Haploid plants regenerated from androgenic cell cultures of Digitalis lanata.

Androgenic callus was obtained from cold treated anthers and pollen of Digitalis lanata. The callus was mixoploid and contained haploid, diploid and tetraploid cells as shown by impulse cytophotometry. Haploid cell lines were selected by colony cloning. They were unstable and selection had to be repeated every 1-2 months. Mixoploid shoot cultures were derived from embryogenic haploid cell lines via somatic embryos. Haploid shoots were selected by explanting shoot tips. The shoots showed wide variability in cardenolide content and profile. Rooting of the haploid shoots resulted in haploid plants. These plants were smaller in size than diploid plants. Often the flowers were morphologically abnormal and showed male sterility due to crippled anthers.

Isolation and characterization of two acyl-CoA-binding proteins from proembryogenic masses of Digitalis lanata Ehrh.

Two acyl-CoA-binding-protein (ACBP) isoforms were isolated from proembryogenic masses of Digitalis lanata Ehrh. by column chromatography and preparative HPLC. The ACBPs had molecular masses of 9926 and 9997 Da, respectively. Partial sequence data indicated high similarity to each other and to ACBPs of other plant species such as Ricinus communis, Brassica napus and Arabidopsis thaliana. The isolated ACBPs bound palmitoyl-CoA with high affinity as determined by isoelectric-point shift.

Stress-induced expression of cyclophilins in proembryonic masses of Digitalis lanata does not protect against freezing/thawing stress.

Using proembryonic masses (PEMs) of Digitalis lanata Erh., it was demonstrated that cold, hormonal or osmotic stress, which increased freezing tolerance during cryopreservation, induced an increasing level of two peptidyl-prolyl-cis/transisomerases (PPIases). The difference in pI (9.2 +/- 0.2 and 9.5 +/- 0.2, +/- SD; n = 3) allowed the separation of the two enzymes by free-flow isoelectrophoresis. Both were inhibited by cyclosporin A and thus belong to the cyclophilin family of PPIases. The enzymes differed slightly in their substrate specificity and their relative molecular masses of 18038 +/- 4 Da (D. lanataCyp18.0) and 18132 +/- 3 Da (D. lanataCyp18.1). Both cyclophilins were blocked N-terminally. Partial internal amino acid sequences from the two cyclophilins, with a length of 34 amino acids, displayed 82% sequence identity to each other. Pretreatment of PEMs with abscisic acid, sorbitol or a combination of both substances led to a 270 +/- 30% elevation of the total cytosolic cyclophilin concentration determined with a cyclophylin affinity sensor. During the first 4 d of pretreatment, the total PPIase activity was enhanced up to 230 +/- SD% compared with the control culture. The lag phase between maximal PPIase concentration after 4 d of pretreatment and maximal effect of freezing tolerance after 10 d of pretreatment indicated that increasing levels of cytosolic PPIases may be necessary to overcome the stress induced by hormones and osmotica during pretreatment but not to protect against freezing/thawing stress.

21'-Di-dehydro-deacetyllanatoside C, a biotransformation product of deacetyllanatoside C from senescent shoot cultures of Digitalis lanata.

Feeding deacetyllanatoside C to senescent shoot cultures of Digitalis lanata resulted in the formation of a new product, which was isolated by semi-preparative HPLC. The molecular structure was elucidated by means of HPLC-mass spectrometry and NMR as 21'-di-dehydro-deacetyllanatoside C.

Cardenolides in Digitalis lanata Cells Transformed with Ti-Plasmids.

Crown galls were induced by transformation of leaves, leaf discs, and shoots of the plant DIGITALIS LANATA with the AGROBACTERIUM TUMEFACIENS strains C58 pTi C58, B6S3 pTi B6S3, and A136 pTi A6NCtmr-338::Tn5. Integration of plasmid DNA in the genome of D. LANATA was demonstrated by hybridization experiments. The transformed cells synthesized opines and showed hormone-autotrophic growth. The crown galls formed on leaves of D. LANATA plants contained digitoxigenin derivatives (up to 0.8 muimol digitoxin equivalents g (-1) dry weight). Transformed cell lines derived from the crown galls built cardenolides IN VITRO (ca. 0.03 mumol digitoxin equivalents g (-1) dry weight). The rate of cardenolide biosynthesis IN VITRO did not decrease during a cultivation period of 12 months.

Batch Cultures of Somatic Embryos of Digitalis lanata in Gaslift Fermenters. Development and Cardenolide Accumulation.

Somatic embryos of DIGITALIS LANATA STRAIN VII were successfully grown as batch cultures in gaslift fermenters. Embryo development and formation of cardenolides in the embryos resembled those of cultures grown in shake flasks. Both processes depended on the developmental stage of the embryos at the time point of inoculation, the homogeneity of the embryo structures, the density of the inoculum, the composition of the nutrient medium, the intensity of irradiation, and the composition of the gas mixtures used for agitation of the embryo suspension. The cultures contained 0.7&-1.0 (micro,mol digitoxin equivalents g (-1) dry wt. (2.6-6.5 micromol digitoxin equivalents l (-1)) after a cultivation period of 28 d.

Formation of Digitalis lanata Clone Lines by Shoot Tip Culture.

Propagation of DIGITALIS LANATA by shoot tip culture made possible (a) the rapid multiplication of elite plants with the formation of plant clones and (b) the long-term cultivation of these plants which under normal growth conditions would die at the end of the second vegetation period. Optimum conditions were established for the regeneration of shoots from shoot tips, for daughter shoot formation and rooting as well as for the adaptation of the regenerated plants to the open ground. Gene banks of valuable clones were built by keeping shoots at 4 degrees C on media with high sucrose concentration (maximum period of storage 2 years) or by growing juvenile clone plants in the greenhouse at temperatures preventing the induction of flowering. The clone plants were in the juvenile state even if they were derived from flowering mother plants. They showed normal growth and development.

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.

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.