Comparative transcriptome study provides insights into acquisition of embryogenic ability in upland cotton during somatic embryogenesis

Background： The conversion from non-embryogenic callus （NEC） to embryogenic callus （EC） is the key bottleneck step in regeneration of upland cotton （Gossypium hirsutum）, and hinders the transgenic breeding of upland cotton. To investigate molecular mechanisms underlying acquisition of embryogenic potential during this process, comparation analysis of transcriptome dynamics between two upland cotton cultivars with different somatic embryogenesis abilities was conducted. Results： Differentially expressed genes involved in the transformation from NEC to EC were detected in the two different cultivars. Principal component analysis based on DEGs showed that the NEC tissues of the two cultivars were highly heterogeneous, whereas the derived EC tissues were similar, which suggested the homogeneousness of EC between different lines. In the highly embryogenic cultivar CCRI 24, more of these genes were down-regulated, whereas, in the recalcitrant cultivar CCRI 12, more were up-regulated. Bioinformatics analysis on these DEGs showed that the vast majority of differentially expressed genes were enriched in metabolism and secondary metabolites biosynthesis pathways. Flavonoid biosynthesis and phenylpropanoid biosynthesis pathways were enriched in both cultivars, and the associated genes were down-regulated more in CCRI 24 than in CCRI 12. We deduced that vigorous secondary metabolism in CCRI 12 may hinder primary metabolism, resulting in tardiness of cell differentiation. Interestingly, genes involved in the plant hormone signal transduction pathway were enriched in the recalcitrant cultivar CCRI 12, but not in CCRI 24, suggesting more radical regulation of hormone signal transduction in the recalcitrant cultivar. Signal transduction rather than biosynthesis of plant hormones is more likely to be the determining factor triggering NEC to EC transition in recalcitrant cotton lines. Transcription factor encoding genes showed differential regulation between two cultivars. Conclusions： Our study provides valuable information about the molecular mechanism of conversion from NEC to EC in cotton and allows for identification of novel genes involved. By comparing transcriptome changes in transformation from NEC to EC between the two cultivars, we identified 46 transcripts that may contribute to initiating embryogenic shift.

Studies on Genetic Transformatiom of Embryogenic Suspension Cultures of Sweetpotato

Genetic transformation of embryogenic suspension cultures of sweetpotato cv. Lizixiang was conducted by using Agrobacterium tumefaciens strain A208SE harboring the binary vectors pROA93 with β-glucronidase(GUS)and neomycin phosphotransferase(NPTⅡ)genes. The results indicated that embryogenic suspension cultures precultured for 1 - 3 d were suitable for the transformation. The optimal cocultivation time was 4 - 5 d. The optimal concentration of kanamycin was 50-75 mg L-1 for suspension culture and 100 mg L-1 for embryogenic callus proliferation and plant regeneration. The optimal concentration of carbencillin was 100 mg L-1. Transgenic plants identified with GUS assays and PCR analyses were obtained.

Production of Embryogenic Callus and Plant Regeneration from Elite Guizhou Waxy Maize Inbred Lines

Immature embryos from three elite Guizhou waxy maize inbred lines （W21019, B7, and QCL5036） were evaluated for their ability of forming callus and regeneration into plants. Immature embryos harvested at different physiological stages were used as explants to initiate callus on N6 basal medium with 0-3.5 mg L-1 of 2,4-dichlorophenoxy acetic acid （2,4-D）. The concentration of 2,4-D, physiological age of immature embryos and genotype had a significant effect （P0.05） on the percentage of embryogenic callus formed. The optimum 2,4-D concentration for the initiation of embryogenic callus was varied among the waxy maize genotypes from 2.0 mg L-1 （B7 and QCL5036） to 3.0 mg L-1 （W21019）. The shoots were generated from embryogenic callus which were transferred into the regeneration medium supplemented with 0-2.5 mg L-1 of 6-benzylaminopurine （6-BA）. 6-BA in the medium significantly promoted the regeneration of embryogenic callus. Embryogenic size was also an important factor that affected regeneration capacity. 0.6-0.7 cm was proved to be the best size for regeneration from embryogenic callus and the mean number of shoots per primary callus in all genotypes achieved the highest number. The ability of the plant regeneration was also affected by genotype. W21019 had the highest number of shoots formed per primary embryogenic callus. With the optimum condition, the highest mean number of shoots per primary callus was up to 12.13, 5.73, and 3.33 in line W21019, B7, and QCL5036, respectively. The successful regeneration of the two inbred lines provides a basis for development of genetic transformation to improve priority traits such as enhanced insects and drought tolerance.

Induction and Genetic Identification of Embryogenic Calli from Hybrids of Shatian Pummelo

Shatian pummelo （Citrus grandis L. Osbeck cv. Shatian） is an elite variety in China, and the regeneration of the embryogenic callus is difficult. Diploid Shatian pummelo was used as the female and crossed with the allotetraploid somatic hybrid NS （Nova Tangelo ＋ Succari Sweet orange）, [ （ C reticulata Blanco x C. paradisi Macf.） cv. Nova ＋ C sinensis L. Osbeck cv. Succari]. About 90 days after pollination, the embryos obtained from crosses were cultured on the solid media of MT ＋ ME （malt extraction, 500 mg L^-1） and MT ＋ GA3 （1 mg L^-1）. The embryogenic callus was initiated from the embryoids and plantlets＇ hypocotyls and could be subcultured. Flow cytometry and SSR analysis verified that the callus was from the triploid hybrids. The callus had embryogenesis capacity and produced a large number of embryoids on MT ＋Lactose （50 g L^-1） medium after being subcultured for two years. It is comparatively easier to obtain the callus from the hybrid embryo than from Shatian pummelo itself. The callus is valuable for the conservation and utilization of Shatian pummelo.

Improved Culture Media for Embryogenic Callus Generation in Sorghum [Sorghum bicolor (L.) Moench]

Many attempts on optimization of sorghum[Sorghum bicolor(L.)Moench]tissue culture induction media have been made,but the culture system remains with some bottlenecks compared to that of other crops.This study aimed at assessing the suitability of various induction media to produce embryogenic callus(yellow and friable)with high induction rates and reduced phenolic exudation.The six culture medium modifications:3 based on Murashige and Skoog(MS)medium and one each based on Chu N6,Gamborg B5 and 190-2 media respectively were applied in the culture of mature embryos from 10 sorghum genotypes.Although there was a genotype influence on the attainment of a yellow callus,friability of the callus was determined to be dependent on the culture medium and not the genotype.Half strength MS medium with 0.2 mg/l 2,4-D with 2.8 g/l Gelrite®as the gelling agent modified with 1.0 g/l KH_(2)PO_(4),1.0 g/l L-proline,1.0 g/l L-asparagine and 0.16 mg/l CuSO_(4)·5H_(2)O(type E)was found to be the most effective resulting in about 60%yellow coloured callus induction with 25%friability.Addition of CuSO_(4)·5H_(2)O,KH_(2)PO_(4),L-proline and L-asparagine significantly reduced the phenolic production.Half strength MS medium was observed to contribute to quality callus production when compared to full strength MS media modified with the compounds.The half strength MS medium was also observed to suppress phenolic production.Medium 190-2 produced the highest regeneration frequency(40%)among the 3-regeneration media tested.The results provide information on a suitable sorghum callus induction medium necessary for embryogenesis.

Geometric triangular chiral hexagon complexes and clonal embryogenic body organization on the Turin Shroud crucified man image: A predictable tissue response to injury

The shroud continues to remain one of the most studied and controversial artifacts in human history. Many tests, X-ray fluorescence, reflectance, spectrometry and low energy/high-resolution X ray transmission have shown that the crucified body is not compatible with a painted image. Researchers confirm that the alleged blood is real blood. We documented the self-assembly of geometric triangular chiral hexagon complex (GTCHC) with structural organization of embryoid bodies in cancer tissues. The identification of these structures is not only limited to malignant tumors but also appears in extreme injured tissues. Our interest is to determine if we can predict and identify these patterns in the Shroud of Turin. Based on pattern recognition image was analyzed over 100 shroud images. We identified a central spectral emission line that exhibits a characteristic signature on a plot of residual electromagnetic radiation, head area narrowing and low extremities broadening, indication of decay energy changes in the velocity of the molecules in the traversal trajectory. This Electromagnetic collision event generates in the cloth stagnant blood areas with patterns identical to those identified for us in cancer damage tissues. Inflammatory cytokines activate stem cells and Notch signaling proteins in cascade of interactions to generate real clonal human embryoid template. Can we predict function from structure? These structures evoke life, regeneration, but not death. Our findings suggest the image of a crucified man on the Shroud of Turin is a real physical electromagnetic collision event in response to extreme tissue injury, with the fact that supports our previous findings in cancer tissues as real and predictable. Proteins derived from these emergent damage tissue derivate stem cells could be used to design biologic templates in regenerative medicine and develop novel strategies in cancer therapy.

Identification and characterization of cell cultures with various embryogenic/regenerative potential in cotton based on morphological,cytochemical,and cytogenetical assessment

Somatic embryogenesis(SE) plays a vital role in genetic transformation and massive propagation of important agronomical and economical crops.Here,we conducted a systematic assessment of the morphological,cytochemical,and cytogenetical characteristics of six culture strains with various embryogenic/regenerative potential during SE process in cotton.Results indicated that the six cell culture strains had stable ploidy levels,and did not reveal any relationship between the cytogenetic state and their morphogenetic potential.Moreover,the six culture strains were compared via double staining with Evans blue and Acetocarmine to efficiently distinguish embryogenic and non-embryogenic cells and determine the embryogenic nature of the calli.In addition,the kind of auxins added in medium affected not only growth property,color,size of cell clumps but also ploidy level and regeneration ability.By combining analysis of morphological,cytochemical,and cytogenetical characteristics of the cell cultures,we are able to obtain and maintain homogeneous cell population with high morphogenic and regeneration ability and establish efficient somatic embryogenesis and regeneration system from short-term cell cultures in upland cotton,which highlight the application of biotechnological approaches in crop breeding,and above all,to better understand totipotency of cells in higher plants.

Transformation and Expression of Choline Monooxygenase(CMO) Gene in Embryogenic Tissue of White Pine(Pinus strobus)

A transformation procedure of choline monooxygenase(CMO) gene, involved in stress tolerance, was established in white pine embryogenic tissue by using A. tumefaciens C58/pMP90. The CMO cDNA fragment(1.3 kb) was generated by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) with primers based on the report sequence of CMO in gene bank. A chimerical gene composed of the cauliflower mosaic virus (CaMV) 35S promoter fused to CMO cDNA and β-glucuronidase (GUS-marker gene) was transferred into Ti-derived disarmed binary vector pBI121. The new vector, p35SCMOp, was transferred into Agrobacterium tumefaciens C58/pMP90 by freeze-thaw method. Somatic embryogenesis (SE) initiation of Pinus. Strobus L. and Pinus.Koraiensis Sieb. et Zucc. depended on the manipulation of plant growth regulator (PGR) concentrations in the GLH culture medium. Transgenic embryos and regenerated plants of two Pine species were produced after co-culture of embryogenic tissue with the disarmed strain of A. tumefaciens C58/pMP90/ p35SCMOp and selected on medium containing 25mg/L kanamycin. The transformed embryogenic tissue was initially confirmed by histochemical GUS assay followed by PCR. One copy of T-DNA was detected by transgenic lines analysis in Pinus. Strobus L. and transgenic plants were regenerated for two species using modified protocols for maturation and germination of somatic embryos.

Histological and Ultrastructural Observation Reveals Significant Cellular Differences between Agrobacterium Transformed Embryogenic and Non-embryogenic Calli of Cotton

Over the past few decades genetic engineering has been applied to improve cotton breeding. Agrobacterium medicated transformation is nowadays widely used as an efficient approach to introduce exogenous genes into cotton for genetically modified organisms. However, it still needs to be improved for better transformation efficiency and higher embryogenic callus induction ratios. To research further the difference of mechanisms for morphogenesis between embryogenic callus and non-embryogenic callus, we carried out a systematical study on the histological and cellular ultrastructure of Agrobacterium transformed calli. Results showed that the embryogenic callus developed nodule-like structures, which were formed by small, tightly packed, hemispherical cells. The surface of some embryogenic callus was covered with a fibrilar-like structure named extracellular matrix. The cells of embryogenic calli had similar morphological characteristics. Organelles of embryogenic callus cells were located near the nucleus, and chloroplasts degraded to proplastid-like structures with some starch grains. In contrast, the non-embryogenic calli were covered by oval or sphere cells or small clusters of cells. It was observed that cells had vacuolation of cytoplasm and plastids with a well organized endomembrane system. This study aims to understand the mechanisms of embryogenic callus morphogenesis and to improve the efficiency of cotton transformation in future.

Factors Affecting Embryogenic Callus Production and Plant Regeneration in Anther Culture of Bupleurum chinense

Objective To evaluate the influences of the genotypes,anther developmental stages,and cultural conditions on the efficiency of embryogenic callus induction and plant regeneration in the anthers culture of Bupleurum chinense.Methods The different effects such as four genotypes,plant growth regulators,and temperature condition were compared in the experiments.The histological study was performed with the process of the anther culture.Results The highest inducing rate of embryogenic calli were achieved for the genotypes Zhongcaiyihao(ZCYH),Z4,and Z5 at the early-to middle-uninucleate stages,except for genotype ZPM1 at the tetrad stage.Cold pretreatment increased the production of the embryogenic callus,in which 4-day cold pretreatment improved the production of embryogenic callus from 0% to 2.2% and 5.0% for genotypes ZPM1 and ZCYH,respectively.No embryogenic callus was induced in the medium containing less than 0.75 mg/L 2,4-dichlorophenoxyacetic acid(2,4-D) .The highest regeneration rate(34.6%) was obtained in 1/2 MS salts regeneration medium supplemented with 0.1 mg/L 6-benzylmaminopurine(BA) .The low concentration of BA was able to promote the embryogenic callus formation and subsequent plantlet regeneration via somatic embryogenesis.Chromosome counting of regenerated plantlets showed mostly diploid plant(2n = 12) with only one haploid plant(n = 6) .Because of the low rate of microspore embryo formation,we only tracked the process of embryogenesis from the connective tissue,instead of microspore by histological observations.Conclusion This study establishes an efficient system for embryogenic callus induction and plant regeneration system.This is the first report on the haploid plantlet through the anther culture of B.chinense.

Vitrificational cryopreservation and subsequently fertile plant regeneration from rice (Oryza sativa L.) embryogenic suspension cells

CRYOPRESERVATION by vitrification is a new ultralow-temperature-storage technique developed in the 1980s. Operationally, procedures for vitrification of biological specimens consist of the following basic steps: treatment of the samples in a highly concentrated vitrification solution and then plunging the specimens into liquid nitrogen. The word 'vitrification' is sometimes used to describe the high moisture of the in vitro cultures or regenerants. However, in this note, this word means a biophysical process in which the cells and the solution are transformed into a homogeneous and amorphous state. The water in cells does not change into ice, but in

大蕉胚性培养物及其原生质体再生植株的染色体分析

采用看护培养法对大蕉胚性细胞悬浮系(Embryogenic cell suspensions,ECS)来源的原生质体进行培养,并研究了不同品种的看护细胞对原生质体培养的影响,以及大蕉ECS及其原生质体再生植株的染色体数目。结果发现,采用贡蕉ECS作为看护细胞时,大蕉原生质体培养15 d后细胞分裂频率达到8.5%,培养30 d后细胞团形成率为0.35%;而以大蕉ECS作为看护细胞时,全部原生质体褐化,不能正常发育。染色体检测结果表明,大蕉ECS的染色体数目不稳定,除含有正常的三倍体染色体数目的细胞(2n=3x=33)外,还出现大量染色体数目异常的细胞。其中绝大部分细胞含有多倍和非整倍的染色体数目,仅14.5%的细胞含正常三倍体染色体数目;但通过ECS原生质体培养获得的再生植株具有正常的三倍体染色体数目(2n=3x=33)。

濒危植物香果树愈伤组织经长期继代后的6种同工酶研究(摘要)(英文)

[目的]研究濒危植物香果树愈伤组织经长期继代后的6种同工酶酶谱的变化。[方法]采用非变性聚丙烯凝胶电泳技术对长期继代后的香果树愈伤组织的酯酶(EST)、酸性磷酸酯酶(ACP)、ATP酶(ATPase)、淀粉酶(AMY)、超氧化物歧化酶(SOD)和过氧化物酶(POD)6种同工酶进行分析。[结果]香果树的胚性愈伤组织和非胚性愈伤组织在6种同工酶水平上均有差异,均可作为鉴别2者的依据。非胚性愈伤组织中的EST、ACP和POD的表达量明显高于胚性愈伤组织。褐化的非胚性愈伤组织与正常非胚性愈伤组织相比,在AMY、SOD和POD同工酶水平上无差别,而EST、ACP和ATPase同工酶含量减少;褐化的胚性愈伤组织与正常胚性愈伤组织相比,EST同工酶含量升高,其他5种同工酶含量降低。[结论]为研究香果树的长期组织培养过程中愈伤组织形态差异及褐化现象提供理论基础。

Screening and verification of the factors influencing somatic embryo maturation of Larix olgensis

With embryogenic callus of Larix olgensisis, we investigated the effects of inositol, glutamine, casein hydrolysate, carbohydrate, abscisic acid and silver nitrate concentration on the maturation of the somatic embryo.Three dominant factors emerged, and we developed a response surface model based on the Box-Behnken design.We defined the optimal conditions for the maturation of somatic embryos. The contents of abscisic acid, silver nitrate, sucrose and casein hydrolysis significantly affected the amount of maturing embryos, but inositol, maltose and glutamine had no effect. By establishing a response surface model with multiple factors, we predicted that the optimal number of L. olgensis somatic embryos was 204 ± 4 gon basal medium, containing 18.28 mg Labscisic acid,5.46 mg Lsilver nitrate and 82.67 g Lsucrose. In the verification experiments, the addition of 20 mg Labscisic acid, 5 mg Lsilver nitrate and 80 g Lsucrose to BM yielded an average of 202.06 somatic embryos per gram. These results should guide large-scale breeding of L. olgensis.

Selection of culture conditions for callus induction and proliferation by somatic embryogenesis of Pinus koraiensis

The induction and proliferation of embryogenic callus are key steps for large-scale propagation of somatic embryogenesis pathway and long-term preservation of coniferous germplasm.Callus can be induced from immature embryos of Korean pine(Pinus koraiensis Sieb.et Zucc.;Pinaceae)as explants,but there are problems,such as low proliferation efficiency,loss of embryogenicity,poor vigor;thus,best conditions for proliferation and culture of immature embryos of Korean pine are not yet clear.To solve the problems with somatic embryogenesis of Korean pine and determine the best culture conditions for callus induction and proliferation,we varied hormone concentration,subculture cycle of proliferation and other plant growth regulators combinations in media to induce callus formation by megagametophytes of three Korean pine families at different developmental stages,then analyzed the effects on embryogenic callus retention and cell proliferation using a quadratic regression orthogonal rotation design.The results showed that the family origin and collection date of explants significantly affected callus induction(induction rate reached 1.67%).Embryogenic maintenance and callus proliferation were best on DCR medium supplemented with 0.25 mg L^(−1)6-benzyl adenine,1 mg L^(−1)naphthaleneacetic acid,30 g L^(−1)sucrose,500 mg L^(−1),L-glutamine,500 mg L^(−1)casein hydrolysis and 6.5 g L^(−1)agar.In addition,the combination of 2,4-dichlorophenoxyacetic acid+6-benzyl adenine also had a better proliferative effect on callus.The effects of different combinations of growth regulators on callus proliferation efficiency were significantly different.Transfer to new medium every 13–15 days not only maintained robust callus vigor,but also yielded a larger proliferation coefficient.The techniques and conditions for embryogenic callus induction and proliferation of Korean determined here will serve as a foundation for establishing a large-scale system for somatic embryogenesis and propagation of Korean pine.

Effects of Auxins and Media on Callus Induction of Chinese Spring Wheat(Triticum aestivum L.)

The effects of auxins and media on callus induction from the mature and immature embryos of Chinese spring wheat （Triticum aestivum L.） varieties were investigated. It was found that genotype, medium, auxin source and concentration had the significant effects on the induction of embryogenic callus, explants germination and the increment of callus fresh weight. For immature embryos cultured on MS medium, 2 mg L^-1 of 2, 4-D was optimal, and the highest frequency of embryogenic callus （33.50%） was observed. For the mature embryos on N6 medium, 4 mg L^-1 of 2, 4-D was optimal. The frequency of embryogenic callus and increment of callus fresh weight on 2, 4, 5-T media were higher than those on 2, 4-D media, and in the presence of 2, 4, 5-T the precocious germination of explants for all genotypes were significantly suppressed. These results indicated that 2, 4, 5-T was superior to 2, 4-D and NAA in the culture of immature embryos. This is the first report about the effect of 2, 4, 5-T and NAA on wheat tissue culture, particularly in comparison with 2, 4-D in detail.

Establishment and Optimization of the Regeneration System of Mature Embryos of Maize (Zea mays L.)

A reliable system was developed for regeneration from mature embryos derived from callus of four maize inbred lines （Liao 7980, Dan 9818, Dan 340, and Dan 5026）. The protocol was mainly based on a series of experiments involving the composition of culture medium. We found that 9 pM 2,4-dichlorophenoxyacetic acid in MS medium was optimum for the induction of callus. The induction frequency of primary calli was over 85% for four inbred lines tested. The addition of L- proline （12 mM） in subculture medium significantly promoted the formation of embryogenic callus but it did not significantly enhance growth rate of callus. Efficient shoot regeneration was obtained on regeneration medium containing 2.22 μM 6- benzylaminopurine in combinations with 4.64 μM Kinetin. Regenerated shoots were rooted on half-strength MS medium containing 2.85 μM indole-3-butyric acid. This plant regeneration system provides a foundation for genetic transformation of maize.

Radiation-induced in vitro mutagenesis system for salt tolerance and other agronomic characters in sugarcane(Saccharum officinarum L.)

Gamma ray-induced in vitro mutagenesis and selection for salt(NaC l) tolerance were investigated in sugarcane(Saccharum officinarum L.). Embryogenic callus cultures were irradiated(10 to 80 Gy) and subjected to in vitro selection by exposure of irradiated callus to NaC l(0, 50, 100,150, 200, and 250 mmol L-1). Increasing NaC l concentrations resulted in growth reduction and increased membrane damage. Salt-selected callus lines were characterized by the accumulation of proline, glycine betaine, and Na+and K+concentration. Higher accumulation of proline and glycine betaine was observed in NaC l stressed callus irradiated at 20 Gy. Na+concentration increased and K+concentration decreased with increasing salt level. Irradiated callus showed50–60% regeneration under NaC l stress, and in vitro-regenerated plants were acclimatized in the greenhouse, with 80–85% survival. A total of 138 irradiated and salt-selected selections were grown to maturity and their agronomic performance was evaluated under normal and saline conditions. Of these, 18 mutant clones were characterized for different agro-morphological characters and some of the mutant clones exhibited improved sugar yield with increased Brix%,number of millable canes, and yield. The result suggest that radiation-induced mutagenesis offers an effective way to enhance genetic variation in sugarcane.

利用悬浮培养和培养基选择改善火炬松的体细胞胚胎发生和植株再生(英文)

Mature zygotic embryos of three families of loblolly pine ( Pinus taeda L.) were cultured on callus induction medium containing 8?mg·L -1 2,4 dichlorophenoxyacetic acid (2,4 D), 4?mg·L -1 6 benzyladenine (BA), 4?mg·L -1 kinetin (KT), 500?mg·L -1 casein hydrolysate, and 500?mg·L -1 glutamine for 9 weeks, callus was formed on cotyledons, hypocotyls, and radicles of mature zygotic embryos. Callus was sub cultured on the callus proliferation medium with 1 6?mg·L -1 2,4 dichlorophenoxyacetic acid (2,4 D), 0 8?mg·L -1 6 benzyladenine (BA), 0 8?mg·L -1 kinetin (KT) for 9 weeks. White translucent, glossy, mucilaginous embryogenic callus containing embryogenic suspensor masses (ESM) and immature somatic embryos was obtained, and the highest frequency of explants forming embryogenic callus was 16 9%. Embryogenic suspension cultures were established by culturing embryogenic callus in liquid callus proliferation medium. Liquid cultures containing embryogenic suspension masses and immature somatic embryos were transferred to medium containing abscisic acid (ABA), polyethylene glycols (PEG), or activated charcoal for enhancing the production of cotyledonary somatic embryos. After mature somatic embryos were cultured on medium containing indole butyric acid (IBA), gibberellic acid (GA 3), BA, and activated charcoal and being lowered sucrose concentration for 4～12 weeks, somatic embryos germinated to form regenerated plantlets. Seventy one regenerated plantlets were transferred to a perlits∶peatmoss∶vermiculate (1∶1∶1) soil mixture, and 23 plantlets survived in the field.

Optimization of the uidA Gene Transfer of Rosa hybrida via Agrobacterium tumefaciens: an Assessment of Factors Influencing the Efficiency of Gene Transfer

To develop a transformation protocol of Rosa hybrida Samantha via Agrobacterium tumefaciens, the authors examined the effect of different factors on T-DNA transfer by measuring transient expression levels of an intron-containing -glucuronidase gene. The results indicate that explant, light condition, salt concentration and acetosyringone (AS) concentration in co-culture medium are the most important factors, and factors like co-culture temperature, co-culture period and bacteria density have a strong effect on the growth of bacteria and then T-DNA transfer. Optimized co-cultivation was performed by inoculation of embryogenic callus with bacteria at a density of OD600= 0.50.8 for 20 min and co-culture in darkness under 23 C on medium with 1/2 MS salts and 300 mol稬1 AS for 3 d.