induce callus on the inducing culture of IAA1.0mg/L and Phytagel agarose 2g/L, and therefrom isolate 6 parts of green embryonal connective tissues, put into and contain MS basic element 4.6g/L, sucrose 30g/L, NAA1.0mg/L, 2, cultivate on the subculture medium of 4-D0.5mg/L, BA0.5mg/L and Phytagel agarose 2g/L.Expand numerously after some, therefrom select 30 block organizations and immerse 2%DMSO solution 45min, imposing energy behind the surface water mark that dries in the air is 20KeV, and dosage is 10.4 * 10
15Ions cm
-2N
+Radiation.Recover to cultivate after 3 months the survival of 8 block organizations.It is changed over to induce on the bud differential medium that contains MS basic element 4.6g/L, sucrose 30g/L, BA3mg/L and Phytagel agarose 2g/L sprout, again bud is downcut, forward on the root differential medium that contains MS basic element 2.3g/L, sucrose 15g/L and Phytagel agarose 2g/L and induce root.Amount to and obtain regrowth strain more than 200.Growth was carried out attributional analysis after 3 months in Nanjing University's intelligent greenhouse, found to have 152 strains one or many index variation to occur, wherein forward variation sample 57 strains, and have 28 samples the DNA variation to occur in the forward variation sample.Have wherein that a strain leaf is long, stem slightly, on the ground/ratio of underground biomass do not have significant change, but plant height, internode are wide apart from, leaf, tiller number, ground biomass, underground biomass and crude protein content are followed successively by original plant 1.7,1.5,1.3,3.1,5.4,5.9 and 1.4 times, primer A
10Two fragments of 1200bp and 700bp in the amplified production of original fox rice grass are disappeared in this strain system simultaneously.This new lines is built the group easily and is considered suitable for the beach wetland and recovers owing to the tall and big stalwartness of plant, and also is kept in the Nanjing University greenhouse as new quality product system.
Claims (1)
1, the method for a kind of fox rice grass mutation breeding is characterized in that utilizing energy to be 20KeV, and dosage range is 2.6 * 10
15Ions cm
-2To 15.6 * 10
15Ions cm
-2N
+Irradiation fox rice grass embryo callus induces cells,primordial to produce sudden change, and in the regrowth that is differentiated to form, improvement of screening quality trait and genetic material change, new quality product system that can genetic stability, and its technical method is:
(1) with the non-embryo tip cut-off of fox rice grass seed to improve seed germination rate, on the subculture medium of improvement, only expand numerous fox rice grass embryo callus;
(2) with the energy be 20KeV, dosage range is 2.6 * 10
15Ions cm
-2To 15.6 * 10
15Ions cm
-2N
+Irradiation fox rice grass embryo callus is induced the sudden change that produces cells,primordial;
(3) on the differentiation culture of improvement, the embryo callus differentiation that rejuvenates after the raying obtains regrowth, and plants in the environment relative closure, in the greenhouse of the artificial control of climatic factors such as illumination, temperature, humidity;
(4) detect regeneration plant plant height, internode apart from, leaf is long, leaf is wide, stem is thick, tiller number, ground biomass, underground biomass, on the ground/ratio and the crude protein content of underground biomass, the strain of screening quality trait improvement is;
(5) in the strain of quality-improving system, detect DNA with RAPD, the screening genetic material morphs, new quality product system that can genetic stability.
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|---|---|---|---|
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|---|---|---|---|
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250873A (en) * | 2011-06-24 | 2011-11-23 | 石春鸿 | Plant induced mutation breeding method for enhancing mutation frequency and mutation spectrum |
| CN102978115A (en) * | 2012-09-21 | 2013-03-20 | 中国科学院青岛生物能源与过程研究所 | Nannochloropsis sp.OZ-1 mutant strain and heavy ion irradiation selection method for the same |
| CN104782480A (en) * | 2015-04-23 | 2015-07-22 | 中国科学院近代物理研究所 | Sweet sorghum breeding method |
-
2003
- 2003-04-16 CN CN03113207.3A patent/CN1442043A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102250873A (en) * | 2011-06-24 | 2011-11-23 | 石春鸿 | Plant induced mutation breeding method for enhancing mutation frequency and mutation spectrum |
| CN102250873B (en) * | 2011-06-24 | 2013-07-03 | 石春鸿 | Plant induced mutation breeding method for enhancing mutation frequency and mutation spectrum |
| CN102978115A (en) * | 2012-09-21 | 2013-03-20 | 中国科学院青岛生物能源与过程研究所 | Nannochloropsis sp.OZ-1 mutant strain and heavy ion irradiation selection method for the same |
| CN102978115B (en) * | 2012-09-21 | 2014-10-01 | 中国科学院青岛生物能源与过程研究所 | Nannochloropsis sp.OZ-1 mutant strain and heavy ion irradiation selection method for the same |
| CN104782480A (en) * | 2015-04-23 | 2015-07-22 | 中国科学院近代物理研究所 | Sweet sorghum breeding method |
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