<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of embryogenic cell suspension cultures of <Emphasis Type="Italic">Santalum album</Emphasis> L.

An efficient method for Agrobacterium-mediated genetic transformation of embryogenic cell suspension cultures of Santalum album L. is described. Embryogenic cell suspension cultures derived from stem internode callus were transformed with Agrobacterium tumefaciens harbouring pCAMBIA 1301 plant expression vector. Transformed colonies were selected on medium supplemented with hygromycin (5 mg/l). Continuously growing transformed cell suspension cultures were initiated from these colonies. Expression of β-glucuronidase in the suspension cultures was analysed by RT-PCR and GUS histochemical staining. GUS specific activity in the transformed suspension cultures was quantified using a MUG-based fluorometric assay. Expression levels of up to 105,870 pmol 4-MU/min/mg of total protein were noted in the transformed suspension cultures and 67,248 pmol 4-MU/min/mg of total protein in the spent media. Stability of GUS expression over a period of 7 months was studied. Plantlets were regenerated from the transformed embryogenic cells. Stable insertion of T-DNA into the host genome was confirmed by Southern blot analysis. This is the first report showing stable high-level expression of a foreign protein using embryogenic cell suspension cultures in S. album. U. K. S. Shekhawat and T. R. Ganapathi contributed equally to this work.     

Stable transformation of peanut callus viaAgrobacterium-mediated DNA transfer

Transformed callus was produced from peanut (Arachis hypogaea L. cv. Okrun) hypocotyl explants after four days of co-cultivation withAgrobacterium tumefaciens strains EHA101, LBA4404 or ASE1 carrying the binary vector pKYLX71GUS on a defined medium followed by selection with kanamycin (200 mg l^–1). Transformed calluses were cultured as independent cell lines potentially derived from a single transformation event. Stable integration and expression of foreign gene(s) in the callus was confirmed by Southern and western blot analyses and enzyme assays. A few cell lines showed a single insert of the foreign gene. Using the above protocol, transformed peanut callus expressing the peanut stripe virus coat protein gene was obtained.     

Transgenic papaya plants from Agrobacterium-mediated transformation of petioles of in vitro propagated multishoots

Summary In order to establish a model system for introduction of foreign genes into papaya (Carica papaya L.) plants by Agrobacterium-mediated transformation, petioles from multishoots were used as explant source and bacterial neomycin phosphotransferase II (NPT II) gene and -glucuronidase (GUS) gene were used as a selection marker and a reporter, respectively. Cross sections of papaya petioles obtained from multishoots micropropagated in vitro were infected with A. tumefaciens LBA4404 containing NPTII and GUS genes and co-cultured for 2 d. The putative transformed calluses were identified by growth on the selective medium containing kanamycin and carbenicillin, and consequently regenerated to plants via somatic embryogenesis. Thirteen putative transgenic lines were obtained from a total of 415 petiole fragments treated. Strong GUS activity was detected in the selected putative transgenic calli or plants by fluorogenic assay. Western blot analysis using GUS antiserum confirmed that the GUS protein was expressed in putative transformed papaya cells and transgenic plants. The presence of the GUS gene in the papaya tissues was detected by PCR amplification coupled with Southern blot.     

CaMV 35S promoter directs β-glucuronidase expression in the laticiferous system of transgenic Hevea brasiliensis (rubber tree)

Hevea brasiliensis anther calli were genetically transformed using Agrobacterium GV2260 (p35SGUSINT) that harboured the β-glucuronidase (gus) and neomycin phosphotransferase (nptII) genes. β-Glucuronidase protein (GUS) was expressed in the leaves of kanamycin-resistant plants that were regnerated, and the presence of the gene was confirmed by Southern analysis. GUS was also observed to be expressed in the latex and more importantly in the serum fraction. Transverse sections of the leaf petiole from a transformed plant revealed GUS expression to be especially enhanced in the phloem and laticifers. GUS expression was subsequently detected in every one of 194 plants representing three successive vegetative cycles propagated from the original transformant. Transgenic Hevea could thus facilitate the continual production of foreign proteins expressed in the latex. Received: 14 February 1997 / Revision received: 16 August 1997 / Accepted: 20 July 1997     

Introduction of Foreign Gene into Small Cell Groups Digested Partial-Enzymically in Rice and Regeneration of Transgenic Plants

Suspension cultures of small cell groups (SCG; ca. 50–100 cells per group) were established from calli of Japonica rice Fang 7 and Hl24. The SCG were partially digested and transformed by plasmid pBll21 harboring the NPT-II (neomycin phosphotransferase) and GUS (betaglucuronidase) genes. Plasmid DNA was introduced into cells' by PEG, electroporation and PEG plus electroporation. NTP-II and GUS activity assay showed that the report genes were expressed in transformed cells. Transgenic plants were regeneiated possessing GUS activity due to the integration of intact foreign DNA into their genome as evidanced by hybridization. The results prove that the partially digested SCG is a potential, feasible system as receptor for gene transfer, especially for plants which are difficult for protoplast culture and plant regeneration from protoplasts.     

High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco.

We have previously shown that yeast scaffold attachment regions (SARs) flanking a chimeric beta-glucuronidase (GUS) reporter gene increased per-copy expression levels by 24-fold in tobacco suspension cell lines stably transformed by microprojectile bombardment. In this study, we examined the effect of a DNA fragment originally identified in a tobacco genomic clone by its activity in an in vitro binding assay. The tobacco SAR has much greater scaffold binding affinity than does the yeast SAR, and tobacco cell lines stably transformed with constructs containing the tobacco SAR accumulated greater than fivefold more GUS enzyme activity than did lines transformed with the yeast SAR construct. Relative to the control construct, flanking the GUS gene with plant SARs increased overall expression per transgene copy by almost 140-fold. In transient expression assays, the same construct increased expression only approximately threefold relative to a control without SARs, indicating that the full SAR effect requires integration into chromosomal DNA. GUS activity in individual stable transformants was not simply proportional to transgene copy number, and the SAR effect was maximal in cell lines with fewer than approximately 10 transgene copies per tobacco genome. Lines with significantly higher copy numbers showed greatly greatly reduced expression relative to the low-copy-number lines. Our results indicate that strong SARs flanking a transgene greatly increases expression without eliminating variation between transformants. We propose that SARs dramatically reduce the severity or likelihood of homology-dependent gene silencing in cells with small numbers of transgenes but do not prevent silencing of transgenes present in many copies.     

Analysis of conditions forAgrobacterium-mediated transformation of tobacco cells in suspension

We have developed anAgrobacterium-mediated transformation system, using tobacco cell suspensions, that permits evaluation of factors affecting transformation within seven days of co-cultivation. Tobacco cell transformation was determined by monitoring -glucuronidase (GUS) activity detected in plant cell extracts. The use of a chimeric gene construct, 35S-GUS/INT, containing a portable intron in theuidA reading frame, assured only plant-specific GUS expression. During the co-cultivation period, induction of the bacterialvir-region was monitored using a heterologous gene construct composed of avirB promoter fragment from pTiC58 fused to the chloramphenicol acetyltranferase (CAT) gene ofTn9. Tobacco cell transformants were confirmed by antibiotic selection of transformed plant cells and by X-Gluc staining. Maximum transformation was obtained when plant suspension cultures were growing rapidly which also was coincidental with elevated levels of bacterialvir-region expression. One week after co-cultivation, the transformed cultures exhibited a stable pattern of GUS activity which remained constant without antibiotic selection. The system was used to compare the virulence of a number ofAgrobacterium strains. GUS activity of plant cells co-cultivated with a strain containing a cointegrate plasmid was 3-fold higher than that of one with a binary configuration of the T-DNA. When the co-cultivatingAgrobacterium strain also carried the plasmid used to monitorvir induction, the frequency of transformation was reduced by as much, as 97%.     

Transformation system of rice suspension-cultured microcolonies by Electroporation

For establishing a transformation system of rice (Oryza sativa), after three days of culture embryogenic suspension-cultured cell clusters were enzymatically macerated for 2 hours in electroporation buffer containing 2% cellulase and filtered through 550, 400, 250 and 100 μm stainless mesh. Filtered embryogenic microcolonies of 100–250 μm with pBI121 were electroporated at 400 V/cm for 1.2 ms. Four weeks after the electroporation, stable transformed calli were obtained at a frequency of 72% on the selection medium containing 100 mg/L kanamycin. GUS gene in the genomic DNA among 20 out of 22 putative transformed calli lines were detected by PCR analysis. The expression of GUS gene into the kanamycin-resistance calli was confirmed by spectrophotometric assay and histochemical assay of GUS activity. In a histochemical study of the transgenic rice regenerants, it was shown that the GUS activity directed by the CaMV 35S promoter was localized mainly in leaf vein and root apex.     

Expression of foreign genes in transgenic yellow-poplar plants

Cells of yellow-poplar (Liriodendron tulipifera L.) were transformed by direct gene transfer and regenerated into plants by somatic embryogenesis. Plasmid DNA bearing marker genes encoding β-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) were introduced by microprojectile bombardment into single cells and small cell clusters isolated from embryogenic suspension cultures. The number of full-length copies of the GUS gene in independently transformed callus lines ranged from approximately 3 to 30. An enzyme-linked immunosorbent assay for NPT II and a fluorometric assay for GUS showed that the expression of both enzymes varied by less than fourfold among callus lines. A histochemical assay for GUS activity revealed a heterogeneous pattern of staining with the substrate 5-bromo-4-chloro-3-indoyl-β-d-glucuronic acid in some transformed cell cultures. However, cell clusters reacting positively (blue) or negatively (white) with 5-bromo-4-chloro-3-indoyl-β-d-glucuronic acid demonstrated both GUS activity and NPT II expression in quantitative assays. Somatic embryos induced from transformed cell cultures were found to be uniformly GUS positive by histochemical analysis. All transgenic plants sampled expressed the two marker genes in both root and shoot tissues. GUS activity was found to be higher in leaves than roots by fluorometric and histochemical assays. Conversely, roots expressed higher levels of NPT II than leaves.     

Transformation of blueberry without antibiotic selection

Transformation of the blueberry cultivar North Country (Vaccinium corymbosum × V. angustifolium) was achieved using the disarmed Agrobacterium tumefaciens isolate LBA4404, containing a binary vector with an intron-containing β-glucuronidase (GUS) marker gene. Plant regeneration was carried out in the absence of antibiotics due to their toxicity to blueberry explants. Agrobacterium contamination was controlled by dipping the explants in antibiotics and rinsing in sterile distilled water. Once regeneration had been achieved, the plantlets were placed on to medium containing the antibiotic ticaricillin at 250 mg litre^-1 to control and try to eliminate any remaining Agrobacterium. Selection of regenerating explants expressing GUS was achieved by growing the plant material for 2 days on a medium containing 4-methyl umbelliferyl glucuronide (MUG), and examining the medium under UV light to detect fluorescent activity. From the 19 explants showing signs of regeneration, seven produced fluorescent patches on MUG medium. From the selected explants, five plantlets were found to express the GUS gene as detected by fluorometric and histochemical analysis. PCR was used to confirm the presence of the GUS and/or NPTII marker genes after 2 years in culture. Bacterial contamination isolated from plant material (which appeared free of contamination) was examined for GUS activity and analysed using PCR with GUS and NPTII specific primers, but no positive results were obtained.     

Programmed Escherichia coli cell lysis by expression of cloned T4 phage lysis genes

Self-disruptive Escherichia coli that produces foreign target protein was developed. E. coli was co-transformed with two vector plasmids, a target gene expression vector and a lysis gene expression vector. The lytic protein was produced after the expression of the target gene, resulting in simplification of the cell disruption process. In this study, the expression of cloned T4 phage gene e or t was used for the disruption of E. coli that produced beta-glucuronidase (GUS) as a model target protein. The expression of gene e did not lead to prompt cell disruption but weakened the cell wall. Resuspension with deionized water facilitated cell lysis, and GUS activity was observed in the resuspended liquid. Expression of gene e at mid logarithmic growth phase was the optimal induction period for GUS production and release. On the other hand, the expression of gene t induced immediate cell lysis, and intracellular GUS was released to the culture medium. Maximum GUS production was obtained when gene t was induced at late logarithmic growth phase.     

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.     

Foreign protein production using plant cell and organ cultures: Advantages and limitations

Plants and plant tissue cultures are used as host systems for expression of foreign proteins including antibodies, vaccines and other therapeutic agents. Recombinant or stably transformed plants and plant cell cultures have been applied for foreign protein production for about 20 years. Because the product concentration achieved exerts a major influence on process economics, considerable efforts have been made by commercial and academic research groups to improve foreign protein expression levels. However, post-synthesis product losses due to protease activity within plant tissues and/or extracellular protein adsorption in plant cell cultures can negate the benefits of molecular or genetic enhancement of protein expression. Transient expression of foreign proteins using plant viral vectors is also a practical approach for producing foreign proteins in plants. Adaptation of this technology is required to allow infection and propagation of engineered viruses in plant tissue cultures for transient protein expression in vitro.     

Mesorhizobium loti increases root-specific expression of a calcium-binding protein homologue identified by promoter tagging in Lotus japonicus

A promoter tagging program in the legume Lotus japonicus was initiated to identify plant genes involved in the nitrogen-fixing symbiosis between legumes and rhizobia. Seven transformed plant lines expressing the promoterless reporter gene uidA (beta-glucuronidase; GUS) specifically in roots and/or nodules were identified. Four of these expressed GUS in the roots only after inoculation with nodule-forming Mesorhizobium loti. In one line (T90), GUS activity was found in the root epidermis, including root hairs. During seedling growth, GUS expression gradually became focused in developing nodules and disappeared from root tissue. No GUS activity was detected when a non-nodulating mutant of M. loti was used to inoculate the plants. The T-DNA insertion in this plant line was located 1.3 kb upstream of a putative coding sequence with strong homology to calcium-binding proteins. Four motifs were identified, which were very similar to the "EF hands" in calmodulin-related proteins, each binding one Ca2+. We have named the gene LjCbp1 (calcium-binding protein). Northern (RNA) analyses showed that this gene is expressed specifically in roots of L. japonicus. Expression was reduced in roots inoculated with non-nodulating M. loti mutants and in progeny homozygous for the T-DNA insertion, suggesting a link between the T-DNA insertion and this gene.     

Direct gene transfer to plants

Evidence for direct, gene-mediated stable genetic transformation of plant cells of Nicotiana tabacum is presented. A selectable hybrid gene comprising the protein coding region of the Tn5 aminoglycoside phosphotransferase type II gene under control of cauliflower mosaic virus gene VI expression signals was introduced into plant protoplasts as part of an Escherichia coli plasmid. The gene was stably integrated into plant genomic DNA and constitutively expressed in selected, drug resistant, protoplast-derived cell clones. The mode of integration of the foreign gene into the plant genome resembled that observed for DNA transfection of mammalian cells. Plants regenerated from transformed cell lines were phenotypically normal and fertile, and they maintained and expressed the foreign gene throughout the development of vegetative and generative organs. Microspores, grown in anther culture, developed into resistant and sensitive haploid plantlets. Genetic crossing analysis of one of the transformed plants revealed the presence of one dominant trait for kanamycin resistance segregating in a Mendelian fashion in the F₁ generation.     

Highly efficient transfer and stable integration of foreign DNA into partially digested rice cells using a pulsed electrophoretic drive

A new method has been developed to introduce foreign DNA into rice cells. Gene delivery occurred when an electrophoretic drive with cycles of intervallic electric field was applied to a mixture containing partially digested small cell groups (SCGs) and plasmid DNAs. Gene transfer efficiency was evaluated by the detection of -glucuronidase (GUS) activity resulting from expression of a chimaeric plasmid DNA. The optimal combination of treatment conditions (3 V/cm, 30 s pulse and 30 min electrophoretic run) produced a frequency of up to 8.2% of blue cells in transformed microcalluses 40 days after culture of treated SCGs without selection for kanamycin resistance. Southern hybridization showed that the foreign gene had integrated into the chromosomal DNA. These results demonstrate that pulsed electrophoretic drive is applicable to the transfer of foreign genes into plant cells.     

Transgenic papaya plants from Agrobacterium-mediated transformation of somatic embryos

Summary Transgenic papaya (Carica papaya L.) plants were regenerated from embryogenic cultures that were cocultivated with a disarmed C58 strain of Agrobacterium tumefaciens containing one of the following binary cosmid vectors: pGA482GG or pGA482GG/cpPRV-4. The T-DNA region of both binary vectors includes the chimeric genes for neomycin phosphotransferase II (NPTII) and ß-glucuronidase (GUS). In addition, the plant expressible coat protein (cp) gene of papaya ringspot virus (PRV) is flanked by the NPTII and GUS genes in pGA482GG/cpPRV-4. Putative transformed embryogenic papaya tissues were obtained by selection on 150 g·ml^–1 kanamycin. Four putative transgenic plant lines were obtained from the cp gene^– vector and two from the cp gene⁺ vector. GUS and NPTII expression were detected in leaves of all putative transformed plants tested, while PRV coat protein expression was detected in leaves of the PRV cp gene⁺ plant. The transformed status of these papaya plants was analyzed using both polymerase chain reaction amplification and genomic blot hybridization of the NPTII and PRV cp genes. Integration of these genes into the papaya genome was demonstrated by genomic blot hybridizations. Thus, like numerous other dicotyledonous plant species, papayas can be transformed with A. tumefaciens and regenerated into phenotypically normal-appearing plants that express foreign genes.Journal Series no. 3757 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Expression of the Pseudomonas syringae avirulence protein AvrB in plant cells alleviates its dependence on the hypersensitive response and pathogenicity (Hrp) secretion system in eliciting genotype-specific hypersensitive cell death.

The nonpathogenic bacteria Pseudomonas fluorescens and Escherichia coli can elicit a genotype-specific hypersensitive response (HR) in plants if they express both the HR and pathogenesis (Hrp) protein secretion system and the HrpZ harpin from P. syringae pv syringae 61 and a P. syringae avirulence (avr) gene whose presence is recognized by a corresponding disease resistance gene in the plant. We have found that the recognition event appears to require transfer of the Avr protein into the plant cell. Elicitation of a genotype-specific HR was observed with avrB+ P. fluorescens in soybean and Arabidopsis plants carrying resistance genes RPG1 and RPM1, respectively, and with avrPto+ E. coll in tomato plants carrying resistance gene PTO, but only if the Hrp secretion system, HrpZ, and the appropriate Avr proteins were produced in the same bacterial cell. The failure of avrB hyperexpression and exogenous AvrB or HrpZ to alleviate these requirements in soybean and Arabidopsis suggests that the site of AvrB action is not in the bacterial cell or plant apoplast. An Arabidopsis rps3 (rpm1) glabrous1 mutant was transformed with constructs expressing avrB and was crossed with an Arabidopsis ecotype Columbia (RPM1 GLABROUS1) plant. F1 seedlings (identified by their kanamycin-resistant, pubescent phenotype) exhibited extensive necrosis on cotyledon leaves 10 days postgermination. Ecotype Columbia and rps3-1 leaves biolistically cobombarded with plasmids expressing the beta-glucuronidase (GUS) gene and avrB failed to produce GUS activity (indicative of cell death) only when RPM1 and avrB were present in the leaf. Thus, both stable and transient expression of avrB in Arabidopsis resulted in RPM1-dependent necrosis, and the only demonstrable site of action for AvrB was inside plant cells.     

Promoterless gus gene shows leaky β-glucuronidase activity during transformation of tomato with bspA gene for drought tolerance

Transformation of tomato (Lycopersicon esculentum Mill.) was carried out using disarmed Agrobacterium tumefaciens strain EHA 105 harboring a binary vector pBIG-HYG-bspA. The plasmid contains the bspA (boiling stable protein of aspen) gene under the control of a CaMV35S promoter and nopaline synthase (NOS) terminator, hygromycin phosphotransferase gene (hpt) driven by nopaline synthase promoter and polyadenylation signal of Agrobacterium gene7 as terminator and a promoterless gus gene. Very strong β-glucuronidase (GUS) expression was observed in transformed tomato plants but never in non-transformed (control). Since GUS expression was observed only in transformed plants, the possibility of the presence of endogenous GUS enzymes was ruled out. Possibility of false GUS positives was also ruled out because the GUS positive explants reacted positively to polymerase chain reaction (PCR) and PCR-Southern tests carried out for the presence of bspA gene, which indicated the integration of T-DNA in tomato genome. The promoterless GUS expression was hypothesized either due to leaky NOS termination signal of bspA gene or due to different cryptic promoters of plant origin. It was concluded that GUS expression was observed in the putative transgenics either due to the read through mechanism by the strong CaMV35S promoter or due to several cryptic promoters driving the gus gene in different transgenic lines.