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.

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).

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.