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

Induction of Haploid Plants From Isolated Pollen of Brassica napus

Although phenolic compounds are present in both the plant and animal worlds, most of them are of plant origin.1 This heterogeneous group of natural compounds is still, in many textbooks, described as “secondary metabolites”, which suggests their less important role in cellular physiology and biochemistry. It includes both simple phenols and polyphenols as well as their derivatives. In general, the term “phenol” can be defined chemically as a substance that possesses an aromatic ring bearing a hydroxyl substituent and functional derivatives. The natural plant phenols arise biogenetically from two main pathways: the shikimate pathway, which directly provides phenylpropanoids such as the hydroxycinnamic acids and coumarins, and the polyketide (acetate) pathway, which can produce simple phenols and also lead to quinones. The flavonoids, by far the largest group of phenolics, are derived by combination of these two pathways. Among plant single-ring phenolics the group of various compounds that has recently shown growth is the derivatives described as phenolic lipids or long-chain phenols.2 They are amphiphilic in nature due to the non-isoprenoid side chains attached to the hydroxybenzene ring and are believed to be also derived from the polyketide (acetate) pathway, as, for example, 6-pentadecylsalicylic acid. Non-isoprenoid phenolic lipids are relatively uncommon and can be considered for simplicity as fatty acids, with the carboxyl group replaced by the hydroxybenzene ring. Therefore, they are derivatives of monoand dihydroxyphenols, namely, catechol, resorcinol, and hydroquinone. The biogenetic pathway of these compounds, described in textbooks and recent reviews,2-5 is based on incomplete experimental data supplemented with chemical considerations. The biosynthetic aspects of resorcinolic lipids Volume 99, Number 1 January 1999

http://www.halsanet.com/pdf/340516159.pdf

Resorcinolic Lipids, the Natural Non-isoprenoid Phenolic Amphiphiles and Their Biological Activity.

Rapeseed plants have been regenerated after fusion between protoplasts bearing cytoplasms of different genera Cybrids combine, in a first experiment Brassica napus chloroplasts and a cytoplasmic male sterility (cms) trait coming from Raphanus sativus, in a second experiment chloroplasts of a triazine resistant Brassica campestris and cms trait from Raphanus sativus Transfer of chloroplasts has been confirmed by restriction cp DNA analysis and two dimensional thylakoid protein electrophoresis These plants may be very useful for Brassica hybrid seed production

Intergeneric cytoplasmic hybridization in cruciferae by protoplast fusion

For several years it has been recognized that introduction of plant cells into culture results in genetic changes. These genetic alterations have been recovered in the plants regenerated from cell cultures. More recently it has been recognized that this method of introducing genetic changes into crop plants could be used to develop new breeding lines. The technology of introducing genetic variation by using cell culture has been termed somaclonal and gametoclonal variation. Somaclonal variation has been detected and documented in tomato. In particular, several single gene mutations induced by somaclonal variation have been mapped to specific loci. As this variation represents a new tool for the plant breeder, breeding strategies for the use of this variation are presented and discussed. Somaclonal and gametoclonal variation are new tools for the geneticist and plant breeder that permit reduction in the time period for new variety development and that permit access to new classes of genetic variation.

Somaclonal and gametoclonal variation

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.

Haploid microspore-derived plants from amphidiploid Brassica napus give rise in culture to very large numbers of secondary stem embryoids from single epidermal cells. The embryoids can be excised, transferred to fresh medium and induced to repeat the process 4–6 times. Eventually the embryoids form larger plantlets which can be grown to maturity. Spontaneous amphihaploid plants or microspore-derived plants growing either in vitro or in vivo have been used as a source of plant protoplasts. Such protoplasts have been induced to produce calluses capable of both root and shoot production. The limited number of plantlets so far obtained from large numbers of calluses have retained their haploidy. The significance of haploid single-cell cultures of such agriculturally important fodder crops is discussed.

Protoplast regeneration and stem embryogenesis of haploid androgenetic rape

In Solanum tuberosum the production by parthenogenesis of 2x plants with 24 chromosomes, and the regeneration of microspores of such dihaploids to yield monohaploid (1x) plants is reproducibly possible, at least for some specific genotypes. Experiments are described using tissue culture techniques in an applied breeding program with the main aim of increasing the level of resistance to the potato cyst nematode Globodera pallida (Stone) and to the potato viruses X, Y and leaf roll. These resistances follow quantitative as well as qualitative modes of inheritance. Using anther culture it is demonstrated that doubled monohaploid clones can be produced which possess the resistance in the homozygous condition. In both ways of inheritance the ratio of resistant clones is rather high. The genotype of the anther donor plant has, however, a strong influence on the total number of androgenetic plants which can be regenerated. Therefore, experiments were initiated with the aim of integrating this capacity for regeneration (tissue culture ability) into valuable genotypes. The results show that the potentiality for regeneration is under genetic control and can be utilized by combination breeding. Its inheritance and physiological basis, as well as the behaviour of complete homozygous clones, is discussed.

Breeding for nematode and virus resistance in potato via anther culture.

Progeny analysis of androgenetic plants from inbred rape-seed (Brassica napus) shows that selective growth of microspores can occur in cultured anthers. The property of privileged growth in culture seems to be linked to such characters as flowering time and seed glucosinolate content which can be analyzed in regenerated plants. This type of selection and the fact that more variability is visible in regenerants from different microspores than in the progeny of the highly inbred anther donor line, demonstrates the higher degree of homozygosity in the doubled amphihaploids of B. napus. Furthermore, it is shown that haploid genomes of rape may be mutable. Thus it is possible to obtain several different homozygous lines from a single microspore. A system of haploid embryoids arising from single cells of the primary microspore regenerant has also been used to produce experimentally induced mutants. It is demonstrated that recessive mutations can be obtained in a homozygous state in doubled haploid regenerants from mutagenized haploid single cells.

Anther culture as a breeding tool in rape : II. Progeny analyses of androgenetic lines and induced mutants from haploid cultures.

Further progress of studies aimed at increasing production of androgenetic Secale cereale plants via the culture of anthers is described. Two culture media initially developed for rice and wheat anther culture have been shown to have pronounced influence on rye. It has been possible to increase the average percentages of responsive anthers (i.e. those producing embryoids or calluses) from 0.26% to 10% with a maximum in certain experiments of over 40 %. Of nearly 400 plants produced in 1976, 1/4 are green and can be grown further by transfer to potting compost; 3/4 are albino. Stable green haploid lines were present amongst the plants, and after vegetative propagation of the lines representative samples have been treated with colchicine resulting in diploid, triploid and tetraploid plants. The influence of the genetic background of the donor plants on the success rate of anther culture and on the percentage of albino formation is discussed.

G. Wenzel

Papers

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

Protoplast regeneration and stem embryogenesis of haploid androgenetic rape

Breeding for nematode and virus resistance in potato via anther culture.

Anther culture as a breeding tool in rape : II. Progeny analyses of androgenetic lines and induced mutants from haploid cultures.

Increased induction and chromosome doubling of androgenetic haploid rye.