ABSTRACT

Oilseed rape (Brassica napus L.) is one of the leading crops which benefit from the application of genetic engineering through recombinant DNA technology. Present rapeseed oil (Canola) is characterized by a high content of unsaturated C18 fatty acids. In order to improve its industrial usefulness the project aims at the genetic modification of saturated fatty acid content and the development of transgenic B. napus accumulating medium-chain triacylglycerols in its seed oil. For this purpose relevant genes from unrelated plant species forming unusual storage oils are isolated and transferred to oilseed rape. Particularly in B. napus the efficiency of Agrobacterium tumefaciens mediated transformation mainly depends on the susceptibility of the starting material to agrobacteria, the ability to select for newly grown tissue derived from the transformed cells, and the potential to regenerate plants from the selected tissue. In the course of a preliminary study we have investigated intraspecific differences towards shoot regeneration by genetically transforming the resynthesized high-erucic acid rapeseed line ’RS 306’ and the spring Canola cultivar ’Drakkar’ with the gene construct pASBnDES1. The latter harbours a chimeric gene based on a Cuphea lanceolata seed-specific promoter (ClFatB4) and the coding sequence from rapeseed Δ9-desaturase in antisense orientation, in order to modify the content of oleic acid, which is the major precusor for subsequent fatty acid pathways (desaturation, elongation) in both rapeseed genotypes.

INTRODUCTION

Modification of the fatty acid composition to make rapeseed oil more competitive in various segments of the food and industrial oil markets has been an important objective of both plant breeding and molecular genetics in recent years (Friedt and Lühs 1998). In order to improve its industrial usefulness in oleochemistry the long-term aim of this study is to develop B. napus which is able to store a large amount of C10-C14 fatty acids in its seed oil. These medium-chain fatty acids (MCFA) are very common for a lot of members of the Lauraceae family, but they are completely absent from rapeseed oil. Obviously, the adjustment of rapeseed oil composition leading to an accumulation of medium-chain triacylglycerols cannot be achieved by conventional breeding methods. As shown in the cases of laurate canola or rapeseed high in myristic and palmitic acid (Voelker et al. 1996, Rudloff and Wehling 1998) for this breeding objective genetic engineering is the most promising route for transferring relevant genes between rapeseed and distant species. The efficiency of Agrobacterium tumefaciens-mediated transformation protocols, as indicated by the percentage of transgenic plants, depends on the following main factors: susceptibility of the B. napus starting material to Agrobacterium; the ability to select for newly grown tissue derived from the transformed cells; and the potential to regenerate plants from the selected tissue (De Block 1993, Poulsen 1996). In a first step, we established the transformation protocol developed by De Block et al. (1989) under our specific laboratory conditions. Regarding the alteration of fatty acid composition we used the spring Canola cultivar ’Drakkar’ and the resynthesized high-erucic acid rapeseed (HEAR) line ’RS 306’ as plant material donor. Due to differences in regeneration response after co-cultivation with Agrobacterium it is assumed, that the optimum growth regulator concentration and combination in the selectable medium must be found for each genotype allowing sufficient regeneration of potentially transformed plants.

MATERIALS AND METHODS

Plant material used for transformation

’RS 306’ is a resynthesized HEAR (high erucic, high glucosinolate) line originating from the interspecific cross B. rapa ssp. trilocularis (’Yellow Sarson’) x B. oleracea conv. botrytis var. botrytis (cv. ’Super Regama’) with moderate vernalisation requirement but without winter hardiness (Weier et al. 1997, Zarhloul et al. 1999). Seeds of cv. ’Drakkar’ were obtained from Norddeutsche Pflanzenzucht Hans-Georg Lembke KG (Hohenlieth, Germany).

Bacterial strain and vector

The A. tumefaciens strain GV3101/pMP90RK (Koncz and Schell 1986) was transformed with the construct pASBnDES1 carrying the B. napus stearoyl-acyl carrier protein (18:0-ACP) desaturase (Δ9-desaturase) gene fused in antisense orientation to the ClFatB4 promoter (N. Martini, unpubl. data). The binary vector used is pLH9000 (L. Hausmann, unpubl. data) with the neomycin phosphotransferase (NPTII) gene as selectable marker.

Transformation procedure

For genetic transformation and the subsequent selection steps the method of De Block and co-workers (1989) was applied with minor modifications, including the use of 1) etiolated rapeseed hypocotyls, 2) ticarcillin/potassium clavulanate (Betabactyl^TM; SmithKline Beecham Pharma, Germany) instead of carbenicillin for elimination of the agrobacteria after stopping co-cultivation and 3) Gelrite^TM (Serva, Heidelberg/Germany) as gelling agent in the tissue culture media. Seeds were surface-sterilized in 70% ethanol for 5 minutes, then for a further 10 minutes in a 3% NaOCl solution. The rape seedlings were germinated on A₁ medium (De Block et al. 1989) in darkness. After 7 days the etiolated hypocotyls were cut in 1 cm segments and co-cultivated with A. tumefaciens for 3 days in a liquid A₃ medium (De Block et al. 1989) using 9 cm petri dishes. Following co-cultivation, the hypocotyl explants were placed on petri dishes (2 cm high and 14.5 cm in diameter; 25 explants per dish) containing A₅ medium (De Block et al. 1989) with Kanamycin (50 mg/l) as a selective agent. Further sub-culturing of the explants was conducted at intervals of two weeks. After 6-8 weeks of selection the first shoots were formed and removed from the hypocotyl explants and transferred to A₆ medium (with 15 mg/l Kanamycin) on which they continued growing until normal phenotypic characteristics appeared. They were transferred to A₈ rooting medium (De Block et al. 1989) and then, following the formation of roots, to the greenhouse.

Testing the hormone composition of the selectable media

Several variants of the A₅ selectable media were used after co-cultivation step differing in the concentration of the supplemented growth regulators as follows: naphthalene acetic acid (NAA) in the range of 0.1-0.5 mg/l and benzyl amino purine (BAP) ranging from 1.0 to 5.0 mg/l. The hormone concentration 0.1 NAA mg/l / 1.0 mg/l BAP corresponds to the one formerly used in the transformation protocol of De Block et al. (1989)

RESULTS AND DISCUSSION

Following a modified protocol of De Block et al. (1989) we have co-cultivated etiolated hypocotyl segments of the B. napus genotypes cv. ’Drakkar’ and the resynthesized rapeseed line ’RS 306’ by using the Agrobacterium strain GV3101/pMP90RK harbouring the gene construct pASBnDES1 with the NPTII gene as selectable marker (Table 1). The results of the experiment revealed a significant difference (p=0.05) between the two genotypes regarding their regeneration response after co-cultivation with A. tumefaciens. A large variation in regeneration efficiency was found within the genotype ’RS 306’ ranging from 1 to 14% (Table 1). The shoot regeneration of ’Drakkar’ was very insufficient and did not exceed 3%, although this spring Canola cultivar is usually appreciated for its superior transformation and regeneration response (cf. Schaffert et al. 1996). As shown in a previous study, ’RS 306’ is clearly more suitable for our laboratory conditions and the modified transformation protocol (cf. Zarhloul et al. 1999).

Table 1: Kanamycin-resistant shoot regenerants of B. napus ’RS 306’ and ’Drakkar’ after co-cultivation with the Agrobacterium strains GV3101 bearing the binary vector pASBnDES1

Genotype	’RS 306’			’Drakkar’
Agrobacterium strain / gene construct	Explants in co-culture	Regenerated shoots		Explants in co-culture	Regenerated shoots
		number	%		number	%
GV 3101 (pASBnDES1)	200	24	12.0	200	0	0
	200	4	2.0	200	1	0.5
	200	13	6.5	200	2	1.0
	200	2	1.0	200	0	0
	200	11	5.5	200	0	0
	200	28	14.0	200	6	3.0
Average	200	13.6	6.8*	200	1.5	0.8

The experiment consisted of 2 combinations of Agrobacterium strain x B. napus genotypes, while each of the six replications comprised 200 hypocotyl explants (8 dishes x 25 explants); * p=0.05.

Antibiotics used after the co-culture step to eliminate agrobacteria may influence the regeneration response of the transformed explants in that they decrease the shoot differentiation, especially if kanamycin is used as selectable agent. On the one hand, it is reported that Betabactyl^TM (ticarcillin/potassium clavunate), unlike the widely used carbenicillin and cefotaxime, is light-stable and resistant to inactivation by β-lactamase (Ling et al. 1998); on the other hand, it is recommended the use of carbenicillin, which prevents the medium from turning brown and eliminates the toxic effects of prolonged use of silver nitrate on plant tissue (De Block et al. 1989). An experiment was conducted to investigate possible differences between the above mentioned antibiotics (carbenicillin and Betabactyl^TM), but the results showed no statistically significant difference between them based on the number of regenerants (Table 2).

Table 2: Influence of antibiotics on the shoot regeneration from co-cultivated B. napus hypokotyls (cv. 'Drakkar')

Antibiotic	Co-cultivated explants/replicat. #	Number of explants (total)	Regenerated shoots (total)	Regeneration rate (%)
BetabactylTM	225	1,125	7	0.6
Carbenicillin	225	1,125	13	1.2

# Experiment consisted of five replications

In order to ameliorate the regeneration of potentially transformed plants from the spring cultivar ’Drakkar’ hypocotyl segments were placed on selectable A₅ medium differing in the concentration of the supplemented growth regulators. A regeneration rate of 12% was achieved through the increase of the NAA concentration from 0.1 mg/l to 0.5 mg/l (Fig. 1). This effect was observed in all combinations with BAP where the NAA concentration amounted to 0.5 mg/l. These results thus demonstrate a clear NAA effect. However, it still must be said that for the ’Drakkar’ genotype, which can reach a regeneration rate of 28% potential transformed plants (Schaffert et al. 1996), further optimization of the transformation protocol will be necessary.

Figure 1: Influence of the hormone composition on the regeneration of the spring cultivar B. napus cv. 'Drakkar' after co-cultivation with Agrobacterium tumefaciens

To investigate if it is possible to further improve the modified transformation protocol by using different hormone concentrations and combinations, the above experiment was repeated using the ’RS 306’ genotype instead of the ’Drakkar’ genotype (Figure 2). The experiment demonstrates that unlike the spring Canola cultivar ’Drakkar’, in which the 0.5 NAA mg/l / 1.0 mg/l BAP hormone concentration gives the best results, in the resynthesized rapeseed line ’RS 306’ the 0.3 NAA mg/l / 3.0 mg/l BAP concentration provides the best regeneration rate. The latter genotype has also shown an excellent regeneration response using the Agrobacterium DNA-delivery system in combination with other gene constructs (Weier et al. 1997, Zarhloul et al. 1999).

Figure 2: Influence of the hormone composition on the regeneration of the resynthesized rapeseed line 'RS 306' after co-cultivation with Agrobacterium tumefaciens

The results of Table 1 as well as Figures 1 and 2 show once again that for each genotype favourable conditions after co-cultivation and medium supplements must be found which allow the regeneration of sufficient numbers of plants from the transformed cells (De Block 1993, Poulsen 1996). The results also indicate the importance of the genotype, the culture conditions after transformation and medium supplements (Ono et al. 1994, Takasaki et al. 1996, Poulsen 1996, Zhang et al. 1998).

CONCLUSION

Medium-chain fatty acids derived from plant oils are preferred for industrial applications, including the area of commercial detergents, surfactants, cleaning agents and cosmetics. Since rapeseed oil does not furnish this need, this biosynthetic capacity has to be introduced by genetic engineering. As plant oil composition can be substantially modified by either increase or reducing the expression of defined enzyme activities (cf. Knutzon et al. 1992, Cartea et al. 1998) the strategy consists of two-steps: firstly, the content of unsaturated C18 fatty acids will be reduced by antisense inhibition of the endogenous Δ9 or 18:0-ACP desaturase leading to an increase in the proportion of stearic acid (and its precursor fatty acids) on the expense of oleic acid in both Canola and HEAR oil. In the course of the transformation experiments a comparable high number of regenerants has been developed, so far. Following the characterization by using PCR or NPTII ELISA assays the transformants have to be analyzed regarding their alteration in fatty acid composition. Finally, the transformed rapeseed plants showing a significant shift to more saturated fatty acids are considered as an ideal material for further Agrobacterium-mediated transformations transferring relevant genes from the Lauraceae family encoding the synthesis of medium-chain fatty acids.

ACKNOWLEDGEMENT

This work was supported by the Bundesministerium für Ernährung, Landwirtschaft und Forsten, Bonn, and the Fachagentur für Nachwachsende Rohstoffe e.V., Gülzow (Grants 97 NR 079-F and 97 NR 108-F). The excellent assistance of Sonja Weber is gratefully acknowledged.

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