Factors influencing <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of monocotyledonous species

Summary Since the success of Agrobacterium-mediated transformation of rice in the early 1990s, significant advances in Agrobacterium-mediated transformation of monocotyledonous plant species have been achieved. Transgenic plants obtained via Agrobacterium-mediated transformation have been regenerated in more than a dozen monocotyledonous species, ranging from the most important cereal crops to ornamental plant species. Efficient transformation protocols for agronomically important cereal crops such as rice, wheat, maize, barley, and sorghum have been developed and transformation for some of these species has become routine. Many factors influencing Agrobacterium-mediated transformation of monocotyledonous plants have been investigated and elucidated. These factors include plant genotype, explant type, Agrobacterium strain, and binary vector. In addition, a wide variety of inoculation and co-culture conditions have been shown to be important for the transformation of monocots. For example, antinecrotic treatments using antioxidants and bactericides, osmotic treatments, desiccation of explants before or after Agrobacterium infection, and inoculation and co-culture medium compositions have influenced the ability to recover transgenic monocols. The plant selectable markers used and the promoters driving these marker genes have also been recognized as important factors influencing stable transformation frequency. Extension of transformation protocols to elite genotypes and to more readily available explants in agronomically important crop species will be the challenge of the future. Further evaluation of genes stimulating plant cell division or T-DNA integration, and genes increasing competency of plant cells to Agrobacterium, may increase transformation efficiency in various systems. Understanding mechanisms by which treatments such as desiccation and antioxidants impact T-DNA delivery and stable transformation will facilitate development of efficient transformation systems.     

Desiccation of plant tissues post-<Emphasis Type="Italic">Agrobacterium</Emphasis> infection enhances T-DNA delivery and increases stable transformation efficiency in wheat

Summary Factors influencing the Agrobacterium-mediated transformation of both monocotyledonous and dicotyledonous plant species have been widely investigated. These factors include manipulating Agrobacterium strains and plasmids, growth conditions for vir gene induction, plant genotype, inoculation and co-culture conditions, and the selection agents and their application regime. We report here a novel physical parameter during co-culture, desiccation of plant cells or tissues post-Agrobacterium infection, which greatly enhances transfer DNA (T-DNA) delivery and increases stable transformation efficiency in wheat. Desiccation during co-culture dramatically suppressed Agrobacterium growth, which is one of the factors known to favor plant cell recovery. Osmotic and abscisic acid treatments and desiccation prior to inoculation did not have the same enhancement effect as desiccation during co-culture on T-DNA delivery in wheat. An efficient transformation protocol has been developed based on desiccation and is suitable for both paromomycin and glyphosate selection. Southern analysis showed approximately 67% of transgenic wheat plants received a single copy of the transgene.     

Agrobacterium-mediated genetic transformation of plants: The role of host

Agrobacterium-mediated genetic transformation is the most widely used technology to obtain overexpression of recombinant proteins in plants. Molecular events that occur within Agrobacterium during interactions with host plants have been studied extensively, and now we have a reasonable understanding the key factors involved in the regulation of T-DNA nuclear import and genomic integration. By contrast, very little is known about the events that take place in the host cells during genetic transformation by Agrobacterium. Here, we describe the plant-related factors including genotype, genes, proteins, competency of target tissues and phenolic compounds that participate in Agrobacterium-mediated genetic transformation and discuss their possible roles in this process. Because Agrobacterium probably adapts existing cellular processes for its life cycle, identifying the processes in host cells during Agrobacterium infection might contribute to better understanding of basic biological processes as cell communication, intracellular transport and DNA repair and recombination as well as to expanding the host range of Agrobacterium as a genetic engineering tool.     

BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium

Development of efficient methods to transfer large DNA fragments into plants will greatly facilitate the map-based cloning of genes. The recently developed BIBAC and TAC vectors have shown potential to deliver large DNA fragments into plants via Agrobacterium-mediated transformation. Here we report that BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium. We tested the possible factors that may cause instability, including the insert sizes of the BIBAC and TAC constructs, potato DNA fragments consisting of highly repetitive or largely single-copy DNA sequences, different Agrobacterium transformation methods and different Agrobacterium strains. The insert sizes of the potato BIBAC and TAC constructs were found to be critical to their stability in Agrobacterium. All constructs containing a potato DNA fragment larger than 100 kb were not stable in any of the four tested Agrobacterium strains, including two recA deficient strains. We developed a transposon-based technique that can be used to efficiently subclone a BAC insert into two to three BIBAC/TAC constructs to circumvent the instability problem.Communicated by J. Dvorak     

Plant transformation by microinjection techniques

Several techniques have been developed for introducing cloned genes into plant cells. Vectorless delivery systems such as PEG-mediated direct DNA uptake (e.g. Pasz-kowski et al. 1984), electroporation (e.g. Shillito et al. 1985), and fusion of protoplasts with liposomes (Deshayes et al. 1985) are routinely used in many experiments (see several chapters of this issue). A wide range of plant species, dicotyledonous as well as monocotyledonous, has been transformed by these vectorless DNA transfer systems. However, the availability of an efficient protoplast regeneration system is a prerequisite for the application of these techniques. For cells with intact cell walls and tissue explants the biological delivery system of virulent Agrobacterium species has been routinely used (for review see Fraley et al. 1986). However, the host range of Agrobacterium restricts the plant species, which can be transformed using this vector system. In addition, all these methods depend on selection systems for recovery of transformants. Therefore a selection system has to be established first for plant species to be transformed. The microinjection technique is a direct physical approach, and therefore host-range independent, for introducing substances under microscopical control into defined cells without damaging them. These two facts differentiate this technique from other physical approaches, such as biolistic transformation and macroinjection (see chapters in this issue). In these other techniques, damaging of cells and random manipulation of cells without optical control cannot be avoided so far. In recent years microinjection technology found its application in plant sciences, whereas this technique has earlier been well established for transformation of animal tissue culture cells (Capecchi 1980) and the production of transgenic animals (Brin-ster et al. 1981, Rusconi and Schaffner 1981). Furthermore, different parameters affecting the DNA transfer via microinjection, such as the nature of microinjected DNA, and cell cycle stage, etc, have been investigated extensively in animal cells (Folger et al. 1982, Wong and Capecchi 1985), while analogous experiments on plant cells are still lacking.     

In vitro chili pepper biotechnology

Summary Chili pepper is an important horticultural crop that can surely benefit from plant biotechnology. However, although it is a Solanaceous member, developments in plant cell, tissue, and organ culture, as well as on plant genetic transformation, have lagged far behind those achieved for other members of the same family, such as tobacco (Nicotiana tabacum), tomato (Lycopersicon esculentum), and potato (Solanum tuberosum), species frequently used as model systems because of their facility to regenerate organs and eventually whole plants in vitro, and also for their ability to be genetically engineered by the currently available transformation methods. Capsicum members have been shown to be recalcitrant to differentiation and plant regeneration under in vitro conditions, which in turn makes it very difficult or inefficient to apply recombinant DNA technologies via genetic transformation aimed at genetic improvement against pests and diseases. Some approaches, however, have made possible the regeneration of chili pepper plants from in vitro-cultured cells, tissues, and organs through organogenesis or embryogenesis. Anther culture has been successfully applied to obtain haploid and doubledhaploid plants. Organogenic systems have been used for in vitro micropropagation as well as for genetic transformation. Application of both tissue culture and genetic transformation techniques have led to the development of chili pepper plants more resistant to at least one type of virus. Cell and tissue cultures have been applied successfully to the selection of variant cells exhibiting increased resistance to abiotic stresses, but no plants exhibiting the selected traits have been regenerated. Production of capsaicinoids, the hot principle of chili pepper fruits, by cells and callus tissues has been another area of intense research. The advances, limitations, and applications of chili pepper biotechnology are discussed.     

Agrobacterium-mediated sorghum transformation

Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a `Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T₀ plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T₁ generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.     

Rearrangements of large-insert T-DNAs in transgenic rice

Introduction of large-DNA fragments into cereals by Agrobacterium-mediated transformation is a useful technique for map-based cloning and molecular breeding. However, little is known about the organization and stability of large fragments of foreign DNA introduced into plant genomes. In this study, we produced transgenic rice plants by Agrobacterium-mediated transformation with a large-insert T-DNA containing a 92-kb region of the wheat genome. The structures of the T-DNA in four independent transgenic lines were visualized by fluorescence in situ hybridization on extended DNA fibers (fiber FISH). By using this cytogenetic technique, we showed that rearrangements of the large-insert T-DNA, involving duplication, deletion and insertion, had occurred in all four lines. Deletion of long stretches of the large-insert DNA was also observed in Agrobacterium.     

Whisker-mediated plant transformation: An alternative technology

A number of different methods, involving direct DNA delivery are now available for plant transformation. Here we review the most recently developed technique which involves the mixing of silicon carbide whiskers with plant cells and plasmid DNA. Fertile transgenic plants have now been produced using whisker-mediated transformation, and this method can now be considered as a simple, inexpensive alternative for plant transformation. A brief review on transformation of animal cells andChlamydomonas using whiskers technology is also included.     

Genetic transformation in higher plants

Successful transformation of plant cells has been obtained utilizing vectors and DNA delivery methods derived from the plant pathogen, Agrobacterium tumefaciens. This soil bacterium is capable of transferring a DNA segment (T‐DNA), located between specific nucleotide border sequences, from its large tumor inducing (Ti) plasmid into the nuclear DNA of infected plant cells. The exploitation of the Agrobacterium/Ti plasmid system for plant cell transformation has been facilitated by (1) the construction of modified Agrobacterium strains in which the genes responsible for pathogenicity have been deleted; (2) the design of intermediate vectors containing selectable drug markers for introducing foreign genes into the Ti plasmid and subsequently into plant cells; and (3) the development of efficient in vitro methods for transforming plant cells and tissues with engineered Agrobacterium strains. These modifications have led to the development of a simple, efficient, and reproducible transformation system from which morphologically normal transformed plants can be readily regenerated. The foreign genes are stably maintained and expressed in the resulting plants and are inherited by progeny as typical Mendelian traits. The availability of transformation systems has already facilitated numerous studies on gene expression and regulation in plants and should eventually allow for the modification of various crop species in an agronomically significant manner. The needs and possibilities for the development of alternate vectors and transformation procedures will be discussed.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of plants by the pTF-FC2 plasmid is efficient and strictly dependent on the MobA protein

In the transformation of plants by Agrobacterium tumefaciens the VirD2 protein has been shown to pilot T-DNA during its transfer to the plant cell nucleus. Other studies have shown that the MobA protein of plasmid RSF1010 is capable of mediating its transfer from Agrobacterium cells to plant cells by a similar process. We have demonstrated previously that plasmid pTF-FC2, which has some similarity to RSF1010, is also able to transfer DNA efficiently. In this study, we performed a mutational analysis of the roles played by A. tumefaciens VirD2 and pTF-FC2 MobA in DNA transfer-mediated by A. tumefaciens carrying pTF-FC2. We show that MobA+/VirD2+ and MobA+/VirD2– strains were equally proficient in their ability to transfer a pTF-FC2-derived plasmid DNA to plants and to transform them. However, the MobA–/VirD2+ strain showed a DNA transfer efficiency of 0.03% compared with that of the other two strains. This sharply contrasts with our results that VirD2 can rather efficiently cleave the oriT sequence of pFT-FC2 in vitro. We therefore conclude that MobA plays a major VirD2-independent role in plant transformation by pTF-FC2.     

A quick method to estimate the T-DNA copy number in transgenic plants at an early stage after transformation,using inverse PCR

Agrobacterium-mediated transformation of plants is known to result in transgenic plants with a variable number of integrated T-DNA copies [1, 2, 3, 7]. Our aim was to obtain transgenic tobacco plants containing one integrated T-DNA copy per genome. Therefore, a quick method was developed to estimate the T-DNA copy number of young transgenic plantlets within 10 weeks after transformation. Inverse polymerase chain reaction (IPCR) was used to amplify junction fragments, i.e. plant genomic DNA sequences flanking the known T-DNA sequences [5].     

Direct DNA transfer to plant cells

A range of somatic cell and molecular techniques are now available to supplement conventional plant breeding. The introduction and expression of foreign DNA has been used to modify basic aspects of physiology and development, to introduce commercially important characteristics such as herbicide and insect resistance into plants and to insert genes suitable as dominant selectable markers for somatic hybridisation. Several techniques for direct DNA delivery are available, ranging from uptake of DNA into isolated protoplasts mediated by chemical procedures or electroporation, to injection and the use of high-velocity particles to introduce DNA into intact tissues. Direct DNA uptake is applicable to both stable and transient gene expression studies and utilises a range of vectors, including those employed for gene cloning. Although the frequency of stable transformation is low, direct DNA uptake is applicable to those plants not amenable to Agrobacterium transformation, particularly monocotyledons.     

Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment

We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T₀, T₁ and T₂ transgenic plants. Oryza sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hyg^r), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T₁ and T₂ generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T₁ lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression.     

Agrobacterium-mediated transformation of the winter jujube (Zizyphus jujuba Mill.)

Winter jujube, a species that originated in China, is the most prominent elite variety of jujube (Zizyphus jujuba Mill.). Due to its economic value and its recalcitrance to improvements through traditional plant breeding approaches, genetic transformation techniques may have a great potential in providing the means to transfer one or more selected desirable traits into the plant genome. We reported here an improved protocol for the Agrobacterium-mediated transformation of shoot tips of winter jujube. We have identified a set of optimum transformation conditions that take into account Agrobacterium inoculum density, Agrobacterium incubation period, co-cultivation conditions, and vacuum (use of a vacuum pump to create a negative-pressure environment). The highest transformation frequency (5.2%) was obtained when the shoot-tip explants were infected for 10 min and co-cultured for 4 days with Agrobacterium at OD₆₀₀ 0.8 under a negative pressure of 0.5 × 10⁵ Pa. PCR and southern blot analyses confirmed the presence of transgenic plants and the stable integration of the target gene into the genome of regenerated plants. A histochemical staining analysis for GUS activity in the transgenic shoot tips also validated the efficiency of the transformation system.     

Genetic transformation of wheat: progress during the 1990s into the Millennium

Wheat transformation technology has progressed rapidly during the past decade. Initially, procedures developed for protoplast isolation and culture, electroporation- and polyethylene glycol (PEG)-induced DNA transfer enabled foreign genes to be introduced into wheat cells. The development of biolistic (microprojectile) bombardment procedures led to a more efficient approach for direct gene transfer. More recently, Agrobacterium-mediated gene delivery procedures, initially developed for the transformation of rice, have also been used to generate transgenic wheat plants. This review summarises the considerable progress in wheat transformation achieved during the last decade. An increase in food production is essential in order to sustain the increasing world population. This could be achieved by the development of higher yielding varieties with improved nutritional quality and tolerance to biotic and abiotic stresses. Although conventional breeding will continue to play a major role in increasing crop yield, laboratory-based techniques, such as genetic transformation to introduce novel genes into crop plants, will be essential in complementing existing breeding technologies. A decade ago, cereals were considered recalcitrant to transformation. Since then, a significant research effort has been focused on cereals because of their agronomic status, leading to improved genetic transformation procedures (Bommineni and Jauhar 1997). Initially, the genetic transformation of cereals relied on the introduction of DNA into protoplasts and the subsequent production of callus from which fertile plants were regenerated. More recently, major advances have been accomplished in the regeneration of fertile plants from a range of source tissues, providing an essential foundation for the generation of transgenic plants. This review summarises procedures, vectors and target tissues used for transformation, high-lights the limitations of current approaches and discusses future trends. The citation of references is limited, where possible, to the most relevant or recent reports.     

pBECKS

A series of binary T-DNA vectors (pBECKS) has been created for use in theAgrobacterium-mediated genetic transformation of plants. The pBECKS series has corrected the undesirable features of the popular pBIN19 vector; the deleterious mutation within the coding sequence ofnptII has been amended and the cloning sites are now adjacent to the right border repeat in order to reduce the possibility of producing truncated sequences of novel genes within transformants. One set of vectors incorporates various combiantions of the marker genesgusA,C1/Lc,nptII,hph, andbar, for pursuit of early and stable transformation events. A set of constructs which contain deleted T-DNA borders in various combinations and display predictably altered efficacies for gene transfer has also been created. A modular set of vectors has been designed to facilitate the insertion and transfer of novel gene sequences by providing anptII-linked plant expression cassette orlacZ-multiple cloning site. A range of antibiotic resistance genes has been incorporated into the non-T-DNA part of the vectors in order to facilitate their selection across the range ofAgrobacterium virulence strains.     

Floral Spray Transformation Can Efficiently Generate Arabidopsis

In this study, floral spray and floral dip were used to replace the vacuum step in the Agrobacterium-mediated transformation of a superoxide dismutase (SOD) gene into Arabidopsis. The transgene was constructed by using a CaMV 35S promoter to drive a rice cytosolic CuZnSOD coding sequence in Arabidopsis. The transgene construct was developed in binary vectors and mobilized into Agrobacterium. When Arabidopsis plants started to initiate flower buds, the primary inflorescence shoots were removed and then transformed by floral spray or floral dip. More than 300 transgenic plants were generated to assess the feasibility of floral spray used in the in planta transformation. The result indicates that the floral spray method of Agrobacterium can achieve rates of in planta transformation comparable to the vacuum-infiltration and floral dip methods. The floral spray method opens up the possibility of in planta transformation of plant species which are too large for dipping or vacuum infiltration.     

Simple binary vectors for DNA transfer to plant cells

Summary Cosmid binary vectors for the introduction of DNA into plant cells have been constructed. These vectors are derived from the replicon of the broad host range plasmid pRK2 and contain the T-DNA border regions between which have been placed a chimaeric gene conferring resistance to kanamycin in plant cells. Appropriate restriction endonuclease targets have also been placed between the border regions. These binary vectors, in conjunction with appropriate Agrobacterium strains, are capable of delivering DNA to plant cells in cocultivation experiments with very high efficiency. The transformation frequency is shown to be somewhat dependent on the replicon used. re]19850121 rv]19850506 ac]19850513