CN1491537A - Method for cultivating salt-resistant tomato plant - Google Patents
Method for cultivating salt-resistant tomato plant Download PDFInfo
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- CN1491537A CN1491537A CNA031507840A CN03150784A CN1491537A CN 1491537 A CN1491537 A CN 1491537A CN A031507840 A CNA031507840 A CN A031507840A CN 03150784 A CN03150784 A CN 03150784A CN 1491537 A CN1491537 A CN 1491537A
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Abstract
The process of cultivating salt tolerant tomato plant includes the steps of: taking hypocotyl of aseptic seedling as explant to inoculate and culture; inoculating the callus tissues culture after twice subculture to culture medium with one third of artificial compounded sea water; inoculating green callus tissues to culture medium without sea water for 7 or 8 subcultures before transferred to culture medium with one third of artificial compounded sea water to establish salt tolerant cell line; transferring the proved salt tolerant callus tissues to differentiating culture medium without sea water to obtain reproduction seedling; pot cultivating the seedling while irrigating with one third of artificial compounded sea water for salt tolerant screening to obtain salt tolerant plant. Compared with modern genetic engineering breeding method, the present invention has low cost, simple technology, less hidden safety danger and other obvious advantages.
Description
Technical field:
The present invention relates to a kind of salt resistance tomato plant breeding method, be used for solanberry class rearing new varieties such as tomato, eggplant, capsicum, belong to the crop breeding technical field.
Technical background:
Tomato is strict self-pollinated plant, and long-term domestication and sexual hybridization make its genetic background more and more narrow, and some genes of anti-the adverse circumstance have been difficult in its cultivated species and have found.
Though there is some difference for the salt resistance between crop varieties, its salt resistance also can not adapt to far away large-area saline land and the development and use requirement that accounts for the seawater of the global water yield 97.3%.Plant breeder, physiologist and pedologist are carrying out cooperating research, improve the salt resistance of crop varieties from different approach.
Along with totipotency of plant cell is confirmed, the kind that obtains whole plant by cell and tissue culture is increasing, simultaneously prove that also there is spontaneous variation in the plant cultured in vitro, infer that this is the basis that produces somaclonal variation (somaclonal variation), many researchers think that the genetic variation that can obtain enriching is from tissue culture.Therefore, utilize plant cell or the callus cultivated, adopting or do not adopting under the situation of mutagen, influence by selection pressure, can on purpose select desired adverse-resistant characteristic, so cell and tissue culture technique and traditional breeding technique and genetic engineering be the same to be regarded as carrying out plant improvement and to improve anti-adverse circumstance ability, one of main path of particularly anti-(resisting) salt ability.Utilize cell culture technology to obtain the existing many reports of salt tolerant cell-line, relate to 12 sections, 24 belong to, and have 30 kinds at least.Up to the present, the research of relevant tomato vitro Screening salt-tolerant mutant all uses single salt NaCl as selection pressure, and the salt-tolerant plants of cultivation not necessarily is fit to seawater irrigation or is SO in main component
- 4The secondary salinization soil of (sulfate ion) and main component are HCO
- 3Large-area saline land, (bicarbonate ion) China great Northwest.(Tal M.In vitro methodology for increasing salt tolerance in crop plants.Acta Horticulturae, 1993,336,69～78), (Xu Naiyu. the cell/tissue of cultivating resistance plant is cultivated approach. Wuhan botany research, 1987,5 (3), 299～302).
Summary of the invention:
The present invention is directed to the deficiencies in the prior art, a kind of new salt resistance tomato plant breeding method is provided, method is simple, and the salt resistance of crop varieties can effectively be provided, and is adapted to develop the requirement of seawater, satisfies the demand of production and people life.
For realizing such purpose, in the technical scheme of the present invention, the extensive variation that utilizes the plant tissue culture technique process to exist, with 1/3 artificial composite seawater and 1/2 artificial composite seawater is that the directed salt tolerant of selecting of selection pressure makes a variation, and coerce cultivation by the nothing in 7-8 generation, to get rid of the physiological adaptability cell, the variation by AFLP (amplified fragments length polymorphism) (amplified fragment length polymorphism) molecular labeling checking the invention at last occurs on the dna level.Created the tomato salt-tolerant new germ plasm simultaneously, the line stabilization screening of going forward side by side.
Method of the present invention comprises following concrete steps:
1. the foundation of aseptic seedling system
Behind the seed-soaking 24 hours, carry out surface sterilization, use aseptic water washing again.Under aseptic condition, after sprouting 48 hours, be inoculated on 1/2MS (minimal medium of the Murashige and Skoog) medium and obtain aseptic seedling.
2. the foundation of salt tolerant cell-line
Seedling age was got its hypocotyl as explant in the time of 10～12 days, is inoculated on the agar medium of MS+BA (benzyladenine) 2.0mg/L+IAA (heteroauxin) 0.2mg/L+30g/L sucrose+6.5-7.0g/L, and at 25 ± 1 ℃, light intensity 25 μ mol.m
-2.s
-1, 14 little time/10 a hour dark photoperiod cultivates down.Subculture was 1 time in per 15 days, and the callus culture thing of subculture 2 times is inoculated in the medium of additional 1/3 artificial composite seawater, and every liter of full concentration artificial seawater consists of CaCl
2(calcium chloride) 510mg, MgCl
2(magnesium chloride) 3420mg, MgSO
4(magnesium sulfate) 2100mg, KCl (potassium chloride) 750mg and NaCl (sodium chloride) 26.75mg, used solvent is 2 parts of redistilled waters and 1 part of artificial composite seawater when promptly disposing medium.After about 20 days, brownization is dead gradually under the coercing of 1/3 artificial composite seawater for most culture, and the culture of indivedual brownization can have little green point.The cell of under 1/3 artificial composite seawater stress, surviving, may be the salt tolerant cell, also might be the physiological adaptability in the cultured in vitro process, in order to get rid of the cell of physiological adaptability, greeny callus is inoculated in the medium of no seawater, subculture changes the medium of additional 1/3 artificial composite seawater and 1/2 artificial composite seawater over to, again to set up the cell-line of anti-1/3 artificial composite seawater and anti-1/2 artificial composite seawater after 7～8 generations.
3. the acquisition of salt-tolerant plants
Recover to cultivate 7-8 generation at no sea water medium, still the callus of survival is real salt tolerant callus on the medium of additional 1/2 artificial composite seawater, but not physiological adaptability cell-line.The callus that proves salt tolerant is gone on the differential medium that does not contain seawater, obtain regrowth.Regeneration seedling potted plant, and with 1/3 artificial composite seawater as irrigation water, carry out selection of salt tolerance, the acquisition salt-tolerant plants.
4. the molecule of salt-tolerant plants checking
The tomato plant that the present invention obtains can confirm to have taken place sudden change with the AFLP molecular labeling on dna level.Confirmed validity of the present invention simultaneously.
Salt tolerant tomato plant of the present invention is cultivated and is compared with traditional breeding technology, certain advantage is arranged: utilize plant cell or the callus cultivated, adopting or not adopting under the situation of mutagen,, can on purpose select desired adverse-resistant characteristic by the influence of selection pressure; Plant tissue is handled with accurate selection pressure on the medium of regulation, and grows under the condition that strictness is controlled, and can obtain clearer and more definite result, and can not be subjected to the restriction etc. of season, time.The present invention compares with modern genetic engineering breeding method, have tangible advantages such as cost is low, technology is easily grasped, safety hidden danger is low, the crop varieties of cultivating has significantly improved salt resistance, can be adapted to develop the requirement of seawater, satisfies the demand of production and people life.
Embodiment:
Below further specify technical scheme of the present invention by a specific embodiment.
Embodiment 1
Parent material ' bright rich ' (Lycopersicon esculentum ' xianfeng ') with the main cultivar in Shanghai is the mutagenesis object, descend seed soaking after 24 hours with clear water in room temperature condition, immersed 70% alcohol 20 seconds, use 10%～13% bleaching powder saturated solution surface sterilization 20 minutes then, use aseptic water washing again 4～5 times.Under aseptic condition, after sprouting 48 hours, be inoculated on the 1/2MS medium and obtain aseptic seedling.
Seedling age is in the time of 12 days, its hypocotyl is cut into the long segment (removing cotyledon, stem apex and radicle) of 6～8mm as explant, be inoculated on the agar medium of MS+BA (benzyladenine) 2.0mg/L+IAA (heteroauxin) 0.2mg/L+30g/L sucrose+6.5-7.0g/L, at 25 ± 1 ℃, light intensity 25 μ mol.m
-2.s
-1, 14 little time/10 a hour dark photoperiod cultivates down.Healing rate can reach 100% after about 15 days.Subculture was 1 time in per 15 days, and the callus culture thing that subculture is 2 times is inoculated in additional 1/3 artificial composite seawater, and (every liter of full concentration artificial seawater contains CaCl
2510mg, MgCl
23420mg, MgSO
42100mg, KCl750mg and NaCl26.75mg) medium.After about 20 days, brownization is dead gradually under the coercing of 1/3 artificial composite seawater for most culture, and the culture of indivedual brownization can have little green point.Greeny callus is inoculated in the medium of no seawater, and subculture changes the medium of additional 1/3 artificial composite seawater and 1/2 artificial composite seawater over to after 7～8 generations again, to get rid of the physiological adaptability cell, sets up salt tolerant cell-line.
Will be on the medium of additional 1/2 artificial composite seawater assay certificate keep the callus of salt resistance, go on MS+ZT (zeatin) the 1.0mg/L+IAA0.2mg/L differential medium that does not contain seawater, obtain 12 strain regrowths.The regeneration seedling is potted plant, and to be that selection pressure is directed with 1/3 artificial composite seawater select the salt tolerant variation, promptly uses 1/3 artificial composite seawater as irrigation water, carries out selection of salt tolerance, obtain 8 strain salt-tolerant plants, and confirm on dna level, to have taken place sudden change with the AFLP molecular labeling.But has only the 2 strains knot of normally blooming.
According to the investigation of comprehensive agronomy proterties, with wherein a strain as the seed selection object.Selective nursery is arranged at marine real group soil salinity reaches 0.8% area on the Chongming Island of Shanghai, and uses seawater irrigation.Each generation and check variety ' bright rich ' carry out the screening of salt resistance stability in the whole growth phase that field planting is later with seawater irrigation.Until F5 is 0% colony for 3 blossom-end rot incidences of disease occurring, and the blossom-end rot incidence of disease of contrast is 100%.Owing to consider the seawater salt content instability of Chongming Island, and by substrate culture and utilize and to contain 1/2 artificial composite seawater and irrigate selection, and in the screening of field, carry out the repetitive identified of individual plant salt resistance with side shoot cuttage (1/3 artificial composite seawater is irrigated selection), screen 1 of the neat substantially salt tolerant colony of economical character.The salt tolerant colony that obtains except that do general cultivation tomato with, be expected on coastal farm, cultivations such as naval base, booth that the soil salinization is serious, greenhouse, area use, or available 1/3～1/2 seawater irrigation.
Claims (1)
1, a kind of salt resistance tomato plant breeding method is characterized in that comprising:
1) foundation of aseptic seedling system: seed-soaking 24 hours, use aseptic water washing after the surface sterilization, after under aseptic condition, sprouting 48 hours, be inoculated on the 1/2MS medium and obtain aseptic seedling;
2) foundation of salt tolerant cell-line: seedling age was got its hypocotyl as explant in the time of 10～12 days, is inoculated on the medium of MS+ benzyladenine 2.0mg/L+ heteroauxin 0.2mg/L+ sucrose 30g/L+ agar 6.5-7.0g/L, and at 25 ± 1 ℃, light intensity 25 μ mol.m
-2.s
-1, a hour dark photoperiod of 14 little time/10 cultivates down, and subculture was 1 time in per 15 days, and the callus culture thing of subculture 2 times is inoculated in the medium of additional 1/3 artificial composite seawater, and every liter of full concentration artificial seawater consists of CaCl
2510mg, MgCl
23420mg, MgSO
42100mg, KCl750mg and NaCl26.75mg, after 20 days, greeny callus is inoculated in the medium of no seawater, subculture is after 7～8 generations, change the medium of additional 1/3 artificial composite seawater and 1/2 artificial composite seawater again over to, to set up the cell-line of anti-1/3 artificial composite seawater and anti-1/2 artificial composite seawater;
3) acquisition of salt-tolerant plants: the callus that proves salt tolerant of still surviving on the above-mentioned medium is gone on the differential medium that does not contain seawater, obtain regrowth, regeneration seedling potted plant, and with 1/3 artificial composite seawater as irrigation water, carry out selection of salt tolerance, obtain salt-tolerant plants;
4) molecule of salt-tolerant plants checking: the tomato plant that obtains is confirmed to have taken place sudden change with the amplified fragment length polymorphism molecular labeling on dna level.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101294943B (en) * | 2008-06-05 | 2011-08-31 | 上海交通大学 | Device and brine solution for fast appraising salinity and alkalinity resistance of tree |
| CN103430783A (en) * | 2013-08-22 | 2013-12-11 | 江苏省农业科学院 | Method for authenticating and evaluating salt tolerance of seedling-stage wheat |
| CN103869058A (en) * | 2014-03-21 | 2014-06-18 | 上海交通大学 | Rapid evaluation method for salt tolerance of tomatoes |
| CN105104193A (en) * | 2015-08-19 | 2015-12-02 | 单县绿丰种业有限公司 | Method for raising salt-tolerant tomato plants |
| CN109105235A (en) * | 2018-08-17 | 2019-01-01 | 河北省农林科学院昌黎果树研究所 | A kind of identification method of grape rootstock salt tolerance |
| CN110583395A (en) * | 2019-09-11 | 2019-12-20 | 宁夏农林科学院农作物研究所（宁夏回族自治区农作物育种中心） | Method for evaluating salt tolerance of japonica rice seeds under salt stress |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102106259B (en) * | 2009-12-24 | 2013-07-17 | 上海市农业科学院 | Method for improving salt endurance of cereal crops |
-
2003
- 2003-09-04 CN CN 03150784 patent/CN1203755C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101294943B (en) * | 2008-06-05 | 2011-08-31 | 上海交通大学 | Device and brine solution for fast appraising salinity and alkalinity resistance of tree |
| CN103430783A (en) * | 2013-08-22 | 2013-12-11 | 江苏省农业科学院 | Method for authenticating and evaluating salt tolerance of seedling-stage wheat |
| CN103430783B (en) * | 2013-08-22 | 2015-04-08 | 江苏省农业科学院 | Method for authenticating and evaluating salt tolerance of seedling-stage wheat |
| CN103869058A (en) * | 2014-03-21 | 2014-06-18 | 上海交通大学 | Rapid evaluation method for salt tolerance of tomatoes |
| CN105104193A (en) * | 2015-08-19 | 2015-12-02 | 单县绿丰种业有限公司 | Method for raising salt-tolerant tomato plants |
| CN109105235A (en) * | 2018-08-17 | 2019-01-01 | 河北省农林科学院昌黎果树研究所 | A kind of identification method of grape rootstock salt tolerance |
| CN110583395A (en) * | 2019-09-11 | 2019-12-20 | 宁夏农林科学院农作物研究所（宁夏回族自治区农作物育种中心） | Method for evaluating salt tolerance of japonica rice seeds under salt stress |
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