Expression of the β-subunit of β-conglycinin in seeds of transgenic plants

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein -conglycinin, encoded by a multigene family. -Conglycinin consists of three subunits; , , and . A genomic clone for a -subunit of -conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this -subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The -subunit expressed in seeds of petunia and tobacco was recognized by anti--conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the -subunit were produced. There was approximately a twofold variation in the accumulation of the -subunit protein in the mature seeds of transgenic petunia plants, each containing a single -subunit gene. However, the level of protein accumulation in mature seeds and the amount of -subunit mRNA in developing seeds was not correlated. Accumulation of the -subunit protein in transgenic seeds was less than the -subunit protein that accumulated in transgenic petunia seeds containing a single -subunit gene and less than the amount of the -subunit in mature soybean seeds which contain 8–13 -subunit genes. In transgenic tobacco plants, the accumulation of the -subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.Abbreviations kb kilobase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Selective inhibition of photosystem II in spinach by tobacco mosaic virus: an effect of the viral coat protein

Leaves of Spinacia oleracea inoculated with tobacco mosaic virus (TMV) strain PV230 develop mild chlorotic and mosaic symptoms of infection. Thylakoid membranes isolated from these infected leaves showed a reduced Fv/Fm ratio for chlorophyll fluorescence kinetics, at 25 degrees C. The photosystem II (PS II)-mediated electron-transport rate was inhibited 50%, whereas PS I activity was unaffected by virus infection. Protein analysis indicated that TMV coat protein was associated with thylakoids, in particular with the PS II fraction. The results demonstrate that TMV-infected S. oleracea shows inhibition of photosynthetic electron transport through PS II. We propose that the inhibition of photosynthetic activity results from the association of viral coat protein with the PS II complex.     

Nucleotide homologies between the glycosylated seed storage proteins ofGlycine max andPhaseolus vulgaris

We have compared the partial nucleotide and derived amino acid sequences of a phaseolin seed storage protein gene ofPhaseolus vulgaris (1) and a conglycinin storage protein gene ofGlycine max (2). Although these proteins are not antigenically related to one another, the architecture of the genes is similar throughout the sequences compared here. Intervening sequences interrupt the same amino acid positions in both genes. Within the 28% of theG. max gene and the 38% of theP. vulgaris gene represented in this comparison, 73% of the nucleotides in the coding and intervening sequences are identical, excluding the insertions and deletions. The nucleotide mismatches found in the coding sequences are distributed throughout the three codon positions with little bias towards the third codon position. In addition to the single nucleotide differences, six insertions or deletions, ranging from three to twenty-seven nucleotides in length, occur in this portion of the coding region and these are partially responsible for the molecular weight differences of the conglycinin α′-subunit and the phaseolin subunit.     

Local and systemic spread of tobacco mosaic virus in transgenic tobacco.

Expression of a chimeric gene encoding the coat protein (CP) of tobacco mosaic virus (TMV) in transgenic tobacco plants confers resistance to infection by TMV. We investigated the spread of TMV within the inoculated leaf and throughout the plant following inoculation. Plants that expressed the CP gene [CP(+)] and those that did not [CP(-)] accumulated equivalent amounts of virus in the inoculated leaves after inoculation with TMV-RNA, but the CP(+) plants showed a delay in the development of systemic symptoms and reduced virus accumulation in the upper leaves. Tissue printing experiments demonstrated that if TMV infection became systemic, spread of virus occurred in the CP(+) plants essentially as it occurred in the CP(-) plants although at a reduced rate. Through a series of grafting experiments, we showed that stem tissue with a leaf attached taken from CP(+) plants prevented the systemic spread of virus. Stem tissue without a leaf had no effect on TMV spread. All of these findings indicate that protection against systemic spread in CP(+) plants is caused by one or more mechanisms that, in correlation with the protection against initial infection upon inoculation, result in a phenotype of resistance to TMV.     

Selection of large quantities of embryogenic calli from indica rice seeds for production of fertile transgenic plants using the biolistic method

The microprojectile bombardment of immature embryos has proven to be effective in transforming many indica rice varieties. One of the drawbacks of using immature embryos is the requirement of a large number of high quality immature embryos, which itself is a tedious and laborious process. To circumvent these problems, we have developed a procedure, using indica variety TN1 as a model that generates highly homogenous populations of embryogenic subcultured calli by selectively propagating a small number of regeneration-proficient calli derived from seeds. Thousands of embryogenic calli were produced from 50 seeds within 10 weeks. Ten to 20 independent R0 transgenic lines were regenerated per 500 embryogenic calli bombarded. The convenience and reliability offered by this transformation system has made transformation of indica rice a routine procedure.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA naphthalene acetic acid - BAP 6-benzylaminopurine - kb kilobase - GUS -glucuronidase - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronide - HPH hygromycin B phosphotransferase     

Regeneration of fertile transgenic indica (group 1) rice plants following microprojectile transformation of embryogenic suspension culture cells

Regenerable embryogenic suspensions of elite Indica (group 1) rice varieties IR24, IR64, IR72 and an advanced Indica rice breeding line IR57311-95-2-3 were established within 6–8 weeks from 3–4 week old calli derived from mature seeds. Transgenic rice plants were obtained by introducing a plasmid carrying genes encoding hygromycin phosphotransferase (hph, conferring resistance to hygromycin B) and ß-glucuronidase (uidA), both driven by the CaMV 35S promoter, via particle bombardment of embryogenic suspensions. The effect of osmotic conditioning on transformation was evaluated. Regenerated plants were resistant to hygromycin B and expressed the uidA (GUS) gene. The growth of mother plants (R₀) was normal and seeds were produced. Southern blot analysis of R₀ and R₁ plants showed that hygromycin resistant plants contained intact hph genes that were inherited in a Mendelian fashion. A protocol for a simple, efficient, repeatable, genotype- and environment-independent Indica rice transformation system is described.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA -naphthalene acetic acid - kb kilobase - GUS ß-glucuronidase - hph hygromycin B phosphotransferase