Agrobacterium tumefaciens and related Agrobacterium species have been known as plant pathogens since the beginning of the 20th century. However, only in the past two decades has the ability of Agrobacterium to transfer DNA to plant cells been harnessed for the purposes of plant genetic engineering. Since the initial reports in the early 1980s using Agrobacterium to generate transgenic plants, scientists have attempted to improve this “natural genetic engineer” for biotechnology purposes. Some of these modifications have resulted in extending the host range of the bacterium to economically important crop species. However, in most instances, major improvements involved alterations in plant tissue culture transformation and regeneration conditions rather than manipulation of bacterial or host genes. Agrobacterium-mediated plant transformation is a highly complex and evolved process involving genetic determinants of both the bacterium and the host plant cell. In this article, I review some of the basic biology concerned with Agrobacterium-mediated genetic transformation. Knowledge of fundamental biological principles embracing both the host and the pathogen have been and will continue to be key to extending the utility of Agrobacterium for genetic engineering purposes.

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Agrobacterium-Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

Section A 1 Somatic Embryogenesis in White Spruce: Studies of Embryo Development and Cell Biology L Kong, et al 2 Proliferative Somatic Embryogenesis in Woody Species K Raemakers, et al 3 Somatic Embryo Germination and Desiccation Tolerance in Conifers EI Hay, PJ Charest 4 Performance of Conifer Stock Produced Through Somatic Embryogenesis SC Grossnickle 5 Apoptosis During Early Somatic Embryogenesis in Picea spp L Havel, DJ Durzan 6 Water Relation Parameters in Conifer Embryos: Methods and Results N Dumont-Beboux, et al 7 Image Analysis for Sorting Somatic Embryos Y Ibaraki 8 Somatic Embryogenesis in Woody Legumes RN Trigiano, et al 9 Cold Storage and Cryopreservation of Camellia Embryogenic Cultures AM Vieitez, A Ballester 10 Cryopreservation of Embryogenic Cultures of Conifers and Its Application to Clonal Forestry DR Cyr 11 Commercialization of Plant Somatic Embryogenesis BCS Sutton, DR Polonenko Section B 12 Somatic Embryogenesis in Myrtaceous Plants JM Canhoto, et al 13 Somatic Embryogenesis Induction in Bay Laurel (Laurus nobilis L) JM Canhoto, et al 14 Somatic Embryogenesis in Simarouba glauca Linn GR Rout, P Das 15 Somatic Embryogenesis in Magnolia spp SA Merkle 16 Somatic Embryogenesis and Evaluation of Variability in Somatic Seedlings of Quercus serata by RAPD Markers K Ishii, et al 17 Somatic Embryogenesis from Immature Fruit of Juglans cinerea PM Pijut Section C 18 Somatic Embrygonesis in Pinus patula Scheide et Deppe NB Jones, J van Staden 19 Somatic Embryogenesis in African Cycads (Encephalartos) AK Jager, J van Staden 20 Somatic Embryogenesis in Picea wilsonii Y Yang, Z Guo 21 Somatic Embryogenesis in Jack Pine (Pinus banksiana Lamb) YS Park, et al 22 Somatic Embryogenesis in Hybrid Firs J Jasik, et al 23 Somatic Embryogenesis in Taxus SR Wann, et al

Somatic embryogenesis in woody plants

Terpenoid biomaterials

The role of activated charcoal in plant tissue culture.

transgenic plant overexpressing a recombinant polynucleotide, wherein said transgenic plant has improved compared production with a non-transgenic plant or wild type plant, wherein the polynucleotide encodes recombinant encodes a polypeptide having at least 95% identity in amino acid sequence over the entire length of SEQ ID nO: 328, said polypeptide providing improved compared to a non-transgenic plant that does not overexpress the polypeptide production.

Polynucleotides and polypeptides in plants

During 2002–2004, three laboratories in Canada and France collaborated to improve initiation of somatic embryogenesis (SE) in jack pine (Pinus banksiana Lamb.), eastern white pine (P. strobus L.), maritime pine (P. pinaster Ait.), and Scots pine (P.␣sylvestris L.), giving particular attention to the effects of (1) N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) versus various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA), (2) differences in basal nutrient media, i.e., macro- and microelements, and (3) gelling agent concentration. The work was carried out separately at␣each laboratory, but the details of media compositions were shared and tested on their respective species. Results indicate that the developmental stage of the zygotic embryo (ZE) and genotype effects had a large influence on SE initiation, and that genetic effects were consistent over time. Different species responded differently to PGR types and concentration, basal nutrient media, trace elements, and their combinations. Currently, our best initiation rates based on a selected group of genotypes, optimal development stage of ZE, and medium are 3.9% for jack pine, 54.6% for eastern white pine, 76.2% for maritime pine, and 19.7% for Scots pine.

Initiation of somatic embryogenesis in Pinus banksiana, P. strobus, P. pinaster, and P. sylvestris at three laboratories in Canada and France

Adult conifers are notoriously recalcitrant in vegetative propagation and micropropagation that would result in the regeneration of juvenile propagules through somatic embryogenesis (SE) has not been demonstrated to date. Because SE-derived material is more amenable in subsequent tissue culture experiments compared with seed-derived material, a multi-year study was conducted to investigate induction of SE from primordial shoot (PS) explants that were excised from shoot buds of somatic embryo-derived white spruce. The SE induction experiments were carried out first with greenhouse-grown and later with field-grown trees each year from 2002 (2-year-old) to 2010 (10-year-old). Of the four genotypes tested, 893-2 and 893-12 never responded, 893-1 responded up to year 4 and 893-6 consistently responded every year. In 2010, for the first time, three of the 17 893-6 clonal trees produced male strobili as well as SE from cultured PS explants. SE induction was associated with formation of a nodule on the surface of an elongated needle primordium or in callus. Early somatic embryos were detectable after about 3 weeks of culture. Of 11 genes whose expression profiles were followed during the PS cultures, CHAP3A, VP1, WOX2 and SAP2C were expressed exclusively in the early stages of SE, and could potentially be used as markers of embryogenecity. Mature somatic embryos and plants were produced from the explants of responding genotype. Implication of these results for future research on adult conifer recalcitrance in micropropagation is discussed.

Initiation of somatic embryos and regeneration of plants from primordial shoots of 10-year-old somatic white spruce and expression profiles of 11 genes followed during the tissue culture process

Excised, immature zygotic embryos of Larix laricina (Du Roi) K. Koch (tamarack) gave rise to embryogenic cultures on modified Murashige and Skoog (MSG) medium supplemented with growth regulators. Three lines of embryonal masses were maintained for 1 year by biweekly subcultures prior to the maturation experiments. All of these lines showed the ability to produce mature somatic embryos. Both elevated medium osmolality (315.0n543.6 mmol·kg n1 ) and presence of abscisic acid (ABA) at 40 μM stimulated the maturation process when applied simultaneously. Sucrose was most effective at 0.4 M, and polyethylene glycol (PEG) at 5 or 10% was effective only in combination with 0.2 or 0.4 M sucrose. The germination frequency of somatic embryos depended on both osmolality and ABA concentration in the maturation medium. Over 90% of mature somatic embryos were capable of secondary somatic embryogenesis when placed on the induction medium. This particular ability was exploited in order to achieve genetic transformation. Four vectors were delivered to the embryonal masses and somatic embryo cells via bombardment of DNA-coated gold particles. The vectors pBI426 and pRT99gus carried a gene encoding resistance to kanamycin, pRT66gus to hygromycin, and pRT55gus to methotrexate. All vectors carried the gene coding for β-glucuronidase (GUS) and were over 6 kilobases in size. Assays for both transient and stable transformation were carried out. The only vector that yielded two transgenic lines was pBI426. These lines of embryonal masses, designated as 2D1 and 2D2, were analysed by hybridization of the plasmid to the genomic Southern blots and revealed several insertions of the vector. Line 2D1 gave rise to young germinants that expressed the GUS gene uniformly throughout the root, hypocotyl, and cotyledons but failed to develop further. Line 2D2 gave rise to transgenic plants that displayed random and ipatchyi expression of the GUS gene. Polymerase chain reaction (PCR) amplification of the GUS insert in 6-month-old 2D2 transgenic plants showed the presence of diagnostic fragment in all parts of the plants. Resume : Des embryons zygotiques immatures de Larix laricina (Du Roi) K. Koch (mOlze laricin) ont donnO du tissu embryogne sur un milieu Murashige et Skoog modifiO (MSG) par liajout de rOgulateurs de croissance. Trois lignOes embryognes ont OtO maintenues pendant 1 an par sous-cultures bihebdomadaires avant les expOriences de maturation. Ces trois lignOes ont dOmontrO leur aptitude ‡ produire des embryons somatiques matures. Une osmolalitO OlevOe (315,0n

Larix laricina (tamarack): somatic embryogenesis and genetic transformation.

This review compiles research results published over the last 14 years on conifer somatic embryogenesis (SE). Emphasis is placed on the newest findings that affect the response of seed embryos (typical explants) and shoot primordia (rare explants) to the induction of SE and long-term culture of early somatic embryos. Much research in recent years has focused on maturation of somatic embryos, with respect to both yield and quality, as an important stage for the production of a large number of vigorous somatic seedlings. Attempts to scale up somatic embryo production numbers and handling have resulted in a few bioreactor designs, the utility of which may prove beneficial for an industrial application. A few simplified cryopreservation methods for embryonal masses (EM) were developed as a means to ensure cost-efficient long-term storage of genotypes during clonal field testing. Finally, recent long-term studies on the growth of somatic trees in the field, including seed production yield and comparison of seed parameters produced by somatic versus seed-derived trees, are described.

Advances in Conifer Somatic Embryogenesis Since Year 2000.

Pinus pinaster (Ait.) somatic embryogenesis (SE) has been developed during the last decade, and its application in tree improvement programs is underway. Nevertheless, a few more or less important problems still exist, which have an impact on the efficiency of specific SE stages. One phenomenon, which had been observed in embryogenic tissue (embryonal mass, EM) initiated from immature seed, has been the loss of the ability to produce mature somatic embryos after the tissue had been cultured for several months. In an attempt to get insight into the differences between young cultures of EM (3-mo-old since the first subculture) of P. pinaster that produced mature somatic embryos and the same lines of significantly increased age (18-mo-old, aged EM) that stopped producing mature somatic embryos, we analyzed in both types of materials the levels of endogenous hormones, polyamines, the global DNA methylation, and associated methylation patterns. In addition, we included in the analysis secondary EM induced from mature somatic embryos. The analysis showed that the two tested genotypes displayed inconsistent hormonal and polyamine profiles in EM cultures of a similar phenotype and that it might be difficult to attribute one specific profile to a specific culture phenotype among genotypes. Experiments were also undertaken to determine if the global DNA methylation and/or the resulting methylation pattern could be manipulated by treatment of the cultures with a hypomethylating drug 5-azacytidine (5-azaC). An aged EM was exposed to different concentrations and durations of 5-azaC, and its response in culture was established by fresh mass increases and somatic embryo maturation potential. All of the analyses are new in maritime pine, and thus, they provide the first data on the biochemistry of EM in this species related to embryogenic potential.

Krystyna Klimaszewska

Papers

Initiation of somatic embryogenesis in Pinus banksiana, P. strobus, P. pinaster, and P. sylvestris at three laboratories in Canada and France

Initiation of somatic embryos and regeneration of plants from primordial shoots of 10-year-old somatic white spruce and expression profiles of 11 genes followed during the tissue culture process

Larix laricina (tamarack): somatic embryogenesis and genetic transformation.

Advances in Conifer Somatic Embryogenesis Since Year 2000.

Biological characterization of young and aged embryogenic cultures of Pinus pinaster (Ait.).