Expression pattern of the pre-prothaumatin II gene under the control of the CaMV 35S promoter in transgenic cucumber (Cucumis sativus L.) flower buds and fruits.

Thaumatin II is an extremely sweet-tasting protein produced by fruits of the West African shrub Thaumatococcus daniellii Benth, so it can be used in biotechnology to improve the tastes of various plant products. This study is concerned with the spatial and temporal aspects of expression of the 35S-pre-prothaumatin II chimeric gene in flower buds and fruits of transgenic cucumber (Cucumis sativus L.) line 225. The activity of the 35S promoter in organs of line 225 was compared with its activity in 2 other transgenic lines. The accumulation of recombinant thaumatin varied spatially in flower bud tissues of transgenic lines. We found that these differences in the spatial accumulation of transgenic protein concerned the ovary of female buds and the perianth of male buds. In contrast to flower parts, recombinant thaumatin was found in nearly all parts of the young fruit from the transgenic plants. The pre-prothaumatin II gene expression was detected at a very early developmental stage in male buds, and its pattern was rather conserved as the buds aged. The expression of the transgene was also detected in vascular tissues of examined organs but was undetectable in pollen grains, in agreement with the generally held view that the CaMV 35S promoter is virtually silent in pollen. Immunocytochemical analyses of sections of control organs revealed endogenous homolog(s) of thaumatin when using polyclonal antisera, but not when using monoclonal antibodies for recombinant thaumatin detection in transgenic cucumber.

Changes in the organization of the tubulin cytoskeleton during the early stages of Solanum lycopersicoides Dun. protoplast culture.

Changes in the tubulin cytoskeleton during protoplast culture and plant regeneration of Solanum lycopersicoides Dun. were analyzed using an immunodetection method. Directly after isolation, four groups of protoplasts were distinguished: (1) mononuclear, (2) polynuclear, (3) homogeneous, (4) anuclear. The tubulin cytoskeleton of the protoplasts underwent rearrangements, correlating to the number and structure of cell nuclei in the protoplast. All protoplast groups with the exception of mononuclear were characterized by perturbations in the organization of the tubulin cytoskeleton. Anuclear and homogeneous protoplasts did not have a tubulin cytoskeleton. Polynuclear protoplasts had cortical microtubules, but were not capable of re-forming their original arrangement and did not possess a radial or perinuclear cytoskeleton. Irregularities in microtubule arrangement of these three groups of protoplasts caused their inability to regenerate a cell wall and to divide. Anuclear, polynuclear and homogeneous protoplasts were eliminated from the culture. Mononuclear protoplasts rearranged their cortical microtubules and reestablished the radial and perinuclear tubulin cytoskeleton. Re-formation of the cell suspension and subsequent regeneration of plants occurred exclusively from mononuclear protoplasts, which were able to regenerate cell walls and to divide.

A major deletion in the cucumber mitochondrial genome sorts with the MSC phenotype.

The cucumber mitochondrial genome is unique because of its large size, paternal transmission, and the existence of a paternally transmitted mosaic (MSC) phenotype spontaneously appearing after independent tissue culture experiments. Transmission studies eliminated paternal imprinting as the genetic basis for the MSC phenotype. We identified a 13 kb region (JLV5-DEL) in the wild-type mitochondrial genome that was absent from three MSC lines. This deleted region was paternally transmitted with the MSC phenotype through the F3 and test-cross generations. Southern hybridizations and PCR amplifications using primers within the JLV5 region revealed that rare wild-type sorters possessed the wild-type region. MSC plants possess the wild-type region at levels (approximately 0.002) below detection by standard PCR reactions and Southern hybridizations, using genomic DNA. Sequence analysis of the wild-type contig revealed no homologies to mitochondrial genes and no open reading frames within JLV5-DEL. PCR amplifications across JLV5-DEL using MSC mtDNA revealed that the loss of this region was not a simple deletion and may be associated with a rearrangement. Because no genic regions were identified within the wild-type JLV5 region, the specific lesion conditioning the MSC phenotype may be associated with the putative rearrangement or another mutation that occurred during tissue culture or exists substoichiometrically in the parental line and is transmitted together with JLV5 within the same mitochondrion.

Structural and ultrastructural analysis of root primordia in vitro cultures (RPC) of Solanum lycopersicoides Dun.

The structure and ultrastructure of cell aggregates and derived root primordia were analysed in established suspension cultures of Solanum lycopersicoides Dun. A total of four modified Murashige and Skoog (1962) (MS) media were used, two containing alpha-naphthaleneacetic acid (NAA) and two containing 2,4-dichlorophenoxyacetic acid (2,4-D). Considerable differences were observed in the size and structure of the aggregates regardless of the medium. The largest aggregates had a four-zone structure (cover, starch, dividing and differentiation zones) with distinct ultrastructural organization. The degree of histological and ultrastructural differentiation in the aggregates indicated rhizogenesis initiation. It begins with the protrusion of mounds of root primordia, which are the result of radial growth of cells in defined zones of the aggregate. Further growth from the primordium forms a root meristem with three tiers initial centre and wholly distinguished histogens (dermatogen, periblem, plerome). Rhizogenesis was more regular on media with NAA than on media with 2,4-D. Primordia and fully organized roots were liberated due to fragmentation of the aggregate and underwent changes in the permanently active developmental cycle of the culture which gave rise to successive generations of aggregates. Growth regulators had different influences on the organellar composition and phenolic compounds presence in the different zones of aggregates.

Ultrastructural determination of the early stage of conversion from root primordia to shoot primordia in vitro cultures of Solanum lycopersicoides Dun.

The object of the research were in vitro cultures of root primordia and the early stage of conversion from root to shoot primordia cultures (ESCRS) of Solanum lycopersicoides Dun. The cell ultrastructure of both culture types was studied in order to determine processes underlying the root and shoot morphogenesis type. It was found that a change of a morphogenesis type occurs together with reorganization of aggregate structure and the cell ultrastructural organization. There were 4 zones detected in root primordia culture aggregates (the surface, the starch, the division and the differentiation zone) and 3 zones in ESCRS culture aggregates (the surface, the inner and the differentiation zone). The differences were observed also in storage substances accumulated in a culture (starch in root primordia cultures; proteins, lipids and starch in ESCRS ones).

Genetic factors influencing regeneration ability in rye (Secale cereale L.). I. Immature inflorescences.

Immature inflorescences of ten rye inbred lines (inbred degree S10 and S11) were cultured on solidified MS medium supplemented with 3.0 mg/dm(3) of 2,4-D. According to their capability for callus production explants were classified into two groups : responsive (giving weak or intensive callus production) and non responsive (lack of callus formation). After transferring responsive material into hormone-free medium the regeneration of roots or shoots from the intensive growing callus was observed. Consistent differences between lines in the portion of explants with a certain response were found. They were divided into five groups reacting in the same way. Lines with different in-vitro response were crossed in an incomplete diallel. F1, F2 and F3 generations were analyzed and the following conclusions drawn: the ability for plant regeneration from immature inflorescences in rye is determined by numerous loci, has a recessive character, and both callus production and regeneration suppression may be controlled by complementary genes.

In vitro culture of Cucumis sativus L. : 7. Genes controlling plant regeneration.

The ability to regenerate plants from leaf explants has been tested for three highly inbred cucumber lines (B, G, S), their reciprocal hybrids, F2 and BC1 generations. The lines differed from each other in their regenerating ability, which was expressed by the percentage of explants regenerating embryoidal callus and mean number of plantlets per plant. Thus, the lines could be classified as frequently (B), intermediately (G) or occasionally regenerating ones (S). There were no reciprocal cross differences in the regeneration. It was found that the intermediately and intensively regenerating lines contain two pairs of dominant genes responsible for plant regeneration, characterized by complementary and probably additive interaction. The frequently regenerating line differed from the intermediately regenerating in the effect of one gene. It is supposed that the above-mentioned genes belong to three different loci. The ability to regenerate plants from leaf expiants had high heritability.

In vitro culture of Cucumis sativus L. V. Stabilizing effect of glycine on leaf protoplasts.

A method for leaf mesophyll protoplast isolation and plant regeneration of cucumber (Cucumis sativus L.) is described. Using an isolation solution complemented with 0.1 M glycine, 8.2·10(6) viable protoplasts were isolated from 1 g of fresh leaves. The effect of the growth substances indole-3-acetic acid, naphthalene acetic acid, 2,4,-dichlorophenoxy acetic acid, 6-benzylaminopurine, 2-isopentenyladenine and kinetin at concentrations from 0.5 to 5 mg·1(-1) was studied using the multi-hanging drop technique. The optimal growth substance combination, namely 5 mg·1(-1) naphthalene acetic acid and 3 mg·1(-1) 2-isopentenyladenine, together with agarose medium in a so-called bead culture resulted in a plating efficiency of 21%. Some of the colonies obtained regenerated to plantlets which developed to plants.