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.

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

Potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum) are members of the Solanaceae (nightshade family) and have the same basic chromosome number (x = 12). However, they cannot be cross-hybridized and, until now, it was unknown how conserved the gene order might be between these two species. We report herein the construction of a genetic linkage map of potato chromosomes based on genomic and cDNA clones from tomato. The potato map was drawn from segregation data derived from the interspecific cross S. phureja X (S. tuberosum X S. chacoense) (2n = 2x = 24), and consists of 135 markers defining 12 distinct linkage groups. Nearly all of the tomato probes tested hybridized to potato DNA, and in most cases, the copy number of the employed clones was the same in both species. Furthermore, all clones mapped to the same linkage group in both species. For nine chromosomes, the order of loci appears to be identical in the two species, while for the other three, intrachromosomal rearrangements are apparent, all of which appear to be paracentric inversions with one breakpoint at or near the centromere. These results are consistent with cytogenetic theory, previously untested in plants, which predicts that paracentric inversions will have the least negative effect on fitness and thus be the most likely form of chromosomal rearrangements to survive through evolutionary time. Linkage maps based on a common set of restriction fragment length polymorphism markers provide a basis for uniting the previously separate disciplines of tomato and potato genetics. Using these maps, it may now be possible to test theories about homologies or orthologies of other genes, including those coding for disease resistance and stress tolerances.

https://www.genetics.org/content/genetics/120/4/1095.full.pdf

RFLP Maps Based on a Common Set of Clones Reveal Modes of Chromosomal Evolution in Potato and Tomato.

The considerable amount of activity in the field of electrofusion and electropermeabilization is very promising from the point of view of new insights into biomembranes and new technologies in the future for the production of new compounds and modification of cell systems for nutrition, energy production and the removal of waste products. It is particularly gratifying to see how basic science has provided the foundation for a useful technology, although in some cases the time needed to develop an application is very long. In other cases, it is necessary to overcome the difficulties posed by existing schools of thought which have been shown to be wrong. It is fascinating to observe the many developments and discoveries in the areas of physics, material science, space technology and electronics which are just waiting to be applied to biological systems. An increased interdisciplinary collaboration between physicists and biologists could provide considerable impetus to biology and its application in technology. However, this can only be achieved if basic research into biological membranes is accelerated. The techniques for electrical breakdown, electropermeabilization and electrofusion could be an important tool in this process, since we cannot rule out the possibility that the high electrical fields occurring naturally in the membrane play an important role in the selective transport of substances across the membrane as well as in natural regulatory processes.

Electrical breakdown, electropermeabilization and electrofusion

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.

https://www.pnas.org/content/pnas/71/9/3672.full.pdf

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

Plant protoplasts show physiological perceptions and responses to hormones, metabolites, environmental cues, and pathogen-derived elicitors, similar to cell-autonomous responses in intact tissues and plants. The development of defined protoplast transient expression systems for high-throughput screening and systematic characterization of gene functions has greatly contributed to elucidating plant signal transduction pathways, in combination with genetic, genomic, and transgenic approaches.

/pdf/signal-transduction-in-maize-and-arabidopsis-mesophyll-4b65wac81t.pdf

Signal Transduction in Maize and Arabidopsis Mesophyll Protoplasts

Mesophyll protoplasts of Lycopersicon esculentum Mill. var. cerasiforme (Dunal) Alef, mutant yellow green 6, Rick and protoplasts of a liquid callus culture of the dihaploid strain HH258 of Solanum tuberosum L. were prepared and many fusion products were visible after the protoplasts were incubated together first in the presence of polyethylene glycol and then with a high Ca2+ ion concentration. The protoplasts were transferred to a rich medium and the resultant calli were cultured. Some calli regenerated normal green shoots which were transferred to soil or grafted onto a tomato stock. The subunit polypeptide pattern of ribulose 1,5-bisphosphate carboxylase prepared from leaf material of four regenerated plants was analyzed by isoelectric focusing. The ribulose bisphosphate carboxylase enzyme oligomer in the four plants contained the small subunit products resulting from the expression of both tomato and potato nuclear genes proving these plants to be somatic hybrids between tomato and potato. In three of the four plants the large subunit polypeptides and hence the functional chloroplast DNA were from tomato whereas in the fourth the large subunit and therefore the chloroplast DNA was derived from potato. The plant material was insufficient to establish the chromosome numbers precisely, however counts close to 50 which is near to the expected 48 were obtained for three of the hybrids whereas in the fourth a number close to 72 was observed. In the absence of a selection system against the potato parent, the analysis of ribulose bisphosphate carboxylase provides a convenient marker to demonstrate the hybrid nature of the plants.

/pdf/somatic-hybrid-plants-of-potato-and-tomato-regenerated-from-1vewf7v12d.pdf

Somatic hybrid plants of potato and tomato regenerated from fused protoplasts

(1963). [5] v. Sengbusch, R., in: Vortragsveranstaltung fiber neue Methoden der Fischzflchtung und -haltung. Hamburg: MPI f. KulturpflanzenzClchtung t 9 6 7 . [6~ Langhans, V. : NachrichtenbI. Fischzucht Fischerei 1, 122 (t928). [7] Krupauer, V.: Ryb~stvi H. 6, 85 (t963). [8] v. Sengbusch, R., Meske, Ch., Szablewski, W.: Experientia 21, 614 (1965). --. [9] v. Sengbusch, R., et al.: Z. Fiscberei 15 N. F., 45 (1967). [10]Meske, Ch.: Fischwirt 16, 309 (1966). [il~ I(oBmann, H.: ibid. 20, 255 ( 1 9 7 0 ) . ~t2~ SchS~perclaus, W.: Lehrbuch der Teichwirtschaft. Berlin-Hamburg: Paul Parey 1961. [13] Jancarik, A.: Z. Fischerei 12 N. t~'., 602 (1964). [t4~ Lflhr, B., in : Vortragsveranstaltung fiber neue Methoden der Fischzflchtung und -haltung. Hamburg: MPI f. t,~ulturpflanzenzflchtung 1 9 6 7 . [15~ Meske, Ch.: Fischwirt 18, 310 (t968). [t6] 3/Ieyer-Waarden, P. F., in: Der Aal. Hamburg: A. Keune 1 9 6 5 . [171 M~ller, H. : Dtsch. Fischerei-Z. 14, t (t967). r t81 Meske, Ch.: Arch. Fischereiwiss. 20, 26 (1969). [191 Suworow, I~.: Grundlagen der Ichthyologie. Moskau 1 9 4 8 . [20] Ihering, G.: Zool. Anz. 3 (t935). [2t] Gerbilski, H. L.: Vesti. Leningrad un.-ta Mr. (zitiert nach Woyn~rovich 1963). (t951). [22] Woyn~rovich, E.: Beitr. Gew/~sserforsch. 4, 210 ( 1 9 6 4 ) . [23] Meske, Ch., et al.: Theoret. Appl. Genetics 38, 47 ( t 9 6 8 ) . [24~ Kof3mann, H.: Zuchthygiene, im Druck. [25] Meske, Ch., Lflhr, B., Szablewski, \"vV.: Naturwissenschaften 54, 291 (1967). [26] v. Sengbusch, R., Meske, Ch.: Zflchter 37, 271 ( 1 9 6 7 ) . [27] KoBmann, H., Szablewski, W.: Fischwirt 21, 49 (1971): ~281 Denkschrift der DLG-Tierzuchtabteilung ( t 9 6 7 ) . [29] Meske, Ch. : Fischwirt 19, 244 (t969).

Regeneration of whole plants from isolated mesophyll protoplasts of tobacco

The techniques of microspore and protoplast regeneration starting from dihaploid Solanum tuberosum plants has been improved to such an extent that the production of more than 2000 microspore derived A1 plant lines and of several hundred protoplast derived plantlets has become possible. Further, from the dihaploid Solanum species S. phureja the regeneration of microspores to plants, and from the species S. infundibuliforme, S. sparsipilum and S. tarijense the regeneration of protoplasts to calluses, has been achieved. The plants descending from the two single cell culture systems are compared with reference to phenotypic markers and economic qualities. Some principles characteristic for either microspore or protoplast derived plants are examined and their significance is discussed. The results are compiled into an extended analytical synthetic breeding scheme based on a stepwise reduction of the autotetraploid to the monohaploid level and a subsequent controlled combination to a new synthetic completely heterozygous tetraploid potato.

Comparison of single cell culture derived Solanum tuberosum L. plants and a model for their application in breeding programs.

Mesophyll protoplasts of “haploids” produced by “anther culture” of two chlorophyll deficient, light sensitive varieties of Nicotiana tabacum, sublethal (s) and virescent (v) are fused with the method of Keller and Melchers (1973). The double heterozygote hybrid of the two recessive genes for light sensitivity complements to normal leaf colour and resistance to high light intensity. By using this complementation obviously 12, possibly 21 independently arisen somatic hybrid plants are selected. Since leaf colour mutations are frequent and light sensitivity of leaf colour mutants is not too rare in higher plants, it is possible to use generally this selection method.

Somatic hybridisation of plants by fusion of protoplasts

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.

Georg Melchers

Papers

Somatic hybrid plants of potato and tomato regenerated from fused protoplasts

Regeneration of whole plants from isolated mesophyll protoplasts of tobacco

Comparison of single cell culture derived Solanum tuberosum L. plants and a model for their application in breeding programs.

Somatic hybridisation of plants by fusion of protoplasts

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