Abstract:To explore the controlling effect of different dosages of gibberellin(GA3) contained in the foliar fertilizer mingled by potassium dihydrogen phosphate and gibberellin(GA3) and the spraying times of gibberellin(GA3) at heading stage on the resistance of rice to low temperature stress during the flowering period,two rice cultivars matured lately and sensitive to low-temperature stress at heading stage,hybrid ‘Peiliangyou 537’ and conventional ‘Ezhong 5’,were used as experimental materials in this study.Twenty four adjustment patterns including the supplement of 0.0（A0），7.5(A1)，15.0（A2），22.5(A3)，30.0(A4)，37.5 g(A5) of GA3 based on 3.0 g/hm2 potassium dihydrogen phosphate and the spraying time at spike initial appearance stage (B1),initial heading stage (B2),heading stage (B3),full heading stage (B4) were adopted,thereafter heading dynamics,uniformity of panicle layer,yield and yield components of rice were investigated.The results showed that the 1 000-grains weight,grains per panicle,rate of seed setting,heading time and yield of rice under the patterns with spraying dosage of GA3 at 15.0-22.5 g/hm2 and the spraying time at B1 or B2 were better than those under other patterns and spraying times.However,under spraying the foliar fertilizer at B3 or B4,high rice yield requires the increase of spraying dosage.Increasing of spraying dosage or earlier spray time during heading period could improve the uniformity of rice panicle layer,but the former would diminish grain-filling and decrease 1000-grain weight.The addition of GA3 in the fertilizer can increase the resistance of rice to low temperature,in which the appropriate dosage of GA3 is 15.0-22.5 g/hm2.The increased dosage of GA3 may be useful to improve rice yield when the foliar fertilizer spraying time is delayed due to rain.

Abstract:Several dwarf plants occurred in potato lines transformed with StERF3 over expression vector and the influencing factors of the dwarfism was investigated.Gene expression test revealed that StERF3 expression level in dwarf plants was lower than that of control.StERF3 gene expression decreased in the interference transgenic lines,but no dwarf plants appeared.The results indicated that the dwarfism was not related with the expression of StERF3 gene.Medium supplemented with 0.5 mg/mL exogenous GA3 could improve the growth of dwarf plants in vitro to some extent.Most of the dwarf lines contained 3-4 copies.Multi-copy integration always led to transgenic silencing.In conclusion,the dwarf might be caused by multiple copies integration into the genomic DNA which interrupted the genes associated with GA3 pathway.

Abstract:Key factors affecting the digestion-ligation,pre-amplification and selective amplification in amplified fragment length polymorphism(AFLP) analysis in sesame (Sesamum indicum L.) were optimized.Results showed that the best protocol was 200 ng/μL template DNA，digestion-ligation at 37℃ for 6 h,1.0 mmol/L Mg2+,0.3 mmol/L dNTP,0.5 U Taq DNA polymerase in the pre-amplification system and pre-amplification products diluted by 10 times,1.0 mmol/L Mg2+, 0.2 mmol/L dNTP,and 1.0 U Taq DNA polymerase,0.6 mmol/L primers in the selective amplification.

Abstract:One new record species of genus Lactarius is reported in China: Lactarius purpureus R. Heim ex R. Heim. Materials were collected from Forest Farm of Xiaokeng,Shaoguan City,Guangdong Province. It is characterized by its orange-brownish zonate and adpressed scales, globose to broadly ellipsoid basidiospores with ridges or warts, subfusiform to subcylindrical-cone pleuromacrocystidia with acute or moniliform apex, fusiform cheilomacrocystidia and pileus surface hyphae being flat-lying.

Abstract:Endo-β-1,4-glucanase gene (with or without signal peptide coding sequence ) was amplified from Sulfolobus islandicus REY15A genomic DNA with PCR and inserted into the Sulfolobus expression vector pZC2,constructing the recombinant plasmid pZC2-eng-YS and pZC2-eng-WS,respectively.Then the recombinant plasmids were electro-transformed into the host strain S.islandicus E233S (△pyrEF△lacS).After induced by D-arabinose and purified with the Ni2+-nitrilotriacetate column,two obvious protein bands with molecular weight about 43 ku and 41 ku appeared on the SDS-PAGE.The following analysis showed the recombinant protein (ENG-W) without signal peptide does not have endo-β-1,4-glucanase activity.However,the activity of recombinant protein (ENG-SP) with signal peptide was 103.4 U/L.The optimal temperature and pH for the recombinant protein (ENG-SP) was 90℃ and 4.0,respectively.Further research indicated that after incubating at 90 °C for one hour,ENG-SP still had 40% of the highest activity.Mn2+ could increase the activity of ENG-SP by 50%,while Ca2+ could inhibit the activity of ENG-SP by 50%.

Abstract:The genetic polymorphisms of partial sequence of OSTF1（osteoclast stimulating factor 1）gene in 227 Holstein cows were detected by PCR-SSCP with five pairs of primers P1-P5.Two polymorphic sites,A45416G and C45436T of primer P3 were found,and A45416G mutation caused amino acid change (methionine→valine).Five genotypes (AA,AB,BB,AC and CC) were found in Holstein cows,and the genotype frequencies of AA,AB,BB,AC and CC were 0.515,0.366,0.075,0.035 and 0.009,respectively.The relationship between polymorphisms of OSTF1 gene and somatic cell score (SCS) in Holstein cows was analyzed by least squares linear model.Least squares mean of SCS for BB genotype was significantly lower than that for AA,AB or AC (P<0.05),and no significant difference was found in least squares mean of SCS between other genotypes (P>0.05).So genotype BB was mastitis resistant and it may be used as a molecular genetic marker to improve the phenotype of anti-mastitis in Chinese Holstein dairy cows.

Abstract:The ultrastructural characteristics of root,stem,leaf,pollen in three kinds of Tengcha (Ampelopsis grossedentata) coming from the province of Jiangxi,Hunan,Hubei in the same ecological environment were investigated using light microscopy (LM) and scanning electron microscopy (SEM).The results showed that the cross section of root and stem had little difference among the three kinds of Tengcha,the parenchyma cells of root and stem contex all existed calcium oxalate needle crystal.There were some nonglandulars hairs in the stem epidermal cell of Hubei and Jiangxi Tengcha,calcium oxalate needle crystal also existed in the mesophyll tissue and the parenchyma cells of leaf midrib.The characteristics of lower epidermal surface of leaf including nonglandular hairs,the stripe number of the outer stomatal rim,the density,length and open of stoma were different; there were much nonglandulars at upper epidermal surface along leaf midrib,but the density and shape were different among the three kinds of Tengcha; the characteristics of upper epidermal ornamentation were different as well,the upper epidermal surface existed granular or flaky waxiness at Jiangxi Tengcha leaf,pinshripes at Hunan Tengcha leaf,but nothing at Hubei Tengcha leaf; the antheral characteristics were the same but large.The pollens of Hubei and Jiangxi were spheroidicity but pollen of Hunan Tengcha was global and the largest.The results described the special ultrastructural characteristics of the three kinds of Tengcha and the difference maybe result from its genetic diversity.

Abstract:This paper described the female Chaorthippus taibaieusis Zheng et al,2009 from Gansu Province,which is the first record of the female of the world. The specimens are deposited in the Institute of Zoology,Shannxi Normal University.

Abstract:Heat shock protein 90 genes (hsp90s) are multifunctional genes, which are related to biological development, biological defense reactions, environmental stress tolerance and so on. In this research,a heat shock protein 90 gene (Dd-hsp90-1; GenBank Accession: HQ901595) was cloned from Ditylenchus destructor by RT-PCR and RACE. The cDNA sequence consisted of a 2 160 bp open reading frame (ORF) encoding 719 amino acid residues that was franked by a 70 bp 5′ -untranslated region (UTR) and a 117 bp 3′-UTR. The result of intron-exon structure analysis showed that the genomic sequence of Dd-hsp90-1 also contained 9 introns. And all the introns obeyed the GT/AG rule in the splice-site junctions. The molecular weight of Dd-hsp90-1 deduced protein was about 82.79 ku. Dd-hsp90-1 was a single copy gene was highly homologous to other hsp90s. There were consensus amino acid sequences and motifs of HSP90s in the amino acid sequence of deduced protein Dd-HSP90-1. Furthermore,the deduced protein Dd-HSP90-1 also contained five signature sequences and MEEVD motif specific to cytoplasmic HSP90s. All of these demonstrated that Dd-hsp90-1 was an cytoplasmic hsp90. Phylogenetic dendrogram indicated that Dd-hsp90-1 was most closely related to Baursaphelenchus xylophilus hsp90 (ACY01918) with 86.65% sequence identity. It also revealed the differences in the feeding behaviors of plant parasitic nematodes.

Abstract:Cylindrocladium parasiticum was a very important invasive alien species in China. The biological characteristics,such as mycelial growth and sporulation,etc. of C.parasiticum were observed. The results showed that C.parasiticum could grow in different media,such as Potato Dextrose Agar (PDA), V8 juice (V8), Yeast Peptone Dextrose Agar (YPDA),Oatmeal Agar (OMA),and Synthetic Fungal(SF). Among them,OMA was most suitable for mycelial growth, whereas V8 was most suitable for microsclerotium, conidium, and perithecium formation. C.parasiticum could utilize carbon sources, such as dextrose, fructose, sucrose, maltose, lactose, and starch for growth. Among them, dextrose, lactose and starch were most suitable for mycelial growth and microsclerotium formation. However, all of the above carbon sources were unsuitable for conidium and perithecium formation. In the case of nitrogen sources,C.parasiticum could utilize sodium nitrate,urea,ammonium sulphate,yeast,and peptone for growth. Among them, sodium nitrate was most suitable for mycelial growth and microslerotium formation,whereas,urea was most suitable for conidium formation. However,perithecium was sparsely formed only in sodium nitrate. C.parasiticum could grow under temperature condition between 10-30 ℃. The optimum temperature for both of growth and conidia sporulation was between 25-30 ℃. Although the mycelial growth of C.parasiticum was not impacted by period the light,but a12 hours alternation of light and dark was suitable for conidia sporulation. C.parasiticum could grow between pH values of 4.0-10.0. But the favorable pH value for mycelial growth was neutral to partial acid.

Abstract:Polygalacturonase-inhibiting protein (PGIP) is a cell wall binding protein that can specifically inhibit the activity of fungal polygalacturonase. A PGIP was purified from healthy banana seedlings by a series of column chromatography: ConA affinity column, CM-Sepharose ion exchange column and Sephadex S-100 gel filtration column. The 38.2 ku PGIP was shown as a single band, as determined by SDS-PAGE. Its activity is sensitive to different temperatures. The inhibition of the PGIP to Fusarium oxysporum f.sp.cubense race 4 (Foc4) is higher than its inhibition to Aspergillus niger PG, which suggested the banana PGIP activity might specifically inhibit certain PG.

Abstract:With the methods of in vivo and in vitro,the potential enhancement of Kluyveromyces marxianus by ammonium molybdate (NH4Mo) to control green mold caused by Penicillium digitatum on citrus fruit was investigated.Meanwhile,the effect of NH4Mo on growth and proliferation of K.marxianus was tested.The results showed that NH4Mo at different concentration did not inhibit the growth and proliferation of K.marxianus in culture medium or wounds of citrus fruits.The combination of K.marxianus and NH4Mo had suppressive effect on green mold caused by P.digitatum.Treatment of citrus fruits with K.marxianus plus NH4Mo (1 mmol/L) resulted in a consistent biocontrol activity,the disease incidence after 3 and 6 days were 28.33% and 60.00%,respectively.While the control treatment resulted in a disease incidence as high as 93.33% and 100%,suggesting that the combination of K.marxianus with NH4Mo may have great potential against green mold of citrus fruits.

Abstract:The effects of straw mulching on red soil N mineralization at different temperature,soil moisture,N fertilization were investigated through laboratory aerobic incubation.The results showed that if the soil was mulched by straw,its net N mineralization was lower than that of the non-mulched soil during the incubation time at 20，30℃.At 40℃ the net N mineralization of soil in mulching was lower than that of non-mulching during the first 40-day but higher than that of non-mulching after incubating 60 days.When the soil moisture was at 0.20 cm3/cm3 or 0.25 cm3/cm3,the difference between the mulching and non-mulching was more remarkable than that at 0.15 cm3/cm3.When the temperature was raised,the difference was narrowed and even disappeared.The straw mulching also had effects on soil N nitrification and N ammonification.When the temperature was 20℃ and soil moisture was 0.15 cm3/cm3,N nitrification was lower and N ammonification was higher in the mulching treatment.When the temperature was 40℃ and soil moisture was 0.25 cm3/cm3,N nitrification was higher and N ammonification was lower in mulching treatment than that of non-mulching treatment.These results suggest that it is essential to adjust the application of straw mulching in red soil fields according to the soil moisture and N fertility.

Abstract:Huashuang No.4 was selected to study the effects of potassium fertilizer on the whole stage of rapeseed growth including indexes of seedling stage,yield and yield components and finally to test the potassium uptake and use efficiency in the lab in Wuxue City,Hubei Province during 2008-2009.The optimum fertilizer application was calculated by establishing the model of economic region potassium.The results showed that the suitable application rate of potassium was helpful for the over-wintering stage of rapeseed seedling and significantly improved effective branches and pod numbers per plant.The results demonstrated that when the application of potassium was 180 kg/hm2,the total dry matter,seed yield and accumulation of potassium reached the largest which were 10 410,2 791 and 235.7 kg/hm2, respectively,and the net profit of K was 1 967 Yuan/hm2.Combining the model of fertilizer efficiency with the field trial,the optimum potassium fertilization rate of 160 kg/hm2 was recommended under the experimental field condition.

Abstract:Soil culture and pot experiments of bio-ash mixed with soil were employed to investigate the effects of bio-ash on soil properties and the seedling growth of rape.The results showed that the contents of soil effective nutrients increased with the increase of bio-ash application amount.Particularly the contents of available P and K in soil increased significantly.With the increase of ash amount,pH of soil increases and tends to neutral.The average increase is 7.78%.In low level of fertilizer,the increase of ash application amount is more conducive to the improvement of the soil available P,and the maximum increase is up to 4.2 times.The effect of changes in fertilizer application amount on the P absorption of rape is little.But with the increase of ash application amount,the content of total P in rape emerges the trend of increase first and then decrease.In middle level of fertilizer,the ash enhances the capacity of K absorption of rape plants,and the maximum increase of the content of total K in rape is up to 44.43%.In high level of fertilizer,the ash does not affect the capacity of K absorption of rape plants.The manifestation of antagonism between K and Ca,Mg in rape can be changed by the ash application.With the increase of ash application amount,the content of Ca and Mg absorption of rape emerges the trend of increase first and then decrease.All in all,a reasonable proportion of integrated fertilization with bio-ash and chemical fertilizers can promote the migration and transformation of nutrient elements in soil-plant system and the seedlings growth of rape.

Abstract:Based on plot data of poplar forestland soil (Ⅰ1,9 a; Ⅰ2,6 a; Ⅰ3,4 a; Ⅰ4,2 a),garden-land soil (vegetable soil and fruit soil) (Ⅱ),nonirrigated farmland soil (Ⅲ1,cotton soil; Ⅲ2,sugar cane soil; Ⅲ3,maize soil),irrigated farmland soil (rice soil) (Ⅳ) and wasteland soil (Ⅴ) located at Qianlianghu polder,Dongting Lake Area,a research was carried out to investigate the distribution and pollution of soil heavy metals (Cu,Zn,Mn,Pb,Cd,As and Hg) in five typical land-use patterns mentioned above.The results showed that in the soil layer of 0 to 25 cm,the highest contents of Cu,Zn,Pb,As and Hg all occurred in Ⅰ1,and the highest contents of Mn and Cd in Ⅰ4 and Ⅳ,respectively.In the soil layer of 25 to 50 cm,the highest contents of Cu,Mn and Hg occurred inⅠ1,Zn and As in Ⅳ,Pb and Cd in Ⅲ2 and Ⅰ3,respectively.Soils of pattern Ⅰ2,Ⅰ3,Ⅲ2 and Ⅳ were polluted with Cd,and the highest integrated pollution index occurred in Ⅳ and the lowest one occurred in Ⅴ.The interrelate regularities between heavy metals and soil physical and chemical properties were found through principal component analysis and double sieving stepwise regression analysis.

Abstract:A case study of Gucheng County was conducted to explore the influence of the four different types of land-use (villages,towns,rural and highways) on the basic farmland from 2006 to 2009.First,the levels of easy-encroached land factor and encroached regions were identified.Then,the changing percent and rates of basic farmland was used to check out the divided result.The results showed that the basic farmland factor can be divided into two classes and the encroached region can be divided into four grades.The easy-encroached land of different years was stable at the same level,and basic farmland occupied varies according to the grades.This research provides some valuable insights for protecting basic farmland.

Abstract:Based on the meteorological data of Hubei Province dated from 1961 to 2008, the changing trends of primary heat resource elements of rice including the beginning and terminal day of ambit temperature, the interval days and accumulated temperatures between them, the daily temperature discrepancy during rice growing seasons were analyzed in detail and its impacts on rice production were also discussed. The results showed that the beginning day with daily temperature above 8℃,10℃ and 12℃ stably was advanced and the terminal day of 20℃ and 22℃ was delayed. The interval between them was extended and the accumulated temperatures were increased. The interval days and accumulated temperatures from the beginning day of 10℃ to the terminal day of 20℃ and 22℃ were extended and increased in most areas of Hubei Province. It indicated that the suitable stage of rice planting was extended. The daily temperature discrepancy during rice growing season and rice grain-filling stages was decreased, and the decreasing trend of eastern Hubei Province was more remarkable than that of western Hubei Province. Based on the analysis above, measures can be taken to improve unit rice yield by changing early maturing rice with medium or late maturing rice having long growth period and high yield. The area of double-crop rice cultivation can be extended to northern Hubei Province with abundant water and good irrigation, which will effectively increase the total rice production of Hubei Province.

Abstract:Primers were designed according to glutathione S-transferase-rho gene from EST database of grass carp.The PCR products were sequenced for detecting polymorphisms in cultured population and two SNPs sites were found:C+129T in exon 1 and C+192T in exon 2.Both SNPs are synonymous mutation.The polymorphisms were genotyped by PCR-RFLP methods.The percentage of CC,TC and TT genotype in C+129T site was 0.69%,18.75% and 80.56%,respectively.The percentage of CC,TC,and TT genotype in C+192T site was 25.4%,48.6% and 26.1%,respectively.A general linear model was used to analyze relationship between GSTR gene polymorphisms and 6 major growth traits.The results indicated that the average value of growth character TT genotype was higher than that of TC genotype in C+129T site,although no significant difference was observed.The average body weight of TT genotype was higher than that of TC and CC genotype in C+192T site (P<0.05).Six diplotypes were constructed based on two SNPs in the experiment population and the average body weight of diplotype D1 was higher than that of D3 diplotypes and D6 diplotypes（P＜0.05).GSTR gene could be recommended as a candidate gene for characterizing grass carp growth traits which could be useful for molecular breeding.

Abstract:Aeromonas is one of the major pathogens which frequently cause the bacterial diseases in freshwater fish terrapin and crustaceans. Aeromonas hydrophila caused economic losses widely. In order to detect Aeromonas spp. and Aeromonas hydrophila,two pairs of specific primers were designed according to the glycerophospholipid-cholesterol acyltransferase (GCAT) gene of Aeromonas spp. and the 16S rRNA gene of Aeromonas hydrophila. So a duplex PCR assay was developed to amplify the 16S rRNA gene and the GCAT gene. The reaction conditions of the duplex PCR were optimized and PCR products were sequenced. Meanwhile,the specificity of duplex PCR was studied. Five Aeromonas hydrophila strains,seven Aeromonas strains and the other five strains of non Aeromonas were tested by duplex PCR. The results showed that the GCAT gene and the 16S rRNA gene could be amplified from Aeromonas hydrophila strains,but only the GCAT gene from the other Aeromonas species and neither from the other five strains. It could be concluded that the duplex PCR was specific and could be used in quick diagnose Aeromonas hydrophila and Aeromonas spp.

Abstract:Rhus chinensis Mill (RCM)， Rheum palmatum Linn (RPL)， Rhizoma polygoni Cuspidati (RPC)，and Punica granatum Linn (PGL)，were first ground into 5-10 μm superfine powder with ultra-pulverization.Then they were mixed one by one based on chessboard experimental method to get four kinds of compounds of Chinese herbs.To study inhibitation activity of the compounds，the minimum inhibitory concentration （MIC） and the minimum bactericidal concentration （MBC） of single drug and the triple compounds against nine strains of pathogenic bacteria from eels were measured with agar dilution method.The results showed that four kinds of Chinese herbs had certain antibacterial effects on nine bacteria.RCM had the best antibacterial effect，followed by PGL and RPL，and RPC had little antibacterial effect.Four triple combinations had synergism antibacterial effects on pathogenic bacteria，and the value of FIC was from 0.38 to 0.76.Antibacterial synergistic effect of prescription No.1 was the best，whose value of FIC is less than or equal to 0.5 against nine strains of pathogenic bacteria.The inhibitory concentration of each triple compounds reduced at least 79%，and the cost calculated in aquaculture production was lower than single Chinese herbs at least 45%.

Abstract:The biodiversity of zooplankton and analysis of water in 34 locations of the two river systems in Haihe River basin were investigated in 2009.Results obtained in this study showed that:in the Yongding River system and Luanhe River system,the protozoa were the most abundant zooplankton,accounting for 36.84% and 45.07%,respectively; the second richest category was rotifer,accounting for 31.58% and 39.44%,respectively; the third richest ones were cladocera,accounting for 21.05% and 8.45%,respectively,and the least were copepods,accounting for 10.53% and 7.04%,respectively.Analysis results of Shannon-Weaver diverstiy and Simpson index demonstrated that:the middle and lower reaches of the Yongding River and the Yongding New River were moderately polluted while Sanggan River,Yang River and upstream of Yongding River were heavily polluted; downstream of Luanhe River and the Qinglong River were moderately polluted while the upper and middle reaches of the Luanhe River and the Wulie River were heavily polluted.The quantity and biomass of the four categories of zooplankton in 34 locations of the two river systems were analyzed by hierarchical cluster analysis using SAS software.The results revealed that:in Yongding River system,sample locations of the middle and lower reaches of Yongding River and the Yongding New River were moderately polluted while sample locations of the Sanggan River,Yang River and upstream of Yongding River were heavily plluted; in Luanhe River system,sample locations of the downstream of Luanhe River and the Qinglong River were moderately polluted while sample locations of the upper and middle reaches of the Luanhe River and the Wulie River were heavily polluted.The principal component analysis with SPSS was used to analyze the quantity and biomass of four categories of zooplankton of 34 locations in the two river systems.Two main factors were used for producing interactive graph.The results obtained from the principal component analysis were coincident with those of the hierarchical cluster analysis.

Abstract:Based on different planting conditions and sowing time in the same place and according to different requirements of rape seeding density,the mechanism for variable sowing on 2BFQ-6 rape precision combined direct planter has been designed, depending on the theory that adjusting the engaging relations of gears can change the input shaft’s speed of the metering device. Its structure and working principle were studied in this paper, and it has the regulatory function with four planting densities. At the same time some main structural and performance parameters were analyzed and presented. Modal analysis showed that the rigidity of the output gear shaft is sufficient. The bench tests approved that the error ratio between the sowing amount of the metering device when the mechanism for variable sowing is not installed and the one when it is installed is less than 7% within the same time and under the same condition. It can satisfy the requirements of rape’s variable sowing. 

Abstract:In the present study,cytoplasmic inheritance of Citrus somatic hybrids and cybrids from 38 intergeneric and interspecific fusion combinations was analyzed with restriction fragment length polymorphisms (RFLPs),cleaved amplified polymorphic sequence (CAPS) and chloroplast simple sequence repeat (cpSSR) markers.The experimental procedures were modified and optimized.A novel marker,cpSSR,was developed in Citrus and other tropical fruit crops.Meanwhile,genetic variations of organelle from 23 Citrus calli were analyzed.The main results were as follows：1.A simple and efficient method for genomic DNA extraction from woody fruit crops containing high level of polysaccharides was developed.This method involves a modified CTAB or SDS procedure employing a purification step to remove polysaccharides by using water saturated ether at the presence of 1.25-1.30 mol/L NaCl.The quality of DNA samples extracted with this method was suitable for PCR and RFLPs analysis and for long-term storage.In addition,this procedure was successfully applied in DNA isolation from the freezed or withered or senile leaves of Citrus and more than 20 kinds of tropical and subtropical fruit crops.2.Five universal pairs of chloroplast DNA (cpDNA) primer and 3 universal pairs of mitochondrial DNA (mtDNA) primers amplified monomorphic fragments among 4 intergeneric hybrids and 3 interspefic fusion combinations.After digested by restriction endonuclease,polymorphic mitochondrial CAPS markers were displayed in the 4 intergeneric combinations,while polymorphic chloroplast CAPS markers were found in 3 intergeneric and 2 interspecific fusion combinations.The results showed that the specific bands of mitochondria among all the hybrids were identical with embryogenic suspension parents,while the chloroplast were identical either with the mesophyll parents or with the embryogenic suspension parents.3.Detailed RFLPs analysis of mitochondrial genome among the hybrids of 38 fusion combinations were performed through digesting the genomic DNA with 5 restriction endonucleases and then hybridizing with 12 mitochondrial probes,results revealed that the mitochondrial genome of Citrus somatic hybrids and cybrids was biasly derived from the embryogenic suspension parent with commonly occurred rearrangements.The characterization of mitochondrial genome recombination was influenced by nuclear background of the hybrids,and mtDNA composition usually existed in allotetraploid somatic hybrids was more complicated than that in diploid cybrids.The common mtDNA fragments in the fusion parents could be usually detected in its offsprings.Mitochondrial inheritability was correlated with the genotype of the fusion parents.The ancient ancestor had stronger mitochondrial transferability than its offspring had.When both fusion parents are evolutionarily later than C.reticulata,there had the possibilities of C.reticulata like mitochondrial RFLP banding pattern displayed in their hybrids.When modern genotype acts as embryogenic suspension parent and ancient genotype as mesophyll parent,the mitochondria in the fusion hybrids may derived biasly from the mesophyll parent.4.Leaves from the somatic hybrid of ‘Valencia’ orange and ‘Meiwa’ kumquat were harvested monthly for a whole year and analyzed with RFLPs marker combined with the field observation.Results revealed that the loss of the mtDNA fragments was correlated with the plant died back,suggesting that the genetic instability of mitochondrial genome is one possible reasons for the growth abnormality of the somatic hybrids.5.Combined the data of CAPS with RFLPs in chloroplast genome analysis,14 out of the 38 fusion combinations showed some degree of polymorphisms,chloroplasts were randomly segregated among these combinations.To further study the origin of chloroplast genome among other combinations,new markers are required to be developed.6.To characterize the organelle at the expression level,RT-PCR and Northern hybridization were used in mitochondria.Results showed that expression of mitochondrial genes in somatic hybrids was higher than that in their corresponding fusion parents,while no clear difference existed among cybrids and their parents.7.Thirty-three pairs of cpSSR primers designed according to the chloroplast genomes of pine (Pinus thunbergii),rice (Oryza sativa),tobacco (Nicotiana tabacum) and Arabidopsis thaliana were amplified with Citrus genomic DNA.After strictly screened,fourteen specific primers were obtained and named as SPCC1 to SPCC14,and subsequent studies were performed as follows:1)Thirty-four representative accessions of Citrus,Fortunella,Poncirus and some of their hybrids were selected and amplified with 14 SPCC primers.Loci among the 34 genotypes ranged from 1 to 8 with an average of 3.54,and the mean PIC value was 0.356.Results of neighbor-joining cluster based upon the cpSSR data showed that all the samples were divided into 7 clusters,namely citron (C.medica),lime (C.aurantifolia),pummelo (C.grandiss),Yichang papeda:(C.ichangensis),kumquat (Fortunella spp.),trifoliate orange (Poncirus spp) and mandarin (C.reticulata) clusters.2)Seventeen fragments of SPCC1 and 21 of SPCC11 were cloned,sequenced and aligned withsequences of the original primer motif from tobacco and Arabidopsis ,the outcomes revealed that all the cloned sequences were from the chloroplast genomes and the polymorphism was caused by mononucleotide repeat of poly (A) or poly (T).3)According to the cpSSR classification results,suitable SPCC primers were selected to analyze the chloroplast genomes among the hybrids of the 38 fusion combinations,random segregation of chloroplast in Citrus fusion hybrids was further proved,and 1∶1 segregation ratio were revealed in the fusion combinations of Kinnow tangerine+Jincheng orange and Bonanza navel orange+Goutou sour orange,respectively.4)Sixty-four DNA samples belonging to 31 kinds of tropical and subtropical fruit crops were amplifiedwith the synthesized cpSSR primers,and a set of wide adaptable universal primers were screened.For instance,SPCC1 was rich in polymorphism among intergenera or interspecies,such as persimmon (Diospyros kaki ) and dateplum persimmon (Diospyros lotus),banana (Musa spp.),guava (Psidium spp.),papaya (Carica spp.) and grape (Vitis spp.).8.Twenty-three genotypes of calli adequately representing the Citrus species and related genera were selected,and corresponding leaves were used as the control to analyze the stability of mitochondrial and chloroplast (plastome) genome.RFLPs,histological section,cpSSR and mtDNA transferring from the cytoplasm to the nuclei were analyzed.The results were as follows:1)Low frequency of mtDNA mutation existed in Citrus calli,while the mtDNA contents increased in individual mitochondria,and no such phenomena were observed in chloroplast genome.2)Analyzed with cpSSR,novel bands were detected among most of the calli.Both Southern hybridization and sequencing proved that the bands were amplified uniquely,suggesting that short sequences insertion occurred frequently in Citrus calli plastid genomes.3)Dot blotting analyses were performed by using nuclear DNA hybridized with mitochondrial probes.Results showed that mitochondrial DNA transfering from cytoplasm into nuclei occurred during long-term subculture.

Abstract:In light of the critical need to increase genetic diversity in the gene pool,cotton improvement programs are increasingly turning to the application of molecular approaches to breeding and germplasm utilizations.The abundant species of wild cotton (Gossypium spp.) are an important renewable resources and have been the valuable genetic germplasms for cotton genentic improvement,and which has been significant in the reality and theory,potential in the application.It is very difficult to widecross between cultivars and wild species for the distant relationship,or no fertility of F1 hybrids,or very low fertility.Application of biotechnology is an effective way for developing new germplasm in cotton,and we aim to develop new sources of cotton germplasm via somatic cell culture,protoplast culture and protoplast fusion.Our studies involved somatic embryogenesis and plant regeneration in wild cotton species,protoplast culture in Gossypium hirsutum L.and wild species,protoplast fusion between cultivars and wild species.The main results of this research were as follows:1.Calli were induced from 9 wild cotton species.Among them,the normally regenerated plants were obtained from G.davidsonii,G.klotzschianum, G.raimondii and G.stocksii via somatic embryogenesis,regenerated plants with abnormal morphology from G.aridum.Only non-embryogenic calli were obtained from G.anomalum,G.africanum,G.thurberi and G.bickii.We studied the methods and factors for embryogenic callus induction and conservation,improving somatic embryos maturation and germination,plant regeneration in detailed,and then a new and elementary protocol has been developed for somatic cell culture,mainly somatic embryogenesis and plant regeneration in wild cotton species.The combination of 2,4-D/KT was very useful for callus induction in all tested wild species.Different combinations of PGR,sugar sources,suspension culture and environmental stress etc improved the formation of embryogenic callus,the maturation and germination of somatic embryos,and plant regeneration to some degree.Embryogenic calli of wild species subcultured and conserved on MSB semi-solid medium supplementing with IBA 0.984 μmol/L,KT 0.232 mol/L for 4 years still have the capability of differentiation and provide a mass of materials.It is the first report of regeneration of plants via somatic embryogenesis in many wild cotton species.2.Protoplasts were isolated from different explants of 2 species (Coker 201 and YZ1) in Gossypium hirsutum L.(embryogenic cell suspension culture,embryogenic callus,immature somatic embryos,hypocotyls,young roots and leaves).Plants regenerated from cultured protoplasts of 6 explants in Coker 201,but the plating frequencies of protoplasts from different explants varied significantly.The plating frequency of suspension culture-protoplast,embryogenic callus-and somatic embryo-protoplast,hypocotyl-young root-and leaf-protoplast was 10%,6%,less than 2%.The plating frequencies of plants regenerated form protoplast cultures isolated from embryogenic suspension cultures,somatic embryos and embryogenic callus in YZ1 were lower (1%-2%) than that of plants regenerated from same explants in Coker 201.Plants regenerated from protoplasts isolated from somatic embryos and embryogenic suspension cultures in wild cotton G.klotzschianum with the plating frequencies ranging 6% to 8%.RAPD analysis demonstrated that the regenerated plants were genetically homogeneous.This study emphasized on enzyme combinations for protoplast isolation,the influences of culture density and PGR combinations etc for protoplasts sustained division,callus formation,and then a practical protocol for protoplast culture in cotton is established.3.In this research,symmetric fusion including 8 combinations mediated by electricity was carried out.Plants regenerated from Coker 201+G.klotzschianum，Coker 201+G.davidsonii，Coker 201+G.bickii,Coker 201+G.stockii,which were morphologically intermediated fusion parents,apt to wild cotton parent.Cytological examinations and flow cytometric analysis showed that all of the tested plants were hexaploids with chromosomes (2n=2x=78),or aneuploids nearing 78 chromosomes,the sum of that of parents,and were somatic hybrids.Analysis of RAPD and other molecular markers demonstrated that the majority of regenerated plants had its parents’ specific bands.Somatic hybrid plants of Coker 201+G.klotzschianum，Coker 201+G.davidsonii flowered and set bolls in green house,but difficultly flowered outside in the field.Somatic hybrid plants of Coker 201+G.bickii,Coker 201+G.stockii flowered and set bolls at fall in the field and at winter in the green house.The fertility of four somatic hybrids was comparatively high,perhaps related with photoperiod response.Somatic hybrid callus was obtained from G.arboreum + G.stockii,but this callus differentiated very difficultly.It is the first report on production of somatic hybrid plants between cultivars and wild species via protoplast fusion in cotton.4.In this research,we studied various factors in efforts to improve somatic embryogenesis and plant regeneration in wild cottons through 3 aspects of somatic cell culture,protoplast culture in wild cotton species and regeneration of somatic hybrids between wild speciesand cultivars,in order to improve the efficiency of regeneration in wild cotton species and broarden the range of wild species in which plant regenerated via somatic embryogenesis.