M. D. Sacristán

Bio: M. D. Sacristán is an academic researcher from Max Planck Society. The author has contributed to research in topics: Crepis capillaris & Callus. The author has an hindex of 8, co-authored 8 publications receiving 525 citations. Previous affiliations of M. D. Sacristán include University of Hohenheim.

The caryological analysis of plants regenerated from tumorous and other callus cultures of tobacco

Abstract: 1. Large numbers of tumorous (auxin-autotrophic) and non-tumorous (auxin-heterotrophic) tobacco callus cultures were transferred from their culture media in darkness to media with high kinetin content in light for regeneration. There were two lines of tumorous cultures, Tc and Ta; they were started in 1946 from the tobacco variety “White Burley”. Tc had been transformed by infection with Agrobacterium tumefaciens, Ta spontaneously by “habituation”. Non-tumorous cultures (Tn1 and Tn2) were started in 1961 from an unknown variety of tobacco. All the four types of cultures we received from the laboratory of Prof. R. Gautheret in Paris, in 1965. 2. Regeneration from tumorous cultures to fully developed plants was largely successful. These plants, however, were always sterile and none represents a genuine specimen of “White Burley”. Regeneration from non-tumorous, so-called “normal”, cultures was on the contrary very poor. The number of plants regenerated from Tn2 was very small. The plants were more or less uniform, growing well only as scions on other tobacco plants. Only a few “teratomata” from Tn1 developed and, in one case only, a plantlet with roots, shoots and leaves. In grafting experiments with Tn1 teratoma as scion on “Xanthi” tobacco stock, all regenerating plants originated from material with the gene N, probably from the “Xanthi” stock. 3. A comparison of the chromosome numbers of the callus cultures with those of the plants regenerated from them shows that selection plays an important part in the process of regeneration. Plants regenerated from tumorous cultures had extreme aneuploid chromosome numbers and this fact supports the assumption that abnormal chromosome numbers of tumorous cells (in the original callus cultures) are not the cause of tumor growth. On the other hand, the high aneuploidy of the regenerated plants suffices to explain their abnormal morphology and sterility. These abnormal features should not be regarded as a consequence of the tumor condition of the original cultures. The poorly regenerating non-tumorous Tn1 and Tn2 plants, which were always abnormal and aneuploid, underline this viewpoint as do our own observations (Melchers, 1965). 4. TMV in the callus cultures (Tc and Tn2) does not influence the chromosome numbers or the ability to regenerate normal plants. In Tc nearly all regenerated plants are free of TMV, whereas all Tn2-regenerates contain TMV. This interesting difference remains unexplained. 5. “Secondary” callus cultures started from regenerated plants of Tc and Ta behave as control material of “White Burley”: on a medium with auxin, this material grows as callus, without auxin it regenerates to plantlets. 6. The experiments of Braun (1959) and his arguments against the interpretation of “recovery” (from Agrobacterium transformed cells to completely normal plants) excluding the possibility of a selection process, are discussed. His work is compared with our own and that of Lutz (1966) who described the isolation of two single cell clones, one with high, the other with low tumorous activity, from habituated material.

Karyotypic changes in callus cultures from haploid and diploid plants of Crepis capillaris (L.) Wallr

Abstract: Two auxin-heterotrophic callus cultures of Crepis capillaris, one coming from an haploid plant and the other from a diploid one, were studied in regard to karyotypic changes for over a year. The degree of polyploidisation of the originally haploid culture was considerably higher than that of the diploid culture. The frequency of chromosome rearrangements was significantly higher in polyploidised karyotypes than in not polyploidised karyotypes and correspondingly greater in the “haploid” culture. However, the cytogenetical stability of the cultures cannot be measured only through their degree of polyploidisation: it has been found that new karyotypes also originate through chromosome rearrangements at the same ploidy level as the original explant.

Regeneration of plants from “habituated” and “Agrobacterium-transformed” single-cell clones of tobacco

Abstract: One “habituated” and three “Agrobacterium-transformed crown gall” callus strains of tobacco, all hormone-autotrophic, were cloned and tested for regeneration to plants. The crown gall strains originated from “unorganized” tumors induced by highly virulent strains of Agrobacterium tumefaciens. The regeneration of complete plants from a great number of habituated clones as well as from three fully transformed single-cell clones isolated from a young crown gall strain definitely proves that both processes, habituation and Agrobacterium-transformation, are reversible.

Transformation to auxin-autotrophy and its reversibility in a mutant line of Crepis capillaris callus culture

Abstract: From a callus culture which was started from explants of a single plant of Crepis capillaris, a line was isolated which could grow on auxin- and kinetin-free medium (in contrast to the other lines of the culture). The calluses of this habituated line grew on hormone-free medium as “teratomas”. Regenerated plants were obtained from the habituated line and from other lines of the culture as control. In the secondary callus cultures which originated from these plants, the subcultures belonging to the initially transformed line no longer had the ability to grow on hormone-free medium. Since this line was also a chromosome mutant and the same mutation was present in its regenerated plants and in the secondary culture line of them, the loss of the auxin-autotrophy cannot be explained in this case on the basis of a selection of cells with karyotypes different from those of the primary culture.

Tumorous cultures of Crepis capillaris : chromosomes and growth

Abstract: Different crown-gall culture strains of Crepis capillaris derived from haploid and diploid tumors were studied karyologically during the period of 18 transfers (18–20 months). The very low proportion of mitotic anomalies observed in the first transfers is in agreement with the generally accepted assumption that chromosomal changes are not involved in the initiation of tumors. However, it has been established that an intimate relationship exists between chromosome aberrations and the further development of the tumors. Morphological and physiological changes which occurred in some strains and substrains were invariably associated with cytologieal changes. The results are discussed in connection with similar phenomena described for animal tumors.

Somaclonal variation — a novel source of variability from cell cultures for plant improvement

Abstract: It is concluded from a review of the literature that plant cell culture itself generates genetic variability (somaclonal variation). Extensive examples are discussed of such variation in culture subclones and in regenerated plants (somaclones). A number of possible mechanisms for the origin of this phenomenon are considered. It is argued that this variation already is proving to be of significance for plant improvement. In particular the phenomenon may be employed to enhance the exchange required in sexual hybrids for the introgression of desirable alien genes into a crop species. It may also be used to generate variants of a commercial cultivar in high frequency without hybridizing to other genotypes.

The host range of crown gall

Abstract: Crown gall is a plant tumor disease caused by the specific action of the bacteriumAgrobacterium tumefaciens. In the current literature its host range is not clearly defined or is thought to be restricted to the dicotyledonous class of the angiosperms.

Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors

Abstract: Renaturation kinetics of labeled Agrobacterium tumefaciens DNA are not influenced by addition of 10(4)-fold excess of crown gall tumor DNA. Reconstruction experiments demonstrated that 0.01% added bacterial DNA produces a detectable increase in rate of renaturation of labeled DNA. Crown gall tumor DNA therefore cannot contain as much as 0.01% A. tumefaciens DNA (one entire bacterial genome per three diploid tumor cells). By the same technique, PS8 bacteriophage DNA is not detected in crown gall tumor DNA under conditions that allow detection of 0.0007-0.001% added phage DNA. Crown gall tumor DNA cannot contain as much as one entire phage genome per diploid tumor cell. The presence of a small fraction of either genome (less than 5%), even if present as multiple copies, would escape detection by this method.

Plant Callus: Mechanisms of Induction and Repression

Abstract: Plants develop unorganized cell masses like callus and tumors in response to various biotic and abiotic stimuli. Since the historical discovery that the combination of two growth-promoting hormones, auxin and cytokinin, induces callus from plant explants in vitro, this experimental system has been used extensively in both basic research and horticultural applications. The molecular basis of callus formation has long been obscure, but we are finally beginning to understand how unscheduled cell proliferation is suppressed during normal plant development and how genetic and environmental cues override these repressions to induce callus formation. In this review, we will first provide a brief overview of callus development in nature and in vitro and then describe our current knowledge of genetic and epigenetic mechanisms underlying callus formation.

Plating of isolated tobacco mesophyll protoplasts on agar medium

Abstract: A technique was developed to derive cell and plant clones from isolated mesophyll protoplasts of tobacco. The protoplasts, plated on a fully defined agar medium, divided and grew actively forming visible colonies after one month of culture. Efficiency of colony formation depended on cell density and light condition during incubation. Under standard conditions, 60% of plated protoplasts formed colonies. Upon transfer onto suitable media, these colonies differentiated shoots and roots, and eventually regenerated whole plants. Advantages of mesophyll protoplasts as the source of clones as well as implication of the plating technique for genetical studies are discussed.