ABSTRACT

In the past, transgenic Brassica napus has been developed mainly using cv. Westar. The cultivar is not grown any longer and has been replaced by many new cultivars. The use of old cultivars requires that the transgene must be backcrossed into elite lines, a process which is time-consuming and expensive. In order to develop efficient transformation protocols for elite breeding material, we tested four different regeneration protocols and Agrobacterium tumefaciens co-cultivation with new yellow-seeded B. napus lines. The test lines had been selected for differences in seed oil content and seed size, subsequent to initial selections for yellow seed colour. The yellow-seeded material originated from interspecific crosses between cv. Westar and yellow-seeded Brassica juncea and yellow-seeded Brassica carinata, followed by one backcross to Westar. Transformation efficiencies similar to those of a cotyledonary petiole explant method used for cv. Westar were achievable using hypocotyl explants of the yellow B. napus seeds and several of the regeneration protocols. Transformation efficiencies of yellow seeds segregating from a brown B. napus sibling line were lower than yellow seeds from the selected B. napus lines and similar to those of the parent yellow-seeded B. juncea line and to a brown-seeded B. juncea line.

INTRODUCTION

In the past, protocols for the transformation of Brassica napus have been developed mainly using the Canadian canola-quality summer rape variety, Westar. Westar is not grown any longer and has been replaced by higher yielding cultivars. Its use as a recipient of transgenes requires that the transgene must be backcrossed into elite lines after it has been introduced into Westar. In order to avoid this time-consuming and expensive process, we initiated a study to develop efficient transformation protocols for new elite yellow-seeded B. napus. This germplasm was developed through interspecific hybridization between Westar and B. juncea which carries recessive alleles for yellow-seed and B. carinata which carries a single dominant yellow-seed gene (Rashid et al., 1994).. Yellow-seeded B. napus cultivars will have a low content of seed meal fibre and a high seed oil content and may replace many of the black-seeded cultivars currently grown in Canada. Hence, the new cultivars will be sought in the future as background germplasm for the introduction of new transgenes.

MATERIALS AND METHODS

Yellow-seeded B. napus lines were derived from interspecific crosses between B. napus Westar and yellow-seeded Brassica juncea ZYR-6 and yellow-seeded Brassica carinata PGRC-21164, followed by one backcross to Westar and six generations of selection for yellow-seeded B. napus phenotypes (Rashid et al., 1994). Several yellow-seeded B. napus lines which were not true-breeding for the yellow-seeded trait were selected from this cross at the time this experiment was initiated.

Hypocotyl explants (7-10 mm long) were excised from five-day-old germinating yellow seeds segregating from the selected B. napus lines. Replicated trials consisted of treating 330-450 explants to each of four protocols (A-D) as follows. First, the explants were co-cultivated for three days on media BCH1 with Agrobacterium tumefaciens containing the 35S-enhanced GUS:nptII fusion plasmid pHS723 (Table 1) (Hirje et al., 1996). Explants were then transferred for seven days onto one of two types of recovery media to stimulate non-selective growth and expression of the transgene (Table 1). Subsequently, explants were regenerated on one of two selection protocols (Table 1). The protocol used in A and C was originally developed for brown-seeded B. juncea in which shoots were induced from hypocotyl explants in 10-20 days (Hammerlindl and Keller, unpublished data). The protocol used in B and D was a modification of a method used to transform cotyledonary petioles of B. napus cv. Westar (Moloney et al., 1989). In this method, dark green transgenic shoots with thick, odd-shaped leaves are induced after 14-21 days on cytokinin-supplemented media, followed by recovery of normal shoot morphology on a shoot elongation media with no cytokinins. Well-developed leafy green shoots from all four protocols were transferred to a common root-stimulating media (Table 1), and planted into soil when roots had formed. The occasional shoot which formed callus on the rooting media was transferred directly to soil after removal of the callus.

Parental lines used to develop the yellow-seeded B. napus lines were also transformed with plasmid pHS723 for comparative purposes. In total, 990 hypocotyl explants and Protocol A were used with yellow-seeded B. juncea, and 910 cotyledonary petioles and Protocol D were used with B. napus Westar. The protocol for 250 yellow-seeded B. carinata cotyledonary petioles included media containing AgNO₃ and phloroglucinol to stimulate shoot development (Babic et al., 1998). Brown-seeded B. juncea Blaze (765 hypocotyl explants) and B. carinata S67 (720 cotyledonary petioles), which are genetically related to the yellow-seeded parental lines, were also transformed using protocols for their corresponding yellow-seeded lines. Agrobacterium-free control experiments were included for each trial.

$-glucuronidase (GUS) activity and Southern hybridization were used to confirm transformation of Km^r plantlets and to determine the frequency of transgene rearrangements. In most cases, transformation efficiencies were calculated conservatively by only including plants which grew to produce seed in the greenhouse. Southern hybridization was also used to determine copy number of the nptII gene.

Table 1: Protocols Used to Regenerate Yellow-Seeded Brassica napus

All media contained sucrose (3%) and Timentin (300 mg.L-1). Recovery media (BCH1 and M2), shoot regeneration media (SR and M3), and shoot elongation media (M4) contained Phytagar (0.7%). Root-inducing media (R) contained normal agar (0.7%) to reduce costs. MS, Murashige and Skoog salts, MES, morpholine sulphonic acid; 2,4-D, 2,4-diphenoxyacetic acid; NAA, naphthalene acetic acid; BA, benzyl adenine; IBA, indole butyric acid; Km, kanamycin; NA, not applicable.

^amethod to transform hypocotyl explants of Brassica juncea (Hammerlindl and Keller, unpublished)

^bmethod to transform cotyledonary petioles of Brassica napus cv. Westar (modification of Moloney et al., 1989 by Hammerlindl and Keller)

RESULTS AND DISCUSSION

The yellow-seeded trait, which normally behaves as a single dominant gene in the B. carinata background and as two recessive genes in B. juncea, did not behave in the same fashion in the new yellow-seeded B. napus material. Instead, three yellow-seeded lines selected out of this material segregated for seed colour in a 1:1 ratio when tested after six generations of self-pollination (Table 2). A black B. napus sibling line segregated in a fashion indicative of a single recessive yellow colour gene.

Germination rates for yellow seed from the four new B. napus lines varied substantially, and the rates were low compared with parental lines (Table 2). Consequently, hypocotyl explants, rather than cotyledonary petioles, were used to generate the large number of explants required for transformation assays. Shoot regeneration potential varied with the new B. napus lines in the absence of kanamycin. Development to the pre-rooting leafy stage under kanamycin selection was substantially slower for the new lines than for the parent B. napus Westar. Similar delays in growth have also been observed under field conditions with other yellow-seeded B. napus lines derived from this cross (Rakow, unpublished results). In addition, yellow seeds from the low oil line appear to have developed a higher tolerance to kanamycin than other progeny or parental lines, since 10% of the uninfected control explants from this line developed green shoots. Plants appeared to regenerate normally from all B. napus and B. juncea lines. However, the B. carinata parent tended to develop roots rather than undergo normal shoot regeneration.

Table 2. Characterization of yellow-seeded Brassica napus

NB: Other parental lines had germinated at frequencies of 92% (B. juncea ZYR-6) and 74% (B. carinata PGRC-21164 three days after imbibition).

*Yellow = yellow to light brown; Black = dark brown to black.

In general, transformation frequencies for yellow seeds segregating from the new B. napus lines were similar to those of the parental B. napus (Table 3) and B. carinata lines (data not shown). However, significantly higher rates were observed with the high oil line when protocols A and B were tested. The high oil line may respond more favorably to the hormone and vitamin composition present in the nonselective recovery media BCH1. The lower transformation frequencies of cv. Westar and B. carinata observed in this study compared with the experience of others (9.1% and 30-48%, respectively) may reflect our conservative way of estimating transformation frequencies (Babic et al., 1998; Selvaraj et al., personal commun.).

Transformation frequencies for yellow seeds segregating from the new black-seeded B. napus sibling line tended to be similar to the lower frequencies observed for the parental yellow-seeded B. juncea line and for the brown-seeded B. juncea Blaze, except where the yellow seeds were grown on media used for B. juncea. These data suggest that some of the genes from B. napus Westar that normally facilitate plant transformation and regeneration may be masked in this line. The recessive nature of the yellow seed colour in the new black-seeded line also suggests that this line has retained a yellow gene from B. juncea, but lost the yellow gene from B. carinata.

Table 3. Transformation frequencies for yellow seeds from yellow-seeded and black-seeded B. napus

*based on GUS activity or Southern hybridization with the nptII gene

Approximately 1.9% of all Km^r yellow-seeded B. napus regenerates were not transgenic. In addition, a comparison of GUS activity and Southern hybridization with the nptII gene indicated that both genes were present in only 90.7% of the tested yellow-seeded transgenic plants. Incomplete transfer of the T-DNA had occurred in 3.3% of the remaining transgenic plants and DNA rearrangements in 6%.

Single insertions of nptII were more frequently observed with B. juncea ZYR-6 and B. carinata S67 than with yellow-seeded B. napus lines, B. napus Westar and B. carinata 21164. A tendency towards multiple gene insertions has been observed by others when transforming B. napus and B. juncea with other types of transgenes (Barfield and Pua, 1991; DeBlock et al., 1989; Moloney et al., 1989; Fu and McGregor, personal commun.). The data points to the necessity for obtaining larger numbers of transgenic B. napus plants, in order to assess the effects of stable single gene insertions. Our finding of multiple insertions into B. carinata is contrary to observations by others with this species (Babic, 1998).

Table 4. Frequency of Single Insertions of the nptII gene in transgenic Brassica

CONCLUSIONS AND FUTURE DEVELOPMENTS

From 1996-1998, plant transformation protocols were successfully tested on new lines of yellow-seeded B. napus using a GUS:nptII gene. In general, these protocols stimulated transgene insertion into the new lines with a profile similar to Westar, a standard B. napus cultivar used for plant transformation and one of the parents used to develop this material. However, the introgression of B. carinata and B. juncea into B. napus introduced colour instability and problems with vigour into the B. napus genome. As well, genes for high glucosinolate and high erucic acid levels and for undesirable oil profiles were introduced (data not shown).

In 1998, the first set of stable lines of yellow-seeded B. napus were tested in the field. These lines are now being backcrossed several times to black B. napus lines to re-construct suitable canola oil composition. As the stabilized lines become available, our experience with their predecessor lines suggest they will be good candidates as background germplasm for molecular breeding strategies.

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