Karin Sonntag, Eicke Rudloff, Youping Wang
Federal Centre for Breeding Research on Cultivated Plants, Institute of Agricultural Crop Groß Lüsewitz, Germany
Development of Brassica napus
with improved seed oil quality
Hodowla Brassica napus o ulepszonym składzie jakościowym
oleju z nasion
Key words: Brassica napus, hybridization, fatty acids, microspores, mutagenesis, genetictransformation
Rapeseed is one of the most important annual oilseed crops in the world, ranking third with respect to oil production after soybean and palm. Modification of the fatty acid composition to make oilseed rape more competitive in various segments of the industrial oil markets has recently been an important objective of molecular genetics and in plant breeding. One of the most important goals of rapeseed breeding is the modification of seed oil by maximising the proportion of specific fatty acids.
The present report demonstrates the use of different methods for the modification of oil quality via microspore culture combined with in vitro mutagenesis, symmetric and asymmetric hybridisation and genetic transformation especially to obtain a high level of erucic acid.
Słowa kluczowe: Brassica napus, hybrydyzacja, kwasy tłuszczowe, mikrospory, mutageneza transformacja genetyczna
Rzepak jest jedną z najważniejszych jednorocznych roślin oleistych w świecie, zajmuje trzecie miejsce po soi i palmie oleistej. Ważnym zadaniem dla hodowców i genetyków molekularnych wydaje się być modyfikacja składu kwasów tłuszczowych pod kątem wykorzystania oleju rzepako-wego w przemyśle. Szczególnie ważnym celem jest modyfikacja oleju nasion poprzez maksyma-lizację proporcji specyficznych kwasów tłuszczowych.
Niniejsza praca przedstawia wykorzystanie różnych metod: połączenia kultury mikrospor z muta-genezą in vitro, symetrycznej i niesymetrycznej hybrydyzacji oraz transformacji genetycznej, w celu modyfikacji składu jakościowego oleju w kierunku wysokiej zawartości kwasu erukowego.
Microspore mutagenesis in transgenic rapeseed
for the modification of fatty acid composition
The aim of this project was to develop germplasms for the breeding of rapeseed with high myristic acid (C14:0) content and a lower palmitic acid
/myristic acid (C16/C14) ratio in the seed oil by use of the haploid techniques
C14:0 content and a lower C16/C14 ratio are of economical importance for the
production of oleochemical compounds.
Six transgenic lines of cv. ‘Drakkar’ which had been transformed with the thioesterase gene ClFatB4 (Martini et al. 1999) were used as microspore donors. Isolation of microspores was done as described by Lichter (1982). Microspores were treated with different intensity (0.005; 0.02; 0.03 J/cm2) of ultraviolet (UV)
light to induce mutation. Mutagenic treatment of microspores by means of UV irradiation is performed to induce heritable changes in the fatty acid biosynthesis of transgenic lines. Plants regenerated in vitro were transferred in the greenhouse, where they were selfed by bagging after colchicin treatment of the haploid plants. Seeds of each plant were harvested separately. Fatty acid composition was analysed using one cotyledon after half-seed technique according to Thies (1971) and growing the remaining embryo for seed production.
The first cell divisions in the isolated microspores were visible after four days, followed rapidly by further divisions within the next few days and the development to globular and heart-shaped stage and finally to embryos.
There were differences for embryogenic capability among the six transgenic lines of cv. ‘Drakkar’ after UV treatment. The embryogenic capability varied between 0.03 and 2.6 embryos per bud in the control and between 0.1 and 0.4 after UV treatment. After UV treatment only three of six transgenic lines regenerated to homozygous plants for use in breeding programs. Applying an UV dose of 0.005 J/cm2 resulted in 74 regenerants from which flow cytometry revealed that 23%
doubled their chromosomes spontaneously.
The fatty acid composition was analysed in 9 doubled-haploid lines (Table 1). These had been derived from plants regenerated from microspores of transgenic lines #6 and #39, respectively. Eight lines were from haploid regenerants and one from a diploid regenerant. The seven DH lines of #6 displayed significant differences in both mean myristic acid content and C16/C14 ratio, whereas the two
DH lines of #39 did not so. There was no difference between the #6-derived DH lines (20.3%) and the #39-derived lines (19.8%) with respect to the mean C14:0
content, and the C16/C14 ratio (1.18 vs. 1.13), respectively. The mean content of the
lines #6 varied between 14.5 and 23.8%. There was a relatively high variability within the DH lines as demonstrated by the coefficient of variability (C.V.)
Fifty-two half-seed individuals of the 145 analysed seeds were selected regarding high C14:0 content and low C16/C14 ratio, respectively. The high variability
in the myristic acid showed that there were differences between lower than 10% to higher than 30% (Fig.1A). There was a great variability in the C16/C14 ratio, too.
Table 1 Fatty acid composition in the offspring of regenerated plants resulting from UV mutagenesis of microspores from transgenic rapeseed lines — Skład kwasów tłuszczowych potomstwa
roślin uzyskanych drogą mutagenezy UV mikrospor linii transgenicznych rzepaku
C14:0 [%] C16:0/C14:0
Offspring
Pokolenie Ploidy a
Ploidalność Nb mean
średnia significanceistotność C.V. średnia mean significance istotność C.V.
39A 1n 27 19.9 n.s.* 23.0 1,15 n.s.* 16,6 39B 1n 38 19.6 n.s.* 19.1 1,12 n.s.* 20,8 6A 1n 25 21.6 a** 17.9 1,04 be** 15,4 6B 1n 5 17.5 ac** 23.1 1,27 ade** 16,3 6C 1n 9 20.5 a** 16.3 1,47 ac** 14,8 6D 1n 9 23.8 a** 16.7 1,12 bd** 15,2 6E 2n 10 19.3 ac** 18.1 1,35 ad** 14,0 6F 1n 16 20.1 a** 17.5 1,13 bd** 19,4 6G 1n 6 14.5 bc** 29.2 1,23 ade** 22,6 * — F-test: Fobs.= 1.48 (C14:0%) Fobs.= 1.49 (C16/C14) Ftab.(P = 0.95; 26/37 FG) = 1.86
** — means followed by an identical letter are not significantly different, (Tukey-test; P = 0.95) średnie oznaczone tymi samymi literami nie różnią się istotnie
a — in vitro plants — rośliny in vitro
b — number of analysed seeds — liczba analizowanych nasion
0 5 10 15 20 25 30 35 6A 6B 6C 6D 6E 6F 6G 39A 39B 39C C 14: 0-G e ha lt (% ) 0,0 0,5 1,0 1,5 2,0 2,5 6A 6B 6C 6D 6E 6F 6G 39A 39B 39C C 16/ C 1 4 -Q u ot ie n t A B
Fig. 1. Half-seed analysis of the fatty acid composition of doubled-haploid lines resulting from in vitro mutagenesis of transgenic microspores by UV irradiation; mean of the lines, maximum and minimum values, respectively, of A) C14:0 content and B) C16/C14 ratio — Analiza składu kwasów tłuszczowych wykonana metodą połówek nasion podwojonych haploidów uzyskanych z poddawanych mutagenezie promieniami UV transgenicznych mikrospor; średnia dla linii, maksymalna i minimalna zawartość: A) C14:0; B) stosunek C16/C14
The microspore-derived plants were grown in the greenhouse, showing a normal and homogeneous development. The fatty acid analysis of their offspring will show whether the action of environmental influences on the plant development is an appropriate explanation for the observed variability.
Development of rapeseed with high erucic acid content
by asymmetric somatic hybridisation
The aim of this project is to develop germplasm with high erucic acid content through asymmetric hybridisation of Brassica napus and Crambe abyssinica.
C. abyssinica is an annual cruciferous oilseed crop with a high erucic acid content
of 55–60% in the seed oil.
Our approach was to fuse UV-irradiated mesophyll protoplasts of C.
abys-sinica cultivar ‘Carmen’ and ‘Galactica’ as a tool for production of asymmetric
somatic hybrids with hypocotyl protoplasts of B. napus cv. ‘Maplus’ and breeding line ‘11502’ (Wang et al. 2003). The protoplast of C. abyssinica were exposed to different doses of UV: ranging from 0.05 J/cm2 to 0.3 J/cm2. Confirmation
of hybrids was carried out by flow cytometry, AFLP analysis, and morphological observation. Further GISH analysis was used for the identification of C. abyssinica chromosomes in the hybrids and the CAPS analysis of the fae1 gene indicated hybrid progenies with different band patterns compared to both fusion parents (Wang et al. 2004).
The first division of the cultured protoplast was observed on the 3rd–4th day
after fusion. Microcalli were formed after 3 weeks of culture. Shoots emerged 3–4 weeks later and were transferred to the greenhouse. Shoot regeneration frequency varied between 5.7 and 20.8%, and a total of 124 shoots were regenerated. The frequency of asymmetric hybrids varied between 20 and 33%. After pre-selection by flow cytometry, 42 putative hybrids were obtained. AFLP analysis was carried out to confirm their hybridity. Of the 42 putative hybrid plants 22 plants displayed only the B. napus AFLP-DNA pattern and 20 showed the B.
napus pattern containing at least one unique band characteristic of C. abyssinica,
thus proving that the hybrid carried part of the genome from C. abyssinica (Fig. 2). Additional bands from C. abyssinica were found, too.
The effect of different doses of UV irradiation showed that high levels of DNA were present when UV dose was low. Also low pollen viability and low seed set were obtained when UV irradiation was low. This indicates that increasing UV-irradiation doses result in higher pollen viability and higher seed set (Fig. 3). Chromosome number was higher with low UV irradiation (0.005 J/cm2). It was
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
*
* * *
*
Fig. 2. AFLP banding patterns using the primer pair E-AAC/M-CGA of B. napus plant (lane 1), somatic hybrids (lane 2–21) and C. abyssinica plant (lane 22). 14 bands characteristic of C. abyssinica are indicated with arrows, specific bands from C. abyssinica in the hybrids are indicated with an asterisk (*) — Wynik analizy AFLP z wykorzystaniem praimerów E-AAC/M-CGA dla roślin Brassica napus
(ścieżka 1), mieszańce somatyczne (ścieżki 2–21) oraz roślina C. abyssinica (ścieżka 22). 14 prążków charakterystycznych dla C. abyssica jest zaznaczone strzałkami, charakterystyczne dla C. abyssinica prążki w mieszańcach wskazuje gwiazdka (*)
The hybrids showed differences in the erucic acid level. 15 out of 20 of the hybrids had an erucic acid content higher than B. napus (47.9%), ranging between 48.0% and 51.2%. In 6 hybrids the average erucic acid content differed significantly from the parental B. napus. However, 5 hybrids showed lower erucic acid content than B. napus, ranging from 42.6% to 47.8%. Among the 24 plants of the F2 and BC1 analysed cytologically, 11 had 38 chromosomes, the other 13 had
more than 38 chromosomes constituting a complete chromosomal set from
a b c
f
d e
Fig. 3. Pollen viability and seed set of B. napus (+) C. abyssinica asymmetric hybrid plants after ripening in the greenhouse. a–c: various levels of pollen viability and d-f: corresponding seed set —
Żywotność pyłku oraz zawiązywanie nasion u mieszańca asymetrycznego Brassica napus (+) C. abyssinica po dojrzewaniu w szklarni, a–c różny poziom żywotności pyłku oraz d–f odpowiadające im zawiązywanie nasion
Most of the F2 and BC1 plants had erucic acid levels higher than B. napus
(50.3%). One individual plant was characterised by 55.0% of erucic acid. Twelve plants contained significantly (P < 0.05) greater amounts of erucic acid than
B. napus, whereas six plants produced erucic acid levels lower than that of B. napus.
Analysis of cleaved amplified polymorphic sequence derived from the fae1 gene showed novel patterns different from the B. napus parent in some hybrid offspring. As shown in Table 2 some plants e.g. S1/2, B2/2 and S4/1 with specific bands from C. abyssinica exhibited a higher erucic acid content in the seed compared to B. napus. This may indicate that the genetic system involved in the synthesis of erucic acid was transferred from C. abyssinica into B. napus. However, some plants e.g. S3/2 and S4/2 had a lower erucic acid level although they possessed one or more specific bands from C. abyssinica. One of the reasons is assumed to be physiological irregularities due to meiotic instability and intergenomic recombination in the plants resulting in disorders in the formation of gametes as well as seeds, and thus leading to the change of fatty acid composition. The other reason might be explained by the fact that not every compound of the
Table 2 Chromosome number, pollen fertility, seed set and erucic acid content in selected F2 and BC1 plants derived from six asymmetric hybrids of B. napus cv. ‘Maplus’ (+) C. abyssinica cv. ‘Galactica’ — Liczba chromosomów, płodność pyłku, liczba zawiązanych nasion oraz
zawartość kwasu erukowego w wyselekcjonowanych roślinach F2 i BC1 otrzymanych z sześciu
asymetrycznych mieszańców Brassica napus cv. „Maplus” (+) C. abyssinica cv. „Galactica” Line Linia Chromosome number in Liczba chromosomów w F1 Plant-ID Symbol rośliny Chromosome number in Liczba chromosomów w F2/BC1 Pollen fertility Płodność pyłku [%] Seed set Liczba zawiązanych nasion [n] Erucic acid Kwas erukowy (mean %, ± SD średnia % ± SD) 1 69 B1/1 38 63 8.7 51.2 ± 0.3 B1/2 50 56 8.5 53.5 ± 0.1* S1/1 53 59 4.1 53.3 ± 0.1* S1/2 64 54 3.6 53.2 ± 0.3* 2 78 B2/1 59 46 7.5 51.1 ± 0.3 B2/2 64 39 3.0 53.1 ± 0.2* B2/3 66 43 4.1 52.1 ± 0.5* S2/1 68 37 3.0 49.7 ± 0.3 3 50 B3/1 38 67 6.2 49.6 ± 0.4 B3/2 38 63 5.9 50.8 ± 0.3 S3/1 39 66 7.5 53.1 ± 0.1* S3/2 48 52 4.6 50.2 ± 0.6 4 46 B4/1 38 67 6.9 53.4 ± 0.5* B4/2 38 70 8.8 50.2 ± 0.6 S4/1 39 64 4.8 53.0 ± 0.3* S4/2 40 52 2.9 48.6 ± 0.1 15 50 B15/1 38 78 5.0 52.4 ± 0.6* B15/2 38 65 7.0 51.6 ± 0.7 S15/1 40 73 4.8 53.1 ± 0.2* S15/2 50 68 4.7 53.6 ± 0.5* 18 38 S18/1 38 >94 28.0 52.5 ± 0.4* S18/2 38 >94 21.8 51.3 ± 0.5 S18/3 38 >94 21.5 51.6 ± 0.2 S18/4 38 >94 22.7 49.9 ± 0.1 20 38 S20/1 38 >94 18.7 53.2 ± 0.2* S20/2 38 >94 15.2 51.9 ± 0.7* S20/3 38 >94 20.2 50.9 ± 0.5 S20/4 38 >94 20.0 52.1 ± 0.5* B. napus ‘Maplus’ >94 14.9 50.3 ± 0.8 C. abyssincia ‘Galactica’ 57.6 ± 0.4
S — self-pollinated — samozapylanie (F2); B — backcrossed with — krzyżowanie wsteczne Brassica napus
* — significant differences to B. napus cv. ‘Maplus’ (P < 0.05)
by the growing conditions in the greenhouse, such as temperature or maturity level of the seed (Wilmer et al. 1996). In B. napus, genetic variation in erucic acid content corresponds to individual fae1 alleles, which can be distinguished on the molecular level.
The chromosomes of B. napus and C. abyssinica origin could be clearly discriminated by genomic in situ hybridisation in mitotic and meiotic cells. These experiments were made together with the group of Prof. W. Friedt at the university of Gießen. In backcross-progeny plants with 2n = 50 chromosomes were discriminated the 38 B. napus chromosomes from 12 chromosomes of C.
abys-sinica. Furthermore, meiotic GISH enabled identification of intergenomic chromatin
bridges and of asynchrony between the B. napus and C. abyssinca meiotic cycles. Lagging, bridging and late disjunction of univalents derived from C. abyssinica were observed, too. It is known that small introgressions can often be observed among species with larger chromosomes (Parokonny et al. 1994, Xia et al. 2003) and strong GISH signals only at centromeric regions in Brassica (Skarzinskaya et al. 1996, Snowdon et al. 2000).
The present study confirms that UV irradiation aided asymmetric somatic hybridisation is able to produce genetically different hybrids with good fertility in their offspring. Thus it is potentially useful to facilitate the introgression of desired exotic germplasm into B. napus. Some progenies with increased erucic acid content could be created. The chromosomes of B. napus and C. abyssinica origin could be clearly discriminated by GISH in mitotic and meiotic cells. Analysis of CAPS markers derived from the fae1 gene showed novel patterns different from the
B. napus recipient. Further investigations with extended materials will demonstrate
that the content of erucic acid in progenies of the asymmetric hybrids between
B. napus and C. abyssinica can be increased. Sexual progenies of the asymmetric
hybrids that exhibit a high erucic acid content combined with good agronomic performance will be of particular value for further breeding.
Development of rapeseed with high erucic acid content
by symmetric somatic hybridisation
Symmetric hybridisation of B. napus (+) Brassica juncea, B. napus (+)
Sinapis alba and B. napus (+) Raphanus sativus was carried out with the aim
to modify the seed-oil composition. The most common method for somatic hybridisation of Brassica protoplasts is chemical fusion with polyethylene glycol (PEG). In our experiments were established an electrofusion method with B. napus and so both methods were used (Müller and Sonntag 2000a, b).
Independent fusion experiments have been carried out with mesophyll protoplast of B. napus on the one side and B. juncea, R. sativus and S. alba on the other side (Müller et al. 2001, Sonntag et al. 2003). The protoplast culture conditions
are similar to somatic hybridisation with C. abyssinica (described in chapter 2), the confirmation of hybridity of fusion product was estimated on the basis of micro-satellites and the fatty acid content was analysed with capillar gas chromatography according to Thies (1974).
After fusion in the combination of B. napus (+) R. sativus 91 plants were produced and the shoot frequency reached 6.8%. From these plants 24% were confirmed as hybrids.
After transfer of the hybrids into greenhouse self-pollination was not possible for all plants due to pollen sterility. After backcrosses progenies showed an intermediate erucic acid as well as oleic acid content. This results in decreasing both linoleic and linolenic acid content (Fig. 4).
6,8 10,6 13,3 3,5 11,7 8,9 25,9 25,4 11,5 44,9 30,7 49 0 10 20 30 40 50 60 70 80 90
Hybrid R. sativus B. napus
C22:1 C18:1 C18:3 C18:2 6,9 : 2,5 : 2,7 : 1,0 1,0 1,0
Fig. 4. Relation of unsaturated fatty acids in symmetric hybrids of B. napus (+) R. sativus in comparison to the parents — Proporcje kwasów nienasyconych w symetrycznych mieszańcach B. napus (+)
R. sativus w porównaniu do form rodzicielskich
Until now a low amount of seeds was obtained from backcrosses (BC1) with
hybrids of B. napus (+) R. sativus and the first BC1 plants were transferred into the field.
Here were found plants with two different flower colors: white and yellow (Fig. 5). Two combinations were made between B. napus and S. alba. The shoot regeneration efficiency in these combinations ranged from 12.6% to 41%. Twelve plants were identified as hybrids. The symmetric hybrids of B. napus cv. ‘Lisandra’ and S. alba cv. ‘Mustang’ were pollen-sterile while B. napus ‘11502’ and S. alba cv. ‘Litember’ were not.
Fatty acid composition was compared between somatic hybrids backcrossed with either the B. napus cv. 'Maplus' or the high-erucic acid B. napus line '11502'. The results demonstrated that backcross progeny involving two high-erucic parents had considerably higher erucic acid contents. Four of five BC1 plants had erucic
acid contents of more than 62%. The erucic acid content was very heterogeneous in the offspring of somatic hybrids between B. napus cv. ‘Lisandra’ and S. alba cv. ‘Mustang’ (Fig. 6).
Fig. 5. BC1 plants of B. napus (+) R. sativus with yellow and white flowers — Rośliny BC1 B. napus
(+) R. sativus z żółtymi i białymi kwiatami
0 10 20 30 40 50 60 70 A5 + I4 x A 5 A5 + I4 x A 6 A5 + I4 x A 13 A6 + I6 A6 + I6 x A 6 A6 + I6 x A 13 Eruc ic -ac id c ontent (% )
A5 — B. napus cv. ‘Lisandra’; A6 — B. napus line ‘11502’; A13 — B. napus cv. ‘Maplus’; I4 — S. alba cv. ‘Mustang’; I6 — S. alba cv. ‘Litember’
Fig. 6. Erucic acid contents of F1 and BC1 plants of symmetric hybrids between B. napus and S. alba
The data based on half-seed analyses of symmetric somatic hybrids of
B. napus line ‘11502’ and S. alba cv. ‘Litember’ indicated increased erucic acid
contents up to 65% in the offspring when backcrossed with high-erucic acid lines. This is consistent with the results obtained by Heath and Earle (1995) from fusions between B. oleracea and B. rapa. Based on these results further back-crossings are planned for stabilising the high-erucic acid level in following generations.
Majority of hybrids, altogether 136, were produced in the combination of
B. napus and B. juncea with a shoot frequency of 11% and hybrid frequency of 22%.
Two types of B. juncea plants were used in these experiments: type E – a European type with lower erucic acid content and type A – an Asian type with more than 50% erucic acid. According to the results of a ten-seed sample we were able to distinguish between low-erucic-hybrid plants with 0–23% erucic acid and high-erucic acid hybrids with at most 57.5% high-erucic acid depending on the B. juncea type used. The back-cross generation showed similar results. The erucic acid content reached a maximum of 63%. The comparison of the F2 and F3 generation in one
somatic hybrid after half-seed selection demonstrates that the erucic acid can be further increased.
In summary, these results support the hypothesis that variation in erucic acid is mainly induced by different alleles, which can be detected on a molecular level as homologues of fae1 gene. That will be the matter of further investigations. The results in fusion hybrids of B. napus (+) S. alba indicate an increase of the erucic acid content from 52% in the B. napus-parent up to 65% in the F1 seed of hybrids.
Asian type of B. juncea resulted in somatic hybrids with higher erucic acid than combinations with the European type. Back-crosses showed that combining Asian type hybrids with high-erucic varieties can reach up to 62.7% erucic acid. Fatty acid composition of B. napus and R. sativus, respectively, appears to be combined in the fusion hybrid. Polyunsaturated fatty acids are markedly decreased compared to both parents.
Development of transgenic rapeseed
with modified fatty acid composition
Delivery of foreign DNA into Brassica has been established by many different methods (Moloney 1989, Pechan 1998, Thomzik 1990, Poulsen 1996). Rapeseed is one of the more easily transformable plants with a high susceptibility to Agrobacterium tumefaciens. Thus, attempts have been made to increase the erucic acid content by Agrobacterium mediated transformation of hypocotyls or protoplasts (Wang et al. 2004).
A variety of genes affecting agriculturally important traits have been transferred into B. napus. The first successful alteration of fatty acid composition in B. napus
concerning desaturation was accomplished by Knutzon et al. 1992. To date, a wide range of various genes encoding enzymes involved in plant storage-lipid synthesis have been isolated and cloned and thus are available to genetically engineer the fatty acid composition of seed oil.
In our experiments the cultivars ‘Drakkar’ and ‘Maplus’ were involved. The genes used were designed at the university of Aachen (Prof. M. Frentzen) for modification of erucic acid. In addition to hypocotyls used widely as explants, we used protoplasts also. To our knowledge, there has been so far no report on successful transformation of B. napus protoplasts by co-cultivation with an
A. tumefaciens bearing agronomically important genes. The construct encoding the
condensing enzyme ß-ketoacyl-CoA-synthase (KCS) which confers the first and rate-limiting step of the microsomal fatty acid elongation reaction and determines the acyl chain length of the very long-chain-fatty acids (VLCFA) and lyso-phosphatidic acid acyltransferase (LPAAT) are the key enzymes for the improvement of the erucic acid content in the seeds.
Mesophyll protoplasts from winter-type B. napus cv. ‘Maplus’ were used for the protoplast transformation with Agrobacterium tumefaciens. The construct involved the napin promoter and kanamycin as selection marker. Transformed protoplasts were regenerated to fertile and morphologically normal transgenic plants. Transformants were confirmed by PCR and southern blot analysis of the nptII gene.
For transformation experiments, hypocotyls from 6-day-old etiolated seedlings of cv. ‘Drakkar’ were cut into 7 mm-segments and incubated with the
Agro-bacterium strain C58 ATHV bearing the same gene construct as described above.
Transformation was performed according to De Block et al. (1989) with minor modifications in the duration of the co-cultivation and in the intensity of the kanamycin selection. Regenerated transgenic plants were cultivated in the greenhouse for harvest of seeds. Fatty acid composition of the seed oil of transformants was analysed with gas chromatography using the half-seed technique according to Thies (1971).
The seeds of the transformants of cv. ‘Maplus’ showed a changed fatty acid profile, and two transformants had a high erucic acid level which differed significantly from control plant B. napus. Interestingly, the content of oleic acid was decreased (Table 3). This can be explained by KCS-over expression in the transformants which facilitates the fatty acid elongation reaction from oleolyc CoA to erucoyl CoA. The seeds of the hypocotyl transformants of cv. ‘Drakkar’ showed at most 30% C22:1.
Our findings suggest that further investigations are necessary to increase the erucic acid synthesis.
Our opinion is that the microspore mutagenesis, the somatic hybridisation and the genetic transformation presented may offer a chance to produce plants with modified oilseed quality as renewable resources.
Table 3 Comparison of pollen fertility, seed set and fatty acid composition in transformed plants of cv. ‘Maplus’ — Porównanie żywotności pyłku, liczby zawiązywanych nasion oraz składu
kwasów tłuszczowych w transformowanych roślinach cv. „Maplus”
Fatty acid — Kwas tłuszczowy [%] Transformant
Transformant Żywotność pyłkuPollen fertility
Seed set Liczba zawiązanych nasion C18:1 C22:1 1 92 16.3 12.7 51.0 2 84 10.1 9.9 a 52.6 a 3 94 17.5 11.3 a 53.8 a Control plant Roślina kontrolna 97 15.3 12.9 50.3
a — significant difference compared to B. napus cv. ‘Maplus’ istotne różnice w porównaniu do B. napus cv. „Maplus”
Acknowledgements
The project was funded by the Fachagentur für Nachwachsende Rohstoffe. We wish to thank M. Frentzen and D. Weier for providing of plasmid and strain. The authors thank J. Gramenz and I. Groeneveld for carrying out somatic hybridisation with B. juncea,
R. sativus and S. alba, B. Hackauf for support of the molecular analysis and P. Wehling
for helpful comments on this manuscript. I. Müller, H. Dreier, A. Schuldt, M. Gödecke, W. Butzmann, N. Hecht are acknowledged for the excellent technical assistance.
References
De Block M., De Brouwer D., Tenning P. 1989. Transformation of Brassica napus and Brassica
oleracea using Agrobacterium tumefaciens and the expression of the bar and neo genes in the
transgenic plants. Plant Physiol., 91: 694-701.
Knutzon D.S., Thomson G.A., Radke S.E., Johnson W.B., Knauf V.C., Kridl J.C. 1992. Modification of Brasssica seed oil by antisense expression of a stearolyl-acyl carrier protein desaturase gene. Proc. Natl. Acad. Sci. USA, 89: 2624-2628.
Lichter R. 1982. Induction of haploid plants from isolated pollen of Brassica napus L. Plant Cell Rep., 1: 209-211.
Martini N., Schell J., Abbadi A., Spener F., Töpfer R. 1999. Rapsöl mit mittelkettigen Fettsäuren. In: Brauer D., Röbbelen G., Töpfer R. (Hrsg.): Bioengineering für Rapssorten nach Maß-Ergebnisse eines BMBF-Forschungsverbunds, Vortr. Pflanzenzüchtg., 45: 133-154.
Müller J., Sonntag K. 2000a. Electrofusion of protoplasts in Brassicaceae. Cruciferae Newsletter, Eucarpia, INRA Rennes (Ed.), 22: 25-26.
Müller J., Sonntag K. 2000b. Somatic hybrids of Brassica napus and Brassica juncea based on different fusion methods. Vortr. Pflanzenzüchtg., 47: 51.
Müller J., Sonntag K., Rudloff E. 2001. Somatic hybridisation between Brassica spp. and Raphanus
sativus. Acta Hort, 560: 219-220.
Moloney M.M., Walker J.M., Sharma K.K. 1989. High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Rep., 8: 238-42.
Parokonny A.S., Kenton A., Gleba Y.Y., Bennett M.D. 1994. The fate of recombinant chromosomes and genome interaction in Nicotiana asymmetric somatic hybrids and their sexual progeny. Theor. Appl. Genet., 89: 488-497.
Pechan P.M. 1998. Successful cocultivation of Brassica napus microspores and embryos with
Agrobacterium. Plant Cell Rep., 12: 462-467.
Poulsen G.B. 1996. Genetic transformation of Brassica. Plant Breed., 115: 209-225.
Snowdon R.J., Winter H., Diestel A., Sacristán M.D. 2000. Development and characterisation of Brassica napus – Sinapis arvensis addition lines exhibiting resistance to Leptosphaeria
maculans. Theor. Appl. Genet., 101: 1008-1014.
Skarzhinskaya M., Landgren M., Glimelius K. 1996. Production of intertribal somatic hybrids between Brassica napus L. and Lesquerella fendleri (Gray) Wats. Theor. Appl. Genet., 93: 1242-1250.
Sonntag K., Rudloff E., Groeneveld I., Weier D., Frentzen M., Lehmann L., Zarhloul K., Lühs W. 2003. Development of rapeseed plants with optimised oilseed quality. Proc. 11th Int. Rapeseed Congr. 06-10.07, Kopenhagen, Dänemark, 1: 232-234.
Thies W. 1971. Schnelle und einfache Analysen der Fettsäurezusammensetzung in einzelnen Raps-kotyledonen. I. Gaschromatographische und papierchromatographische Methoden. Z. Pflanzen-züchtg., 65: 181-202.
Thies W. 1974. New methods for the analysis of rapeseed constituents. Proc. 4th Intl. Rapeseed Conf., Giessen, 275-281.
Thomzik J.E., Hain R. 1990. Transgenic Brassica napus plants obtained by cocultivation of proto-plasts with Agrobacterium tumefaciens. Plant Cell Rep., 9: 233-236.
Wang Y.P., Sonntag K., Rudloff E. 2003. Development of rapeseed with high erucic acid content by asymmetric somatic hybridization between Brassica napus and Crambe abyssinica. Theor. Appl. Genet., 106: 1147-1155.
Wang Y.P., Snowdon R.J., Rudloff E., Wehling P., Friedt W., Sonntag K. 2004. Cytogenetic characterization and fae1 gene variation in progenies from asymmetric somatic hybrids between
Brassica napus and Crambe abyssinica. Genome, 47: 724-731.
Wang Y.P., Sonntag K., Rudloff E., Han J. 2004. Production of fertile transgenic Brassica napus by Agrobacterium-mediated transformation of protoplasts. Plant Breeding (in press).
Wilmer J.A., Helsper J.P.F.G., Van der Plas L.H.W. 1996. Effect of growth temperature on erucic acid levels in seeds and microspore-derived embryos of oilseed rape Brassica napus L. J. Plant Physiol., 147: 486-492.
Xia G., Xiang F., Zhou A., Wang H., Chen H. 2003. Asymmetric somatic hybridisation between wheat (Triticum aestivum L.) and Agropyron elongatum (Host) Nevishi. Theor. Appl. Genet., 107: 299-305.
