Cryopreservation of Digitalis lanata Shoot Tips.

A method for the preservation in liquid nitrogen of shoot tips (meristems) of D. LANATA is described. It includes the following steps: (a) hardening of shoots by cultivation at 4 degrees C for 8 weeks, (b) treatment of the explanted shoot tips with cryoprotectors, e.g., 2 mol DMSO l (-1) for 2 h, (c) either ultrarapid cooling (ca. 4000 K min (-1)) of the shoot tips by submerging in liquid nitrogen or slow cooling (ca. 0.5 K min (-1)) of the shoot tips to -40 degrees C using a suitable freezer, (d) storage of the shoot tips at -196 degrees C in liquid nitrogen, (e) ultrarapid rewarming of the ultrarapidly cooled shoot tips by placing them directly into nutrient medium or rapid rewarming of the ampoules containing the slowly cooled shoot tips with water at 40 degrees C, and (f) recultivation of the shoot tips at the surface of a solidified nutrient medium containing 2.5 micromol BA 1 (-1). About 70% of the shoot tips survived this procedure and about 30% of the shoot tips regenerated shoots.

Introduction of a nitrogen-fixing cyanobacterium into tobacco shoot regenerates.

Tobacco (Nicotiana tabacum L.) shoots associated with the nitrogen-fixing cyanobacterium Anabaena variabilis Kütz. (ATCC 29413) were regenerated in mixed cultures of tobacco callus and the cyanobacterium. The cyanobacteria were localized inside the tissues as well as on the surface of regenerated shoots, formed heterocysts, and were capable of acetylene reduction.

Diversity of Vicia faba circular mtDNA in whole plants and suspension cultures.

A comparative analysis of the Vicia faba mitochondrial genome in whole plants and in longterm suspension culture has been conducted. Restriction fragment patterns of the mtDNA isolated from these two sources were notably different. Electronmicroscopic analysis also revealed significant differences. Large circular mtDNA patterns shifted from a 37-80 kb subpopulation, which was predominant in whole plants, to 18-34 kb subpopulations although in both classes notable quantities of circular molecules of 80 to 120 kb and more were also found. Both in whole plant and suspension culture cells very large circular DNAs were observed. Some of them had lengths nearly 290 kb and could be considered as evidence of the existence of master chromosomes. The minicircular DNA population was also altered. In the suspension culture we observed a notable increase of percentage of minicircles with sizes near 1 kb. Simultaneously, the percentage of minicircles with sizes near 3.5-10 kb significantly increased in suspension culture cells. In addition, a new peak (10-12 kb) of minicircles appeared. Copy number alterations for some sequences homologous to CCC1A, CCC1B and CCC2 (Negruk et al. 1982, 1985) were shown. Southern hybridization revealed the existence of a family of minicircles having sizes 1.4-2 kb with predominance of CCC1A, CCC1B and CCC2. The copy numbers of CCC1B and some minor minicircles was changed in the suspension culture when compared with the whole plants.

Cryopreservation of Digitalis lanata cell cultures.

Suspension cultures of Digitalis lanata strain I were grown in a medium containing 3% mannitol. For cryopreservation cell suspensions were treated with a mixture of sucrose-glycerol (20%/20 V%), cooled slowly (about 1 degrees C/min) till -100 degrees C and then were transferred to liquid nitrogen. After storage in liquid nitrogen the cells were thawed rapidly in a water bath of 40 degrees C and spread on the surface of a solidified nutrient medium. After 7 days of regrowth the cells were suspended in liquid nutrient medium for further cultivation. About 50% of the cells survived freezing and thawing. However, also the apparently surviving cells showed signs of injury (membrane vesicles outside the plasmalemma, dilated ER cisternae and separation of the nuclear membranes). The cultures derived from the surviving cells had the same growth rate and biochemical activity relative to the transformation of cardenolides, e.g., digitoxin, as the parent cultures. The frequency distribution of the nuclear DNA content in the cell cultures was the same before and after cryopreservation. These results indicate that there is no selection of a special cell type during freezing and thawing.