James A. Saunders

Identification of molecular markers in soybean comparing RFLP, RAPD and AFLP DNA mapping techniques

Three different DNA mapping techniques—RFLP, RAPD and AFLP—were used on identical soybean germplasm to compare their ability to identify markers in the development of a genetic linkage map. Polymorphisms present in fourteen different soybean cultivars were demonstrated using all three techniques. AFLP, a novel PCR-based technique, was able to identify multiple polymorphic bands in a denaturing gel using 60 of 64 primer pairs tested. AFLP relies on primers designed in part on sequences for endonuclease restriction sites and on three selective nucleotides. The 60 diagnostic primer pairs tested for AFLP analysis each distinguished on average six polymorphic bands. Using specific primers designed for soybean fromEco RI andMse I restriction site sequences and three selective nucleotides, as many as 12 polymorphic bands per primer could be obtained with AFLP techniques. Only 35% of the RAPD reactions identified a polymorphic band using the same soybean cultivars, and in those positive reactions, typically only one or two polymorphic bands per gel were found. Identification of polymorphic bands using RFLP techniques was the most cumbersome, because Southern blotting and probe hybridization were required. Over 50% of the soybean RFLP probes examined failed to distinguish even a single polymorphic band, and the RFLP probes that did distinguish polymorphic bands seldom identified more than one polymorphic band. We conclude that, among the three techniques tested, AFLP is the most useful.

The characterization of defense responses to fungal infection in alfalfa

The enzyme activity and transcript level of three enzymes involved in flavonoid biosynthesis, phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (CA4H), and isoflavone reductase (IFR), were monitored in alfalfa seedlings (Medicago sativa) that had been challenged with the fungal pathogen Colletotrichum trifoliii. In many legumes, a pathogen–host infection leads to an induced synthesis of fungitoxic phytoalexins produced via these key enzymes. For example, when alfalfa is exposed to an avirulent fungal type, phytoalexins are produced by the plant providing protection from additional exposure to a virulent fungal race. This defensive plant protection response was accompanied by increases in transcript levels of PAL, CA4H and IFR gene expression, by increases in PAL enzyme activity, and by production of the end product phytoalexins, medicarpin and sativan. The expression of these defense genes and PAL enzyme activity were significantly greater in seedlings responding to combined inoculations of avirulent and virulent fungi, compared to plants inoculated with the avirulent fungus alone. Inoculation with the virulent race alone elevated the gene transcripts compared with control plants but these levels were less than found in plants inoculated with the avirulent race or those inoculated with both. Production of gene transcripts reached its peak in treated plants after 49 h. The phytoalexins medicarpin and sativan increased rapidly and reached maximal levels 97 h after inoculation with the avirulent fungal race. The plants challenged with combined inoculations of the avirulent and the virulent fungi showed significantly greater accumulation of medicarpin than in any other treatment. These results suggest that the increased medicarpin accumulation produced in induced resistant tissues, following challenge inoculation with virulent race, is attributable to increased expression of genes of flavonoid biosynthesis.

DNA uptake during electroporation of germinating pollen grains.

Abstract Electroporation of germinating pollen grains of Nicotiana gossei (L.) Domin under a variety of conditions showed that DNA was taken up by the pollen without detrimental effects on the viability of the pollen. By optimizing both the field strength of the electroporation pulse and the DNA concentration in the electroporation medium up to 6% of the donor DNA can be taken up by the germinating pollen while maintaining a pollen viability of 90%. Field strengths as high as 9 kV/cm could be applied to germinating pollen grains without detrimental effects on viability. Southern hybridizations demonstrated that DNA encoding the marker enzyme β-glucuronidase (GUS) was incorporated into electroporated pollen. Germinating pollen, treated in this manner, is capable of producing 300–400 seeds per capsule of viable seed when applied to the stigmas of compatible flowers of N. gossei which has been emasculated 4 days earlier.

Plant Gene Transfer Using Electrofusion and Electroporation

In recent years, the rapidly developing fields of electrofusion of somatic cells and electroporation of selected cell types have become an extremely important tool in the production and modification of hybrid somatic gene pools. In plants, these procedures have thus far been restricted to protoplasts in which the thick secondary cell wall has been stripped off usually through enzymatic digestion of the cellulosic and pectic components of the cell wall. Protoplasts with a diameter of 35–55 μm can be predictably isolated from normal mesophyll leaf tissue or from cell suspension cultures. These cells are ideally suited for the simple, rapid, and consistent procedures that have been developed for both electrofusion and electroporation (Bates et al., 1987). These procedures are very effective for cells of this size and have made electromanipulation of cell membranes the method of choice for gene transfer due to the simplicity, convenience, and ease of operation.

Expression of a foreign gene in electroporated pollen grains of tobacco

SummaryThe incorporation of genetically engineered DNA into pollen and subsequent fertilization of eggs by the transformed pollen would be a convenient method for producing genetically engineered seed. This method of pollen transformation would circumvent the need for other types of gene transfer methods such as the use of Agrobacterium tumefaciens, which has a limited host range and thus a limited capability for genetically engineering plants. It would also avoid the problems associated with the regeneration of some plants from tissue, cell, or protoplast culture after receiving foreign DNA. To this end, the genetically engineered plasmid DNA vector pBI221 containing the gene encoding β-glucuronidase (GUS) was introduced by electroporation into germinating pollen grains of tobacco (Nicotiana gossei L.). Transient expression of the GUS gene was demonstrated by the presence of GUS activity in fluorometric assays of pollen extracts 24 h after the introduction of pBI221 via electroporation. Intact pBI221 was detected by Southern blotting procedures as a distinct DNA band in pollen extracts 1 h after electroporation. In addition, pBI221 was detected as a diffuse band of higher molecular weight DNA 24 h after electroporation, suggesting that some of the pBI221 was incorporated into the genome of the pollen.

Rapid optimization of electroporation conditions for plant cells, protoplasts, and pollen

The optimization of electroporation conditions for maximal uptake of DNA during direct gene transfer experiments is critical to achieve high levels of gene expression in transformed plant cells. Two stains, trypan blue and fluorescein diacetate, have been applied to optimize electroporation conditions for three plant cell types, using different square wave and exponential wave electroporation devices. The different cell types included protoplasts from tobacco, a stable mixotrophic suspension cell culture from soybean with intact cell walls, and germinating pollen from alfalfa and tobacco. Successful electroporation of each of these cell types was obtained, even in the presence of an intact cell wall when conditions were optimized for the electroporation pulse. The optimal field strength for each of these cells differs, protoplasts having the lowest optimal pulse field strength, followed by suspension cells and finally germinating pollen requiring the strongest electroporation pulse. A rapid procedure is described for optimizing electroporation parameters using different types of cells from different plant sources.

Reduction of nuclease activity released from germinating pollen under conditions used for pollen electrotransformation

Nucleases released from germinating tobacco (Nicotiana gossei L. Domin), corn (Zea mays, hybrid golden queen) and alfalfa (Medicago sativa L. var. Saranac) pollen degrade added plasmid pBI221 DNA. Isolated DNA from pollen/pBI221 suspensions showed increased degradation of pBI221 as the incubation time with the pollen was increased. Electroporation immediately after the addition of pBI221 had little effect on the amount of degradation. The addition of increasing amounts of ethylenediaminetetraacetic acid (EDTA) or MgSO4 to the pollen suspension after germination but before the addition of plasmid, followed by electroporation, resulted in deceasing nuclease activity. Changing the germination medium prior to electroporation also decreased nuclease activity.

Expression of GUS and CAT activities using electrotransformed pollen

Abstract Two plasmid constructs, carrying either the gene encoding β-glucuronidase (GUS) or chloramphenicol acetyltransferase (CAT) have been successfully incorporated into electroporated pollen from tobacco. The procedure employs the electroporation of germinating pollen in the presence of foreign DNA using an exponential discharge pulse. When the pollen tube membrane is permeabilized by the electroporation pulse, it allows the influx of DNA that has been added to the electroporation medium. Species-specific modifications of the electroporation medium maintain the viability of germinating pollen while optimizing the DNA uptake. Transient assays of CAT and GUS expression in pollen were used to monitor changes in uptake of DNA to optimize DNA expression by electroporated pollen. One DNA construct, pLAT52-7, contained a pollen-specific promoter, which enhanced GUS expression in pollen as compared with experiments using pBI221 plasmid with the CaMV 35S constitutive promoter. Production of transformed plants by pollination with electroporated pollen was confirmed by genomic DNA Southern hybridization, PCR amplification and hybridization, fluorometric GUS assays, and tissue level histological localization of GUS expression. The introduction of genetic material into the pollen and the production of transformed plants produced from seed formed after fertilization with treated pollen could have a tremendous impact on the improvement of economically important crops.

Reporter Genes and Transient Assays for Plants

The introduction of DNA into plant cells, protoplasts, or intact tissue s has been accomplished by a variety of mechanisms, including electropo-ration, electrofusion, particle bombardment, liposome transfer, the us e of bacterial vectors, polyethylene glycol treatment, and other procedures. As new techniques are developed or modified, it is necessary to use a reliable gene-expression system to monitor DNA uptake, transcription , and translation. A series of DNA plasmids containing reporter genes encoding readily assayed enzymes are available for this purpose. Several reporter gene systems have been used in experiments to transform plant s and to perform transient assays with plant material. In_ general, thes e reporter genes encode enzymes whose activities can be detected through assays and stains, thus facilitating the identification of transformed cell s and quantification of the transformation process. Reporter genes also provide a method for analyzing regulatory characteristics of promoters , such as promoter strength and tissue specificity, when the promoter fro m a gene of interest is coupled to the reporter gene. Reporter gene constructs are comprised of a reporter gene togethe r with active promoter and terminator regions cloned into a plasmid vector. Sometimes these constructs lack promoter regions, so different pro

Successful Gene Transfer in Plants Using Electroporation and Electrofusion

Electroporation and electrofusion of plant tissues are rapid, reliable, techniques for genome modification. To accomplish these procedures two different wave forms have been developed and successfully used in plants during the last decade. We have shown that both square and exponential wave pulses are capable of incorporating genetic material into tobacco protoplasts. The production of an insect resistant potato hybrid, verified by phenotypic and genetic traits, has demonstrated the utility of electrofusion by the combination of two separate genomes. Specific genomic modifications are also possible as shown by the electroporation of marker genes into germinating tobacco pollen. The impact of these continuing investigations into the biological effects of electrical fields on plant cells, will enhance our ability to genetically manipulate biological systems.