Specificity of a promoter from the rice tungro bacilliform virus for expression in phloem tissues.

The major promoter region for the transcription of the genome of rice tungro bacilliform virus (RTBV), a newly described badnavirus, has been identified. Fragments of the RTBV genome upstream of the site of transcription initiation were isolated and tested for promoter activity using a beta-glucuronidase receptor gene (gusA). Assays of transient gusA expression were performed following introduction of the chimeric gene into protoplasts via electroporation. The chimeric RTBV-promoter: gusA gene was more active in rice protoplasts than in maize or tobacco protoplasts, but was weaker than gusA controlled by an enhanced 35S promoter from cauliflower mosaic virus. Analysis of gusA gene expression following introduction of chimeric reporter genes into intact leaves via micro-projectile bombardment indicated that the GUS activity is present primarily in vascular tissues. Transgenic rice plants carrying the chimeric gusA gene had GUS activity only in the phloem of the vascular bundles in the leaf. Tissue printing studies demonstrated that RTBV accumulates in the vascular bundles of infected rice leaves. The results of our study indicate that phloem-specific expression from the RTBV promoter is an intrinsic property of the viral promoter.

Subdomains of the octopine synthase upstream activating element direct cell-specific expression in transgenic tobacco plants.

Previous work has shown that the octopine synthase (ocs) gene encoded by the Agrobacterium tumefaciens Ti-plasmid contains an upstream activating sequence necessary for its expression in plant cells. This sequence is composed of an essential 16-bp palindrome and flanking sequences that modulate the level of expression of the ocs promoter in transgenic tobacco calli. In this study, we have used RNA gel blot analysis of RNA extracted from transgenic tobacco plants to show that the octopine synthase gene is not constitutively expressed in all plant tissues and organs. This tissue-specific pattern of expression is determined, to a large extent, by the 16-bp palindrome. Histochemical analysis, using an ocs-lacZ fusion gene, has indicated that the 16-bp palindrome directs the expression of the ocs promoter in specific cell types in the leaves, stems, and roots of transgenic tobacco plants. This expression is especially strong in the vascular tissue of the leaves, leaf mesophyll cells, leaf and stem guard cells, and the meristematic regions of the shoots and roots. Sequences surrounding the palindrome in the upstream activating sequence restrict the expression of the ocs promoter to fewer cell types, resulting in a reduced level of expression of beta-galactosidase activity in the central vascular tissue of leaves, certain types of leaf trichomes, and the leaf primordia.

Transgenic indica rice plants.

We have established a system to genetically engineer indica rice plants. In order to obtain transgenic plants, genes were introduced into protoplasts isolated from suspension cells of the indica rice var 'IR54' with the aid of polyethylene glycol (PEG). The neo gene was on pKAN and the gusA gene was on pPUR. The promoter for both genes was CaMV35S. Transformed calli were readily recovered from medium supplemented with G-418. In contrast, kanamycin interfered with plant regeneration from protoplast-callus. Transgenic plants were regenerated from calli resistant to G-418 in several separate experiments and grown to maturity in a growth chamber. Southern blot analysis of DNA isolated from leaves of T0 plants verified the presence of the transferred neo and gusA genes in the plant genome. A study of gene expression showed that the CaMV35SgusA gene was active in all of the organs examined. Mendelian inheritance of the introduced gusA gene was observed in progeny obtained by backcrossing the T0 plants to untransformed plants.

Changes in nucleus, nucleolus and cell size accompanying somatic embryogenesis of Theobroma cacao L. I. Relationship between DNA and total protein content and size of nucleus, nucleolus and cell.

There was a linear relation between an increase in DNA content and size of nuclei, nucleoli and cells in callus and proembryos (Theobroma cacao L.). In callus the increase of DNA content was accompanied by proportional increase in nuclear size whereas in proembryos the increase in nuclear size did not match the increasing amount of DNA. The stimulation of embryogenesis by 10(-2) mg/l 2,4-D was associated with increase in nuclear and nucleolar size and with decrease in cell sizes. Inhibition of embryogenesis by 1.0 mg/l 2,4-D+10% coconut water did not change nuclear size, but increased cell size in relation to the control. The process of embryo formation was accompanied by changes in relationship between nuclear, nucleolar and cell size and the total (DNFB-stained) proteins content. In callus as well as in proembryo the increase in total protein content in nucleus was not equivalent to the increasing sizes of nuclei which leads to the decrease in nuclear protein concentration. Similar situation was observed for nucleoli. Differences were found in the concentration of cytoplasmic proteins between the callus and proembryo cells. The stimulation of embryogenesis by low concentration of 2,4-D resulted in decrease in concentration of total proteins in nuclei and nucleoli and the increase in cytoplasm.

Changes in nucleus, nucleolus and cell size accompanying somatic embryogenesis of Theobroma cacao L. II. Relation between basic protein content and size of nucleus, nucleolus and cell.

Embryo formation from callus of Theobroma cacao L. was associated with the changes in relationship between nuclear, nucleolar and cell sizes and the content of basic proteins (FG-FCF-stained). Together with the increase in nuclear size of callus and proembryo cells the increase in the amount of nuclear basic proteins was found. In the callus cells the increase in nucleolar protein content exceeded that in nucleolus size, which led to the rise in basic protein concentration in the nucleolus. However, in the early stage of embryogenesis the increase in protein content was not so marked as that in callus, which indicated that embryogenesis involved a decrease in concentration of nucleolar basic proteins. Differences between callus and proembryo cells were also observed in the concentration of cytoplasmic proteins. The increase in size of callus cells was the same as the increasing amount of cytoplasmic proteins. In proembryos a significant increase in cell size was accompanied by only slight changes in cytoplasmic proteins. The stimulation of embryogenesis by 2,4-D resulted in an increase of nuclear concentration of basic proteins in proembryos. The intensification of embryogenesis involved the decrease of the concentration of nucleolar proteins together with the increase in concentration of basic cytoplasmic proteins.

Cytomorphological and biochemical changes in dwarf pea shoots induced by gibberellic acid.

Treatment of 8-day-old pea plants for 15 and 24 hr with gibberellic acid resulted in: 1. 2-3 Fold increase of the cell length. 2. About 20% increase of the nuclei and nucleoli size in meristematic zone of plant shoots. 3. Increase of endomitotic and mitotic synthesis of DNA. 4. Increased ability of the cells to bind [3H]Actinomycin D. 5. Higher rate of RNA synthesis. 6. Increase of the fresh weight of apical parts of the green pea shoots. 7. These results indicated that gibberellic acid accelerates the growth and differentiation of plant cells.

Cytological study of inhibitory effects of IAA on the root growth in the Pinus silvestris.

It has been found that IAA at the concentrations 0.1--10.0 ppm retards the pine root growth and decreases mitotic activity. All the applied concentrations of this hormone cause a decrease of the 3H-thymidine incorporation index and inhibit endomitotic polyploidization in suprameristematic segments. The mean time of the cell cycle prolongs and the heterogeneity of cellular populations increases in parallel with the increase of IAA concentration. The template activity of DNA decreases under the influence of the applied concentrations of IAA; this effect is being particularly strong in the meristematic root segment. IAA exerts also an inhibitory influence on protein synthesis, especially reducing the synthesis of histones.