CN105284608A - Culture medium for watermelon flower callus induction and method for inducing watermelon flower callus by adopting culture medium - Google Patents
Culture medium for watermelon flower callus induction and method for inducing watermelon flower callus by adopting culture medium Download PDFInfo
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Abstract
The invention discloses a culture medium for watermelon flower callus induction and a method for inducing watermelon flower callus by adopting the culture medium. After treatment for 2d-3d at the low temperature of 4 DEG C, the effective callus induction rate reaches 18.33%; after culture for 2d-3d at a high temperature, the effective callus induction rate reaches 14%; the inducing effect of NAA is better than that of 2,4-D; after 500mg.L<-1> of acid hydrolyzed casein (CH) is added to the culture medium, the effective callus induction rates respectively reach 19.00% and 20.33%; at 6:00-8:00 in a sunny day, flower buds in a mid-late uninucleate stage are taken, are treated at the low temperature for 2d-3d, and then are inoculated onto the MS culture medium added with NAA with the concentration of 1.0mg.L<-1>, 6-BA with the concentration of 2.0mg.L<-1>, KT with the concentration of 1.0mg.L<-1> and CH with the concentration of 500mg.L<-1> for culturing at the high temperature for 2d-3d, and thus, the effective callus induction rate is highest.
Description
Technical field
The present invention relates to watermelon induction of anther callus method and the medium for inducing, be specifically related to for watermelon induction of anther callus medium and utilize the method for this medium inducing watermelon anther callus.
Background technology
Anther culture obtains haploid effective way.At present, obtain the embryogenetic crop of high gamete by anther culture, mainly on cereal, Cruciferae and some Solanaceae class crops.Anther culture induction monoploid by the impact of several factors, as inducing culture, microspore development period, process in early stage, condition of culture, donor plant growth conditions, genotype etc.As far back as 1981, Xue Guangrong etc., on variety of watermelon " fine jade is crisp ", successfully induced pollen plant, subsequently again on " thoughtful red " variety of watermelon, obtain pollen plant, but its callus induction rate and differentiation frequency are all lower, breeding is difficult to utilize, is not promoted, Yuan Wanliang " YUANWanliang, FURuiming, LEIBaolin.Thepreliminaryreportofwatermelonantherculture [J] .ShanxiAgriculturalSciences, 1995, (1): 29-30. Yuan ten thousand is good, Fu Runmin, Lei Baolin etc. watermelon flower training Preliminary Report on Experiment [J]. Shaanxi agricultural science, 1995, (l): 29-30. ", Wei Ying " WEIYing.Theeffectsoflow-temperaturepretreatmentonantherc allusinduction [J] .AgriculturalScienceandTechnology, 1999, (9): 34-36. Wei Ying. Cold pretreatment is on the impact [J] of watermelon flower pesticide evoked callus. agriculture of gansu science and technology, 1999, : 34-36. " (9) research such as, the anther cultural callus induction rate of watermelon is increased, but there is no follow-up report, to the quality of callus also without clearly stating, the gorgeous rosy clouds of Gou " [GOUYanxia.Thetechnologyresearchofwatermelonantherculture [D] .Hangzhou, ZhejiangUniversity, the gorgeous rosy clouds of 2006. Gou. watermelon vitro anther culture technical research [D]. Hangzhou: Zhejiang University, 2006. " Li Juan " [13] LIJuan.Thestudyofwatermelonantherandmicrosporeculture [D] .Yaan, SichuanAgriculturalUniversity, 2006. Li Juan. watermelon flower pesticide and microspores culture research [D]. Yaan: Sichuan Agricultural University, 2008. " etc. watermelon flower pesticide and microspores culture are studied, but do not obtain remarkable effect.
Summary of the invention
In order to solve the deficiencies in the prior art, the invention provides for watermelon induction of anther callus medium and utilize the method for this medium inducing watermelon anther callus, propose the suitable effective callus induction method of watermelon flower pesticide, for the foundation of watermelon anther culture regeneration techniques system lays the foundation.
Technical scheme of the present invention is: for the medium of watermelon induction of anther callus, and described medium adds NAA, 6-BA, KT and CH on the basis of MS medium, and the concentration of described NAA, 6-BA, KT and CH is respectively 0.2-1.0mgL
-1, 2.0mgL
-1, 1.0mgL
-1and 300-500mgL
-1.
Further improvement of the present invention comprises:
Medium adds NAA, 6-BA, KT and CH on the basis of MS medium, and the concentration of described NAA, 6-BA, KT and CH is respectively 1.0mgL
-1, 2.0mgL
-1, 1.0mgL
-1and 500mgL
-1.
Another object of the present invention is to the method for the medium inducing watermelon anther callus provided described in utilization, comprise the following steps: get mid-late uninucleate stage bud, low temperature treatment 2d-3d, be inoculated on described medium, High Temperature Pre cultivates 2-3d.
Described mid-late uninucleate stage bud access time is fine day 6:00-8:00 in morning.
Described mid-late uninucleate stage bud is selected from 5-6 month bud and indulges footpath 2-4mm, transverse diameter 3-4mm, and sepal is close to petal, petals close, and in bottle green, anther color is faint yellow to yellow green.
Described low temperature treatment comprises: get bud, with tap water 3 times, then wraps with wet gauze, processes, then, on aseptic operating platform, pretreated bud is thrown in the alcohol of 75% 30s that sterilizes, then use the HgCl of 0.1% at being placed in 4 DEG C
2sterilize 8 minutes, finally use aseptic water washing 3-5 time; After having sterilized, the aseptic knife tweezers strip off of aseptic bud, take out flower pesticide, be seeded on inducing culture and cultivate.
Described High Temperature Pre is cultivated and is comprised: postvaccinal flower pesticide is put into 35 DEG C of constant incubators respectively and cultivates 2d.
Present invention also offers the application of above-mentioned medium in watermelon induction of anther callus.
Watermelon in described application is HJ001, No. 8, western agriculture and the black beauty of large fruit.
The present invention with " HJ001 " (crossbreed), No. 8, western agriculture, the black beauty of large fruit for test material, carry out Different hypothermia pretreatment time, different High Temperature Pre incubation time, plant growth regulator, different additives, different genotype, Different growing environment and sampling time of explant respectively to the impact of watermelon induction of anther callus, effective callus induction rate of statistics different disposal and invalidly organize inductivity.Through 4 DEG C of low temperature treatment 2d-3d, effective callus induction rate reaches 18.33%; High Temperature Pre cultivates 2d-3d, and effective callus induction rate reaches 14%; The inducing effect of NAA is good compared with 2,4-D; Medium adds acid hydrolyzed casein (CH) 500mgL
-1effective callus induction rate reaches 19.00% and 20.33% respectively; At the beginning of 6 months late Mays, the bud vigor of collection is higher, and bud indulges footpath 2-4mm, transverse diameter 3-4mm, and sepal is close to petal, petals close, and in bottle green, anther color is faint yellow to mostly being microspore mid-late uninucleate stage during yellow green, slightly difference between different cultivars; Effective callus induction rate and total callus induction rate of 3 kinds have larger difference, and difference is maximum reaches 16% and 46.34%.At fine day 6:00-8:00 in morning, get mid-late uninucleate stage bud, low temperature treatment 2d-3d, be seeded in MS+NAA1.0mgL
-1+ 6-BA2.0mgL
-1+ KT1.0mgL
-1+ CH500mgL
-1high Temperature Pre cultivates 2d-3d, and effective callus induction rate is the highest.
Accompanying drawing explanation
Fig. 1 is microspore viability examination result figure.
Fig. 2 is microspore monokaryotic stage microscopy figure.
Fig. 3 affects result figure to the effective callus induction rate of watermelon the low temperature treatment time.
Fig. 4 affects result figure to the invalid callus induction rate of watermelon the low temperature treatment time.
Fig. 5 affects result figure to the effective callus induction rate of watermelon the high temperature treatment time.
Fig. 6 affects result figure to the invalid callus induction rate of watermelon the high temperature treatment time.
Fig. 7 is white, flocculence callus.
Fig. 9 is the hard callus closely of quality.
Fig. 8 is yellow open-textured callus.
Figure 10 is the hard callus closely of quality.
Figure 11 is the impact of variable concentrations additives for watermelon callus induction rate.
Figure 12 is that various seasons affects result figure to the effective callus induction rate of watermelon.
Figure 13 is that various seasons affects result figure to the invalid callus induction rate of watermelon.
Embodiment
Below in conjunction with accompanying drawing, the present invention is elaborated.
The present invention is used for the medium of watermelon induction of anther callus, and described medium adds NAA, 6-BA, KT and CH on the basis of MS medium, and the concentration of described NAA, 6-BA, KT and CH is respectively 0.2-1.0mgL
-1, 2.0mgL
-1, 1.0mgL
-1and 300-500mgL
-1.As the preferred embodiments of the present invention, described medium adds NAA, 6-BA, KT and CH on the basis of MS medium, and the concentration of described NAA, 6-BA, KT and CH is respectively 1.0mgL
-1, 2.0mgL
-1, 1.0mgL
-1and 500mgL
-1.
Another object of the present invention is to the method for the medium inducing watermelon anther callus provided described in utilization, comprise the following steps: get mid-late uninucleate stage bud, low temperature treatment 2d-3d, be inoculated on described medium, High Temperature Pre cultivates 2-3d.
Described mid-late uninucleate stage bud access time is fine day 6:00-8:00 in morning.Described mid-late uninucleate stage bud is selected from 5-6 month bud and indulges footpath 2-4mm, transverse diameter 3-4mm, and sepal is close to petal, petals close, and in bottle green, anther color is faint yellow to yellow green.
Described low temperature treatment comprises: get bud, with tap water 3 times, then wraps with wet gauze, processes, then, on aseptic operating platform, pretreated bud is thrown in the alcohol of 75% 30s that sterilizes, then use the HgCl of 0.1% at being placed in 4 DEG C
2sterilize 8 minutes, finally use aseptic water washing 3-5 time; After having sterilized, the aseptic knife tweezers strip off of aseptic bud, take out flower pesticide, be seeded on inducing culture and cultivate.Described High Temperature Pre is cultivated and is comprised: postvaccinal flower pesticide is put into 35 DEG C of constant incubators respectively and cultivates 2d.
Comparative trial
1.1 material
Test material has 3.HJ001 is this seminar autogamy crossbreed, the shallow white of fruit, flesh white, fruit development period 60 days; The black beauty of large fruit and No. 8, Xi Nong are commodity kind, and the black beauty of large fruit is provided by diploid watermelon seminar of Zhengzhou Fruit-tree Inst., Chinese Agriculture Science Academy; No. 8, western agriculture is provided by Yang Ling agricultural high-tech Development stock Co., Ltd.
Test and carry out in key lab of Henan Province in 2010-2011.Test material adopts the mode of interval sowing to carry out.Vernalization sowing is carried out respectively in 2010.03.05,2010.04.10,2010.07.15,2011.03.10,2011.04.15,2011.07.17, wherein 2010.03.05,2010.07.15,2011.03.10,2011.07.17 are greenhouse gardenings, and all the other are open country plantation.Material is planted in Zhengzhou Fruit-tree Inst., Chinese Agriculture Science Academy's Experimental Base.
1.2 method
1.2.1 draw materials time and period
The bud that 6:00-7:00,7:00-8:00,8:00-9:00,9:00-10:00 gather different size morning carries out the observation in Pollen Activity and microspore development period.Pollen Activity and microspore development all adopt FDA method to observe period.
1.2.2 low temperature treatment
Get the bud that microspore is in monokaryotic stage, with tap water 3 times, then wrap with wet gauze, processing 1d, 2d, 3d respectively at being placed in 4 DEG C, is contrast with what do not process, then, on aseptic operating platform, pretreated bud is thrown in the alcohol of 75% 30s that sterilizes, then use the HgCl of 0.1%
2sterilize 8 minutes, finally use aseptic water washing 3-5 time.After having sterilized, the aseptic knife tweezers strip off of aseptic bud, take out flower pesticide, be seeded on inducing culture and cultivate, each process inoculation 5 bottles, every bottle graft 20 pieces of flower pesticide, if 3 times are repeated, add up effective callus (white and quality closely hard) inductivity and invalid callus (yellow, transparent, quality loosen and white, loosen, flocculence) inductivity.
Flower pesticide number × 100% of effective callus induction rate (%)=induce flower pesticide number/inoculation of effective callus
Flower pesticide number × 100% of invalid callus induction rate (%)=the induce flower pesticide number/inoculation of invalid callus
1.2.3 High Temperature Pre is cultivated
Postvaccinal flower pesticide is put into 35 DEG C of constant incubators respectively, cultivate 1d, 2d, 3d, with without high temperature treatment for contrast, each process inoculation 5 bottles, every bottle graft 20 pieces of flower pesticide, if 3 times repeat, statistics callus induction rate.
1.2.4 plant growth regulator
At MS+6-BA2.0mgL
-1+ KT1.0mgL
-1on medium, add 2,4-D and NAA, three concentration levels are set respectively, 0.2,0.5 and 1.0mgL
-1, all the other condition of culture are identical.Each process inoculation 5 bottles, every bottle graft 20 pieces of flower pesticide, if 3 times are repeated.Statistics callus induction rate.
1.2.5 medium additives
At MS+NAA1.0mgL
-1+ 6-BA2.0mgL
-1+ KT1.0mgL
-1on medium, add acid hydrolyzed casein (CH).Each process inoculation 5 bottles, every bottle graft 20 pieces of flower pesticide, if 3 times are repeated.Statistics callus induction rate.As shown in Table 2 below.
The different additives process of table 1
Table1ThetreatmentofdifferentOrganicstype
Note :-for not add any material, lower same.
Note:-fornotaddinganymaterial,thesamebelow
1.2.6 growing environment
Under the prerequisite that condition of culture is identical, compare the difference of two kinds of environmental conditions (open country and greenhouse cultivation) callus induction rate, each process repeats 3 times.
2 results and analysis
2.1 sampling time of explants and time are on the impact of watermelon induction of anther callus
At fine day 6:00-8:00 in morning, (now microspore vigor is higher, as shown in Figure 1), for " HJ001 ", get bud and indulge footpath 2-4mm, transverse diameter 3-4mm, sepal is close to petal, petals close, in bottle green, anther color is faint yellow to yellow green, and microscopy shows, now pollen mostly is microspore mid-late uninucleate stage, as shown in Figure 2, effective callus induction rate is higher.Different materials is difference slightly.
2.2 low temperature treatment are on the impact of watermelon induction of anther callus
Same material, 4 DEG C of Cold pretreatment times are different, and callus induction rate is different.Found out by Fig. 3, effective callus induction rate of " HJ001 " 4 DEG C of low temperature treatment 2d and 3d is significantly higher than process 1d and contrast, effective callus induction rate of 4 DEG C of low temperature treatment 1d is significantly higher than contrast, without significant difference between effective callus induction rate of process 2d and 3d, and being up to 18.33% in effective callus induction rate of 4 DEG C of low temperature treatment 2d, untreated effective callus induction rate only has 2.0%; Effective callus induction rate of western agriculture No. 84 DEG C of low temperature treatment 2d and 3d is significantly higher than process 1d and contrast, without significant difference between effective callus induction rate of process 2d and 3d, also without significant difference between process 1d and effective callus induction rate of contrast; Effective callus induction rate that the black beauty of large fruit processes 3d is significantly higher than contrast, and remarkable with the difference of low temperature treatment 1d and 2d, reaches be up to 8.56% in effective callus induction rate of process 3d.As can be seen here, 4 DEG C of low temperature treatment are beneficial to the induction of effective callus, and general 4 DEG C of low temperature treatment 2d-3d, effective callus induction rate reaches the highest.
Found out by Fig. 4, the invalid callus induction rate of " HJ001 " 4 DEG C of low temperature treatment 3d, be significantly higher than process process 2d, 1d, 0d, reach 30.67%, there was no significant difference between process 1d and the invalid callus induction rate of contrast; The invalid callus induction rate of No. 8, western agriculture process 2d is significantly higher than contrast, and there are no significant difference between all the other 3 process; The performance of the black beauty of large fruit is with No. 8, western agriculture.As can be seen here, 4 DEG C of low temperature treatment are equally also conducive to the induction of invalid callus.
2.3 high temperature treatment are on the impact of watermelon induction of anther callus
Found out by Fig. 5, " HJ001 " is all significantly higher than contrast through effective callus induction rate of 35 DEG C of high temperature treatment 1d, 2d, 3d, there was no significant difference between effective callus induction rate of 35 DEG C of high temperature treatment 1d, 2d, 3d; No. 8, western agriculture and the black beauty of large fruit are through there was no significant difference between 35 DEG C of high temperature treatment and contrast.
Fig. 6 shows, " HJ001 " is all significantly higher than process 3d through the invalid callus induction rate of 35 DEG C of high temperature treatment 2d, invalid callus induction rate through 35 DEG C of high temperature treatment 3d is significantly higher than contrast again, through there was no significant difference between 35 DEG C of high temperature treatment 1d, 3d and contrast invalid callus induction rate; No. 8, western agriculture is significantly higher than through 35 DEG C of high temperature treatment 1d and contrast through the invalid callus induction rate of 35 DEG C of high temperature treatment 2d, and not remarkable with the difference of process 3d; The black beauty of large fruit, the invalid callus induction rate of 35 DEG C of high temperature treatment 3d, significantly processes 2d, 1d and contrast, reaches 14.33%, processes 1d and contrasts there was no significant difference between invalid callus induction rate; As can be seen here, 35 DEG C of high temperature treatment, also can significantly improve invalid callus induction rate.
In sum, 35 DEG C of high temperature treatment are beneficial to the induction of the effective callus of watermelon flower pesticide, and when 35 DEG C of high temperature treatment 2d, effective callus induction rate is higher.
2.42,4-D and NAA is on the impact of watermelon induction of anther callus
Table 2 shows, and 2,4-D concentration is 0.2mgL
-1, NAA concentration is 1.0mgL
-1time, the callus induction rate of " HJ001 " reaches 56.67% and 59.67% respectively, and between the two without significant difference.2,4-D concentration is 0.5mgL
-1, NAA concentration is 1.0mgL
-1time, the callus induction rate of No. 8, western agriculture reaches 60.33% and 61.33% respectively, and significant difference is not remarkable between the two.2,4-D concentration is respectively 0.2,0.5,1.0mgL
-1time, " HJ001 " callus induction rate increases along with 2,4-D concentration and reduces, and shows as 2 of low concentration, 4-D and promotes calli induction, and 2,4-D of high concentration suppress the induction of callus, and the performance of NAA is then in contrast.No. 8, western agriculture is performance same " HJ001 " on the medium adding NAA, and interpolation 2,4-D time, concentration is 0.5mgL
-1time callus induction rate reach and be up to 60.33%, reach significant difference with all the other two concentration levels.
Although 2,4-D and NAA is when suitable concn, without significant difference between callus induction rate, but Callus morphology feature difference is very large, add the medium of 2,4-D, callus shows as loose type (invalid callus) more, loose type can be divided into two kinds again, is yellow, transparent, open-textured callus (Fig. 7), is separately white, loose, flocculence callus (Fig. 8).Add the medium of NAA, callus shows as white (full light culture) and hard closely (Fig. 9,10) (the effective callus) of quality more.
Table 22,4-D and NAA is on the impact of induction of anther callus
Table2Effectof2，4-DandNAAwithanthercallusinduction
2.5 different additives are on the impact of watermelon callus induction rate
Being found out by Figure 11, add acid hydrolyzed casein (CH) in inducing culture, is 100mgL when adding concentration
-1time, effectively slightly low compared with the control with invalid two class callus induction rate, but difference is not remarkable; Be 300mgL when CH adds concentration
-1time, invalid callus induction rate is higher by 16.34% than contrast, and difference reaches significance level, and effective callus induction rate is identical with contrast, is 6.33%; When CH addition is 500mgL
-1shi Youxiao callus is the highest, and difference is extremely remarkable compared with the control for the inductivity of two class anther callus, is respectively 46.33% and 19.00%.
As can be seen here, in inducing culture, add acid hydrolyzed casein (CH) 300-500mgL
-1callus induction rate reaches the highest.
2.6 growing environments are on the impact of watermelon induction of anther callus
As can be seen from Figure 12,13, same kind, various seasons, invalid callus induction rate differs greatly, effective callus induction rate difference is less, and also clearly, 3 kinds are all high than the callus induction rate of greenhouse cultivation in open country plantation for total callus induction rate difference.
2.7 genotype are on the impact of watermelon induction of anther callus
Found out by table 3,4, when material is without low temperature treatment, between 3 kinds, effective callus induction rate is without significant difference; And when low temperature treatment 1d, 2d, 3d, effective callus induction rate of HJ001 is significantly higher than No. 8, western agriculture and the black beauty of large fruit.3 materials are consistent with the performance of low temperature treatment through the performance of high temperature treatment.
Table 3 different cultivars is effective callus induction rate under low temperature treatment
Table3Differentvarietiesunderthelowtemperaturetreatmenteffectivecallusinductionrate
Table 4 different cultivars is effective callus induction rate under high temperature treatment
3 discuss
Research in recent years shows, many factors significantly affect anther cultural success.The most important thing is genotype and the growing environment of donor plant.And microspore development period, medium component, particularly hormone kind and concentration, also have condition of culture, also determine anther culture whether success.Find in test, the genotype of donor plant, growing environment, microspore development period, pretreatment in early stage, different plant growth regulator and additives etc. all produce larger impact to watermelon induction of anther callus rate.
3.1 microspore development periods are on the impact of watermelon induction of anther callus
Can the process of response external, depend on the suitable age of pollen development.6 periods can be divided into through reduction division to the process of mature pollen from pollen mother cell: monokaryon is early stage, monokaryon mid-term, the monokaryon later stage, m period, two core pollen periods and three core pollen periods.Find in the test of eggplant microspores culture, can there is separating phenomenon in the microspore of monokaryotic stage, and obtain a large amount of callus.The anther cultural best period of solanaceous vegetables be monokaryotic stage to dicaryotic phase, especially mid-late uninucleate stage, anther culture preferably.Karakullukcu etc. " KarakullukcuS; AbakK.Studiesonanthercultureofeggplant.I..Determinationo fthesuitablebudstage [J] .TurkishJournalofAgricultureandForestry; 1993; 17:801-810. " research finds that the bud being about to cracking front and back with sepal is comparatively suitable, the long 22.4-24.7mm of bud, bud diameter 10.8-12.mm.In this test, for material " HJ001 ", get bud and indulge footpath 2-4mm, transverse diameter 3-4mm, sepal is close to petal, petals close, in bottle green, anther color is faint yellow to yellow green, during microscopy, pollen mostly is microspore mid-late uninucleate stage, effective callus induction rate and total callus induction rate all higher, different materials is difference slightly.This and SibiM etc. are pointed out, the size correlation of sepal and petal is that microspore is in the morphology mark in monokaryon late period and matches, but this correlation is not go for all genotype
[26].Thus, in test, should first the Pollen stage of concrete test material be found to draw materials again by microscopy.
The impact of process in 3.2 early stages on induction of anther callus
Before cultivating, suitable intermittent warming is carried out to flower pesticide and can significantly improve culture effect.Cold pretreatment can improve Pollen Activity, plays a driving role to startup microspore nuclear division and Callus formation; High temperature heat shock can promote cell symmetrical fissions, changes microspore development direction.Thus, pretreatment is the anther cultural important step of many crops.
In this test, preculture 2d-3d under 4 DEG C of Cold pretreatment 2d-3d and 35 DEG C hot conditions, the inductivity of the effective callus of flower pesticide comparatively contrasts and is significantly increased, the intermittent warming of this and Li Juan have similarity " LIJuan.Thestudyofwatermelonantherandmicrosporeculture [D] .Yaan; SichuanAgriculturalUniversity; 2006. Li Juan. watermelon flower pesticide and microspores culture research [D]. Yaan: Sichuan Agricultural University, 2008. ".Someone thinks, callus produces when flower pesticide does not expand, low temperature treatment makes the cell of anther wall sustain damage, lose the activity of division growth, but the inner pollen cell of flower pesticide still has activity, then likely callus is formed during further induction, so, still keep the feature of color and luster pale green, quality relative close in squamous subculture.The height of temperature and the length in processing time all can produce significant impact to the inductivity of callus.Model suitable " FANShi.Thestudyofeggplantmicrosporeculture [D] .Changsha:HunanAgriculturalUniversity; 2003; 1-33. model is fitted. eggplant microspores culture research [D]. Changsha: Agricultural University Of Hunan; 2003; 1-33. " etc. research discovery, through heat shock in 6 days eggplant microspore colony expand that rate is the highest, vigor is the highest, Callus induction rate also significantly improves.Liu Gongshe etc. then think that High Temperature Pre is cultivated and not work at any time, only within the 24h of initial cultivation the most responsive [29]; The low temperature treatment of the use such as Phippen-5 DEG C-10 DEG C swims in the wheat anther on medium, finds that treatment temperature is lower or the processing time longer inhibitory action to cultivating reaction is very large.Rotino " RotinoGL; FalavignaAandRestainoF.Productionofanther-derivedplantle tsofeggplant [J] .CapsicumNewsletter; 1987.6:89-90. " studies discovery, heat shock makes the conversion of microspore generation from gametophyte to sporophyte, can promote the growth starting arrhenokaryon.This have also been obtained confirmation in many microspores culture.
Also find in test, the difference that the temperature and time of different pretreatments produces anther culture effect is larger.Not only the time of callus generation is different, and callus quality is also not quite similar.Also show in the test of the gorgeous rosy clouds of Gou: in watermelon anther culture, through the anther callus of low temperature treatment from the callus of the light green of splitting in the middle of flower pesticide, quality relative close, there is very large difference compared with the calli induction process obtained under normal temperature condition.Different Crop, pretreatment is different on anther cultural impact, but all creates certain impact, and therefore, pretreatment needs further to study to the anther cultural mechanism of action.
3.3 plant strain growth Auto-regulators are on the anther cultural impact of watermelon
Plant growth regulating substance is the micro substance that plant produces at privileged site, not only plays a very important role growing of plant, and also plays decisive action to Plant Tissue Breeding success or not.For most plants, exogenous auxin forms callus to inducing cell dedifferentiation and growth has decisive role, and particularly 2,4-D are considered to start the necessary condition that microspore cells division forms callus.But in this test, in interpolation 2, on the medium of 4-D, callus shows as incompact callus more, and on the medium adding NAA, callus shows as white and the hard callus closely of quality more, this and many reports are not too consistent such as DoiH, TakahashiR, HikageT, etal.Embryogenesisanddoubledhaploidproductionfromantherc ultureingentian (Gentianatriflora) [J] .PlantCell, TissueandOrganCulture (PCTOC), 2010, 102 (1): 27-33.This is because 2,4-D is the antagonistic substance of other plant hormone, itself have the effect stimulating and organize cambial activity, although can promote calli induction when concentration is lower, the differentiation capability of consequent callus significantly reduces.And NAA is broad spectrum type plant growth regulator, cell division and expansion can be promoted, the regeneration capacity improving plant callus generating ability and plant plays very important effect
[35,31].Much research shows that NAA and 6-BA is combined, and can significantly improve callus induction rate, and the differentiation capability of callus is also comparatively strong, and the antagonism between this and hormone has certain relation.This is similar to this result of the test.The growth conditions of 3.4 plant is on the impact of induction of anther callus
Have report display, haploid induction is determined by the growth conditions of donor plant.The growth conditions of plant and physiological status etc. all can have influence on the growth of its arrhenokaryon, and no matter be greenhouse or the plant at field planting, the pollen of eugonic healthy plant all obtains good inducing effect.Find in the test of eggplant Isolated microspore, low by 42% than under open country condition of the callus induction rate of greenhouse condition, refer to FANShi.Thestudyofeggplantmicrosporeculture [D] .Changsha:HunanAgriculturalUniversity, 2003,1-33. model is fitted. eggplant microspores culture research [D]. and Changsha: Agricultural University Of Hunan, 2003,1-33.This development test result shows, in Zhengzhou area, the test material induction of anther callus rate gathered at the beginning of 6 months is by the end of May high, and the test material induction of anther callus rate gathered at the beginning of 9 months is by the end of August lower, and had a strong impact on follow-up differentiation cultivation, be consistent with the research of forefathers.Also there are some researches show, first batch of material etheogenesis ability be eager to excel, health and the flower pesticide of eugonic plant has larger etheogenesis ability.In this test, at fine day 6:00-8:00 in morning, the bud microspore vigor taked is higher, and callus induction rate is higher.But in Brassicas
[37]and cabbage type rape
[38]in, the pollen seeming not only old but also weak has higher product embryo rate on the contrary, and same result, also has performance in leaf mustard, and the flower pesticide in shepherd's purse late period is higher than the flower pesticide germ extraction rate of normal flowering time
[39].Find in this test, the material of open country plantation is high compared with the callus induction rate of greenhouse cultivation.Cause the reason of this phenomenon may be different planting environment, different female bloom dates its pollen viability of bud may be different, inductivity then has notable difference.The test of the gorgeous rosy clouds of Gou shows, at the watermelon Microsporogenesis initial stage, all day is cloudy and drizzly for days on end, pollen abortion can be caused, the activity of microspore reduces, and induction of anther callus rate is low and shoot regeneration frequency is low, and transfers on differential medium also easily dead, Li Juan etc. also illustrate suitable growth conditions under study for action, are just more conducive to watermelon anther culture embryonal induction and plant regeneration.Heberle is by using NAA, ethephon process donor plant, change the growth conditions such as the duration of day, temperature, the change of plant embryo generation pollen grain quantity is also not obvious, although this shows that the growing environment of donor plant is very large to the generation effect of embryo's generation pollen grain, also do not find at present and improve by controlling donor plant growing environment the effective ways that plant anther cultivates inductivity.Therefore, set up an efficient anther-cultural system, the cultivation condition exploring and grasp examination material is also very necessary.
3.5 genotype are on anther cultural impact
Induction of Anther Culture Callus inductivity has very large difference because testing the difference of kind used.
On watermelon, Wei Yue etc. think that suitable genotype is the key factor of watermelon flower pesticide Fiber differentiation, and different genotype is different to the susceptibility of Hormone change, and optimum medium is also different; In the test of the gorgeous rosy clouds of Gou, the order of watermelon induction of anther callus rate is " by bit ", " spring scenery " and " happiness all " reduces successively; Li Juan research shows, in watermelon flower pesticide and microspores culture, wild material is higher than the callus induction rate of cultivar.On other crops, in 46 kinds and 9 species hybrids of Solanum, 19 species and 4 hybrids are only had to produce pollen plant.Cao Yu and Yuan Mei carries out anther cultural result show different triticale, peanut, and between different genotype, Callus induction rate exists notable difference, and the research of Eudes etc. also shows that haploid generation has genotype-independent.
In this test, different process, effective callus induction rate of material " HJ001 " is all significantly higher than No. 8, western agriculture and the black beauty of large fruit.And the effective callus induction rate between No. 8, western agriculture and the black beauty of large fruit is without significant difference.As can be seen here, between different genotype, effective callus induction rate differs greatly, and is consistent with the research of forefathers.
The Jing such as Afele cross and think into Yi Walk research, and the induction of embryo shape callus may be due to the difference on different cultivars mrna expression.But, with regard to genotype the research that androgenesis affects also mainly rested at present and cultivates observation aspect, though there are some researches show, germ extraction rate can be improved by what should not go out embryo with the genotypic crossing exceeding embryo.But less to deep study mechanism, therefore, in watermelon anther culture process, the Genetic Mechanisms of research genotype effect factor is particularly urgent.
4 conclusions
At fine day 6:00-8:00 in morning, get mid-late uninucleate stage bud, low temperature treatment 2d-3d, be seeded in MS+NAA1.0mgL
-1+ 6-BA2.0mgL
-1+ KT1.0mgL
-1+ CH500mgL
-1high Temperature Pre cultivates 2d-3d, and effective callus induction rate is the highest, and this kind of method is the best approach of suitable watermelon induction of anther callus.
The present invention with " HJ001 " (crossbreed), No. 8, western agriculture, the black beauty of large fruit for test material, carry out Different hypothermia pretreatment time, different High Temperature Pre incubation time, plant growth regulator, different additives, different genotype, Different growing environment and sampling time of explant respectively to the impact of watermelon induction of anther callus, effective callus induction rate of statistics different disposal and invalidly organize inductivity.Through 4 DEG C of low temperature treatment 2d-3d, effective callus induction rate reaches 18.33%; High Temperature Pre cultivates 2d-3d, and effective callus induction rate reaches 14%; The inducing effect of NAA is good compared with 2,4-D; Medium adds acid hydrolyzed casein (CH) 500mgL
-1effective callus induction rate reaches 19.00% and 20.33% respectively; At the beginning of 6 months late Mays, the bud vigor of collection is higher, and bud indulges footpath 2-4mm, transverse diameter 3-4mm, and sepal is close to petal, petals close, and in bottle green, anther color is faint yellow to mostly being microspore mid-late uninucleate stage during yellow green, slightly difference between different cultivars; Effective callus induction rate and total callus induction rate of 3 kinds have larger difference, and difference is maximum reaches 16% and 46.34%.At fine day 6:00-8:00 in morning, get mid-late uninucleate stage bud, low temperature treatment 2d-3d, be seeded in MS+NAA1.0mgL
-1+ 6-BA2.0mgL
-1+ KT1.0mgL
-1+ CH500mgL
-1high Temperature Pre cultivates 2d-3d, and effective callus induction rate is the highest.
More than show and describe general principle of the present invention and principal character and advantage of the present invention.The technical staff of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (9)
1. for the medium of watermelon induction of anther callus, it is characterized in that, medium adds NAA, 6-BA, KT and CH on the basis of MS medium, and the concentration of described NAA, 6-BA, KT and CH is respectively 0.2-1.0mgL
-1, 2.0mgL
-1, 1.0mgL
-1and 300-500mgL
-1.
2. the medium for watermelon induction of anther callus according to claim 1, is characterized in that, medium adds NAA, 6-BA, KT and CH on the basis of MS medium, and the concentration of described NAA, 6-BA, KT and CH is respectively 1.0mgL
-1, 2.0mgL
-1, 1.0mgL
-1and 500mgL
-1.
3. utilize the method for the medium inducing watermelon anther callus described in any one of claim 1-2, it is characterized in that, comprise the following steps: get mid-late uninucleate stage bud, low temperature treatment 2d-3d, be inoculated on described medium, High Temperature Pre cultivates 2-3d.
4. method according to claim 3, is characterized in that, described mid-late uninucleate stage bud access time is fine day 6:00-8:00 in morning.
5. method according to claim 3, is characterized in that, described mid-late uninucleate stage bud is selected from 5-6 month bud and indulges footpath 2-4mm, transverse diameter 3-4mm, and sepal is close to petal, petals close, and in bottle green, anther color is faint yellow to yellow green.
6. method according to claim 3, it is characterized in that, described low temperature treatment comprises: get bud, with tap water 3 times, then wrap with wet gauze, process at being placed in 4 DEG C, then, on aseptic operating platform, pretreated bud is thrown in the alcohol of 75% 30s that sterilizes, then use the HgCl of 0.1%
2sterilize 8 minutes, finally use aseptic water washing 3-5 time; After having sterilized, the aseptic knife tweezers strip off of aseptic bud, take out flower pesticide, be seeded on inducing culture and cultivate.
7. method according to claim 3, is characterized in that, described High Temperature Pre is cultivated and comprised: postvaccinal flower pesticide is put into 35 DEG C of constant incubators respectively and cultivates 2d.
8. the application of the medium as described in any one of claim 1-2 in watermelon induction of anther callus.
9. application according to claim 8, is characterized in that, described watermelon is HJ001, No. 8, western agriculture and the black beauty of large fruit.
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