Identification and fine mapping of a thermo-sensitive chlorophyll deficient mutant in rice (<Emphasis Type="Italic">Oryza sativa </Emphasis>L.)

A thermo-sensitive chlorophyll deficient mutant was isolated from more than 15,000 transgenic rice lines. The mutant displayed normal phenotype at 23°C or lower temperature (permissive temperature). However, when grown at 26°C or higher (nonpermissive temperature) the plant exhibited an abnormal phenotype characterized by yellow green leaves. Genetic analysis revealed that a single nuclear-encoded recessive gene is responsible for the mutation, which is tentatively designed as cde1(t) (chlorophyll deficient 1, temporally). PCR analysis and hygromycin resistance assay indicated the mutation was not caused by T-DNA insertion. To isolate the cde1(t) gene, a map-based cloning strategy was employed and 15 new markers (five SSR and ten InDels markers) were developed. A high-resolution physical map of the chromosomal region around the cde1(t) gene was made using F₂ and F₃ population consisting of 1,858 mutant individuals. Finally, the cde1(t) gene was mapped in 7.5 kb region between marker ID10 and marker ID11 on chromosome 2. Sequence analysis revealed only one candidate gene, OsGluRS, in the 7.5 kb region. Cloning and sequencing of the target region from the cde1(t) mutant showed that a missense mutation occurred in the mutant. So the OsGluRS gene (TIGR locus Os02 g02860) which encode glutamyl-tRNA synthetase was identified as the Cde1(t) gene.     

Transgenic broccoli expressing aBacillus thuringiensis insecticidal crystal protein: Implications for pest resistance management strategies

We usedAgrobacterium tumefaciens to transform flowering stalk explants of five genotypes of broccoli with a construct containing the neomycin phosphotransferase gene and aBacillus thuringiensis (Bt) gene [CryIA(c) type] optimized for plant expression. Overall transformation efficiency was 6.4%; 181 kanamycin-resistant plants were recovered. Of the 162 kanamycin-resistant plants tested, 112 (69%) caused 100% morality of 1st-instar larvae of aBt-susceptible diamondback moth strain. Southern blots of some resistant transformants confirmed presence of theBt gene. Selected plants that gave 100% mortality of susceptible larvae allowed survival of a strain of diamondback moth that had evolved resistance toBt in the field. F₁ hybrids between resistant and susceptible insects did not survive. Analysis of progeny from 26 resistant transgenic lines showed 16 that gave segregation ratios consistent with a single T-DNA integration. Southern analysis was used to verify those plants possessing a single T-DNA integration. Because these transgenic plants kill susceptible larvae and F₁ larvae, but serve as a suitable host for resistant ones, they provide an excellent model for tests ofBt resistance management strategies.     

Inheritance and tagging of gene regulating flowering time in the green manure crop <Emphasis Type="Italic">Sesbania rostrata</Emphasis> (Bremek. &; Obrem.)

This is the first report on genetic studies and molecular tagging of a gene regulating flowering time in the stem nodulating legume crop Sesbania rostrata (Bremek. & Obrem.). An F₂ segregating population was developed from a cross between Trombay Sesbania rostrata-1 (TSR-1, a radiation induced late flowering mutant) and S. rostrata. A phenotypic segregation ratio of 3 (normal flowering):1 (late flowering) in the F₂ generation indicated that the late flowering is governed as a monogenic recessive trait. A genotypic ratio of 1:2:1 in the F₂ generation, determined from phenotypic segregation patterns in 73 F₃ families, confirmed the monogenic inheritance of the late flowering trait. Inter Simple Sequence Repeat (ISSR) and Amplified Fragment Length Polymorphism (AFLP) marker techniques were evaluated for their applicability as genetic marker systems in this green manure crop. Using the F₂ segregating population, an ISSR marker (UBC 881₁₀₀₀) tightly linked to the trait was identified. Two linked AFLP markers GCTG₅₀₀ and CCAT₃₅₀ were also identified. They were found to be at a distance of 1.4 ± 0.034 cM and 8.0 ± 0.047 cM flanking the flowering locus respectively. The ISSR marker UBC 881₁₀₀₀ was converted into a Sequence Characterized Amplified Region (SCAR) marker. The single recessive mutation regulating the late flowering trait and the availability of tightly linked, flanking markers will help in identification and isolation of the gene controlling the flowering time trait.     

The Nir1 locus in barley is tightly linked to the nitrite reductase apoprotein gene Nii

pBNiR1, a cDNA clone encoding part of the barley nitrite reductase apoprotein, was isolated from a barley (cv. Maris Mink) leaf cDNA library using the 1.85 kb insert of the maize nitrite reductase cDNA clone pCIB808 as a heterologous probe. The cDNA insert of pBNiR1 is 503 by in length. The nucleotide coding sequence could be aligned with the 3 end of other higher plant nitrite reductase apoprotein cDNA sequences but diverges in the 3 untranslated region. The whole-plant barley mutant STA3999, previously isolated from the cultivar Tweed, accumulates nitrite after nitrate treatment in the light, has very much lowered levels of nitrite reductase activity and lacks detectable nitrite reductase cross-reacting material due to a recessive mutation in a single nuclear gene which we have designated Nir1. STA3999 has the characteristics expected of a nitrite reductase apoprotein gene mutant. Here we have used pB-NiR1 in RFLP analysis to determine whether the mutation carried by STA3999 is linked to the nitrite reductase apoprotein gene locus Nii. An RFLP was identified between the wild-type barley cultivars Tweed (major hybridising band of 11.5 kb) and Golden Promise (major hybridising band of 7.5 kb) when DraI-digested DNA was probed with the insert from the partial barley nitrite reductase cDNA clone, pBNiR1. DraI-digested DNA from the mutant STA3999 also exhibited a major hybridising band of 11.5 kb after hybridisation with the insert from pBNiR1. F₁ progeny derived from the cross between the cultivar Golden Promise and the homozygous nir1 mutant STA3999 were heterozygous for these bands as anticipated. Co-segregation of the Tweed RFLP band of 11.5 kb and the mutant phenotype (leaf nitrite accumulation after nitrate treatment/loss of detectable nitrite reductase cross-reacting material at M_r 63000) was scored in an F₂ population of 312 plants derived from the cross between the cultivar Golden Promise and the homozygous mutant STA3999. The Tweed RFLP band of 11.5 kb and the mutant phenotype showed strict co-segregation (in approximately one quarter (84) of the 312 F₂ plants examined). Only those F₂ individuals heterozygous for the RFLP pattern gave rise to F₃ progeny which segregated for the mutant phenotype. We conclude that the nir1locus and the nitrite reductase apoprotein gene Nii are very tightly linked.     

<Emphasis Type="Italic">pgd1</Emphasis>, an <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> deletion mutant,is defective in pollen germination

An Arabidopsis deletion mutant was fortuitously identified from the alpha population of T-DNA insertional mutants generated at the University of Wisconsin Arabidopsis Knockout Facility. Segregation and reciprocal crosses indicated that the mutant was a gametophytic pollen sterile mutant. Pollen carrying the mutation has the unusual phenotype that it is viable, but cannot germinate. Thus, the mutant was named pollen germination defective mutant 1 (pgd1), based on the pollen phenotype. Flanking sequences of the T-DNA insertion in the pgd1 mutant were identified by thermal asymmetric interlaced (TAIL) PCR. Sequencing of bands from TAIL PCR revealed that the T-DNA was linked to the gene XLG1, At2g23460, at its downstream end, while directly upstream of the T-DNA was a region between At2g22830 and At2g22840, which was 65 genes upstream of XLG1. Southern blotting and genomic PCR confirmed that the 65 genes plus part of XLG1 were deleted in the pgd1 mutant. A 9,177 bp genomic sequence containing the XLG1 gene and upstream and downstream intergenic regions could not rescue the pgd1 pollen phenotype. One or more genes from the deleted region were presumably responsible for the pollen germination defect observed in the pgd1 mutant. Because relatively few mutations have been identified that affect pollen germination independent of any effect on pollen viability, this mutant line provides a new tool for identification of genes specifically involved in this phase of the reproductive cycle.     

The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation

This paper describes a so-called ternary transformation system for plant cells. We demonstrate that Agrobacterium tumefaciens strain LBA4404 supplemented with a constitutive virG mutant gene (virGN54D) on a compatible plasmid is capable of very efficient T-DNA transfer to a diverse range of plant species. For the plant species Catharanthus roseus it is shown that increased T-DNA transfer results in increased stable transformation frequencies. Analysis of stably transformed C. roseus cell lines showed that, although the T-DNA transfer frequency is greatly enhanced by addition of virGN54D, only one or a few T-DNA copies are stably integrated into the plant genome. Thus, high transformation frequencies of different plant species can be achieved by introduction of a ternary plasmid carrying a constitutive virG mutant into existing A. tumefaciens strains in combination with standard binary vectors.     

Non-antibiotic,efficient selection for alfalfa genetic engineering

A selectable marker gene (SMG), usually conferring resistance to an antibiotic or herbicide, is generally introduced into the plant cells with the gene(s) for the trait of interest to allow only the cells that have integrated and express the foreign sequences to regenerate into a plant. The availability of several SMGs for each plant species is useful for both basic and applied research to combine several genes of interest in the same plant. A selection system based on gabaculine (3-amino-2,3-dihydrobenzoic acid) as the selective substance and the bacterial hemL gene [encoding a mutant for of the enzyme glutamate 1-semialdehyde aminotransferase (GSA-AT)] as the SMG was previously used for genetic transformation of tobacco. The hemL gene is a good candidate for a safe SMG, because GSA-AT is present in all plants and is likely involved in one metabolic step only, so that unintended effects of its overexpression in plants are not probable. In this work, we have compared this new selection system with the conventional, kanamycin-based system for alfalfa Agrobacterium-mediated transformation. The hemL and NptII genes were placed together into a T-DNA under the control of identical promoters and terminators. We show that the gabaculine-based system is more efficient than the conventional, kanamycin-based system. The inheritance of hemL was Mendelian, and no obvious phenotypic effect of its expression was observed.     

High Efficiency Transgene Segregation in Co-Transformed Maize Plants using an Agrobacterium Tumefaciens 2 T-DNA Binary System

For regulatory issues and research purposes it would be desirable to have the ability to segregate transgenes in co-transformed maize. We have developed a highly efficient system to segregate transgenes in maize that was co-transformed using an Agrobacterium tumefaciens 2 T-DNA binary system. Three vector treatments were compared in this study; (1) a 2 T-DNA vector, where the selectable marker gene bar (confers resistance to bialaphos) and the -glucuronidase (GUS) reporter gene are on two separate T-DNA's contained on a single binary vector; (2) a mixed strain treatment, where bar and GUS are contained on single T-DNA vectors in two separate Agrobacterium strains; (3) and a single T-DNA binary vector containing both bar and GUS as control treatment. Bialaphos resistant calli were generated from 52 to 59% of inoculated immature embryos depending on treatment. A total of 93.4% of the bialaphos selected calli from the 2 T-DNA vector treatment exhibited GUS activity compared to 11.7% for the mixed strain treatment and 98.2% for the cis control vector treatment. For the 2 T-DNA vector treatment, 86.7% of the bialaphos resistant/GUS active calli produced R₀ plants exhibiting both transgenic phenotypes compared to 10% for the mixed strain treatment and 99% for the single T-DNA control vector treatment. A total of 87 Liberty herbicide (contains bialaphos as the active ingredient) resistant/GUS active R₀ events from the 2 T-DNA binary vector treatment were evaluated for phenotypic segregation of these traits in the R₁ generation. Of these R₀ events, 71.4% exhibited segregation of Liberty resistance and GUS activity in the R₁ generation. A total of 64.4% of the R₀ 2 T-DNA vector events produced Liberty sensitive/GUS active (indicating selectable-marker-free) R₁ progeny. A high frequency of phenotypic segregation was also observed using the mixed strain approach, but a low frequency of calli producing R₀ plants displaying both transgenic phenotypes makes this method less efficient. Molecular analyses were then used to confirm that the observed segregation of R₁ phenotypes were highly correlated to genetic segregation of the bar and GUS genes. A high efficiency system to segregate transgenes in co-transformed maize plants has now been demonstrated.     

Molecular genetic mapping of a high-lysine mutant gene (<Emphasis Type="Italic">opaque-16</Emphasis>) and the double recessive effect with <Emphasis Type="Italic">opaque-2</Emphasis> in maize

The lysin content in maize endosperm protein is considered to be one of the most important traits for determining the nutritional quality of food and feed. Improving the protein quality of the maize kernel depends principally on finding a mutant with a higher lysine content. Two high-lysine mutant lines with opaque endosperm, QCL3024 and QCL3021, were isolated from a self-cross population derived from Robertsons Mutator stocks. The gene controlling this mutation is temporarily termed opaque-16 (o16). In order to illuminate the genetic locus and effect of the o16 gene, two F_2:3 populations, one developed from a cross between QCL3024 and QCL3010 (a wild type line) and another from a cross between Qi205 (opaque-2 line) and QCL3021, were created, and F₃ seeds from the F₂ plants in the two populations were evaluated for lysine content. The distributions of lysine content and tests for their normality indicate that the lysine content in the two populations is regulated by the major gene of o16 and genes of o2 and o16, respectively. Based on two data sets of the linkage maps of the F₂ plant marker genotypes and the lysine content of F₃ seeds originating from the two F_2:3 populations, the o16 gene was located within 5 cM, at either 3 or 2.2 cM from umc1141 in the interval between umc1121 and umc1141 on the long arm of chromosome 8, depending on the recombination rate in the two populations as determined by composite interval mapping. According to the data of the F_2:3 population constructed from the o2 and o16 lines, the double recessive mutant effect was analyzed. The average lysine content of the F₃ o2o2o16o16 families identified by the umc1066 and umc1141 markers was approximately 30% higher than that of the F₃ o2o2 and o16o16 families, respectively. The lysine content of seven F₃ families among nine F₃ double recessive mutant families showed different increments, with an average increase of some 6% compared with that of the maternal o2 line. The potential application of the o16 mutant for maize high-lysine breeding may be to combine it with the o2 mutant bearing modifier genes, thus obtaining a mutant with much higher lysine content. For the purpose of pyramiding the o16 with o2 genes, the availability of closely linked markers of the o16 and o2 loci will facilitate marker-assisted selection and greatly reduce breeding time and effort.     

Reducing the tetraploid non-nodulating alfalfa (Medicago sativa) MnNC-1008(NN) germ plasm to the diploid level

MnNC-1008(NN) (referred to as MN-1008) is a tetraploid alfalfa mutant with two recessive genes (nn ₁ and nn ₂ )conditioning the non-nodulating trait. The tetraploid level (2n=4x=32) of this Medicago sativa germ plasm was reduced to the diploid (2n=2x=16) level using the 4x-2x genetic cross originally described as a workable method for the induction of haploidy in alfalfa by T. E. Bingham. In our experiments more than 7000 emasculated flowers of a single non-nodulating MN-1008 mutant alfalfa plant with purple petals were cross-pollinated with pollen from a single, diploid, yellow-flowered alfalfa plant. Mature seeds from these crosses were collected and germinated, after which the plants were subjected to morphological and cytogenetic analyses as well as to DNA fingerprinting. Out of 26 viable progeny, 6 were hybrid plants, 19 proved to be self-mated derivatives of MN-1008, while one descendant turned out to be a diploid (2n=2x=16), purple flowered, non-nodulating plant denoted as M. sativa DN-1008. This diploid, non-nodulating alfalfa plant can serve as starting material to facilitate the comprehensive morphological, physiological and genetic analysis (gene mapping and cloning) of nodulation in order to learn more about the biology of the symbiotic root nodule development. To produce diploid, nodulating hybrid F₁ plants, DN-1008 was crossed with a diploid, yellow-flowered M. sativa ssp. quasifalcata plant. An F₂ population segregating the nn ₁ and nn ₂ genes in a diploid manner, in which the genetic analysis is more simple than in a tetraploid population, can be established by self-mating of the F₁ plants.     

Chromosomal localization of foreign genes in Petunia hybrida

Summary A F1 hybrid of Petunia hybrida, heterozygous for at least one marker on each of the seven chromosomes, was transformed with a modified strain of Agrobacterium tumefaciens in which the phytohormone biosynthetic genes in the transferred DNA (T-DNA) were replaced with a NOS/NPTII/NOS chimeric gene and a wildtype nopaline synthase (NOS) gene. The chimeric gene, which confers kanamycin resistance, was used as selectable marker during the transformation process and the NOS gene was used as a scorable marker in the genetic studies. After plants had been regenerated from the transformed tissues, the transgenic plants that expressed both of these markers were backcrossed to the parental lines. The offspring were examined for the segregation of the NOS gene and the Petunia markers. Genetic mapping was thus accomplished in a single generation.By Southern hybridization analysis we confirmed the presence of the expected T-DNA fragments in the transformed plants. Four out of the six plants presented here, had just one monomeric T-DNA insertion. The sizes of the plant/T-DNA junction fragments suggest that the integration occurred in different sites of the Petunia genome. One transformant gave a more complicated hybridization pattern and possibly has two T-DNA inserts. Another transgenic plant was earlier reported (Fraley et al. 1985) to have two, possibly tandemly repeated T-DNAs.Data is presented on the genetic localization of the T-DNA inserts in six independently obtained transgenic plants. The T-DNA inserts in three plants were mapped to chromosome I. However, the distances between the NOS gene and the marker gene on this chromosome were significantly different. In another transgenic plant the NOS gene was coinherited with the marker on chromosome IV. Two other transgenic plants have the T-DNA insert on chromosome III. A three point cross enabled us to determine that both plants have the NOS gene distally located from the peroxidaseA (prxA) marker and both plants showed about 18% recombination. However, Southern hybridization analysis shows that the sizes of the plant/T-DNA junction fragments in these transgenic plants are different, thus suggesting that the integrations occurred in different sites.     

拟南芥ABA敏感突变体asm1的分离与鉴定

以拟南芥(Arabidopsis thaliana)为研究材料,从T-DNA突变体库中筛选分离得到1株脱落酸(ABA)敏感突变体asm1(ABA sensitive mutant 1,asm1),在含有ABA的培养基中,与野生型相比,asm1突变体的根伸长明显受到抑制,且其种子萌发结果显示asm1对ABA同样表现出敏感特性。在生长发育方面,asm1突变体抽苔时间提前,植株矮化,并且荚果长度明显小于野生型。利用远红外成像系统分析发现,在干旱胁迫下asm1突变体叶面温度高于野生型;失水率分析显示突变体失水率降低以及水分散失减少。遗传学分析表明,asm1是单基因隐性突变且与一个T-DNA插入共分离;通过图位克隆成功获得候选基因ASM1。RT-PCR结果显示,在突变体中ASM1的表达受到抑制,并且能够调控多种ABA信号通路和胁迫应答基因的表达水平。研究结果表明,ASM1可能参与调控ABA信号转导并应答干旱胁迫。     

The use of the two T-DNA binary system to derive marker-free transgenic soybeans

Summary A binary vector, pPTN133, was assembled that harbored two separate T-DNAs. T-DNA one contained a bar cassette, while T-DNA two carried a GUS cassette. The plasmid was mobilized into the Agrobacterium tumefaciens strain EHA101. Mature soybean cotyledonary node explants were inoculated and regenerated on medium amended with glufosinate. Transgenic soybeans were grown to maturity in the greenhouse. Fifteen primary transformants (T₀) representing 10 independent events were characterized. Seven of the 10 independent T₀ events co-expressed GUS. Progeny analysis was conducted by sowing the T₁ seeds and monitoring the expression of the GUS gene after 21 d. Individual T₁ plants were subsequently scored for herbicide tolerance by leaf painting a unifoliate leaf with a 100 mgl⁻¹ solution of glufosinate and scoring the leaf 5 d post application. Herbicide-sensitive and GUS-positive individuals were observed in four of the 10 independent events. Southern blot analysis confirmed the absence of the bar gene in the GUS positive/herbicide-sensitive individuals. These results demonstrate that simultaneous integration of two T-DNAs followed by their independent segregation in progeny is a viable means to obtain soybeans that lack a selectable marker.     

Evaluation of a cotyledonary node regeneration system forAgrobacterium-mediated transformation of pea (Pisum sativum L.)

Summary A rapid regeneration system was used for studies ofAgrobacterium-mediated transformation inPisum sativum L. Cotyledonary node explants were inoculated withAgrobacterium tumefaciens strains containing binary vectors carrying genes for nopaline synthase (NOS),β-glucuronidase (GUS), and neomycin phosphotransferase (NPTII) and placed on selection medium containing either 75 or 150 mg/liter kanamycin. A GUS encoding gene (uidA) containing an intron was used to monitor gene expression from 6 to 21 days postinoculation. GUS activity could be observed 6 days after inoculation in the area of the explant in which regeneration-occurred. Regenerating tissue containing transformed cells was observed in explants on selection medium 21 days postinoculation. Using this system, a single transgenic plant was obtained. Progeny of this plant, which contained two T-DNA inserts, demonstrated segregation for the inserts and for expression of the NOS gene in the selfed R₁ progeny. NPTII activity was observed in the R₂ generation, indicating inheritance and expression of the foreign DNA over at least two generations. Attempts to repeat this procedure were unsuccessful.     

Location of the recessive gene ym (yellow margin) on chromosome 12 of diploid Solatium tuberosum by means of trisomic analysis

Summary Ten out of twelve primary trisomics of dip-loid S. tuberosum were crossed as females with a recessive mutant for yellow margin (ym ym) obtained from S. phureja. All primary trisomics used proved to be homozygous dominant. Trisomic plants from all ten F₁'s were backcrossed with the mutant and trisomics from eight F₁'s were crossed also with a disomic heterozygous f₁ plant from triple 10 X mutant.In both BC₁ and half sib progeny of each trisomic type the mutant plants were easily identified because of their typical small roundish leaflets with yellow or reddish margins. The observed segregation ratios for normal to mutant were tested against the expected non-critical ratios and against various expected critical ratios.From the results of these tests it is concluded that the gene ym is located on chromosome 12 of the potato. A hypothesis of linkage between ym and a gene l _x for lethality is put forward. It is concluded that l _x is not identical with a previously detected recessive gene l ₂ which is responsible for yellow cotyledons and lethality.     

拟南芥叶形态建成基因ABL1的图位克隆及鉴定

前期研究表明ABL1可能在植物叶发育过程中扮演重要的角色,其突变表现为叶片生长迟缓、成熟叶片叶缘缺刻明显等生长缺陷特征。该研究利用图位克隆及其精细定位技术,将ABL1基因锁定在2个SSLP标记T23K8和T8F5之间,该区间包含44个基因。通过生物信息学成功找到ABL1突变基因为拟南芥FAS1,该基因编码染色质组装因子CAF1的一个亚基,在植物顶端分生组织生长调控中扮演重要角色。RT-PCR结果显示,该基因表达受阻,功能互补实验证实abl1突变体的确是FAS1基因的一个新等位突变。研究结果暗示,ABL1/FAS1在植物叶形态建成中也起着重要作用。     

Isolation and characterization of a novel semi-lethal Arabidopsis thaliana mutant of gene for pentatricopeptide (PPR) repeat-containing protein

A novel Arabidopsis thaliana mutant of one member of the pentatricopeptide repeat (PPR) gene family has been identified among T-DNA insertion lines. Tagging of the At1g53330 gene caused the appearance of a semi-lethal mutation with a complex phenotypic expression from embryo lethality associated with the abnormal pattern of cell division during globular to heart transition to fertile plants with just subtle phenotypic changes. The PPR protein At1g53330.1 was predicted to be targeted to mitochondria by TargetP and MitoProt programs. Complementation analysis confirmed that the phenotype is a result of a single T-DNA integration. A thorough functional analysis of this mutant aimed at finding a particular organelle target of At1g53330.1 protein will follow.     

Inheritance of annual habit in celery: cosegregation with isozyme and anthocyanin markers

Summary Vernalization response was determined in an annual and two biennial celery strains, Apium graveolens L. and their F₂ hybrids. Although the annual strain did not require vernalization to bolt, plants exposed to 10°C for 7 days bolted 2 weeks earlier than non-treated plants. Inheritance studies based on F₂ and backcross segregations demonstrate that annual habit in celery is partially dominant over biennial and determined by a single gene designated Hb. Cosegregation studies of this trait with nine isozyme loci and a gene determining petiole anthocyanin pigmentation disclosed the following linkage relationships: Adh-1-Sdh-1-Mdh-1, and Got-1-Mdh-2-Hb-A. The recombination frequency observed for Hb and Mdh-2 was too large to use the latter as a useful marker for annual habit.     

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.