Nigel W. Scott

Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.)

Molecular approaches to sugar beet improvement will benefit from an efficient transformation procedure that does not rely upon exploitation of selectable marker genes such as those which confer antibiotic or herbicide resistance upon the transgenic plants. The expression of the green fluorescent protein (GFP) signal has been investigated during a program of research that was designed to address the need to increase the speed and efficiency of selection of sugar beet transformants. It was envisaged that the GFP reporter could be used initially as a supplement to current selection regimes in order to help eliminate “escapes” and perhaps eventually as a replacement marker in order to avoid the public disquiet associated with antibiotic/herbicide-resistance genes in field-released crops. The sgfp-S65T gene has been modified to have a plant-compatible codon usage, and a serine to threonine mutation at position 65 for enhanced fluorescence under blue light. This gene, under the control of the CaMV 35S promoter, was introduced into sugar beet via Agrobacterium-mediated transformation. Early gene expression in cocultivated sugar beet cultures was signified by green fluorescence several days after cocultivation. Stably transformed calli, which showed green fluorescence at a range of densities, were obtained at frequencies of 3–11% after transferring the inoculated cultures to selection media. Cocultivated shoot explants or embryogenic calli were regularly monitored under the microscope with blue light when they were transferred to media without selective agents. Green fluorescent shoots were obtained at frequencies of 2–5%. It was concluded that the sgfp-S65T gene can be used as a vital marker for noninvasive screening of cells and shoots for transformation, and that it has potential for the development of selectable marker-free transgenic sugar beet.

The plant cell cycle in context.

Biological scientists are eagerly confronting the challenge of understanding the regulatory mechanisms that control the cell division cycle in eukaryotes. New information will have major implications for the treatment of growth-related diseases and cancer in animals. In plants, cell division has a key role in root and shoot growth as well as in the development of vegetative storage organs and reproductive tissues such as flowers and seeds. Many of the strategies for crop improvement, especially those aimed at increasing yield, involve the manipulation of cell division. This review describes, in some detail, the current status of our understanding of the regulation of cell division in eukaryotes and especially in plants. It also features an outline of some preliminary attempts to exploit transgenesis for manipulation of plant cell division.

Efficient somatic embryogenesis in sugar beet ( Beta vulgaris L.) breeding lines

Efficient regeneration via somatic embryogenesis (SE) would be a valuable system for the micropropagation and genetic transformation of sugar beet. This study evaluated the effects of basic culture media (MS and PGo), plant growth regulators, sugars and the starting plant material on somatic embryogenesis in nine sugar beet breeding lines. Somatic embryos were induced from seedlings of several genotypes via an intervening callus phase on PGo medium containing N6-benzylaminopurine (BAP). Calli were mainly induced from cotyledons. Maltose was more effective for the induction of somatic embryogenesis than was sucrose. There were significant differences between genotypes. HB 526 and SDM 3, which produced embryogenic calli at frequencies of 25–50%, performed better than SDM 2, 8, 9 and 11. The embryogenic calli and embryos produced by this method were multiplied by repeated subculture. Histological analysis of embryogenic callus cultures indicated that somatic embryos were derived from single- or a small number of cells. 2,4-dichlorophenoxyacetic acid (2,4-D) was ineffective for the induction of somatic embryogenesis from seedlings but induced direct somatic embryogenesis from immature zygotic embryos (IEs). Somatic embryos were mainly initiated from hypocotyls derived from the cultured IEs in line HB 526. Rapid and efficient regeneration of plants via somatic embryogenesis may provide a system for studying the molecular mechanism of SE and a route for the genetic transformation of sugar beet.

PlantID – DNA-based identification of multiple medicinal plants in complex mixtures

BackgroundAn efficient method for the identification of medicinal plant products is now a priority as the global demand increases. This study aims to develop a DNA-based method for the identification and authentication of plant species that can be implemented in the industry to aid compliance with regulations, based upon the economically important Hypericum perforatum L. (St John’s Wort or Guan ye Lian Qiao).MethodsThe ITS regions of several Hypericum species were analysed to identify the most divergent regions and PCR primers were designed to anneal specifically to these regions in the different Hypericum species. Candidate primers were selected such that the amplicon produced by each species-specific reaction differed in size. The use of fluorescently labelled primers enabled these products to be resolved by capillary electrophoresis.ResultsFour closely related Hypericum species were detected simultaneously and independently in one reaction. Each species could be identified individually and in any combination. The introduction of three more closely related species to the test had no effect on the results. Highly processed commercial plant material was identified, despite the potential complications of DNA degradation in such samples.ConclusionThis technique can detect the presence of an expected plant material and adulterant materials in one reaction. The method could be simply applied to other medicinal plants and their problem adulterants.

Green fluorescent protein as a visual selection marker for coffee transformation

The green fluorescent protein (GFP) was used as a visual selectable marker to produce transgenic coffee (Coffea canephora) plants following Agrobacterium-mediated transformation. The binary vector pBECKS 2000.7 containing synthetic gene for GFP (sgfp) S65T and the hygromycin phosphotransferase gene hph both controlled by 35S cauliflower mosaic virus CaMV35S promoters was used for transformation. Embryogenic cultures were initiated from hypocotyls and cotyledon leaves of in vitro grown seedlings and used as target material. Selection of transformed tissue was carried out using GFP visual selection as the sole screen or in combination with a low level of antibiotics (hygromycin 10 mg/L), and the efficiency was compared with antibiotics selection alone (hygromycin 30 mg/L). GFP selection reduced the time for transformed somatic embryos formation from 18 weeks on a hygromycin (30 mg/L) antibiotics containing medium to 8 weeks. Moreover, visual selection of GFP combined with low level of antibiotics selection improved the transformation efficiency and increased the number of transformed coffee plants compared to selection in the presence of antibiotics. Molecular analysis confirmed the presence of the sgfp-S65T coding region in the regenerated plants. Visual screening of transformed cells using GFP by Agrobacterium-mediated transformation techniques was found to be efficient and therefore has the potential for development of selectable marker-free transgenic coffee plants.

DNA to PCR: computer-aided learning software for DNA sequencing and amplification techniques of molecular biology

Computer-aided learning software designed to illustrate the structure and replication of DNA, and the principles and applications of DNA sequencing and PCR is described. Tailoring the software to individual needs enhances its effective use over a wide range of higher education levels by students with disparate backgrounds. The computer-based library provides ample support for study in an independent mode.

Strategies for Manipulation of Sugar Beet Storage Organ Morphology

ABSTRACTA strategy has been identified for the production of a low soil tare sugar beet by genetic manipulation of the table beet. This approach will involve increasing the sucrose storage capacity of the table beet by manipulating cell division in the cambial rings of the storage organ. Research aimed at the isolation of suitable cell division regulatory genes and storage organ specific promoter sequences is described. Sugar beet cdc 2 and cyclin sequences have been isolated by PCR techniques. Two root specific cDNA sequences have been characterized; one belongs to a group of hybrid proline-rich/hydrophobic proteins and the other resembles a gene induced during potato tuberisation.

EXPRESSION OF CELL DIVISION CYCLE RELATED GENES IN Beta vulgaris L. STORAGE ORGANS AND CELLS IN SUSPENSION CULTURES

The sucrose storage capacity of the sugar beet, Beta vulgaris, storage organ has been related to its anatomy. A transverse section of the storage organ reveals a pattern of concentric rings which is caused by the alternation of vascular zones with parenchymatous zones. Sucrose is transported to the storage organ via the phloem and moves laterally to the adjacent parenchymatous tissues, where it is stored in the cell vacuoles. The storage organ of sugar beet has 12–15 cambia, the divisions of the 6 inner rings provide the cells which make up some 75% of the storage root while the outer rings (9 and above) make almost no contribution to the bulk of the storage root. It has been found that the small parenchymatous cells close to the phloem accumulate sucrose to a higher concentration than that achieved by large (older) cells remote from the phloem. Hence, it is predicted that for optimum sucrose accumulation the storage organ should have a large number of parenchymatous zones, each of which offers a short diffusion path from the phloem to predominately small storage cells. In order to manipulate sugar beet development toward this optimum it is necessary to have an understanding of the processes that regulate cell division, cell expansion and differentiation. Towards this end we have begun to isolate and characterise cell division cycle related genes from sugar beet and we have developed a cell suspension culture system for analysis of their roles (Elliott et al. 1996).

The entry of sugar beet cells into the G0 state involves extensive re-programming of gene expression mechanisms via transcriptional and translational controls

Sugar beet cells grown in liquid suspension culture entered the stationary phase on day 17 of the batch culture cycle as a consequence of nutrient depletion. Between days 17 and 19 the cells exhibited a number of marked changes in RNA and protein metabolism characteristic of entry into a quiescent or G0 state. Protein synthesis declined considerably and very few polypeptides incorporated sufficient radioactively labelled methionine to be detected by fluorography following polyacrylamide gel electrophoresis (SDS-PAGE). There was a marked decrease in protein half life during the stationary phase and this was accompanied by a rise in protease activity. RNA synthesis also decreased to <10% of the level in dividing cells and the residual transcription was insensitive to inhibition by 0.4 μg ml−1 actinomycin D. However, rRNA remained intact in the quiescent cells, only minor changes were detected in the profiles of stained polypeptides separated by SDS-PAGE, and the cells remained viable. Despite the marked decline in protein synthesis in the stationary phase, a pool of translatable mRNA remained in the cells which produced similar translation products in vitro to the abundant mRNAs from dividing cells. On the other hand, comparison of the mRNA populations of cell samples taken from cells in the stationary phase and after subculture into fresh medium by the techniques of differential display and Northern blotting showed that there were a substantial number of changes in gene expression during the transition from quiescence to division. Only one-third of mRNA sequences were expressed constitutively, a number of stationary phase-specific mRNAs were detected whilst others appeared only after subculture. In conclusion, the G0 state which results from nutrient depletion in these sugar beet cells is characterised by the transcription of a number of G0-specific genes during a period in which there is large overall reduction in transcriptional and translational activity.

Arabidopsis CDC2a and Cyclin Gene Promoter::gusA Constructs as Markers of Cell Growth and Division in Heterologous Plants

Cell growth and division are integrated with a programme of differentiation to determine the form of the plant body. In recent years the molecular controls of cell division and growth have been elucidated. Progression through the cell cycle is controlled by a family of serine/threonine protein kinases termed CDKs (cyclin-dependent kinases) that act in association with regulatory proteins termed cyclins. Specific CDKs associate with specific cyclins in order to bring about both the gene expression changes and the physical modifications that are necessary to complete the cell cycle. The nature and interactions of CDKs and cyclins have been studied in detail in mammalian and yeast cells but there is still relatively little information about such systems in plants. Much of the information is drawn by inference. Little is known about the coupling of cell division with cell growth and the integration of these processes into development.