Cotransformation of aspen and birch with three T-DNA regions from two different replicons in one Agrobacterium tumefaciens strain

The cointegration rate into the aspen and birch genomes of foreign genes from a binary vector and a disarmed Ti plasmid pCBE21 carried by the same Agrobacterium tumefaciens strain was studied. The cotransformation rate for the genes within the Ti plasmid varied from 30 to 100%; while the transformation rate for the gene from T(L) region was twofold higher as compared with the T(R) region. On the average, the gene transfer from all three T-DNAs was recorded in 10.9% of the transgenic lines. For the vector pBI121, the cotransformation rates for the genes from both regions of pCBE21 T-DNA were higher as compared with the vector pGS. In addition, a concurrent transfer of the genes from the Ti plasmid T(L) and T(R) regions was recorded only after the transformation with the vector pBI121. These results can be used for constructing woody plants containing several genes.     

Genetic studies on the role of octopine T-DNA border regions in crown gall tumor formation

Summary Crown gall tumors result from transfer and integration of the T-DNA from the Ti plasmid of Agrobacterium tumefaciens into plant nuclear DNA. In the present study, recombinant plasmids containing deletion and rearrangement deriviatives of the T-DNA region of the octopine Ti plasmid pTiA6 were tested in a binary tumorigenesis system (Hoekema et al. 1983) to determine the requirements for T-DNA border regions in tumor formation. Since two defined segments of the T-DNA region of octopine Ti plasmids can be detected in tumor DNA (the left (T_L-) and right (T_R-) DNA), four border regions exist in this Ti plasmid. Agrobacteria harboring plasmid constructs which contain a T-DNA gene capable of inciting tumors (gene 4, the tmr gene, which is involved in cytokinin biosynthesis) and various T-DNA border regions were tested for ability to cause tumors on Nicotiana glauca and other host plants. Such tmr constructs containing as their only border region the right border of either the T_L-DNA or the T_R-DNA are fully tumorigenic. Analogous tmr constructs containing only the T_L-DNa left border region are not tumorigenic. These results do not depend on the orientation or position of the single border with respect to the tmr gene; furthermore, the T_R-DNA right border can confer tumor-forming ability despite the presence of an intervening copy of the T_L-DNA left border.These results for relatively small plasmids are contrasted with previously determined requirements for border regions in tumorigenesis by intact Ti plasmids. A model previously proposed by Wang et al. (1984) for the role of border regions in DNA transfer to plant cells is extended in order to explain the tumor-forming ability of plasmid constructs containing a single border region. The results of this study interpreted according to the model suggest that the octopine T_L-DNA left border is defective in this DNA-transfer process.     

Octopine Ti-plasmid deletion mutants of agrobacterium tumefaciens with emphasis on the right side of the T-region

One hundred and twelve Agrobacterium tumefaciens mutants with a deleted octopine Ti plasmid were isolated. They originated from four insertion mutants, each of which carried the transposon Tn904 at a different position in the Ti plasmid. The deletion mutants were selected on the basis of loss of the capacity encoded by the Ti plasmid to degrade octopine. They were tested for the expression of other Ti-plasmid coded functions: tumor induction, presence of lysopine dehydrogenase activity in the tumor, and exclusion of phage Ap-1. For 21 mutants affected in at least one of these functions, the map position of the deletions was determined. It was found that deletions at two separated loci give rise to an Occ^? phenotype. Genes for Ap-1 exclusion were mapped on a small region just outside and to the right of the T_L + T_R region. Most of the T_R region, present as T_R-DNA in a limited number of crown gall tissues only, was shown to be unnecessary for tumor formation, since it could be deleted without affecting virulence (tested on various plant species) of the mutants. However, if the T_R region together with a small part of the adjacent T_L region, which is always present as T_L-DNA in normal crown gall tissues, was deleted the mutants became weakly virulent on Kalanchoë and Nicotiana rustica and avirulent on tomato. We hypothesize that in this case a region necessary for T-DNA integration has been deleted. The same region was found to be essential for lysopine dehydrogenase activity in the tumors.     

Expression of GUS in primary transformants and segregation patterns of GUS,TL- and TR-DNA in the T1 generation of hairy root transformants ofLotus corniculatus

Agrobacterium rhizogenes was assessed as a vehicle for transformation ofLotus corniculatus. Plants were co-transformed usingA. rhizogenes strain LBA 9402 harbouring the bacterial plasmid pRi1855 and the binary transformation vector pJit 73. pRi 1855 transfers both T_L and T_R sequences, while pJit 73 encodes β-glucuronidase (GUS) and also two selectable marker genes giving resistance to the antibiotics kanamycin and hygromycin. Three primary transformants (lines 1,6 and 12) were subjected to detailed morphological and biochemical analysis and lines 6 and 12 were also analysed at the molecular level. Tissues of both lines 6 and 12 were resistant to hygromycin and expressed GUS. Analysis of various tissues of each line showed a significantly lower GUS activity in line 6 than in line 12. Genetical analysis of progeny produced between control plants and lines 6 and 12 indicated that line 6 had one dose of theuid gene while line 12 had two or more independently segregating doses of the gene. Both lines 6 and 12 contained multiple copies of T_L-DNA, while only line 6 was T_R positive. In the progeny of lines 6 and 12 there was no evidence for linkage of T_L-DNA withuid, while in the progeny of line 6, T_R-DNA was under-represented. GUS-positive progeny which were free of both T_L and T_R sequences were identified from both lines.     

Improvement of Agrobacterium-mediated transformation and rooting of black cherry

An improved protocol for Agrobacterium-mediated transformation of an elite, mature black cherry genotype was developed. To increase transformation efficiency, vacuum infiltration, sonication, and a combination of the two treatments were applied during the cocultivation of leaf explants with Agrobacterium tumefaciens strain EHA105 harboring a PsAGAMOUS RNAi plasmid (pART27-PsAGRNAi). The effects of Agrobacterium culture density and cocultivation duration on transformation efficiency were examined using EHA105 harboring either pBI121-MDL4 or pBI121-PsTFL1. In addition, the effect of the binary vector on transformation efficiency was also studied. Fifteen-minute vacuum infiltration without sonication produced the highest transformation efficiency (21.7%) in experiments using pART27-PsAGRNAi. OD₆₀₀ values of 1.0 and 1.5 resulted in a transformation efficiency of 5% when pBI121-PsTFL1 was used for transformation. Transformation efficiency of 5% was also obtained from 3-d cocultivation using construct pBI121-MDL4 whereas no shoots regenerated after 4-d cocultivation. The binary vectors used also impacted transformation efficiency. PCR and quantitative-PCR analyses were used to confirm the integration of transgenes and determine the copy number of the selectable marker gene, neomycin phosphotransferase II, in 18 putative transgenic lines. Rooting of transgenic black cherry shoots was achieved at a frequency of 30% using half-strength Murashige and Skoog medium supplemented with 2% sucrose, 5 μM naphthaleneacetic acid, 0.01 μM kinetin, and 0.793 mM phloroglucinol, and the resulting transgenic plants were successfully acclimatized.     

A protocol for efficient biolistic transformation of mothbean<Emphasis Type="Italic">Vigna aconitifolia</Emphasis> L. Jacq. Marechal

A protocol for efficient direct gene transfer by using particle gun bombardment was developed for mothbeanVigna aconitifolia L. Jacq. Marechal. Hypocotyl explants from 2 cultivars of mothbean were transformed with 3 plasmids: pBI121, pHS101, and pHS102. Stable transformants were regenerated on MS medium supplemented with benzyladenine, α-naphthaleneacetic acid, and kanamycin. The helium pressure, plasmid type, and cultivar that were used determined the stable transformation frequency. Complete plants were regenerated and transferred to soil. The integration of the stable transgenes and reporter genes in plant genomes was shown by means of PCR amplification of these genes from plant genomic DNA and Southern blot hybridization with gene-specific probes. This method allows high-efficiency production of transgenic plants in mothbean. Suchita Kamble and Hari S. Misra contributed equally to this work.     

Recovery of phenotypically normal transgenic plants of Brassica oleracea upon Agrobacterium rhizogenes-mediated co-transformation and selection of transformed hairy roots by GUS assay

In this paper we describe the production of transgenic broccoli and cauliflower with normal phenotype using an Agrobacterium rhizogenes-mediated transformation system with efficient selection for transgenic hairy-roots. Hypocotyls were inoculated with Agrobacterium strain A4T harbouring the bacterial plasmid pRiA4 and a binary vector pMaspro::GUS whose T-DNA region carried the gus reporter gene. pRiA4 transfers T_L sequences carrying the rol genes that induce hairy root formation. Transgenic hairy-root production was increased in a difficult-to-transform cultivar by inclusion of 2,4-D in the medium used to resuspend the Agrobacterium prior to inoculation. Transgenic hairy roots could be selected from inoculated explants by screening root sections for GUS activity; this method eliminated the use of antibiotic resistance marker genes for selection. Transgenic hairy roots were produced from two cauliflower and four broccoli culivars. Shoots were regenerated from transgenic hairy root cultures of all four cultivars tested and successfully acclimatized to glasshouse conditions, although some plants had higher than diploid ploidy levels. Southern analysis confirmed the transgenic nature of these plants. T₀ plants from seven transgenic lines were crossed or selfed to produce viable seed. Genetic analysis of T₁ progeny confirmed the transmission of traits and revealed both independent and co-segregation of Ri T_L-DNA and vector T-DNA. GUS-positive phenotypically normal progeny free of T_L-DNA were identified in three transgenic lines out of the six tested representing all the cultivars regenerated including both cauliflower and broccoli.     

Genetic transformation of Medicago truncatula using Agrobacterium with genetically modified Ri and disarmed Ti plasmids

Summary Fertile transgenic plants of the annual pasture legume Medicago truncatula were obtained by Agrobacterium-mediated transformation, utilising a disarmed Ti plasmid and a binary vector containing the kanamycin resistance gene under the control of the cauliflower mosaic virus 35S promoter. Factors contributing to the result included an improved plant regeneration protocol and the use of explants from a plant identified as possessing high regeneration capability from tissue culture. Genes present on the T-DNA of the Ri plasmid had a negative effect on somatic embryogenesis. Only tissue inoculated with Agrobacterium strains containing a disarmed Ti plasmid lacking the T-DNA region or a Ri plasmid with an inactivated rol A gene regenerated transgenic plants. Fertile transgenic plants were only obtained with disarmed A. tumefaciens, and the introduced NPT II gene was transmitted to R₁ progeny.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - NPT neomycin phosphotransferase     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-Mediated Transformation of Large DNA Fragments Using a BIBAC Vector System in Rice

Large DNA fragments were transferred to rice (Oryza sativa L.) by an Agrobacterium-mediated transformation protocol using the binary bacterial artificial chromosome (BIBAC) vector system. Calli derived from mature embryos of japonica rice cultivar H1493 were used as target tissues. LBA4404 with the pCH32 helper plasmid carrying virE and virG was found to be the most efficient strain for the transfer of large DNA fragment into the rice genome. One notable difference between Agrobacterium-mediated transformation using standard binary vectors and that reported herein was that transformation using the BIBAC system required Agrobacterium tumefaciens carrying the virulence helper plasmid with virG/virE. Polymerase chain reaction, Southern blot, and progeny analyses confirmed the integration and inheritance of the insert fragment and marker genes carried by BIBAC in the T₀, T₁, and T₂ generations of transgenic events. To our knowledge, this represents the first report in which fertile, stable transgenic rice has been produced using the BIBAC vector system. The transformation system developed here would be useful for transferring large DNA fragments and for cloning and functional analysis of genes in rice.     

人干扰素α-2b基因在烟草中的表达研究

应用PCR的技术从质粒pAIFN中扩增人干扰素α-2b(Human interferon α-2b,HuIFN α-2b)编码基因,将其连接到pBI121双元载体构建植物真核表达载体pBIFN;用冻融法将该载体转染根癌农杆菌LBA4404;并用叶盘浸染法转化烟草叶片,经转化的烟草叶片的组织培养,诱导愈伤获得再生植株。通过应用PCR,RT-PCR,Wes-tern blot和WISH/VSV方法检测获得的烟草再生植株,结果表明HuIFN α-2b基因已成功整合进烟草核基因组并表达出具有活性的HuIFN α-2b蛋白。本文对HuIFN α-2b基因在烟草核系统中的表达进行了研究,为进一步在烟草叶绿体系统中该基因的表达研究奠定了基础。     

A Cointegrate Ti Plasmid Vector for Agrobacterium tumefaciens - mediated Transformation of indica Rice cv Pusa Basmati 1

An intermediate vector pSSJ1 was constructed by cloning a hph gene and a gus gene with catalase intron in pGV1500. pSSJ1 was cointegrated into a disarmed receptor Ti plasmid pGV2260 harboured in Agrobacterium tumefaciens strain C58C1Rif^R. The resulting A. tumefaciens strain C58C1Rif^R (pGV2260::pSSJ1) stably transformed Oryza sativa L. cv Pusa Basmati 1 scutellum-derived calli at 26% frequency. Introduction of the plasmid pSSJ3 (3′virB, virG and virC of pTiB0542) into A. tumefaciens C58C1Rif^R (pGV2260::pSSJ1) resulted in the elevation of acetosyringone-induced T -strand accumulation. Rice transformation efficiency of the cointegrate plasmid pGV2260::pSSJ1 increased from 26% to 33% in the presence of pSSJ3 and from 26% to 35% in the presence of pToK47 (complete virB, virG and virC). T-DNA integration in To plants was confirmed by Southern hybridization analysis. Inheritance analysis of the T₀ plants with single-copy T-DNA insertions revealed segregation of hygromycin resistance in 3:1 ratio. The feasibility of rice transformation with a cointegrate Ti plasmid vector is clearly established.     

Independent integration and seed-transmission of the TR-DNA of the octopine Ti plasmid pTi Ach5 in Nicotiana plumbaginifolia

Summary After co-cultivation of diploid Nicotiana plumbaginifolia protoplasts with an octopine-type Agrobacterium tumefaciens strain (LBA 4013) putative transformants were selected for hormone-independent growth, and were tested for T-DNA markers. The number of transformants expressing only T_L-DNA markers, i.e. phytohormone autotrophy and octopine synthase, was an order of magnitude higher than that of the cell lines which were simultaneously positive for both T_L- and T_R-DNA markers (the latter being mannopine and agropine). In one transformant, line no. 101, only the T_R-DNA markers were found. Not each of the T_L-, or T_R-DNA markers were expressed in each transformant resulting in a variety of phenotypes. It included the unorganized or the shoot-teratoma type of growth combined with the presence or absence of opines; e.g. agropine was absent from some of the transformants containing its precursor, mannopine. 5-Azacytidine did not induce agropine synthesis in these lines. Southern blot analysis showed that the T_R-DNA region coding for agropine synthesis was rearranged or absent in one of these lines. Similar variation in the expression of agropine and mannopine production was observed in transformants obtained with the leucinopine-type strain A281.From line 101 plants could be easily regenerated with the ability to synthesize agropine and mannopine. The segregation in the self-progeny fitted to a 3:1 ratio, indicating that the T_R-DNA was carried by a single chromosome. The Southern blot analysis showed that only opine-positive plants contained T_R-DNA. It also confirmed the absence of the T_L-DNA, demonstrating the independent integration of the T_R-region of the octopine-type Ti plasmid pTi Ach5.     

用单性结实基因2A11-iaaM转化罗汉果的研究

为了实现罗汉果生产中免除人工授粉和果实无籽化,该研究利用pBI121-Gus构建果实特异启动子2A11与生长素合成相关基因iaaM的嵌合基因(2A11-iaaM)过量表达载体,以罗汉果雌株叶盘为材料,采用农杆菌介导法建立罗汉果高效遗传转化体系,转化和创制单性结实罗汉果种质,通过基因特异引物对的PCR扩增,初步检测出转基因阳性植株,将之移栽大田,观察转基因植株的单性结实性的表现。结果表明:构建罗汉果单性结实性相关的pBAI-Gus植物双元表达载体获得成功;建立了农杆菌介导的罗汉果叶盘遗传转化优化体系,即农杆菌菌液OD_(600)值为0.3～0.5,侵染10 min,最优选择培养基为MS+TDZ 0.7 mg·L~(-1)+IBA 0.5 mg·L~(-1)+Kan 5 mg·L~(-1)+Cef 300 mg·L~(-1);经PCR鉴定共获得4株转基因阳性雌株;将阳性植株扩繁后移栽田间,经田间调查发现,24株阳性扩繁植株中有5株正常开花,占总植株数的20.8%,且其子房未经人工授粉发育成幼果,表现单性结实性。在载体构建和农杆菌介导的罗汉果遗传转化体系优化的基础上,将外源单性结实相关嵌合基因整合进罗汉果基因组并得到表达,为后续研究单性结实罗汉果的遗传生理,创制转基因罗汉果单性结实新种质,以及克服其产业化中需要人工授粉和无籽化提供了理论和应用基础。     

A reliable and efficient protocol for inducing genetically transformed roots in medicinal plant Nepeta pogonosperma

Nepeta pogonosperma is an important medicinal plant with anti-inflammatory effects. An efficient and reliable transformation system for this plant was developed through optimization of several factors which affected the rate of Agrobacterium rhizogenes mediated transformation. Five bacterial strains, A4, ATCC15834, LBA9402, MSU440 and A13, two explant types, leaves and stems, and several co-cultivation media were examined. The maximum rate of hairy root induction was obtained from stem explants using MSU440 and ATCC15834 bacterial strains. A drastic increase in the frequency of transformation (91 %) was observed when MS medium lacking NH₄NO₃, KH₂PO₄, KNO₃ and CaCl₂. Hairy root lines were confirmed by polymerase chain reaction (PCR) using primers of the rolB gene. According to Southern blot analysis, one T-DNA copy was inserted into each of the hairy root lines. In the present study, transgenic hairy roots have been obtained trough genetic transformation by A. rhizogenes harbouring two plasmids, the Ri plasmid and pBI121 binary vector harbouring gus reporter gene. Expression of the gus gene in transgenic hairy root was confirmed by histochemical GUS assay.     

Expression of synthetic genes coding for completely new, nutritionally rich, artificial proteins

Synthetic genes (A, AB and AHB) constructed and cloned into pKK233-2 vector were recloned from the parent plasmid into the new procaryotic expression vectors pGFY221N and pBI052. Gene AF-B (coding for all amino acids besides phenylalanine) was obtained by 'cassette mutagenesis' from gene AB. The plasmid pGFY221N was constructed from pGFY218L by replacing the PstI by an NcoI site; plasmid pBI052 was derived from pGFY221N through replacing the 221-bp EcoRI/NcoI fragment with a synthetic DNA segment of 52 bp representing the Escherichia coli atpE gene translational initiation region. The genes A, AB, AHB and AF-B in the vector pGFY221N were expressed with a six-amino-acid-long leader sequence; in pBI052 the genes were expressed directly. In vitro expression experiments were successfully with all the genes except with the AHB gene integrated into pGFY221N. In the E. coli minicell system expression was demonstrated with the A gene in pGFY221N and the AF-B and AHB genes in pBI052. Complete translation of the expressed genes AB, AF-B and AHB in either the in vitro or in vivo systems could be shown by using 35S-labelled N-terminal methionine and C-terminal cysteine. Both amino acids occur only once in the peptide sequences.     

花粉管通道法介导的铁皮石斛转基因技术

该研究以含有GFP和GUS基因的质粒和农杆菌为载体,采用花粉管通道法对铁皮石斛进行转基因技术研究。结果表明:(1)铁皮石斛种子萌发和原球茎生长对卡那霉素的最低致死浓度分别为90和150 mg·L~(-1)。进一步研究证实,在筛选转化种子和原球茎时,可分别向培养基中添加100和150 mg·L~(-1)的卡那霉素进行选择培养。(2)以携带GFP和GUS基因的质粒(pSuper1300和pBI121)和农杆菌为载体,用无菌去离子水重悬质粒pSuper1300和pBI121至浓度为100 ng·μL~(-1),用2%蔗糖+1/2MS+0.1%silwet-77+0.1%AS或5%蔗糖+0.1%silwet-77+0.1 mmol·L~(-1)AS重悬携带质粒pSuper1300和pBI121的农杆菌至菌液浓度为OD_(600)=0.7~0.8;在授粉后0.5~2.5 h内使用柱头滴加法导入携带外源基因的质粒或农杆菌溶液,收集成熟的转化种子,经选择培养及PCR检测发现,几乎所有处理的转化材料均能检测出外源GFP和GUS基因片段。另外,与农杆菌相比,以质粒为载体进行转化,可获得更高的结实率。该研究结果为铁皮石斛的基因工程育种提供了参考。     

翅碱蓬CMO基因克隆、测序及植物表达载体的构建

从翅碱蓬叶片中提取总RNA,根据相关同源序列设计引物,使用反转录试剂盒进行RT-PCR,扩增得到翅碱蓬胆碱单加氧酶（CMO）cDNA,进行PCR产物测序;然后将CMOcDNAT-A克隆至pMD18-T-simple载体上,经测序正确后亚克隆至pBI121植物表达载体,进行酶切鉴定及克隆测序。     

Developing an <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated genetic transformation for a selenium-hyperaccumulator <Emphasis Type="Italic">Astragalus racemosus</Emphasis>

Agrobacterium tumefaciens strain LBA4404 containing the plasmid pBI121, carrying the reporter gene uidA and the kanamycin resistance gene nptII, was used for gene transfer experiments in selenium (Se)-hyperaccumulator Astragalus racemosus. The effects of kanamycin on cell growth and division and acetosyringone on transformation efficiency were evaluated. The optimal concentration of kanamycin that could effectively inhibit cell growth and division in non-transgenic tissues was 50 mg l⁻¹ and thus all putative transgenic plants were obtained on induction medium containing 50 mg l⁻¹ kanamycin. The verification of transformants was achieved by both histochemical GUS assay and PCR amplification of nptII gene. Southern blot analysis was performed to further confirm that transgene nptII was stably integrated into the A. racemosus genome. A transformation frequency of approximately 10% was achieved using this protocol, but no beneficial effect from the addition of acetosyringone (50 μM) was observed. This transformation system will be a useful tool for future studies of genes responsible for Se-accumulation in A. racemosus.     

Expression of foreign genes in regenerated plants and in their progeny

Chimeric genes comprised of the nopaline synthase promoter and bacterial coding sequences specifying resistance to kanamycin, chloramphenicol or methotrexate, were inserted into the non-oncogenic Ti plasmid vector pGV3850 by recombination (through homologous pBR322 sequences present in the chimeric gene constructs and pGV3850). These co-integrates in Agrobacterium were used to infect single plant protoplasts of Nicotiana by co-cultivation. The resistance traits allowed the selection of transformed calli in tissue culture in the presence of the appropriate antibiotic. Furthermore, as a non-oncogenic Ti plasmid was used for the protoplast transformation, phenotypically normal and fertile plants could be regenerated from the resistant calli. We have shown that these fully differentiated plant tissues exhibit functional expression of resistance traits (Km^R and Cm^R). All plants carrying the chimeric genes developed normally, flowered, and set seeds. The inheritance of several of these resistance traits was analyzed and shown to be Mendelian. These results are model experiments to demonstrate that genes of interest can be systematically transferred to the genome of plants using non-oncogenic Ti plasmid derivatives; and that transformed plants are capable of normal growth and differentiation, thus providing a natural environment for the study of gene expression and development of plant cells.