In order to study the salt-tolerant mechanism of the Salicornia europaea gene Cu/Zn-SOD, conserved sequences of Cu/Zn-SOD from the known plants were designed for degenerate primers in this research. The coding region of Cu/Zn-SOD was amplified by RACE technology. Amino acid sequences and multiple sequences alignment were analyzed by biological software. The gene carried by prokaryotic expression vector pet-duet1 was transferred to Escherichia coli BL21 and expressed after IPTG induction. The status of recombinants cultivated in LB medium with different NaCl concentrations were observed. The salt-tolerant function of Cu/Zn-SOD was assessed by OD600 value. As a result, the Salicornia europaea gene Cu/Zn-SOD was amplified by degenerate primers and RACE technology. The full-length of Cu/Zn-SOD cDNA(GenBank number: JQ074238.2) was 660 bp, containing a 459 bp open reading frame (ORF). The ORF encoded a 152-amino acid polypeptide with a predicted molecular mass of 15.1 kD. The predicted amino acid sequences showed 96% similarity with the corresponding Suaeda salsa gene sequences and 88% similarity with the Capsicum chinense gene sequences. Biological software analysis implied that the Cu/Zn-SOD protein existed in cytoplasm. We constructed a prokaryotic expression vector pETCu/Zn-SOD, contrasted by control pETDuet-1, and transformed them into Escherichia coli BL21. Protein was expressed by IPTG induction. The target protein was successfully expressed, as the molecular mass of it accorded with theoretical value by SDS-PAGE electrophoresis. Salt-tolerance analysis showed that recombinant bacteria BL21 (pET-Cu/Zn-SOD) grew significantly better in the high salinity mediums than that of the control bacteria BL21 (pETDuet-1). It indicates that the Salicornia europaea gene Cu/Zn-SOD may play an important role in the salt stress.

To investigate the serotype distribution, antimicrobial susceptibility and resistant mechanisms of Salmonella isolated from chickens(Gallus domesticus), 110 Salmonella from Beijing and Shanxi were studied. The results of serotyping indicated that S.enteritidis(76.58%) was the predominant serotypes. The susceptibility of 110 Salmonella isolates to 13 kinds antimicrobials was determined by microbroth dilution method. The highest resistant rate was observed by polymyxin E(41.82%), followed by sulfisoxazole(31.82%), ampicillin(29.09%) and doxycycline(20.91%). Resistant rates to other antimicrobials were under 20%. Resistant mechnisms of isolates were detected, and the results showed that integronⅠmainly harbored the resistance genes to aminoglycosides, sulfonamides, lincosamides, which might play a critical role in spresding drug resistance. This study provides basic data for prevalence investigation of Salmonella from chickens and the rational use of antimicrobials for Salmonella infection control in veterinary practice.

Very-long-chain fatty acids(VLCFAs) play an important role in anther development. VLCFAs is catalyzed by the fatty acyl-CoA elongase, a membrane-bound enzymatic complex containing trans-2,3-enoyl-CoA reductase(ECR). To further study the mechanism of male sterility induced by gametocide SQ-1 in wheat(Triticum aestivum L.), primers were designed based on silicon cloning sequence of ECR gene sequences from Brachypodium distachyon, and the open reading frame of the cDNA were obtained, here as designated TaECR that had been registered under GenBank(No. KC222053).Sequence analysis showed the open reading frame of this gene putatively encoding 310 amino acids, and had classical domains of ECR. Analysis showed that TaECR was ubiquitously expressed in anther, glume, leaf and root in wheat, but it was less expressed in root. Compared with fertile lines, the expression levels of TaECR in the physiological male sterile line were much lower than those in fertile line in trinucleate stage, but it showed no significant difference in unicleate and binucleate stage, which very corresponded to trends of wax loads in the development of the anther. These results suggest that the pathway of very long chain fatty synthetic metabolism regulated by TaECR gene may participate in abortion process of sterile line induced by SQ-1.

The aim of this study is to construct a DNA fingerprinting database of 21 annual ryegrass (Lolium multiflorum Lam.) varieties using SSR makers, which is necessary to annual ryegrass varieties identification and germplasm management. The 21 annual ryegrass varieties or strains in the experiment, including the most domestic varieties, introduced varieties and six strains which were cultivated by our research group, were all tetraploid. Since annual ryegrass belongs to the typical cross-pollinated species, the sampling strategies were closely related to the authenticity and efficiency of molecular genetic diversity analysis and the DNA fingerprinting database. On the basis of others' and our previous research experience, we took the bulked DNA samples of 20 individual plants for each group in the experiment. Twelve evenly distributed SSR primer pairs with high polymorphisms and good repeatability were successfully screened out from 100 candidates to construct the fingerprinting database. Among the 21 varieties, 12 primer pairs produced 108 polymorphic genotypes, the ratio of polymorphism was as high as 94.15% and 9 genotypes were detected by each SSR primer pair on an average with the range from 5 to 22; the polymorphic information content (PIC) values ranged from 0.744 to 0.934 with the mean of 0.843. The twelve SSR primer pairs with high polymorphism, good repeatability and specification could be used as the core primer to construct the DNA fingerprinting of more annual ryegrass varieties. Different SSR primer pair could distinguish the different number of annual ryegrass species with the range from 5 to 21. The primer 15-08C amplified 22 polymorphic bands and it could distinguish all the 21 varieties at once, while the primer 07-07G amplified 6 polymorphic bands distinguishing only 5 varieties. The limited combination of different primer pairs could greatly improve the primer pairs' identification ability. Nine varieties had specific genotypes by 7 primer pairs, which couid be identified at the soonest by the feature primer. The genetic similarity coefficient of 21 accessions varied from 0.4956 to 0.8571. UPGMA cluster analysis of genetic similarity showed that all the materials were clustered into three groups at the genetic similarity of 0.69. For distinguishing germplasms more simply, through selection of the primer pairs according to its PIC step by step , we found that the primer 15-08C could distinguish all the germplasms in the experiment separately. At last, we constructed the standard mode image of DNA fingerprinting of 21 annual ryegrass varieties by the primer 15-08C. Each germplasms has its own fingerprinting(bandtype), which provids a basis of the cultivars assessment, germplasms protection and cross breeding of annual ryegrass varieties.

Lolium multiflorum is a crosspollinated plant, there must have the mixtures in hybridizations. The discrimination of true and false hybrids is necessary. The study aimed to select excellent hybrid combinations and progenies, and provided the essential data for the subsequent cross breeding. Parental cultivars we choose were the international good varieties and the leading varieties in southwestern Sichuan. Five parental cultivars of Lolium multiflorum and five cross combinations were used to identify their true hybrids by SSR molecular markers. The SPSS computer software was used to analyze the data to differentiate the differences between parents and progenies on morphological characteristics. The results were as followed:(1) 20 primer pairs produced 426 polymorphic bands with average percentage of polymorphism of 82.41%, each pair of primers amplified 25.9 bands on average, and 21.3 bands had polymorphism on average. Samples had relatively high molecular genetic polymorphisms so it was feasible to identify the parents and hybrids.(2) The hybrid progenies could be divided into two types, including the progenies with paternal characteristic bands and without, the progenies with paternal characteristic were considered as the true, 108 true F1 hybrid individuals were identified as the true hybrids using SSR molecular markers from the 150 hybrid individuals through several pairs of primer. There were 24 true hybrids both in combinations from ChangjiangNo.2×GanxuanNo.1 and GanxuanNo.1×ChangjiangNo.2 which produced the most amount of true hybrids and 19 true hybrids in combinations of Barspectra×GanxuanNo.1 which produced the least amount of hybrids. (3) Significant heterobeltiosis were found in the F1 progenies from the cross combination of ChangjiangNo.2×GanxuanNo.1. The differences in vegetative organ and reproductive organs among the five cross combinations were significant. The coefficient of variance in cross combination of Major×GanxuanNo.1 was the highest while cross combination of Barspectra×GanxuanNo.1 had the lowest coefficient of variance. The phenotypic traits performances of F1 hybrids from ChangjiangNo.2×GanxuanNo.1 was significantly better than the rest of the F1 hybrids produced from other combinations. We finally got the optimal hybrid combinations ChangjiangNo.2×GanxuanNo.1 and its good hybrids in morphological character through SSR molecular markers and analyzing the data to differentiate the differences between parents and progenies on morphological characteristics. This study showed that identifying the true hybrids of five different hybrid combinations by SSR molecular markers indicated the feasibility for Lolium Multiflorum in the assistant identification of SSR molecular. It plays a significant meaningful role in accelerating breeding progress, constructing genetic linkage maps and QTL analysis. The results can provide theoretical basis for cross breeding in Lolium multiflorum and the hybrid progenies can offer potential breeding materials in future.

Caffeic acid O-methyltransferase (COMT) catalyzes the preferential formation of syringyl (S) monolignol subunits, and acts as a key enzyme in lignin synthesis. COMT is implicated in multiple physiological processes in plants, e.g. the functioning of plant vasculature, and defense responses to biotic and abiotic stresses. Up to now, no literature has been available in the cloning and characterization of COMT genes in Hevea brasiliensis (para rubber tree). Previously, we showed that the levels of a COMT protein increased markedly with tapping in the latex of reopened rubber trees. The expressions of this COMT protein correlated well with the patterns of tapping-enhanced latex yields. Here, by searching the assembled latex EST library (20126 high-quality Sanger ESTs, with average length of 575 bp), a contig annotated as COMT was obtained. Based on the sequence of this contig, the full-length cDNA (1 312 bp) that corresponded to a Hevea COMT gene was then cloned by PCR, and named as HbCOMT1(GenBank accession No. GI: 443908530). The genomic clone of HbCOMT1 (1 926 bp) that comprised the complete protein-coding region was also cloned, consisting of four exons and three introns. HbCOMT1 predicted a protein of 368 amino acids with a molecular weight of 40.58 kD and an isoelectric point of 5.46. HbCOMT1 shared the hallmarks of typical plant O-methyltransferases. Phylogenetic analysis put HbCOMT1 and the COMTs of Ricinus communis and Vitis vinifera into one cluster, and the COMTs of the other 11 plant species into the other cluster. Using Real-time quantitative PCR, the expression patterns of HbCOMT1 were examined in different Hevea tissues and in response to a series of treatments. HbCOMT1 mRNA accumulated most abundantly in latex, then in leaf and bark, low in flower and bud, and undetectably in seed. The expression of HbCOMT1 in latex was up-regulated by tapping in re-opened rubber trees, having about 3-fold increase at the fifth tapping. The transcripts of HbCOMT1 were stimulated markedly by wounding, showing an increase of more than 15-fold after 24 h of wounding treatments. Compared with the expression levels in the latex of healthy rubber trees, HbCOMT1 expressions were up-regulated in the trees affected with moderate tapping panel dryness (TPD), but down-regulated in the trees with severe TPD. However, HbCOMT1 expressions were affected little by Ethrel (2-chloroethylphosponic acid, an ethylene releaser) treatment. In brief, the paper reported the cloning and molecular characterization of a COMT gene from H. brasiliensis, which predicted a role in stress response and the regulation of latex flow in the laticifers. The results will be beneficial to further functional characterization of this COMT gene.

Acetochlor is a broad-spectrum and high-efficive chloroacetanilide herbicide. Because of its long degradation period, with the characteristics of difficult for volatilization, photolysis, and easy for residue, the overuse of acetochlor is toxic to human, plants and animals. In order to investigate the microbial degradation mechanisms of acetochlor, an acetochlor-degrading bacterium, named strain M-3 was isolated from acetochlor-contaminated samples using selective culture medium with acetochlor as the sole carbon source. The strain was identified based on its morphological, physiological and biochemical tests, with reference to Bergey’s Manual of Determinative Bacteriology combined with 16S rRNA gene sequence analysis. Strain M-3 was finally identified as Stenotrophomonas sp. . With HPLC and HPLC-MS method, degrading characteristics of strain M-3 were studied under laboratory conditions and the degrading pathway of acetochlor was researched preliminarily. The results showed that strain M-3 could degrade more than 76.6% of 50 mg/L acetochlor within 5 d. The best temperature and pH for the acetochlor degradation by strain M-3 were 30℃ and 7.0, respectively. Based on the identified products, the degrading pathway of acetochlor was speculated. The product was identified as 2-ethyl-6-methyl-2-chloroacetanilide. Our research provides theoretical basis for bioremediation technology of acetochlor pollution.

Haemophilus parasuis is a kind of symbiotic bacteria in upper respiratory tract of pigs, but it can invade the body and cause serious systemic disease under certain conditions. Distinction between virulent strains and avirulent strains is the key point for diagnose of the disease. In order to search for differentially expressed proteins between virulent and attenuated strains of H. parasuis, the proteomics of the cytosolic proteins were compared between H. parasuis virulent 14A7-2 strain and avirulent 20A3-2 strain by two-dimensional gel electrophoresis. The cytosolic proteins of two strains were extracted at late logarithmic phase for 2-DE. After electrophoresis, the gels were analyzed with Imagemaster 2D Platinum 7.0 software, and differentially expressed proteins were identified by MALDI-TOF/MS. By 2-DE and analysis, the results showed that there were 586±10 protein spots in virulent strain and 568±10 in avirulent strain. A total of 80 protein spots were differentially expressed in the two strains. 44 protein spots unique for 14A7-2 or 20A3-2 strain were selected to be identified by mass spectrometry, and 42 protein spots were successfully identified corresponding to 37 proteins. Five proteins were unique for non-virulent strain, while 27 proteins were unique for virulence strains which included ferric uptake regulation protein, trigger factor and 3-dehydroquinate dehydratase associated with virulence. These results provide new information for further studies of virulent factors, pathogenic mechanisms and diagnostic techniques to distinguish different virulence strains of H. parasuis.

In order to further clarify the genetic stability and the target traits of the foreign gene in the progeny of transgenic citrus, the asexual reproduction plants of transgenic Newhall navel orange(Citrus sinensis Osbeck)containing bivalent antibacterial peptide gene(Shiva A-cecropin B) were studied. In this study, the genetic stability of Shiva A gene in T0, T1, T2 and T3 progenies of transgenic citrus varieties were analyzed by PCR, Southern hybridization, Real-time quantitative PCR and greenhouse disease index statistic to Xathomonas axonopodis pv. Citri(Hasse) Dye. The results showed that the antimicrobial peptides Shiva A gene was existed and expressed in T0, T1, T2 and T3 plants. This meant target gene could be stably inherited from one generation to another through asexual propagating. There was difference of Shiva A gene copy number between transgenic T0 and its asexual propagation. Southern bloting analysis showed that T0 generation had two hybridizations, but its progenies had only one hybridization consistent with T0 generation. It could be speculated that the T0 generation plant was a transgenic multicellular mixed cytochimera. In addition, Real-time quantitative PCR results showed that, even if Shiva A gene in the T0 generation genome was double copy numbers, but its expression level was lower than a single copy of the T1, T2 and T3 generation. Therefore, in this study, exogenous gene expression and transgene copy number was a negative correlation. The results of this study provide some basic date and the material for extending the stable phenotype of transgenic citrus strains in carrying out the safety evaluation of transgenic plants.

Leaf is photosynthetic organ of plants which plays an important role in energy fixation and utilization. The study of acting elements and their functions of light-inducible, and stem and leaf-specific expression promoter have important theoretical significance and application value on research for regulation of gene expression. The 1 556 bp sequence of light-inducible, stem and leaf-specific expression promoter ST-LS1 was isolated from potato (Solanum tuberosum L.) genome by PCR assay. The result from sequence analysis showed that the fragment shared 99.68% identity with the reported ST-LS1 promoter (GenBank accession No. X04753.1), and which contained shoot-specific expression and light responsive cis-acting element as-2-box, and cis-acting element G-box with light effect. The plant expression vector pBⅠ121-ST-LS1 was constructed by fusing the fragment with GUS gene, which was transferred to tobacco(Nicotiana tabacum) by Agrobacterium tumefaciens system and obtained the transgenic tobacco plants. Both qRT-PCR and histochemical assay of GUS activity revealed that the GUS gene expression could be detected in the leaf and stem of the transgenic tobacco plants transformed with GUS gene driven by ST-LS1 promoter, while could not be detected in root of the transgenic plant. The result from the transgenic plants under treatment for 20 d with darkness, constant temperature light, natural light culture demonstrated that there was no GUS gene expression in the transgenic plants under darkness treatment, while GUS gene expression was higher in the leaf and stem of the transgenic plants under natural light culture compared with constant temperature light condition. The results could provide theoretical and applied basis for crop improvement using genetic engineering.

Pronucleus microinjection is a commonly used method for the preparation of transgenic mice. The influence of its injection process may be one of the main reasons for low developmental capacity of the pronucleus microinjected embryos. And that the cytoskeleton system is closely related with the pronucleus migration and early cleavage, etc. To observe the dynamic changes of α-tubulin and F-actin, during the embryonic development in KM mouse(Mus musculus) embryos after pronucleus microinjection, and lay the foundation for improving the developmental capacity of the pronucleus microinjected embryos, immunofluorescence and laser confocal microscopy were used to detect the location of α-tubulin and F-actin in the first meiotic division of the microinjected zygotes. The results indicated that: (1)The survival rate was (75.11±2.10)%, the cleavage rate was (79.70±1.75)%, the blastocyst rate was (44.85±3.21)%, and those were highly significant lower (P<0.01) than that of the control group(the cleavage rate: (79.70±1.75)%, the blastocyst rate: (44.85±3.21)% of the microinjected zygotes. (2)At the pronucleus stage of the microinjected zygotes, α-tubulin got a diffuse distribution. Meanwhile, there was no obviously cytaster in cytoplasmic, and fibrous α-tubulin didn't concentrate around the two pronuclei. During the stage of pronucleus migration and fusion, fibrous α-tubulin gradually concentrated around the two pronuclei, and the midbody dissolved gradually. At the beginning of the first mitosis, α-tubulin participated in the formation of spindle. At the 2-cell stage, α-tubulin concentrated in the midbody at the junction of the two blastomeres. At the pronucleus stage of the control mouse zygotes, fibrous α-tubulin composed midbody and cytaster remodeling around male and female pronucleus to pull the pronuclei to get close. With further development, the distribution of α-tubulin had no significant differences from the stage of pronucleus migration and fusion until the 2-cell stage. (3)At the pronucleus stage of the microinjected zygotes, F-actin distributed around the two pronuclei and the cortex regions. Following the pronucleus migration and fusion, F-actin distributed in cytoplasmic gradually decreased, obviously tended to concentrated around the two pronuclei and the cortex regions. At the beginning of the first mitosis, F-actin distributed in cytoplasmic almost disappeared, while the distribution of F-actin was concentrated in the cortex regions. At the 2-cell stage, F-actin mainly distributed in the cleavage furrow. As for the normal mouse zygotes, F-actin concentrated in the cortex regions before the first mitosis. Following the development, the distribution of F-actin had no significant differences from the first mitosis until the 2-cell stage. Our study indicated that the effects of pronucleus microinjection on the microtubule network mainly concentrated at the pronucleus stage. However, it could recover before the stage of pronucleus fusion, while the effects of pronucleus microinjection on the microfilament network mainly concentrate at the beginning of the first mitosis, moreover, it has no significant effect on the distribution of the cortex regions.

Most of the well-characterized promoters that are currently available confer constitutive expression in genetic engineering, however, this expression model may cause plant growth retardation and other problems, so cloning novel tissue-speci?c promoter is needed. Based on microarray data, we identified a rice green-tissue specific expression gene (TIGR Locus: LOC_Os06g21110), a promoter named green-tissue specific promoter(GSP) with 1 951 bp in length of the gene was cloned by PCR from the genomic DNA of rice Minghui 63(O. sativa L ssp. indica). Promoter GSP was further fused with β-glucuronidas (gus) reporter gene and transformed into rice Zhonghua 11(Oryza sativa L ssp. japonica) callus of mature seed through Agrobacterium-mediated transformation. The expression pattern of GSP was identified in transgenic rice plants by histochemical staining. The result verified that GSP was a sheath and leaf specific expression promoter. Then 5' end different length deletions of GSP were fused with gus gene and were transformed into rice respectively. The result of deletion analyses indicated that 592 bp length promoter fragment was sufficient for maintain the sheath and leaf specific expression model, and also identified core functional areas of GSP preliminarily. GSP is a sheath and leaf specific promoter which has not been reported, utilizing tissue specific promoters like GSP will subject to rice improvement and limit the potential of unintended impacts on plant physiology, so it has a good application value in transgenic rice.

Hyriopsis cumingii is an important pearl-breeding mussel in China, and it grows fast and breeds pearls of good quality. In this study, We cloned a full-length cDNA sequence of myosin essential light chain gene(MELC) which was isolated from the mantle of Hyriopsis cumingii according to homology cloning strategy and SMART RACE-PCR technique. The full length cDNA sequence was 1 119 bp, comprising a complete open reading frame of 468 bp which encoded 155 aa of 17.49 kD and pI at 4.53. The GenBank accession number was JX275828. MELC included two consertive EF-hand Ca2+-binding domains which analysed by PROSITE tool, and which belonged to EF-hand Ca2+-binding protein family. The amino acid sequences of MELC gene possessed 69%, 69% and 68% identity with Pinctada fucata, Crassostrea gigas and Haliotis discus discus, respectively. Quantitative Real-time PCR analyses showed that MELC gene was ubiquitously expressed in all tissues of H. cumingi, including adductor muscle, mantle, gill, axe foot, gonad, heart, liver, gut, especially strongly expressed in tissues related with calcium metabolism, such as mantle, gill and axe foot, which were higher than liver and gonad. The result implied MELC gene might take an important part in the calcium metabolic process of H. cumingii. Moreover, the expression of MELC gene in mantle of H. cumingii went up and came down along with the rising of Ca2+ concentration, then reached a climax at 60 mg/L, which demonstrated that the suitable Ca2+ concentration would accelerated the high expression of MELC gene, otherwise, it might inhibit the expression of MELC gene. All these results indicate MELC gene in H. cumingii may play an important role in Ca2+ uptake, transport, storage, and participate in the formation of pearls.

The sealed culture of embryos is the fundation of space embryonic development study. In this special culture system, appropriate oxygen content in culture medium is essential to the development of sealed culture embryos.The purpose of present study is to research the influence of two reference gas containing different percentage of O2 and two different aeration time on mouse(Mus musculus) 2-cell embryos developmental competence in sealed culture system. We firstly aerated culture medium with high purity reference gas(5% O2, 5% CO2, 90% N2 or 7.5 % O2, 5 % CO2, 87.5 % N2) for 120 or 150 min, respectively. Control groups were the embryos cultured in medium of no gas aeration and micro-drop under conventional open condition. Peroxide accumulation in cultured embryos was detected after culture for 24 and 48 h; The expression of hypoxia-inducible factor-1α (HIF-1α) in embryos was detected after culture for 96 and 72 h. Furthermore, the blastocyst rate and hatching rate were statistical analyzed and the total cell number of blastocyst were counted after culture for 72 and 96 h. As the results showed, the peroxide production could be detected in embryos cultured for 24 h in medium aerated with 7.5% O2 for 120 and 150 min. The expression of HIF-1α was detectable after culture for 96 h in medium aerated with 5% O2 for 120 min, 150 min and 7.5% O2 for 120 min group, while the accumulation of HIF-1α in no gas group was detectable since cultered for 48 h. Mouse 2-cell embryo could grow well with a pretty high blastocyst rate and hatching rate in 5% O2 aeration 120 min, 5% O2 aeration 150 min, 7.5% O2 aeration 120 min and 7.5% O2 aeration 150 min group. The blastocyst rate of mouse 2-cell embryo cultured for 72 h with 5% O2 aeration 120 min(92.63±0.89)% was higher than the other three aeration sealed culture group, but no significant difference showed(P＞0.05). The blastocyst rate of no gas group(57.04±10.04)% was significantly lower than each aeration sealed culture group(P＜0.05). The blastocyst rate of micro-drop culture group(98.67±1.33)% was significantly higher than 7.5% O2 aeration 120 min group((87.15±2.35)%, P＜0.05). After culture for 96 h, the blastocyst and hatching rate of each aeration sealed culture group and micro-drop culture group had no significant difference(P＞0.05), but all significantly higher than no gas group(P＜0.05). The total cell number of blastocyst of each aeration sealed culture group cultured for 72 h had no significant difference(P＞0.05). The total cell number of blastocyst in 5% O2 aeration 120 min group(114.12±3.66) at 96 h was significantly higher than other aeration group and no gas group(P＜0.05), but there was no significant difference between micro-drop culture group(110.56±5.24, P＞0.05). Taken together, we could conclude that aerating embryo culture medium with high purity reference gas containing 5% O2, 5% CO2, 90% N2 for 120 ~150 min can support mouse 2-cell embryos developing to blastocyst and hatching in sealed culture system. This study determines the appropriate proportion of O2 in reference gas and appropriate aeration time for sealed culture of mouse 2-cell embryos, and perfects the sealed culture system, and accumulates data for the establishment of a suitable culture system for space embryonic development study.

The iron has a very important role in the growth and development of the plant, iron deficiency causes chlorosis in apple trees. The main producing areas of apple in China just iron deficiency, therefore, filter out iron-efficient resources from rich apple germplasm resources, and breeding new varieties of apple rootstocks through breeding means is fundamental pathway to solve due to iron deficiency affected yield and quality of apple production. Since 1984, the project team screened iron-efficient genotypes from 40 apple stocks and found Malus xiaojinensis Cheng et Jiang, grow normally and do not exhibit symptoms of chlorosisin in conditions of very low Fe content, was considered to be an excellent germplasm with tolerance to iron deficiency. Subsequently, open pollinated hybrids groups were established on the basis of M. xiaojinensis Cheng et Jiang seedlings. In 1990, excellent grades was obtained through original selection from natural seedlings. Then after multiple selection and comparison test, bred apple clonal rootstocks Chistock #1. Chistock #1 is a tetraploid in chromosome number (2n = 4x = 68), with a capacity of apomixis, and setting rate above 85% after emasculation bagged. Then with excellent grafting compatibility, seedling dry good standing and strong solid ground, semi-dwarf, dwarf extent, effects and yield capacity were similar with simi-dwarfing apple rootstock M7. Sweet fruit flavor, palatability, and excellent quality. Resist apple early defoliation disease and branches ring rot, high resistance to apple Chlorotic leaf spot virus(CLSV), Stem pitting virus(SPV) and the Stem groove virus(SGV) and other latent virus. Chistock #1 can effectively prevent etiolation due to iron deficiency as apple rootstock in the lime parent material soil areas.