Breeding of industrial oil crops with the aid of biotechnology: a review

261

Industrial Crops and Products, l(1993) 261-271 0 1993 Elsevier Science Publishers B.V. All rights reserved.0926~6690/93/$05.00

INDCRO PRO33

Breeding of industrial oil crops with the aid of biotechnology: a review A. Thierfelder, W. Liihs and W. Friedt Institute ofAgronomy and Plant Breeding4

Justus-Liebig-University,

Giessen, Germany

(Accepted 1 September 1992)

Abstract Thierfelder, A., Liihs, W. and Friedt, W. 1993. Breeding of industrial oil crops with the aid of biotechnology: a review. Industr. Crops Products 1: 261-271. Different oil crops are characterized by specific fatty acid patterns which can further be optimized by breeding. An impressive example is the elimination of erucic acid from rapeseed oil, which made it possible to use this vegetable oil for human consumption. Furthermore, sunflower (Welianthus annuus L.) cultivars with high oleic acid and linseed (Linum usitatissimum L.) with high linolenic acid contents are available which meet the demand of industry for these specific types of oil. Further progress by breeding can be achieved through the application of biotechnology, i.e. tissue and cell culture and molecular methods. Considerable progress in this field has been reported for rapeseed (Brassica napus L.) and other Brassicaceae, but also for Linum species. In these species and others, it is possible to obtain haploid plants reproducibly through microspore- or anther-culture which can result in a time gain of several years. Interspecific hybridization is another interesting supplementary technique in plant breeding. It can help to create new genetic variability. Interspecific crosses have been successfully used, for example in the Brassicaceae family and in the genus Helianthus, by application of the embryo rescue technique. Consequently, related species can be used as gene sources to improve various agronomically important characters. For extremely recalcitrant species, the protoplast fusion technique may be an elegant method of achieving hybrids, provided that the regeneration of intact plants is feasible. Last but not least, for an application of genetic engineering in plant breeding, necessary prerequisites are principally available, e.g., efficient vectors and transformation systems. Therefore, the transfer of agronomically important genes, e.g. for disease resistance or quality traits, to cultivated species is no longer a utopia - provided that entire plants can be regenerated from the manipulated cell(s) or tissue. Resynthesized rapeseed; Helianthus; Erucic acid; Lipid metabolism

Brassica;

Linum;

Introduction

In recent years, the use of vegetable oils as industrial feedstocks has been greatly inCorrespondence: A. Thierfelder, Institute of Agronomy and Plant Breeding I, Justus-Liebig-University, Ludwigstr. 23, D-6300 Giessen, Germany.

Breeding; Biotechnology; Genetic Engineering;

creased. Besides rapeseed oil, linseed and sunflower oil are of great interest as a source of valuable industrial raw material Particular demands require specific fatty acid profiles which determine the suitability of vegetable oil for technical applications. Different oil crops, i.e. rapeseed, sunflower and

262

linseed, are characterized by specific fatty acid compositions. Extensive breeding efforts have been made in the past to provide oil crops with optimum oil quality. Consequently, a variety of plant species and cultivars are now available which allow the recovery of custom tailored oil (Baumann et al., 1988; Scrowcroft, 1990). Good examples, where impressive effects have been achieved, are the new breeds of rape 20 years ago, where the erucate level in the oil was reduced considerably (Stefansson and Hougen, 1964); mutagenic treatment of linseed has produced strains giving oils with a reduced linolenate content (Green, 1986), and the development of high oleic sunflower types (Soldatov, 1976) represent successful examples of conventional genetic approaches. Whereas food use of vegetable oil requires a precise mixture of saturated and unsaturated fatty acids with chain lengths of 16-18 carbons, an industrial application demands an oil composition with a predominance of a single desirable fatty acid. The wide range of industrially valuable fatty acids is the basis for a number of different breeding targets. Those are, e.g., high erucic acid content in rapeseed oil, high linolenic content in linseed oil and high oleic acid content in sunflower oil (Eierdanz and Hirsinger, 1990; Sonntag, 1991). As well as traditional breeding methods, biotechnologies hold great potential for improving the quality and yield of plant oils (cf. Friedt et al., 1991). The present paper is not intended as an overall recapitulation of previous work on biotechnology methods in breeding oil crops; instead it will focus on some of its potentials and recent applications. Biotechnology

Approaches

The term ‘Biotechnology’ includes a wide range of different cell and tissue culture techniques and molecular methods. Cell and tissue culture techniques are targeted at regenerating plants from isolated plant cells or tissue in vitro, i.e. meristems, immature embryos, anthers, microspores, protoplasts etc. Molecular methods include techniques for an identification, isolation and transfer of genes. Since manipulated cells and tissue must be regenerated to entire

plants, efficient cell and tissue culture methods are an integral part of genetic engineering. New genotypes produced in this way are only the first step towards new cultivars; they are the basic germplasm for further applied breeding programs (for an overview cf. Fig. 1). Haploidy

Technique

The principle advantage of haploidy technique is the rapid fixation of segregating genotypes, occurring in lower frequency, in which recessive genes coding for specific traits are combined in homozygous condition. Studies on introgression of recessive traits, e.g. low erucic acid, low glucosinolate and yellow seed colour, in canola (B. nupus), by haploidy indicate that the number of plants that must be screened can be significantly reduced - compared with traditionally inbred populations - because recessive traits are not masked by dominant genes (Siebel and Pauls, 1989; Henderson and Pauls, 1992). Thus, utilization of microspore culture can allow a substantial abbreviation of a breeding cycle up to four years. B. napus has been shown to be very responsive to haploidy techniques. This is indicated by a regeneration rate of up to 1115 embryos per anther and a spontaneous chromosome doubling rate of 22% (Chuong et al., 1988). Pechan and Keller (1988) have extrapolated an embryogenesis of approximately 50% using a bud of cv. ‘Topas’with 10 600 microspores. Whereas an initial protocol for microspore culture for B. napus was developed by Lichter (1982), advanced contributions from a number of experiments have led to a general protocol summarized by Kott et al. (1990). Nevertheless, further investigations were initiated; e.g., Kott and Beversdorf (1990) optimized the germination rate of microspore-derived embryos up to 94% by exposing 28day-old androgenic embryos to a temperature of 4°C. Takahata et al. (1991) illustrated that, in contrast to previous studies, the average efficiency of embryogenesis from older plants (7 months after sowing) was significantly higher than that of younger donor plants. Similar results have recently been published by Burnett et al. (1992).

263

Biotechnology & Genetic engineering in Rapeseed Breeding HYBRIDS

HAPLOIDS t

pollen .‘\ tissue

+

/

egg cells en.0

A-

/

cells

rapid

\

PROPAGATION

CLONING

I

intacte

callus

foreign

%

genes,

e.g. for quality \

/

/’ GENETIC fN ENGINEERING

0

plasmid

VA

Fig. 1. Biotechnology includes various methods and techniques of cellular and molecular biology which are relevant for plant breeding.

Telmer et al. (1992) reported that microspore samples with a frequency of 1437% binucleate stage contain the maximum of embryogenic late uninucleate or early binucleate microspores. Charne and Beversdorf (1991) compared microspore-derived (MD) and single seed descent populations (SSD) of rapeseed in field experiments: no substantial differences were detected between MD and SSD populations for

economically-important traits. Results of field experiments of Scarth et al. (1991) and Naleczynska and Cegielska (1991) confirmed the data of Charne and Beversdorf (1991) mentioned above. Therefore, they concluded that the important factor responsible for the rate of genetic improvement (gain/cycle) is the length of breeding cycle. Chen and Beversdorf (1990) found that variation in fatty acid profile among doubled haploid rapeseed lines is as great as

264

variation among inbred lines derived via SSD. Consequently, many oilseed rape breeders are actually testing doubled haploids (DH-lines) in their breeding programs and a large number of lines are already in advanced stages of field testing, so that some of them may soon be released as licensed cultivars. The response of anthers of linseed to in vitro culture is comparatively low (Nichterlein et al., 1989a). Whereas direct embryogenesis does usually occur in rapeseed, anthers of linseed primarily respond with callus formation. In a second subculture on anther regeneration media, O-80% of them regenerated shoots (Nichterlein et al., 1991); however, plant regeneration from somatic anther tissue or unreduced gametes cannot be excluded completely in this case. The variable responses observed reflect influences of genotype, growing temperature, induction medium and regeneration medium. Therefore, the anther protocol for linseed may still be improved by optimizing these factors, allowing a more efficient production of haploids. Recently, field trials have been initiated in our institute in order to evaluate performance of linseed lines derived from anther culture. In sunflower, the production of haploids derived from embryogenic microspores is, in principle, possible (Mezzarobba and Jonard, 1988; Giirel et al., 1991). Current progress is certainly demonstrated by recently achieved results from Nenova et al. (1992) who produced more than 300 plants originating from 15 calli derived from anthers of two interspecific hybrids. However, the response as related to the number of anthers cultured is still very low. Interspecific

Gene Transfer

Genetic variability for important traits is sometimes narrow in cultivated crop species. For instance, in the sunflower cultivars presently available, there is only a limited genetic basis for various agronomic traits. For example, most of these varieties are very sensitive to fungal diseases such as Botrytis cinerea and Sclerotinia sclerotiorum. Furthermore, most of the commercial sunflower cultivars represent single cross hybrids and their pro-

duction is solely based on a unique source of cytoplasmic male sterility (CMS), first reported by Leclercq (1969) in the progeny of a cross with Helianthus petiolark. In general, wild Helianthus species are of considerable interest as a source of genetic variation for economically important characteristics, such as CMS (Whelan, 1981; Anaschenko, 1981; Heiser, 1982) and disease resistance (Rogers et al., 1987; Lipps and Herr, 1986). Therefore, wide hybridization can principally help to create new genetic variation. However, it has been difficult to obtain interspecific hybrids by conventional sexual crossing due to incompatibility mechanisms. For instance, the failure of the endosperm to develop normally represents a postfertilization barrier and leads to the abortion of hybrid embryos. The rescue of such hybrid embryos or complete ovules and their cultivation on artificial media in vitro can help to circumvent such barriers and has been greatly successful in many species including sunflower (e.g., Espinasse et al., 1985). The improvement of recovery rates of interspecific hybrids was a prerequisite for obtaining sufficient numbers of offspring. Krauter et al. (1991) optimized the conditions and recovered 481 hybrids, i.e. an average regeneration rate of 41%. Since several of the primitive or wild species involved show partial resistances against fungal pathogens, such as Sclerotinia sclerotiorum, Botrytzk cineria and Verticillium dahliae, differences in susceptibility against these pathogens could be expected. Hammann and Friedt (1992) illustrated significant differences between hybrids and progenies in the degree of susceptibility to all pathogens in an experiment under different environments. In comparison to sunflower and Helianthus species, linseed and Linum species are much more recalcitrant to an application of the embryo rescue method, although early experiments in this field were carried out by Laibach (1925) on interspecific hybrids between L. perenne x L. ostriacum. Nevertheless, hybridizations of L. witatissimum to other highlinolenic Linum species, e.g., L. africanum (42% C18:3), L. angustifolium (57.5%), L. bienne (62.5%), L. crepitans (56.3%) and L. narbonensis (53.3%), were successful and ap-

265

pear to open new possibilities for increasing linolenic content in linseed oil (Nichterlein et al., 1989b). Further investigations in this field have been initiated. Oilseed rape (Brassica napus) is an amphidiploid (AACC) species derived from spontaneous hybridization between B. campes tris (A-genome) and B. oleracea (C-genome) (Prakash and Hinata, 1980). Due to its origin, the gene pool of B. napus only contains the genetic variability of those sub-species or accessions which were involved in the original cross(es). Thus, genetic variation seems to be rather limited in B. napus. Accordingly, interspecific gene transfer might be a means of increasing genetic variability. An excerpt of successful interspecific crosses in Brassica was summarized by Nishiyama et al. (1991) and Plumper (1991). General protocols for embryo culture have been presented by Gland (1983, Chen et al. (1988), for ovary culture by Inomata (1978,199O) and for in ouulo embryo culture by Sacristan and Gerdemann (1986) and Plumper (1991). A particularly impressive strategy in order to broaden the genetic basis of a crop plant is the production of resynthesized rapeseed by crossing the original ancestors ofB. napus, i.e. B. oleracea and B. campestris. The reader is referred to Chen and Heneen (1989a) who mainly focused in their review on the potential of resynthesized B. napus for breeding. More recently, Diederichsen and Sacristan (1991) reported the successful transfer of clubroot resistance from B. oleracea to rapeseed by crossing B. oleracea and B. campestris; the resynthesized rapeseed progeny were included in a practical breeding program. Mithen and Magrath (1992) derived synthetic lines ofB. nupus resistant to kptosphaeria maculans via embryo culture. In order to create new oilseed rape germplasm with an extension of the range of erucic acid content, crosses between B. campestris ssp. trilocularis (‘Yellow sarson’) and several selected B. oleracea convar. botrytk var. botrytis (e.g., cauliflower #2287) have recently been carried out in our laboratory (Liihs et al., 1992). The offspring display desirable variation in the content of the main fatty acids, so that it is possible to produce

lines with an erucic acid content of 60% or more (Table 1). It was shown earlier that erucic acid is under the control of the embryogenic genotype and governed by one gene in monogenomic species such as B. campestris (Dorrell and Downey, 1964) and two genes in amphidiploid species such as B. napus (Harvey and Downey, 1964; Siebel and Pauls, 1989). Multiple alleles occur at each locus acting in largely additive manner. Homozygous genotypes with various alleles produce levels of erucic acid ranging from less than 0.1 to 60%. In different winter rape cultivars various effective alleles are present which can give from 2 to 18% erucic acid in single dose (Jonsson, 1977). In genetic studies with resynthesized rapeseed, Chen and Heneen (1989b) substantiated the hypothesis of digenic segregation concerning erucic-acid heredity mentioned above. Furthermore, the authors emphasized that the effect of responsible alleles for a specific erucic acid content can be greatly influenced by the level of ploidy and genetic background. Since resynthesized rapeseed lines are homozygous, a change in their fatty acid composition, as compared to that of their parents, can be explained by interactions between related (homologous) genes of the A and C genomes. Thus, interTABLE 1 Variation in the erucic acid content of offspring lines derived via hybridization between B. oleraceu (#2287) and Brussica cantpestris (‘Yellow Sarson’) aided by embryo rescue (Ltihs et al., 1992) Oil % WM)

Fatty acids (% of total) C18:l Cl82

Cl83

C2o:l C22:l

‘$2287’

40.1

7.9

10.7

10.7

5.3

57.1

‘Yellow Sarson’

48.8

10.6

10.5

7.6

6.2

56.7

‘Mid-parent’ 44.5

9.3

10.6

9.1

5.8

56.9

‘Resyn’ min.

37.2

9.8

8.2

4.1

4.9

54.2

mean

40.5

13.1

9.7

5.7

6.6

56.8

max.

43.6

16.7

10.5

7.4

8.7

60.1

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genomic gene interactions in the resynthesized rapeseed lines have to be attributed to epistasis rather than dominance (Chen and Heneen, 1989b). Ltihs et al. (1992) suggest achieving recombinants with new allele combinations via introgression of resynthesized germplasm into conventional high erucic acid breeding material. However, the possibility of increasing the erucic acid content by accumulation and combination of desirable alleles is assumed to be limited, since so far no Brassica genotype has been found to produce seed oil with more than 65% erucic acid (Mahler and Auld, 1988). Asexual Hybridization via Protoplast Fusion Somatic hybridization could be an efficient way of achieving wide crosses when sexual hybridization is not possible -even by embryo rescue - due to the large genetic distance between species. In such cases prezygotic incompatibility mechanisms prevent an undisturbed growth of pollen tubes through foreign pistil tissue. Thus, fertilization cannot be accomplished. Basic requirements for interspecific hybridization by protoplast fusion are an efficient rate of protoplast isolation and the capacity to regenerate intact plants from protoplasts. In the genus Brassica progress in protoplast culture and plant regeneration has led to an extensive application of somatic hybridization. For an in-depth review on this subject, see Vamling and Glimelius (1990) and Sundberg and Glimelius (1991). In contrast to rapeseed, protoplast culture of linseed and other Linum species is still in an initial stage of development. Nevertheless, entire plants have already been regenerated from protoplasts of different species and genotypes (Barakat and Cocking, 1983; 1985). Ling and Binding (1987) achieved shoot regeneration from several wild Linum species and eight L. usitatksimum genotypes. The ability to regenerate plants from somatic tissues - such as explants from hypocotyls, leaves or cotyledons - via callus or embryogenesis can be considered a precondition for attempts to regenerate single somatic

cells (protoplasts). Chraibi et al. (1992) described an efficient liquid culture system for sunflower which allows regeneration of fertile plants. They suggested that developmental stage of cotyledons and the phytohormones, naphthalene acetic acid (NAA) and benzylaminopurine (BAP), represent key factors responsible for high efficiency of regeneration. However, in protoplast culture of sunflower and the genus Helianthus in general, many basic problems need to be solved. Among these, pronounced genotypic effects, vitrification and the difficulty of rooting regenerated shoots represent the most prominent obstacles (Guilley and Hahne, 1989). Nevertheless, Chanabe et al. (1991) regenerated plants from H. petiolaris protoplasts for the first time, whereas Burr-us et al. (1991) succeeded in regenerating fertile plants from protoplasts of cultivated sunflower derived from an interspecific hybrid to H. petiolarik. Recently, Krasnyanski et al. (1992) obtained plants from protoplasts of H. giganteus. Although the efficiencies of several steps in the protocols are still low, results encourage continued efforts to develop a system suitable for the transfer of agronomically useful genes into sunflower. Prospects of Genetic Engineering Efficient transformation systems have already been described to be effective in different oil crop species. The most effective method of transferring foreign DNA into a host plant is via the Ti-plasmid of Agrobacterium sp., which inserts part of its DNA into host plants. For instance, for rapeseed (De Block et al., 1989; Thomzik and Hain, 1990), linseed (Basiran et al., 1987; Jordan and McHughen, 1988) and Helianthus (Everett et al., 1987; Schrammeijer et al., 1990) successful transformations by transfection using Agrobacterium tumefaciens were principally achieved. Other major routes by which transgenic tissue can be obtained are: “gene gun approach”, where tiny metal beads coated with DNA are fired at high velocity into living plant tissue, and DNA uptake by protoplasts either directly (Moyne et al., 1989) or after electroporation (Guerche et al., 1987). Recently, Bidney et al. (1992) have demon-

267

&rated that the transformation frequency by Agrobacterium tumefaciens in plants recovered from sunflower apical explants was substantially increased, when the meristems were wounded first by particle bombardment. The authors concluded that Agrobacterium mediation of stable transformation is more effcient than the analogous particle/plasmid protocol alone. Basically, all the enzymes determining specific fatty acid biosynthesis and controlling the triacylglycerol composition are targets for genetic engineering approaches (cf. Knauf, 1987; Ohlrogge et al., 1991). However, it has to be kept in mind that this technology requires detailed knowledge of lipid biochemistry in order to isolate the specific genes; this information is still rather limited. Fatty acid biosynthesis is a complex process -starting with the priming of the “acyl carrier protein” (ACP) with an acetyl group from acetyl-CoA and ending with the final triacylglycerol (TAG) assembly. The whole process is driven by a number of enzymes or enzyme systems; at least 50 different metabolic enzymes must be anticipated involved in the conversion of carbon dioxide to fats, containing saturated and unsaturated fatty acids of different chain length from Cl2 to C22 (Stumpf, 1988). The primary enzymes controlling fatty acid biosynthesis are the acetyl-CoA carboxylase and the fatty acid synthetase (FAS) system. Furthermore, there are elongases, desaturases and specific acyltransferases (AT) involved in the assembly of triacylglycerols typical for each kind of oil plants. For example, certain plants such as rapeseed and mustards C&zssica sp.) contain an enzyme complex that elongates 18:1-CoA to 20:1-CoA and 22:1-CoA for incorporation into the triglyceride fraction (cf. Stumpf and Pollard, 1983; Fehling et al., 1990; Fehling and Mukherjee, 1991; Taylor et al., 1991; see also Stumpf, 1988; Ohlrogge et al., 1991). In rapeseed it is supposed that the microsomal enzyme 1-acyl-sn-glycerol-3-phosphate acyltransferase responsible for the installation of acyl moieties in the central position of the glycerol backbone is not able to accept erucic acid (erucoyl-CoA) as substrate (Bernerth and

Frentzen, 1990; Cao et al., 1990; Taylor et al., 1992). Consequently, rapeseed should not be able to accumulate trierucin (trierucoylglycerol) in its seed oil, as verified by stereospecific analyses (Norton and Harris, 1983; Takagi and Ando, 1991). The screening by HPLC chromatography of the triacylglycerols in our breeding program also supports the suggestion of a theoretical limit of 66% erucic acid in the whole rapeseed genepool (Luhs et al., 1992). In order to increase the erucic acid content, it seems indispensable to alter the selectivity of the sn-2 acyltransferase mentioned above. In other species, such as meadowfoam (Limnanthes alba and L. douglasii), the sn-2 AT possesses a preference for erucoyl-CoA (Cao et al., 1990; Lohden et al., 1990; Taylor et al., 19901, although its seed oil does not contain trierucin, in contrast to Tropaeolum mqjus (Taylor et al., 1990; Liihs et al., 1992). The specificity of the acyltransferase for the sn-2 position makes Limnanthes a prime candidate for genetic engineering experiments. Great efforts are currently expended to isolate this enzyme and transfer the responsible gene(s) to rapeseed (cf. Taylor et al., 1990, 1992; Wolter et al., 1991). After all, the usefulness of genetic engineering concerning target reactions of lipid biosynthesis might be limited by the fact that the expression of chimaeric genes may affect normal seed development; for example, disruptive cell membrane function caused by unusual fatty acids in the polar lipids may be a consequence. Furthermore, the frequency, stability and functionality of foreign genes in progeny of transformants is not always satisfactory. This is partially due to the fact that engineered plants will likely require seed specific promoter sequences which regulate the expression of the structural genes, e.g., coding for enzymes of lipid biosynthesis (cf. Stumpf 1988; Battey and Ohlrogge, 1989; Battey et al., 1989). Finally, the transgenic plants obtained will represent basic material which still needs selection and testing by plant breeders, rather than highly performing varieties which can readily be released as licensed cultivars.

268

Conclusions

The potential of an application of biotechnology methods in plant breeding is certainly indisputable. Therefore, in breeding of sunflower, linseed and oilseed rape for industrial usage particular methods are definitely practical breeding tools. The current state of development in almost all of the biotechniques mentioned above is most advanced in the genus Brassica. For example, the microspore culture technique is nearly optimized in B. napus and its usefulness is justified by field trials. Therefore, the enthusiasm of rapeseed breeders for haploidy technique was stimulated by the impressive efficiency of this method. In addition, embryo rescue techniques such as the in ouzdo embryo culture have been successfully applied in a number of experiments in Brassica for broadening the genetic base by introducing desirable genes. With regard to an industrial usage of new seed oils the production of resynthesized rapeseed could be of particular interest. Protoplast culture and somatic hybridization are ready for practical application in rapeseed; it is a prerequisite of both, somatic hybridization and genetic engineering. In linseed, microspore and anther culture could meanwhile be further improved. However, future investigations will be necessary to identify factors which determine the frequency of embryogenic microspores and to develop conditions which hinder callus formation since this often prevents a normal plant development. If these problems could be solved, haploid technique will also be a promising tool in practical linseed breeding programs. Embryo rescue technique assisted the use of wild L&urn germplasm as a gene source, whereas techniques of protoplast culture in linseed are still at a developmental stage. Great efforts have been invested in establishing an efficient haploidy technique in sunflower. To date, it is possible to induce shoot regeneration out of callus tissue, but the production of entire plants which can be transferred to soil and grown to maturity still remains difficult. A large number of interspecific hybrids were realized in the genus Helianthus

with the aid of the embryo rescue technique. These new hybrids are highly valuable basic material for broadening the genepool of annual cultivated sunflower. In the near future, genetic engineering will certainly reach importance in breeding of oilseed crops with custom tailored oil profiles. The successful development of transformation systems for all major oil crops and the intensive efforts aiming in an isolation and transfer of genes responsible for specific enzymes involved in the triacylglycerol biosynthesis in rapeseed stimulate this expectation.

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