In vitro induction and proliferation of protocorm-like bodies (PLBs) from leaf segments of Phalaenopsis bellina (Rchb.f.) Christenson

An in vitro culture procedure was established to induce protocorm-like bodies (PLBs) from leaf segments of the Phalaenopsis bellina (Rchb.f.) Christenson directly from epidermal cells without intervening callus on ½ strength modified Murashige and Skoog (MS) (in Physiol Plant 15:473–497, 1962) medium supplemented with 1-Naphthaleneacetic acid (NAA; 0, 0.1, 1 mg/l) and Thidiazuron (TDZ; 0, 0.1, 1, 3 mg/l). The best response was established at 3 mg/l TDZ which induced 78% of leaf segments to form a mean number of 14 PLBs per explant after 16 weeks of culture. No PLBs were found when leaf segments were cultured on ½ strength modified MS media supplemented with 0.1 and 1 mg/l NAA. The best induction percentage for auxin: cytokinin combination was at the combination of NAA and TDZ at 1.0 and 3.0 mg/l which gave 72% induction with 9 PLBs per explant. Semi-solid ½ strength MS and liquid Vacin and Went (VW) (in Bot Gaz 110:605–613, 1949) medium were used in order to find the highest survival and number of PLBs proliferation after 3 months in culture. Half strength MS showed an average of 9 PLBs in comparison with VW with an average of 5.3 PLBs per explants. Histological observations revealed that the regenerated PLBs were generally formed from the epidermal layers of the posterior regions of the leaf segments. Scanning electron micrograph of PLBs showed the origin of newly formed PLB from the peripheral region of leaf segments

Supportive development of functional tissues for biomedical research using the MINUSHEET(R) perfusion system

Functional tissues generated under in vitro conditions are urgently needed in biomedical research. However, the engineering of tissues is rather difficult, since their development is influenced by numerous parameters. In consequence, a versatile culture system was developed to respond the unmet needs.Optimal adhesion for cells in this system is reached by the selection of individual biomaterials. To protect cells during handling and culture, the biomaterial is mounted onto a MINUSHEET(R) tissue carrier. While adherence of cells takes place in the static environment of a 24 well culture plate, generation of tissues is accomplished in one of several available perfusion culture containers. In the basic version a continuous flow of always fresh culture medium is provided to the developing tissue. In a gradient perfusion culture container epithelia are exposed to different fluids at the luminal and basal sides. Another special container with a transparent lid and base enables microscopic visualization of ongoing tissue development. A further container exhibits a flexible silicone lid to apply force onto the developing tissue thereby mimicking mechanical load that is required for developing connective and muscular tissue. Finally, stem/progenitor cells are kept at the interface of an artificial polyester interstitium within a perfusion culture container offering for example an optimal environment for the spatial development of renal tubules.The system presented here was evaluated by various research groups. As a result a variety of publications including most interesting applications were published. In the present paper these data were reviewed and analyzed. All of the results point out that the cell biological profile of engineered tissues can be strongly improved, when the introduced perfusion culture technique is applied in combination with specific biomaterials supporting primary adhesion of cells