In vitro plant regeneration and genetic transformation of Dichanthium annulatum

Optimization of in vitro plant regeneration and genetic transformation of apomictic species such as Dichanthium annulatum would enable transfer of desirable genes. Seven genotypes of this grass species were screened through mature seed explant for embryogenic callus induction, callus growth and quality (color and texture), and shoot induction. Genotype IG-1999, which produced highly embryogenic, rapidly growing good-quality callus capable of regenerating at a high frequency, was selected for transformation experiments. Using a binary vector (pCAMBIA1305), frequency of GUS expression was compared between two methods of transformation. Bombardment of embryogenic calli with gold particles coated with pCAMBIA1305 at a distance of 11 cm, pressure of 4 bars, and vacuum of 27 Hg passing through 100 muM mesh produced maximum GUS expression (23%). Agrobacterium infection was maximum at an optical density of 2.0 when cocultured under vacuum for 15 min and cocultivated for 3 days at 28 degrees C in constant dark on MS medium of pH 5.8 with 3 mg/l 2,4-D, and 400 muM acetosyringone. Among two binary vectors used for Agrobacterium-mediated transformation, pCAMBIA1301 showed higher frequency of GUS expression while pCAMBIA1305 recorded more of the GUS spots per callus. Supplementation of acetosyringone in the cocultivation medium was found indispensable for Agrobacterium-mediated transformation. Injuring the calli through gold particle bombardment before their cocultivation with Agrobacterium improved the transformation efficiency. Several transgenic plants were developed using the PIG method, while stable GUS-expressing calli were multiplied during selection on MS medium containing 250 mg/l cefotaxime and 50 mg/l hygromycin, incubated in constant dark. A highly significant difference was observed between two methods of transformation for both frequency of GUS expression and GUS spots per callus. PIG-mediated transformation resulted in higher GUS expression compared to the Agrobacterium method. These results demonstrate that Dichanthium annulatum is amenable to Agrobacterium-mediated genetic transformation using a binary vector.     

Production of herbicide-tolerant zoysiagrass by Agrobacterium-mediated transformation

Herbicide-resistant zoysiagrass (Zoysia japonica Steud.) has been developed by Agrobacterium-mediated transformation. A callus-type transformation system was established by optimizing several factors that affect the rate of transformation, including co-cultivation period and concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), CaCl2 and acetosyringone. Maximal GUS expression was observed when a Type 3 callus was co-cultivated on 2,4-D-free co-cultivation medium for 9 d. In addition, removal of calcium and addition of 50-100 mg/L acetosyringone during co-cultivation enhanced GUS expression. When this optimized protocol was applied to the transformation of the bialaphos resistance gene (bar), four plants per 700 mg of infected calluses survived on the selective medium. DNA gel-blot analysis showed that two copies of the transgene had been integrated. After application of 2 g/L bialaphos for a week the transgenic plants survived herbicide spraying, while untransformed zoysiagrasses and invading weeds died. The herbicide-tolerant zoysiagrass will permit more efficient weed control in this widely cultivated turf grass.     

Particle-inflow-gun-mediated genetic transformation of buffel grass (Cenchrus ciliaris L.): optimizing biological and physical parameters

The present study was conducted to optimize various biological and physical parameters for developing an efficient and reproducible gene transfer method for genetic transformation of buffel grass. Transformation was carried out using a helium-driven particle inflow gun (PIG). Embryogenic calli produced from mature seeds of buffel grass cv. CC-119 were separately bombarded with four plasmids, containing Actin (pAct1DX), Ubiquitin (pAHC-25; pAHC-27) and CaMV-35S (pCaMVGUS) promoters, coated on tungsten and gold particles. The efficiency of transformation was monitored through transient GUS expression. Different parameters, viz., the type of promoter, type and size of microcarrier, helium gas pressure, distance and time of bombardment, were standardized for delivering DNA into embryogenic calli. Bombardment with plasmid DNA carrying the actin promoter coated on 1.6 micro gold particles, at a helium pressure of 4 bars, a distance of 10 cm for 10 micro sec and 28 mm Hg vacuum in the chamber, produced the best result in transient GUS expression. The Actin promoter was found to be more efficient in driving expression of the GUS gene in buffel grass, followed by Ubiquitin and CaMV-35S promoters. Lower helium pressure was found to be sub-optimal, while higher pressure produced a smaller number of blue spots, probably due to excessive damage to the cells. Maximum of 385 blue spots was observed with gold particles of 1.6 micro size, whereas only 213 blue spots were recorded for tungsten particles of 1.0 micro size. The optimized parameters can be employed for genetic transformation of buffel grass with genes of agronomic importance.     

Competence of Arabidopsis thaliana genotypes and mutants for Agrobacterium tumefaciens-mediated gene transfer: role of phytohormones

Many plant species and/or genotypes are highly recalcitrant to Agrobacterium-mediated genetic transformation, and yet little is known about this phenomenon. Using several Arabidopsis: genotypes/ecotypes, the results of this study indicated that phytohormone pretreatment could overcome this recalcitrance by increasing the transformation rate in the known recalcitrant genotypes. Transient expression of a T-DNA encoded ss-glucuronidase (GUS) gene and stable kanamycin resistance were obtained for the ten ARABIDOPSIS: genotypes tested as well as for the mutant uvh1 (up to 69% of petioles with blue spots and up to 42% resistant calli). Cultivation of Arabidopsis: tissues on phytohormones for 2-8 d before co-cultivation with Agrobacterium tumefaciens significantly increased transient GUS gene expression by 2-11-fold and stable T-DNA integration with petiole explants. Different Arabidopsis ecotypes revealed differences in their susceptibility to Agrobacterium-mediated transformation and in their type of reaction to pre-cultivation (three types of reactions were defined by gathering ecotypes into three groups). The Arabidopsis uvh1 mutant described as defective in a DNA repair system showed slightly lower competence to transformation than did its progenitor Colombia. This reduced transformation competence, however, could be overcome by 4-d pre-culture with phytohormones. The importance of pre-cultivation with phytohormones for genetic transformation is discussed.     

Improved production of transgenic Dioscorea zingiberensis (Dioscoreaceae) by Agrobacterium tumefaciens-mediated transformation

The establishment of high-efficiency Agrobacterium-mediated transformation techniques could improve the production of Dioscorea zingiberensis, a medicinal species with a high diosgenin content. We co-cultivated embryogenic calli induced from mature seeds with A. tumefaciens strain EHA105. A binary vector, pCAMBIA1381, which contains the gfp and hpt genes under the control of the ubiquitin promoter and the CaMV 35S promoter, respectively, was used for transformation. Pre-culture, basic medium, acetosyringone, and bacterial density were evaluated to establish the most efficient protocol. The optimal conditions consisted of MS medium without CaCl(2) for pre- and co-cultivation, three days for pre-culture, addition of 200 μM AS, and an OD(600) of 0.5. The transgenic plants grown under selection were confirmed by PCR analysis and Southern blot analysis. This protocol produced transgenic D. zingiberensis plants in seven months, with a transformation efficiency of 6%.     

‘光叶蔷薇’器官间接再生体系的建立及GUS基因转化的初步研究

以‘光叶蔷薇’（Rosa wichuriana ‘Basye’s thornless’）无菌苗的顶生幼嫩小叶为外植体,探讨了其愈伤组织诱导及植株再生的方法。结果表明,高浓度的生长素NAA能诱导外植体产生愈伤组织;由NAA诱导的愈伤组织在附加TDZ的MS培养基上,先暗培养再进行光照培养可直接分化出不定芽。诱导愈伤组织的最佳NAA浓度是7.0 mg/L、暗培养时间为10 d,而最佳分化培养基是MS + 5.0 mg/L TDZ + 30 g/L葡萄糖 + 2.5 g/L GEL,分化率达18.34%。以诱导产生的愈伤组织为侵染受体,初步建立了‘光叶蔷薇’GUS基因转化体系。农杆菌菌液浓度OD₆₀₀值为0.5、侵染30 min、共培养2 d、乙酰丁香酮的浓度为50 μmol/L是'光叶蔷薇’愈伤组织转基因的最优条件。     

高羊茅和黑麦草农杆菌介导转化体系的研究

利用C58C1农杆菌菌系（携带的表达载体上含GUS基因和nptII基因）感染4个草坪草品种追寻者、爱神特、腾跃和守门员成熟胚来源的愈伤组织,共培养后部分愈伤组织进行X-Gluc组织化学染色检测,其余愈伤组织在含G418 10-25 mg/L的MS改良培养上先后筛选抗性愈伤组织和分化抗性再生植株,对移栽成活的144棵抗性再生植株分别进行了ELISA检测、PCR检测和组织化学染色检测。愈伤组织阶段X-Gluc染色检测结果表明,4个草坪草品种GUS基因瞬间表达率8.6%～46.9%,爱神特愈伤组织对农杆菌侵染最为敏感,其次是腾跃和守门员,追寻者最不敏感;ELISA检测结果表明,45株呈现阳性,证明nptII基因已转入草坪草并已表达;PCR检测结果与ELISA检测结果一致,表明nptII基因确实已经整合到了草坪草基因组中,且没有发生沉默现象;转基因植株X-Gluc染色检测结果表明,GUS基因在43株中得到了稳定表达,在2株中发生了沉默现象。4个草坪草品种抗性再生植株分化率0～43.5%,转化率0～21.5 %。结果还表明,GUS基因瞬间表达率与稳定转化率在草坪草上很不一致,不能作为衡量基因型转化效果的指标。     

‘光叶蔷薇’器官间接再生体系的建立及GUS基因转化的初步研究

以‘光叶蔷薇’(Rosa wichuriana‘Basye's thornless’)无菌苗的顶生幼嫩小叶为外植体,探讨了其愈伤组织诱导及植株再生的方法。结果表明,高浓度的生长素NAA能诱导外植体产生愈伤组织;由NAA诱导的愈伤组织在附加TDZ的MS培养基上,先暗培养再进行光照培养可直接分化出不定芽。诱导愈伤组织的最佳NAA浓度是7.0 mg/L、暗培养时间为10 d,而最佳分化培养基是MS+5.0 mg/L TDZ+30 g/L葡萄糖+2.5 g/L GEL,分化率达18.34%。以诱导产生的愈伤组织为侵染受体,初步建立了‘光叶蔷薇’GUS基因转化体系。农杆菌菌液浓度OD600值为0.5、侵染30 min、共培养2 d、乙酰丁香酮的浓度为50μmol/L是‘光叶蔷薇’愈伤组织转基因的最优条件。     

Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium × formolongi

An efficient system for Agrobacterium-mediated transformation of Lilium × formolongi was established by preventing the drastic drop of pH in the co-cultivation medium with MES. Meristematic nodular calli were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm which harbored intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransfease II (NPTII) genes. After three days of co-cultivation on 2 g/l gellan gum-solidified MS medium containing 100 μM acetosyringone, 30 g/l sucrose, 1 mg/l picloram and different concentrations of MES, they were cultured on the same medium containing 12.5 mg/l meropenem to eliminate Agrobacterium for 2 weeks and then transferred onto medium containing the same concentration of meropenem and 25 mg/l hygromycin for selecting putative transgenic calli. Transient GUS expression was only observed by adding MES to co-cultivation medium. Hygromycin-resistant transgenic calli were obtained only when MES was added to the co-cultivation medium especially at 10 mM. The hygromycin-resistant calli were successfully regenerated into plantlets after transferring onto picloram-free medium. Transformation of plants was confirmed by histochemical GUS assay, PCR analysis and Southern blot analysis.     

High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)

Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large- scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene -glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co- cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.     

Genetic transformation of Populus trichocarpa genotype Nisqually-1: a functional genomic tool for woody plants

We report here the Agrobacterium-mediated genetic transformation of Nisqually-1, a Populus trichocarpa genotype whose genome was recently sequenced. Several systems were established. Internodal stem segments from vigorously growing greenhouse plants are the explants most amenable to transformation. For the most efficient system, approximately 40% of the stem segments infected with pBI121-containing Agrobacterium tumefaciens C58 produced transgenic calli, as confirmed by beta-glucuronidase (GUS) staining. The regeneration efficiency of independent transgenic plants was approximately 13%, as revealed by genomic Southern analysis. Some transgenic plants were produced in as little as 5 months after co-cultivation. This system may help to facilitate studies of gene functions in tree growth and development at a genome level.     

Fertile transgenic Brachiaria ruziziensis (ruzigrass) plants by particle bombardment of tetraploidized callus

We have produced transgenic plants of the tropical forage crop Brachiaria ruziziensis (ruzigrass) by particle bombardment-mediated transformation of multiple-shoot clumps and embryogenic calli. Cultures of multiple-shoot clumps and embryogenic calli were induced on solidified MS medium supplemented with 0.5mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 2mg/L 6-benzylaminopurine (BAP) or 4mg/L 2,4-D and 0.2mg/L BAP, respectively. Both cultures were bombarded with a vector containing an herbicide resistance gene (bar) as a selectable marker and the β-glucuronidase (GUS) reporter gene. Sixteen hours after bombardment, embryogenic calli showed a significantly higher number of transient GUS expression spots per plate and callus than multiple-shoot clumps, suggesting that embryogenic callus is the more suitable target tissue. Following bombardment and selection with 10mg/L bialaphos, herbicide-resistant embryogenic calli regenerated shoots and roots in vitro, and mature transgenic plants have been raised in the greenhouse. Polymerase chain reaction (PCR) and DNA gel blot analysis verified that the GUS gene was integrated into the genome of the two regenerated lines. In SacI digests, the two transgenic lines showed two or five copies of GUS gene fragments, respectively, and integration at different sites. Histochemical analysis revealed stable expression in roots, shoots and inflorescences. Transgenic plants derived from diploid target callus turned out to be sterile, while transgenics from colchicine-tetraploidized callus were fertile.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of European chestnut embryogenic cultures

An innovative and efficient genetic transformation protocol for European chestnut is described in which embryogenic cultures are used as the target material. When somatic embryos at the globular or early-torpedo stages were cocultured for 4 days with Agrobacterium tumefaciens strain EHA105 harbouring the pUbiGUSINT plasmid containing marker genes, a transformation efficiency of 25% was recorded. Murashige and Skoog culture medium containing 150 mg/l of kanamycin was used as the selection medium. The addition of acetosyringone was detrimental to the transformation efficiency. Transformation was confirmed by a histochemical -glucuronidase (GUS ) assay, PCR and Southern blot analyses for the uidA (GUS) and nptII (neomycin phosphotransferase II) genes. At present, 93 GUS-positive chestnut embryogenic lines are being maintained in culture. Low germination rates (6.3%) were recorded for the transformed somatic embryos. The presence of the transferred genes in leaves and shoots derived from the germinated embryos was also verified by the GUS assay and PCR analysis.     

Factors influencing the Agrobacterium tumefaciens-mediated transformation of carrot (Daucus carota L.)

To develop an efficient procedure for Agrobacterium tumefaciens-mediated genetic transformation of carrot (Daucus carota L.) the effects of several factors were studied. Parameters which significantly affected the transformation frequency were the variety, the explant type, and the co-cultivation period. Under optimal conditions, using the A. tumefaciens C58C1 containing either pGSTRN943 or pGSGluc1 and 3 days of co-cultivation, the frequency of transformation of petiole explants of the variety Nanco was greater than 45%. This procedure does not require acetosyringone or prolonged precultivation period. Using kanamycin (100 mg l^-1) for selection, a large number of transgenic plantlets developed from the embryogenic calli within 8–10 weeks of culture on hormone-free medium. Transformation was confirmed by histochemical detection of -glucuronidase activity in the transformed cells, by the ability of petiole segments to produce embryogenic calli in presence of kanamycin, and by Southern hybridization analyses.     

Agrobacterium-mediated transformation of Lolium rigidum Gaud.

Lolium rigidum Gaud. is an annual grass grown for forage but also an economically damaging crop weed. A single genotype somatic embryogenic callus line, VLR1-60, was identified from a herbicide susceptible L. rigidum population, VLR1, and proved to be amenable to Agrobacterium tumefaciens-mediated transformation. Somatic embryogenic calli were continuously induced from the meristematic region of VLR1-60 plants multiplied in vitro and the basic tolerance level of VLR1-60 to hygromycin B was determined. A hygromycin phosphotransferase gene was used as a selectable marker for hygromycin B selection. Somatic embryogenic calli derived from in vitro grown vegetative tillers were co-cultivated with the A. tumefaciens strain EHA105 harbouring binary vector carrying reporter genes and selectable marker in the presence of acetosyringone for 3 days. Inoculated calli were recovered on callus proliferation medium containing Timentin? but lacking hygromycin and were then subcultured onto media with hygromycin concentrations increased progressively through time for selection of transformed plant cells. Putative transgenic plants were recovered and integration of transgenes was confirmed by Southern hybridization analysis and by detection of DsRed or GUS activity in transgenic plants. The frequency of plant transformation was 1.3 %. The ability to transform L. rigidum will provide opportunities for functional characterization of genes to improve forage quality and increase our understanding of the evolution of herbicide resistance and of the basic genetics underlying traits that make L. rigidum a damaging crop weed.     

影响农杆菌介导玉米优良自交系遗传转化的因素

以我国玉米骨干自交系9046,齐319,414,Mo17的幼胚为材料,在已经建立的农杆菌介导的玉米幼胚转化体系的基础上,研究了影响农杆菌介导玉米优良自交系遗传转化的因素,建立了优化的玉米优良自交系的遗传转化体系。研究结果表明,1．0—2．0mm的玉米幼胚是最适宜的转化受体;在感染液和共培养基中都加入乙酰丁香酮(200μmol／L)和抗坏血酸(50mg／L),能显著提高农杆菌对玉米的侵染能力;而感染前将幼胚高渗透压预处理未能提高转化率;延迟筛选有利于提高抗性愈伤组织的存活率。应用优化后的转化体系,获得了这4个玉米优良自交系的转基因植株,PCR阳性植株率为1．71％-4．09％。转化植株叶片总DNA的PCR和Southern杂交分析表明,T-DNA上的外源基因已经整合进了玉米基因组,并且在大多数转基因植株(71．4％)中为单位点插入。这一体系的建立,为进一步将有用基因导入玉米优良自交系奠定了基础。     

Genetic transformation of <Emphasis Type="Italic">Agave salmiana</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and particle bombardment

Agave salmiana was transformed using two different protocols: co-cultivation with Agrobacterium tumefaciens and particle bombardment. The uidA (β-glucuronidase) gene was used as a reporter gene for both methods whereas the nptII and bar genes were used as selectable markers for A. tumefaciens and biolistic transformation respectively. Previous reports for in vitro regeneration of A. salmiana have not been published; therefore the conditions for both shoot regeneration and rooting were optimized using leaves and embryogenic calli of Agave salmiana. The transgenes were detected by Polymerase Chain Reaction (PCR) in 11 month old plants. The transgenic nature of the plants was also confirmed using GUS histochemical assays. Transformation via co-cultivation of explants with Agrobacterium harbouring the pBI121 binary vector was the most effective method of transformation, producing 32 transgenic plants and giving a transformation efficiency of 2.7%. On the other hand, the biolistic method produced transgenic calli that tested positive with the GUS assay after 14 months on selective medium while still undergoing regeneration.     

Enhanced resistance against bacterial wilt in transgenic tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis>) lines expressing the <Emphasis Type="Italic">Xa21</Emphasis> gene

To enhance bacterial wilt resistance in tomato plants and simplify the protocol of Agrobacterium tumefaciens mediated gene transfer, parameters affecting transformation efficiency in tomato have been optimized. A. tumefaciens strain EHA101, harboring a recombinant binary expression vector pTCL5 containing the Xa21 gene under the control of the CaMV 35S promoter was used for transformation. Five cultivars of tomato (Rio Grande, Roma, Pusa Ruby Pant Bahr and Avinash) were tested for transformation. Transformation efficiency was highly dependent on preculture of the explants with acetosyringone, acetosyringone in co-cultivation media, shoot regeneration medium and pre-selection after co-cultivation without selective agent. One week of pre-selection following selection along with 400 μM acetosyringone resulted in 92.3% transient GUS expression efficiency in Rio Grande followed by 90.3% in Avinash. The presence and integration of the Xa21 gene in putative transgenic plants was confirmed by polymerase chain reaction (PCR) and Southern blot analyses with 4.5–42.12% PCR-positive shoots were obtained for Xa21 and hygromycin genes, respectively. Transgenic plants of the all lines showed resistance to bacterial wilt. T₁ plants (resulting from self-pollination of transgenic plants) tested against Pseudomonas solanacearum inoculation in glasshouse, showed Mendelian segregation.     

Evaluation of factors influencing Agrobacterium-mediated spinach transformation and transformant selection by EGFP fluorescence under low-selective pressure

We developed an efficient Agrobacterium-mediated transformation protocol for spinach (Spinacia oleracea L.) that uses root-derived callus. Evaluation of this protocol was based on the systematic evaluation of factors that influence transformation efficiency. Four of the five factors that were tested significantly affected the transformation efficiency, including spinach cultivar, Agrobacterium tumefaciens strain and density, and the duration of co-cultivation. Transgenic spinach plants were generated based on optimized conditions, consisting of callus explants of the cultivar Gyeowoonae, A. tumefaciens strain EHA105 with OD₆₀₀ of 0.2, a co-cultivation period of 4 d, and 100 μM acetosyringone supplemented in the inoculation and co-cultivation media. After co-cultivation with A. tumefaciens, explants were cultured in low-selective and then non-selective conditions to enhance the growth of putative transgenic explants. Visualization of the fluorescent marker, enhanced green fluorescent protein (EGFP), was used to select transgenic explants at several stages, including callus, somatic cotyledonary embryo, and plantlet. The best results for fluorescence visualization screening were obtained at the somatic cotyledonary embryo stage. On average, 24.96?±?6.05% of the initial calli regenerated shoots that exhibited EGFP fluorescence. The putative transgenic plants were subjected to β-glucuronidase (GUS)-staining assay, phosphinothricin acetyltransferase (PAT) strip test, and molecular analyses to assess the transgene incorporation into plant genome and its expression. All EGFP-positive plants tested were confirmed to be transgenic by GUS-staining assay, PAT strip test, and molecular analyses. The transformation system described in this study could be a practical and powerful technique for functional genetic analysis and genetic modification of spinach.     

水稻双元细菌人工染色体载体系统转化体系的建立

普通双元载体己被广泛碰用于农杆菌介导的植物转化,但这类载体通常只能转移5～20kb的外源DNA片段;而双元细菌人工染色体（BIBAC）载休可以弥补普通双元裁体的不足,通过它已在烟草、番茄等双子叶植物中实现了大片段DNA（150kb）的转移。BIBAC载体在单子叶植物转化中的应用尚未见报道。面于单、双子叶植物间以及大、小片段转化间的转化体系存在明显差异,常规的农杆菌介导的水稻转化体系不能适应BIBAC系统转化的要求。因此,建立适于BIBAC系统的水稻转化体系是十分必要的。通过比较不同的受体材料,不同的预培养、其培养条件,不同的去除农杆菌及选择阳性愈伤的方式等对转化效率的影响,建矿了适合水稻BIBAC系统的转化体系。该体系的技术要点包括：以水稻品种H1493为转化受体：以含毒性辅助质粒pCH32的LBA4404菌株（HP4404）为侵染菌株;预培养的培养拱pH5．6：以N6A代替AAM悬浮农杆菌：侵染菌液浓度为OD600=1．0;共培养温度为24℃;采用过渡（Resting）培养除去农杆菌;采用二步法进行选择等。基于PCR检测、Southern印迹分析的结果表明,BIBAC载体所携带的插入片段及标记基因已整合到转化植株的基因组中。这个体系的建立为在水稻中利用BIBAC系统进行大片段DNA转化奠定基础。