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A reliable and efficient genetic transformation protocol for various turfgrass species and elite cultivars has been achieved using Agrobacterium tumefaciens. We describe a general protocol for the establishment of embryogenic cell cultures, Agrobacterium tumefaciens-mediated transformation, selection, and regeneration of transgenic turfgrass plants. Embryogenic callus is initiated from mature seeds, maintained by visual selection, and infected with an Agrobacterium tumefaciens strain (LBA4404) that contains either an herbicide-resistant bar gene or an antibiotic-resistant hyg gene driven either by a rice ubiquitin or CaMV35S promoter. Stable transformation efficiencies up to 43.3% were achieved. Southern blot and genetic analysis was used to confirm transgene integration in the turfgrass genomes and normal transmission and stable expression of the transgene in the T1 generation. We demonstrate herein that five elite cultivars of bentgrass can be genetically transformed using this single tissue culture media regime. Additionally, we report the successful Agrobacterium-mediated transformation of an elite tall fescue variety using minor variations in the same transformation protocol. [ABSTRACT FROM AUTHOR]

During the last decade, Agrobacterium-mediated transformation of more than a dozen monocotyledonous plants, including forage and turf grasses, has been achieved. So far, switchgrass is the only warm season grass that has been transformed with A. tumefaciens. We have developed a highly efficient system for transformation of different switchgrass explants utilizing the A. tumefaciens strain AGL1 carrying the binary vector pDM805, containing the phosphinotricin acetyltransferase (bar) and β-glucoronidase (GUS) (uidA or gus) genes. Transformed cultures were selected in the presence of 10 mg/L bialaphos and the resultant plantlets were treated with the herbicide Basta®. The T-DNA delivery frequency was affected by the genotype, explant used, and the presence or absence of acetosyringone during inoculation and cocultivation. The total time required from inoculation to the establishment of plants in soil was 3-4 mo. Stable integration, expression, and inheritance of both transgenes were confirmed by molecular and genetic analyses. Approximately 90% of the tested plants appeared to have only one or two copies of the T-DNA inserts. The transgenes were sexually transmitted through both male and female gametes to the progeny obtained from controlled crosses in the expected segregation ratio of 1∶1 according to a χ2 test at p = 0.05. [ABSTRACT FROM AUTHOR]

Using Eucalyptus camaldulensis as a model system, we describe here a basic Agrobacterium-mediated genetic transformation protocol through organogenesis for the production of transgenic plants. Hypocotyl segments or cotyledon pieces from in vitro seedlings are used as starting materials. The explants are inoculated and cocultivated with a disarmed, binary strain of A. tumefaciens CIB542 harboring a mini Ti plasmid, pBI121. A modified Gamborg's B5 medium is used as the basal culture medium throughout stages of co-cultivation, callus induction and shoot regeneration. The incorporation of neomycin phosphotransferase II (nptII) and β-glucuronidase (gus) genes into the plant nuclear genome are primarily verified by histochemical analysis and polymerase chain reaction (PCR). Modifications of this protocol to use in mature tissues derived from elite trees and other Eucalyptus species are also described. [ABSTRACT FROM AUTHOR]

The yeast Saccharomyces cerevisiae is one of the best characterized eukaryotic organisms. This species has enabled a detailed study of the (genetic) requirements for Agrobacterium-mediated DNA transformation. For instance research with this yeast has led to the recognition that the transforming DNA molecules integrate into the eukaryotic chromosomes either by homologous recombination, which is the preferred pathway in S. cerevisiae, or by nonhomologous end-joining. Based on the protocol for Agrobacterium-mediated transformation of S. cerevisiae methodology has been developed for the transformation of many other yeast and fungal species. [ABSTRACT FROM AUTHOR]

We have devised an easy and effective genetic transformation method for the preeminent edible mushroom, Agaricus bisporus. Our method exploits the T-DNA transfer mechanism in Agrobacterium tumefaciens and relies on the reproductive fruiting body as the recipient tissue. The use of fruiting body explants, particularly the gill, provided highfrequency transformation, overcoming the inefficacy of Agrobacterium-based methods targeting fungal spores or vegetative mycelium. The protocol entails incubation of A. tumefaciens for 3 h with acetosyringone, a signaling molecule that launches the gene transfer mechanism, co-cultivation of the induced bacterium and gill explants for 3 d, and selection for transformants based on an inherited resistance to the antibiotic hygromycin. Between 7 and 28 d on the selection medium, upwards of 95% of the gill explants generate hygromycin-resistant colonies. About 75% of the mushroom transformants show a singlecopy of the hygromycin-resistant gene integrated at random sites in the genome. [ABSTRACT FROM AUTHOR]

Agrobacterium most likely can transform virtually all known plant species, and experimental protocols for Agrobacterium-mediated genetic transformation of yet more plant species, ecotypes, and cultivars are published almost on a daily basis. Interestingly, the Agrobacterium host range is not limited to the plant kingdom, and it has been shown to transform many species of fungi and even prokaryotes. The ability of Agrobacterium to genetically transform HeLa cells further widens the range of potential hosts of Agrobacterium to include humans and perhaps other animal species. Furthermore, because mammalian cells significantly differ from plant cells, they provide a useful experimental system for identification and functional characterization of plant-specific factors involved in the transformation process. Here, we present basic procedures for transfection and Agrobacterium-mediated genetic transformation of mammalian cells. We also demonstrate the use of mammalian cells for studies of the cellular components of the genetic transformation pathway. [ABSTRACT FROM AUTHOR]

This protocol describes the Agrobacterium tumefaciens-mediated nuclear transformation of a microalgae Chlamydomonas reinhardtii, using a gene construct carrying the genes coding for β-glucuronidase (gus), green fluorescent protein (gfp), and hygromycin phosphotransferase (hpt). The transformation frequency with this protocol as revealed by hygromycin resistance was many fold higher (about 50-fold) than that of the commonly used glass bead method of transformation. The simplicity of Agrobacterium-mediated gene transfer and the high transformation frequency as well as the precision of T-DNA integration will enable further molecular dissection of this important model organism as well as other algal systems to understand basic plant metabolic processes as well as to exploit the systems for biotechnological applications. [ABSTRACT FROM AUTHOR]

The genetic transformation of opium poppy, Papaver somniferum, offers the opportunity to study the mechanisms involved in the regulation of benzylisoquinoline and morphinan alkaloid biosynthesis. The development of an efficient transformation protocol for opium poppy has allowed us to transform a range of genotypes from all around the world, including previously recalcitrant high-yielding commercial Australian cultivars. The method involves Agrobacterium tumefaciens infection of hypocotyl explants, followed by the production of antibiotic or herbicide resistant embryogenic callus, the subsequent induction of somatic embryos and development into normal plants. The use of different selective agents, binary vectors, and poppy genotypes has demonstrated the robustness and reliability of this protocol in the production of many hundreds of confirmed transgenic poppies. [ABSTRACT FROM AUTHOR]

Hemp (Cannabis sativa L.) suspension culture cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary plasmid pNOV3635. The plasmid contains a phosphomannose isomerase (PMI) selectable marker gene. Cells transformed with PMI are capable of metabolizing the selective agent, mannose, whereas cells not expressing the gene are incapable of using the carbon source and will stop growing. Callus masses proliferating on selection were screened for PMI expression using a chlorophenol red assay. Genomic DNA was extracted from putatively transformed callus lines and the presence of the PMI gene was confirmed using polymerase chain reaction and Southern hybridization. Using this method, an average transformation frequency of 31.23% ± 0.14 was obtained for all transformation experiments, with a range of 15.1 to 55.3%. [ABSTRACT FROM AUTHOR]

Genetic transformation is an attractive way to improve Panax ginseng C. A. Meyer by introducing new genetic materials and altering metabolic pathways that regulating the production of secondary compounds. In P. ginseng, production of transgenic plants via Agrobacterium tumefaciens is performed via somatic embryogenesis using cotyledon or embryogenic callus as explants. This chapter introduces the protocol of A. tumefaciens mediated-genetic transformation in P. ginseng. [ABSTRACT FROM AUTHOR]

Although rose transformation is successful, it remains difficult to transform myriad rose species as well as different rose genotypes. In this protocol, a detailed description of rose transformation is presented. This protocol relied on Agrobacterium-mediated transfer of embryogenic callus cultures. There are many critical steps that must be followed to achieve successful transformation; however, it is important to keep in mind that these apply to a selected number of genotypes, and as a different genotype is subjected to transformation, modifications of this protocol must be made to achieve successful transformation. [ABSTRACT FROM AUTHOR]

Petunia hybrida genetic transformation continues to be a valuable tool for genetic research into biochemical pathways and gene expression, as well as generating commercial products with varying floral colors. In this chapter, we describe a simple and reproducible genetic transformation protocol for generating transgenic petunia plants harboring a gene of interest and selectable marker. The system utilizes Agrobacterium tumefaciens for transgene integration with plant recovery via shoot organogenesis from leaf explant material. Selection for transgenic plants is achieved using the bar gene conferring resistance to glufosinate or nptII gene for resistance to kanamycin. Transformation efficiencies of around 10% are achievable with shoots being recovered about 8 wk after transgene insertion and rooted plants transferred to the greenhouse about twelve weeks after inoculation. [ABSTRACT FROM AUTHOR]

This chapter provides reproducible methods for the transformation of Dendranthema × grandiflora ‘Shuhou-no-chikara' (standard) and ‘Lineker' (spray) stem internode thin cell layers (TCLs) and conventional stem internode explants using Agrobacterium strains carrying a binary vector with β-glucoronidase (GUS) reporter and nptII selector genes. Transformation efficiencies are reported at the kanamycin selection (callus formation and plantlet rooting), GUS assay, polymerase chain reaction (PCR), and Southern analysis levels. Notes on regeneration improvement through the use of TCLs, as well as the effective use of sonication for both regeneration and generation of transformants, stimulation of Agro-infection and elimination of Agrobacterium, are included. [ABSTRACT FROM AUTHOR]

Carnation is a valuable crop for the cut flower industry and demand for new and improved varieties is growing. However, genetic transformation of carnations is currently limited because of a lack of efficient routine technique. In this chapter, we present an easy and effective protocol for gene transfer to carnation node explants and subsequent adventitious shoot regeneration. For high-adventitious shoot regeneration, node explants from first to third node of 5- to 8-cm long shoots were cultured on Murashige and Skoog (MS) medium, containing 1.0 mg/L thidiazuron (TDZ), 0.1 mg/L α-napthalenoacetic acid (NAA), 20 g/L sucrose, and 2 g/L Gellan gum for 10 d. Then the explants were cut into 8 radial segments and subcultured onto MS medium, containing 1.0 mg/L BA, 0.1 mg/L NAA, 20 g/L sucrose and 2 g/L Gellan Gum. For effective genetic transformation, 3- to 5-d precultured node explants were submerged in an Agrobacerium suspension for 10 min, then cocultivated on filter paper soaked with water and 50 µM acetosyringone (AS). After cocultivation, the explants were cut into eight radial segments and subcultured onto selection medium until transformed shoots regenerated from the explants. [ABSTRACT FROM AUTHOR]

Walnut species are important nut and timber producers in temperate regions of Europe, Asia, South America, and North America. Trees can be impacted by Phytophthora, crown gall, nematodes, and cherry leaf roll virus; nuts can be severely damaged by codling moth and Xanthomonas blight. The long-generation time of walnuts and an absence of identified natural resistance for most of these problems suggest biotechnological approaches to crop improvement. Described here is a somatic embryo based transformation protocol that has been used to successfully insert horticulturally useful traits into walnut. Selection is based on the combined use of the selectable neomycin phosphotransferase (nptII) gene and the scorable uidA gene. Transformed embryos can be germinated or micropropagated and rooted for plant production. The method described has been used to establish field trials of mature trees. [ABSTRACT FROM AUTHOR]

Genetic transformation in strawberry (Fragaria spp.) can be achieved by using the Agrobacterium-mediated procedure on leaves from in vitro proliferated shoots. Regardless of the sufficient regeneration levels achieved from leaf explants of some commercial strawberry genotypes, the regeneration of transformed strawberry plants remains difficult and seems to be strongly genotype dependent. In fact, the main factors that play an important role in the success of strawberry genetic transformation are the availability of both an efficient regeneration protocol and an appropriate selection procedure of the putative transgenic shoots. The strawberry genetic transformation protocol herein described relates to two genotypes resulting from our experience with the highest regeneration and transformation efficiency. The study includes an octoploid Fragaria × ananassa cultivar (Sveva) and a diploid F. vesca cultivar (Alpina W.O.). All the different steps related to the leaf tissue Agrobacterium infection, co-culture, and selection of regenerating adventitious shoots, as well as the following identification of selected lines able to proliferate and root on the selective agent (kanamycin), will be described. [ABSTRACT FROM AUTHOR]

Grapevine (Vitis) is considered to be one of the major fruit crops in the world based on hectares cultivated and economic value. Grapes are used not only for wine but also for fresh fruit, dried fruit, and juice production. Wine is by far the major product of grapes and the focus of this chapter is on wine grape cultivars. Grapevine cultivars of Vitis vinifera L. have a reputation for producing premium quality wines. These premium quality wines are produced from a small number of cultivars that enjoy a high level of consumer acceptance and are firmly entrenched in the market place because of varietal name branding and the association of certain wine styles and regions with specific cultivars. In light of this situation, grapevine improvement by a transgenic approach is attractive when compared to a classical breeding approach. The transfer of individual traits as single genes with a minimum disruption to the original genome would leave the traditional characteristics of the cultivar intact. However, a reliable transformation system is required for a successful transgenic approach to grapevine improvement. There are three criteria for achieving an efficient Agrobacterium-mediated transformation system: (1) the production of highly regenerative transformable tissue, (2) optimal co-cultivation conditions for both grapevine tissue and Agrobacterium, and (3) an efficient selection regime for transgenic plant regeneration. In this chapter, we describe a grapevine transformation system which meets the above mentioned criteria. [ABSTRACT FROM AUTHOR]

Recent advances in plant biotechnology have led to a reliable and reproductive method for genetic transformation of blueberry. These efforts built on previous attempts at transient and stable transformation of blueberry that demonstrated the potential of Agrobacterium tumefaciens-mediated transformation, and as well, the difficulties of selecting and regenerating transgenic plants. As a prerequisite for successful stable transformation, efficient regeneration systems were required despite many reports on factors controlling shoot regeneration from leaf explants. The A. tumefaciens-mediated transformation protocol described in this chapter is based on combining efficient regeneration methods and the results of A. tumefaciens-mediated transient transformation studies to optimize selected parameters for gene transfer. The protocol has led to successful regeneration of transgenic plants of four commercially important highbush blueberry cultivars. [ABSTRACT FROM AUTHOR]

Apple (Malus × domestica) is one of the most consumed fruit crops in the world. The major production areas are the temperate regions, however, because of its excellent storage capacity it is transported to distant markets covering the four corners of the earth. Transformation is a key to sustaining this demand—permitting the potential enhancement of existing cultivars as well as to investigate the development of new cultivars resistant to pest, disease, and storage problems that occur in the major production areas. In this paper we describe an efficient Agrobacterium tumefaciens-mediated transformation protocol that utilizes leaf tissues from in vitro grown plants. Shoot regeneration is selected with kanamycin using the selectable kanamycin phosphotransferase (APH(3)II) gene and the resulting transformants confirmed using the scorable uidA gene encoding the bacterial β-glucuronidase (GUS) enzyme via histochemical staining. Transformed shoots are propagated, rooted to create transgenic plants that are then introduced into soil, acclimatized and transferred to the greenhouse from where they are taken out into the orchard for field-testing. [ABSTRACT FROM AUTHOR]

The key to successful transformation of American chestnut is having the correct combination of explant tissue, selectable and scorable markers, and a reliable regeneration system. Rapidly dividing somatic embryos, growing as proembryogenic masses, are a suitable tissue; the bar gene is a suitable selectable marker in conjunction with 1.0 to 10 mg/L phosphirothricin (PPT); and the mgfp5-ER gene is an effective nondestructive scorable marker. We have also found that the more gently the somatic embryos are treated during the inoculation and co-cultivation steps, the higher the transformation efficiency. The average transformation efficiency that can be expected using the described protocol is approx 20 stable and embryogenic transformation events/g of somatic embryo tissue. Cell line and batch-to-batch deviations both upward and downward should be expected. Finally, somatic embryos can be induced to form shoots, which can then be micropropagated and acclimatized. [ABSTRACT FROM AUTHOR]

We describe the procedures for recovering transgenic sugarcane from co-cultivation of both calli and in vitro plants with Agrobacterium tumefaciens. The correct tissue culture strategies and the use of super-binary vector or super-virulent strain are crucial for the successful sugarcane transformation. Both plant regeneration via calli culture and micropropagation strategies can be optimized to a wide spectrum of sugarcane genotypes, thus the procedures presented here could be applied to genetic engineering of Saccharum spp. after minor modifications. For the case of sugarcane transformation using in vitro plants, four selective micropropagation steps must be sufficient to eliminate chimera plants. [ABSTRACT FROM AUTHOR]

A procedure for pineapple [Ananas comosus (L.) Merr.] genetic transformation is described, which involves temporary immersion bioreactors (TIB) for selection of transgenic plants. Success in the production of transgenic pineapple plants combines tissue culture factors. Firstly, the use of regenerable pineapple callus as starting material for transformation whose cells shown to be competent for Agrobacterium infection. Secondly, the used of filtered callus, resulting in homogeneously sized clusters, thereby increasing the contact between the cell surfaces and A. tumefaciens and releasing phenolic compounds which induce Agrobacterium virulence. Thirdly, regeneration of primary plants without selection pressure, that allowing a massive production of putative transgenic pineapples. Finally, we support that TIB technology is a powerful system to recover nonchimera transgenic plants by micropropagation with the use of an adequate selection agent. [ABSTRACT FROM AUTHOR]

Transgenic papaya plants were initially obtained using particle bombardment, a method having poor efficiency in producing intact, single-copy insertion of transgenes. Single-copy gene insertion was improved using Agrobacterium tumefaciens. With progress being made in genome sequencing and gene discovery, there is a need for more efficient methods of transformation in order to study the function of these genes. We describe a protocol for Agrobacterium-mediated transformation using carborundum-wounded papaya embryogenic calli. This method should lead to high-throughput transformation, which on average produced at least one plant that was positive in polymerase chain reaction (PCR), histochemical staining, or by Southern blot hybridization from 10 to 20% of the callus clusters that had been co-cultivated with Agrobacterium. Plants regenerated from the callus clusters in 9 to13 mo. [ABSTRACT FROM AUTHOR]

Coffee (Coffea sp.) is a perennial plant widely cultivated in many tropical countries. It is a cash crop for millions of small farmers in these areas. As compared with other tree species, coffee has long breeding cycles that make conventional breeding programs time consuming. For that matter, genetic transformation can be an effective technique to introduce a desired trait in an already "elite" variety, or to study a gene function and expression. In this chapter, we describe two Agrobacterium-mediated transformation techniques; the first with A. tumefaciens to introduce an insect resistance gene and the second with A. rhizogenes to study candidate gene expression for nematode resistance in transformed roots. [ABSTRACT FROM AUTHOR]

Since the initial reports on production of transgenic Citrus via Agrobacterium-mediated transformation, significant progress has been made, and many steps of this procedure using the juvenile tissue explants have been improved. Abundant availability of starting material and relative simplicity make this procedure an attractive choice for many researchers despite transformation efficiency that is in the low range of about 1%. Variety of available Agrobacterium strains and reporter/selection genes further facilitate the work by allowing careful planning of experiments in which many steps can be adjusted towards a particular Citrus cultivar. With the use of this procedure, genetically transformed grapefruit, oranges, lime, and rootstock cultivars are routinely produced while some difficulties persist in efforts to transform lemon and especially mandarin cultivars. [ABSTRACT FROM AUTHOR]

Cultivated bananas are vegetatively propagating herbs, which are difficult to breed because of widespread male and female sterility. As a complementary gene transfer method in banana, the described Agrobacterium protocol relies on highly regenerable embryogenic cell cultures. Embryogenic cells are infected and co-cultivated in the presence of acetosyringone with Agrobacterium tumefaciens harboring a binary plasmid vector to obtain a mixed population of transformed and untransformed plant cells. Transformed plant cells are promoted to grow for 2 to 3 mo on a cell colony induction medium containing the antibiotics geneticin or hygromycin as selective agents, while agrobacteria are counterselected by timentin. The whole procedure, including plant regeneration, takes approx 6 mo and results in an average frequency of 25 to 50 independent transgenic plants per plate, which equals 50 mg of embryogenic cells. This method has been applied to a wide range of cultivars and to generate large populations of transgenic colonies and plants for tagging genes and promoters in banana. [ABSTRACT FROM AUTHOR]

Rubber tree (Hevea brasiliensis Muell. Arg.) is an important industrial crop for natural rubber production. At present, more than 9.5 million hectares in about 40 countries are devoted to rubber tree cultivation with a production about 6.5 million tons of dry rubber each year. The world supply of natural rubber is barely keeping up with a global demand for 12 million tons of natural rubber in 2020. Tapping panel dryness (TPD) is a complex physiological syndrome widely found in rubber tree plantations, which causes severe yield and crop losses in natural rubber producing countries. Currently, there is no effective prevention or treatment for this serious malady. As it is a perennial tree crop, the integration of specific desired traits through conventional breeding is both time-consuming and labour-intensive. Genetic transformation with conventional breeding is certainly a more promising tool for incorporation of agronomically important genes that could improve existing Hevea genotype. This chapter provides an Agrobacterium-mediated transformation protocol for rubber tree using immature antherderived calli as initial explants. We have applied this protocol to generate genetically engineered plants from a high yielding Indian clone RRII 105 of Hevea brasiliensis (Hb). Calli were co-cultured with Agrobacterium tumefaciens harboring a plasmid vector containing the Hb superoxide dismutase (SOD) gene and the reporter gene used was β-glucuronidase (GUS) gene (uidA). The selectable marker gene used was neomycin phosphotransferase (nptII) and kanamycin was used as selection agent. We found that a suitable transformation protocol for Hevea consists of a 3-d co-cultivation with Agrobacterium in the presence of 20 mM acetosyringone, 15 mM betaine HCl, and 11.55 mM proline followed by selection on medium containing 300 mg/L kanamycin. Transformed calli surviving on medium containing 300 mg/L kanamycin showed a strong GUS-positive reaction. Upon subsequent subculture into fresh media, we obtained somatic embryogenesis and germinated plantlets, which were found to be GUS positive. The integration of uidA, nptII, and HbSOD transgenes into Hevea genome was confirmed by polymerase chain reaction (PCR) as well as Southern blot analysis. [ABSTRACT FROM AUTHOR]

Although species within the genus Populus are, in general, easier to transform and regenerate in vitro than most other trees, many poplar species are very recalcitrant. Many protocols that previously have been reported were developed for a specific genotype or species. Thus, it has often been necessary to re-optimize a protocol each time research is initiated with a new genotype. The method presented in this chapter has been effective for a wide variety of poplar genotypes. [ABSTRACT FROM AUTHOR]

This chapter describes the transformation of Pinus radiata using organogenic cotyledon explants rather than the more common somatic embryogenesis methods for conifers. The advantages of our method are the year round availability of seed and that over 80% of genotypes can be easily regenerated from the mature cotyledon explants. The transformation efficiency (i.e., the number of transformed shoots regenerated from excised cotyledons) is 1.7% and, as with other Agrobacterium tumefaciens transformation methods, the majority of transgene integrations are single copy. Critical factors for success are survival of the cotyledons, Agrobacterium strain, and selection pressure after cocultivation. [ABSTRACT FROM AUTHOR]

American elm (Ulmus americana) is a valuable and sentimental tree species that was decimated by Dutch elm disease in the mid-20th century. Therefore, any methods for modifying American elm or enhancing disease resistance are significant. This protocol describes transformation and tissue culture techniques used on American elm. Leaf pieces containing the midvein and petiole are used for explants. Agrobacterium tumefaciens strain EHA105 is used for transformation, with the binary vector pSE39, containing CaMV35S/nptII as a selectable marker, ACS2/ESF39A as a putative resistance enhancing gene, and CaMV35S/GUS as a reporter. [ABSTRACT FROM AUTHOR]

A transformation system for selected mature Quercus suber L. trees using Agrobacterium tumefaciens has been established. Embryos obtained from recurrent proliferating embryogenic masses are inoculated with AGL1 strain harbouring the plasmid pBINUbiGUSint, which carries the nptII and uidA genes. Evidence of stable transgene integration is obtained by polymerase chain reaction for nptII and uidA genes, Southern blotting and expression of the uidA gene. The transgenic embryos are germinated and successfully transferred to soil. [ABSTRACT FROM AUTHOR]

Tall fescue (Festuca arundinacea Schreb.) is the predominant cool-season perennial grass in the United States. It is widely used for both forage and turf purposes. This chapter describes a protocol that allows for the generation of large number of transgenic tall fescue plants by Agrobacterium-mediated transformation. Embryogenic calli induced from caryopsis are used as explants for inoculation with A. tumefaciens. The Agrobacterium strain used is EHA105. Hygromycin phosphotransferase gene (hph) is used as the selectable marker and hygromycin is used as the selection agent. Calli resistant to hygromycin are obtained after 4-6 wk of selection. Soil-grown tall fescue plants can be regenerated 4-5 mo after Agrobacterium-mediated transformation. [ABSTRACT FROM AUTHOR]

A protocol that facilitates rapid establishment of Agrobacterium-mediated transformation for perennial ryegrass is described. The synthetic green fluorescent protein (sgfpS65T) reporter gene is introduced in combination with the nptII selectable marker gene into axillary bud derived embryogenic calli of perennial ryegrass (Lolium perenne L.) by co-cultivation with Agrobacterium tumefaciens strain AGL0 harboring binary vector pYF132. Following the co-cultivation calli are cultured for 48 h in liquid callus medium containing timentin at 10°C and 70 rpm, which reduces Agrobacterium overgrowth. Using green fluorescent protein (GFP) as a nondestructive visual marker allows identification of responsive genotypes and transgenic cell clusters at an early stage. GFP screening is combined with paromomycin selection to suppress wild type cells. Transgenic plantlets ready to transfer to soil are obtained within 4 mo of explant culture. Between 8 and 16% of the Agrobacterium-inoculated calli regenerate independent, Southern positive transgenic plants. Reproducibility and efficiency in this perennial ryegrass transformation protocols is controlled by multiple factors including genotype dependent tissue culture and gene transfer response, a short tissue culture-and-selection period and the efficient suppression of Agrobacterium following Agrobacterium-mediated gene transfer. [ABSTRACT FROM AUTHOR]

Potato (Solanum tuberosum L.) is a globally important crop plant producing high yields of nutritionally valuable food in the form of tubers. It has been the focus of substantial study because of its use both as a staple food crop and as a potentially significant source of compounds of interest. This has included the development and application of transgenic technology for introducing novel traits of fundamental and applied interest. This chapter describes a rapid, efficient, and cost-effective system for the routine transformation of this crop plant at rates above 40% efficiency, calculated as the mean number of Southern blot-confirmed independent transgenics per number of internodal explants originally plated. Internodal sections are co-cultivated with Agrobacterium tumefaciens and subjected to a two-stage callus induction/shoot outgrowth system under kanamycin selection. Shoot regeneration rates are high using the described method, and excised independent shoots rooting from the cut end of the stem after two further subcultures on kanamycin are 95% certain to be transformed. The transgenic status can be confirmed by molecular analysis and the plants grown on for tuber production enabling a wide spectrum of further studies. [ABSTRACT FROM AUTHOR]

During the last three years the generation of stably transformed cassava plants having value-added traits has become a reality. Currently, two Agrobacterium-mediated transformation systems are routinely used to engineer cassava. These systems use either somatic embryos or friable embryogenic calli. This paper presents detailed protocols for the transformation of cassava using primary somatic embryos. The effects of explant types, tissue culture conditions, and bacterial and plasmid related factors on transformation efficiency are discussed. [ABSTRACT FROM AUTHOR]

Plants are susceptible to infection by a broad range of fungal pathogens. Many horticulturally important crop species lack adequate genetic resistance to disease. Studies on potential mechanisms of disease resistance in plants have revealed the importance of a range of pathogenesis-related (PR) proteins with antifungal activity in reducing colonization of plant tissues by pathogens. We are evaluating a range of PR-proteins, through heterologous expression in transgenic carrot tissues, for their effects on fungal disease development. The protocols for carrot transformation with a thaumatin-like protein are described. In addition, the use of herbicide resistance as a selectable marker in carrot transformation is illustrated. In this protocol, petiole segments from carrot seedlings are exposed to Agrobacterium for 10-30 min and co-cultivated for 3 d, after which herbicide selection is imposed until embryogenic calli are produced after 8-12 wk. The transfer of the embryogenic calli to hormone-free medium yields transgenic plantlets. This genetic transformation protocol has supported the generation of transgenic carrot plants with defined T-DNA inserts at the rate of between 1 and 3 Southern positive independent events out of 100. [ABSTRACT FROM AUTHOR]

The ability of Agrobacterium to transform plants and other organisms is under highly regulated genetic control. Two Virulence (Vir) proteins, VirA and VirG, function as a two-component regulatory system to sense particular phenolic compounds synthesized by wounded plant tissues. Induction by these phenolic compounds, in the presence of certain neutral or acid sugars, results in activation of other vir genes, leading to the processing of T-DNA from the Ti-plasmid and transfer of T-DNA to recipient host cells. Many plant, and most nonplant, species do not provide sufficient quantities of the correct phenolic compounds to permit efficient Agrobacterium-mediated genetic transformation to occur. In order to transform these species, phenolic inducing compounds must be added to agrobacteria before and/or during cocultivation of recipient cells with the bacteria. This chapter discusses conditions for efficient induction of Agrobacterium virulence genes by phenolic compounds. [ABSTRACT FROM AUTHOR]

Tomato (Lycopersicum esculentum) is an important fruit crop in the Americas, southern Europe, the Middle East, and India, with increasing production in China, Japan, and Southeast Asia. It is amenable to producing pharmaceuticals, particularly for oral delivery; for many of the same reasons, it is a popular vegetable. Its fruit does not contain toxic substances and is palatable uncooked; it is easily processed; the plants are able to be propagated by seed or clonally by tip or shoot cuttings; the plants have a high yield of fruit; there is reasonable biomass and protein content; and they are easily grown under containment. This chapter describes Agrobacterium-mediated transformation of the tomato nucleus using cotyledons as explants. We have used this protocol to generate transgenic lines from several tomato cultivars expressing various genes of interest and selectable markers. We also provide protocols for molecular characterization of transgenic lines and batch processing tomato fruit. [ABSTRACT FROM AUTHOR]

Lettuce is a globally important leafy vegetable with the United States being the largest world producers. The crop is susceptible to a number of viruses that are aphid transmitted and also highly vulnerable to post harvest diseases. Although wild species of lettuce are an important source of disease resistance genes, their introgression into commercial lettuce has been limited owing to sexual incompatibilities. Hence, the development of a gene transfer system for lettuce would be extremely valuable both in improving the genetic diversity of the crop and also for the transfer of useful agronomic traits. This chapter describes an Agrobacterium-mediated gene delivery system that is highly adaptable for the production of transgenic plants using a wide range of lettuce germplasms. The system described, commonly referred to as the genotype-independent transformation system, has been used for the transfer of several agriculturally useful traits into commercial varieties of lettuce. In this case, A. tumefaciens strain LBA4404 carrying a binary vector with supervirulent pToK47 was used for infecting excised cotyledonary explants. The plant selectable marker gene neomycin phosphotransferase II (nptII) was used, and transformed plants were selected using kanamycin in the culture medium. The β-glucuronidase gene with intron (gus-intron) was also used in the gene transfer study to confirm the transgenicity of regenerated plants further. [ABSTRACT FROM AUTHOR]

Agrobacterium is routinely used as a tool for moving genetic constructs into plant cells. The successful use of Agrobacterium as a tool for the genetic engineering of plant cells often requires the manipulation and analysis of nucleic acids present in recombinant Agrobacterium strains. Here we present dependable methods for the isolation of genomic (total) DNA, mega-plasmid DNA, shuttle or binary plasmid DNA, and RNA. In addition, we provide a simple method for the electronic transfer of shuttle plasmids from Agrobacterium to E. coli for use when their low copy number in Agrobacterium impedes plasmid isolation from that strain. [ABSTRACT FROM AUTHOR]

Eggplant is an economically important vegetable crop in Asia and Africa, and although it is grown in Europe and the United States, it does not account for a significant percentage of agricultural production. It is susceptible to a number of pathogens and insects, with bacterial and fungal wilts being the most devastating. Attempts to improve resistance through introgression of traits from wild relatives have had limited success owing to sexual incompatibilities. Therefore, a crop improvement approach that combines both conventional breeding and biotechnological techniques would be beneficial. This chapter describes an Agrobacterium-mediated transformation protocol for eggplant based on inoculation of seedling explants (cotyledons and hypocotyls) and leaves. We have used this protocol to recover transformants from two different types of eggplant, a Solanum melongena L. breeding line, and S. melongena L. var. Black Eggplant. The selectable marker gene used was neomycin phosphotransferase II (nptII) and the selection agent was kanamycin. In vitro grown transformants acclimated readily to greenhouse conditions. [ABSTRACT FROM AUTHOR]

We describe two novel Agrobacterium tumefaciens-based methods of cucumber transformation. The first involves direct regeneration from leaf microexplants selected on kanamycin-containing medium. The second involves regeneration from a long-term established embryogenic suspension culture emitting green autofluorescence (GAF) and selection on medium containing hygromycin. In the latter method, GAF was used as a reporter, thereby allowing a simple and reliable identification of transgenic cells with a high regeneration capacity. (No false positives were observed.) The transformation efficiency in the leaf microexplants fluctuated from 0.8 to 6.5% of the primary explants, whereas in the embryogenic suspension-cultured cells it varied from 6.4 to 17.9% of the aggregates. In the GAF method, the step involving the elimination of the Agrobacterium cells by antibiotics could be omitted; however, this reduced the transformation efficiency to about 3%. The time required from inoculation to regenerated plant in the greenhouse was the same for both methods, but the GAF method required more preinoculation time than the leaf microexplant method. [ABSTRACT FROM AUTHOR]

A better understanding of the genetic basis underlying the genotype dependence of Brassica oleracea transformation is enabling researchers to distinguish between recalcitrant and successful candidate genotypes for routine transformation. In this chapter we outline an A. tumefaciens-mediated transformation method for B. oleracea using 4-d-old cotyledonary explants and a model B. oleracea doubled haploid genotype, AG DH1012. After only 3 wk on kanamycin selection, the first transgenic shoots can be isolated. Transformation efficiencies in the region of 10 to 25% (based on 50-125 PCR-positive independent shoots from 500 infected explants) are typically achieved. For researchers wishing to use their own plant genotype, we highlight the tissue culture phenotypes that are conducive to efficient transformation. [ABSTRACT FROM AUTHOR]

Phaseolus beans are among the major legumes for food consumption, especially in Latin America, Africa, and Asia. Tepary bean (Phaseolus acutifolius L. Gray) is one of the five cultivated species of the genus Phaseolus. This chapter describes an Agrobacterium-mediated transformation protocol for P. acutifolius based on cocultivation of callus, derived from cotyledonary nodes, with Agrobacterium. The selectable marker gene used is neomycin phosphotransferase II (nptII), and the selection agent is geneticin. Selection of transgenic callus material is achieved through four to five passages on geneticin-containing medium, after which shoots are induced on medium without selection agent. The protocol as described here has been applied to transform a cultivated variety of P. acutifolius, TB1, and also with some modifications to a wild genotype, NI576 and another cultivated variety, PI440795. [ABSTRACT FROM AUTHOR]

Agrobacterium tumefaciens-mediated transformation of soybeans can be accomplished using immature zygotic cotyledons as target tissues providing an alternate explant to embryogenic tissue cultures, proliferating meristems, and cotyledonary nodes. The immature cotyledon method includes direct induction of transgenic somatic embryos from the explant plated on selective media after cocultivation, followed by maturation and regeneration of individual somatic embryos into whole plants. Although this method has been improved to be simple, rapid, reproducible, and applicable to a range of cultivars in different maturity groups, the transformation efficiency (Southern-positive, independent plants produced per 100 immature cotyledon explants) is 1.7% and needs to be further increased to make this a robust soybean transformation system. Further refinements of cocultivation conditions, tissue culture, and selection of regenerated transgenic plants will probably result in increases in transformation efficiency. [ABSTRACT FROM AUTHOR]

Agrobacterium tumefaciens-mediated transformation of soybeans has been steadily improved since its development in 1988. Soybean transformation is now possible in a range of genotypes from different maturity groups using different explants as sources of regenerable cells, various selectable marker genes and selective agents, and different A. tumefaciens strains. The cotyledonary-node method has been extensively investigated and across a number of laboratories yields on average greater than 1% transformation efficiency (one Southern-positive, independent event per 100 cotyledonary-node explants). Continued improvements in the cotyledonary-node method concomitant with further increases in transformation efficiency will enhance broader adoption of this already productive transformation method for use in crop improvement and functional genomics research efforts. [ABSTRACT FROM AUTHOR]

Genetic modification of plants by the insertion of transgenes can be a powerful experimental approach to answer basic questions about gene product function. This technology can also be used to make improved crop varieties for use in the field. To apply this powerful tool to red clover, an important forage legume, a population of red clover with a high potential for regeneration in tissue culture has been developed. Here we provide a detailed procedure for Agrobacterium-mediated transformation of genotypes derived from this regenerable population. We have successfully used this methodology to express a β-glucuronidase (GUS) reporter gene and to silence an endogenous polyphenol oxidase gene in red clover. [ABSTRACT FROM AUTHOR]

Pigeonpea [Cajanus cajan (L.) Millsp.], also known as redgram, is one of the major grain legume (pulses) crops grown in the semiarid tropics (SAT) extending between 30°N and 30°S; it is the second most important food legume of India. It is cultivated in about 50 countries of Asia, Africa, and the Americas for a variety of uses (food, fodder, fuel wood, rearing lac insects, hedges, wind breaks, soil conservation, green manure, roofing, and so on). The constraints of enhancing its productivity include the damage caused by various fungi, bacteria, viruses, and insect pests. Conventional plant breeding methods have not been successful for the improvement of pigeonpea because of genetic variation and incompatibility among the wild varieties. Genetic engineering technology can therefore be used as an additional tool for the introduction of agronomically useful traits into established varieties. The development of plant transformation techniques has been a major breakthrough in overcoming constraints to achieve precision in genetic manipulation. The development of efficient plant regeneration protocols is a prerequisite for recombinant technology to carry out genetic transformation. This chapter describes an Agrobacterium-mediated transformation protocol for pigeonpea, a simple, efficient, and reproducible method that is applicable across diverse genotypes of pigeonpea. [ABSTRACT FROM AUTHOR]

Arachis hypogea (peanut, groundnut), an annual oil seed belonging to the Leguminosae family and the Papillionacea subfamily, is a legume native to South America but now grown in diverse environments in six continents between latitudes 40°N and 40°S. Arachis hypogea can grow in a wide range of climatic conditions. The low yields of this crop are mainly attributed to unreliable rainfall patterns with frequent droughts, lack of highyielding adapted cultivars, damage by diseases and pests, poor agronomic practices, and limited use of inputs. Genetic engineering approaches have been shown to be comparatively fast, leading to better isolation and cloning of desired traits for combating the various biotic and abiotic stresses. This chapter describes an Agrobacterium-mediated transformation protocol in peanut using the cotyledon system. The system described here is potentially applicable to a vast range of genotypes with a high transformation frequency of >70% based on the preliminary molecular data, indicating the production of a large number of independently transformed transgenic plants. The method reported here provides opportunities for crop improvement of this important legume crop via genetic transformation. [ABSTRACT FROM AUTHOR]

In this chapter we describe a robust method for transformation of peas that has been successfully used in our laboratory since 1992. Using immature pea seed collected from field- or greenhouse-grown plants, we have produced transgenic lines for over 30 genotypes including named pea cultivars and advanced breeding lines. This method uses immature cotyledons as the explant, and the transformation efficiency is in the range 0.2 to 13.5% of cotyledons producing at least one independently transformed line. Agrobacterium tumefaciens strains AGL1 and KYRT1 are the most successful in our procedure, and kanamycin, phosphinothricin, and hygromycin are reliable selectable markers. Potentially useful genes have been introduced for pest and disease resistance, altering quality traits, and investigating metabolic pathways and are being studied in transgenic pea lines. [ABSTRACT FROM AUTHOR]