A combined use of microprojectile bombardment and DNA imbibition enhances transformation frequency of canola (Brassica napus L.).

Efforts to increase the frequency of recovered homozygous transgenic B. napus plants from direct DNA transformation treatments led to the development of a method of combined microprojectile bombardment and desiccation/DNA imbibition. The combined method was compared to individual treatments in two experiments utilizing microspore-derived embryo hyocotyls as targets for the ß-glucuronidase (GUS) and NPT II genes. Both the transient gene expression of ß-GUS and the stable transformation by NPT II demonstrated that the combined use of microprojectile bombardment and desiccation/DNA imbibition yielded more transgenic plants (at least three-times more) than either individual transformation protocol. In a histochemical analysis for ß-GUS activity, an average of 37% of the hypocotyls receiving the combined treatment displayed a positive response, whereas only 8% of the hypocotyls showed a positive response following microprojectile bombardment alone. The hypocotyls obtained by the joint treatment also showed more multisite expression of the ß-GUS gene per hypocotyl than those treated only with microprojectile bombardment. Southern analysis of NPT II gene integration into subsequently-derived secondary embryos indicated that the transformation efficiency of the combined treatment was 2% in comparison to 0.6% for that of the singular microprojectile bombardment. The number of inserts integrating per transformation event appears to be independent of the transformation methods. Neither of the marker genes was expressed in hypocotyls treated only with desiccation/DNA imbibition. Utilization of hypocotyl regeneration from microspore-derived embryos via a secondary embryogenesis system provided a reliable method for producing transgenic plants. The combined use of microprojectile bombardment and desiccation/DNA imbibition proved to be an efficient approach to obtain homozygous transgenic canola plants.

Identification of an S-locus glycoprotein allele introgressed from B. napus ssp. rapifera to B. napus ssp. oleifera.

Self-incompatible Brassica napus ssp. oleifera lines were generated by introgressing the S-locus from the self-incompatible B. napus ssp. rapifera Z line into the self-compatible cultivars, Topas and Regent, resulting in T2 and R2, respectively. Screening of a cDNA library made from R2 stigma RNA produced several candidate SLG (S-locus glycoprotein) cDNAs. One of the cDNAs, A14, was found to be represented in only the R2, T2 and Z lines. In addition, the corresponding A14 gene was demonstrated to segregate with the T2 self-incompatibility phenotype in an F2 population derived from a cross between T2 and Topas, and to exhibit high mRNA levels in the stigmas prior to anthesis. Sequence analysis of the A14 cDNA revealed close homology to B. oleracea SLG alleles associated with a Class I high activity self-incompatibility phenotype.

Use of the polymerase chain reaction to isolate an S-locus glycoprotein cDNA introgressed from Brassica campestris into B. napus ssp. oleifera.

A self-incompatible canola-quality Brassica napus ssp. oleifera line (W1) was generated by introgressing the S-locus from a self-incompatible B. campestris plant into the Westar cultivar. Using the polymerase chain reaction (PCR) with primers derived from conserved regions in S-locus glycoprotein (SLG) alleles, the central region of the active SLG gene (910) was obtained. The remaining portions of the cDNA for this 910 gene were subsequently cloned using the PCR-rapid amplification of cDNA ends (RACE) procedure. Sequence analysis revealed that the 910 cDNA show a high degree of sequence similarity to SLG alleles associated with Class I self-incompatible lines. The 910 gene was found to be absent in the original self-compatible cv. Westar (B. napus) and segregated with self-incompatibility in a mixed population generated from a cross between self-incompatible W1 and self-compatible Westar. RNA blot analysis indicated that high levels of 910 mRNAs were present in the stigma as buds approached anthesis. Thus, the SLG allele of W1 transferred from B. campestris via backcrosses to a line of cv. Westar has been identified.

Isozyme analysis as a tool for introgression of Sinapis alba germ plasm into Brassica napus.

Isozyme analysis of Brassica napus cv 'Topas' and CGRC5006 as well as of Sinapis alba cv 'Emergo' revealed significant polymorphism between the two species for the isozymes, aconitate hydratase, glucose phosphate isomerase, and diaphorase. F1 hybrids between B. napus '5006' and S. alba cv 'Emergo' were backcrossed to B. napus cv 'Topas', and the S1 progeny of the first two backcrosses were studied isozymically. At the backcross one level the frequency of S. alba or S. alba plus B. napus patterns observed ranged from 18% to 87% across the four lines studied. There were differences between lines for the frequency of S. alba patterns, which could have an impact on the efficiency of selection for subsequent backcrossing. By the backcross two generation in one of the two lines studied, GR86-24, the S. alba patterns for GPI and DIA had been lost, while in the other line, GR86-28, the S. alba pattern for ACO had been lost, resulting in lost opportunity for S. alba gene transfer. In a wide cross such as S. alba x B. napus, which requires an intensive effort to accomplish, the isozymes ACO, GPI, and DIA may serve as useful markers to ensure gene transfer between the two species has occurred. In addition, the identification of lines with divergent isozyme patterns from B. napus will provide the basis for establishing linkages between S. alba traits of interest and isozyme markers.

Desiccation of microspore derived embryos of oilseed rape (Brassica napus L.).

Microspore-derived embryos from Brassica napus L. were dried to less than 15% moisture and stored dry for a minimum of 7 days. Successful plant regeneration was observed when embryos at the cotyledonary stage of development were treated with 50 uM ABA for 7 days prior to desiccation. Solid agar or liquid medium gave similar results. The rate of drying of embryos after ABA pretreatment had only minor effects on embryo survival, but for untreated embryos, slow drying gave a small degree of survival. These results are very comparable to those with alfalfa somatic embryos, suggesting that the ABA treatment of cotyledonary stage embryos may be broadly used as a pretreatment for inducing the expression of desiccation tolerance in plant embryos.

Fatty acid inheritance in microspore-derived Populations of spring rapeseed (Brassica napus L.).

The inheritance of major fatty acids in seed triglycerides was studied in three homozygous microspore-derived populations of spring rapeseed (Brassica napus L.). Crosses were made among parents with contrasting amounts of erucic, oleic, linoleic and linolenic acids. Microspores from F1 plants were cultured, and haploid plants were colchicine-doubled to provide homozygous populations reflecting F1 gametic arrays for fatty acid genotypes. Segregation ratios of the gametic arrays for specific fatty acid contents were compared to hypothetical models by the Chi-square test. Segregation pattern confirmed that erucic acid levels were controlled by two major loci, each having two alleles with additive effects. Oleic acid segregation indicated control of accumulation by at least two segregating genetic systems, one acting on chain elongation and the other involving desaturation. Accumulations of erucic acid and oleic acid were influenced by the same two loci, which control the chain elongation steps leading from oleic acid to erucic acid. Oleic acid was further influenced by at least two additional segregating loci involved in control of desaturation of oleic acid to form linoleic acid. Segregating alleles at loci involved in desaturation had a much smaller influence on oleic acid content than alleles segregating at loci controlling, the elongation of oleic acid to erucic acid. In a population free of erucic acid, the segregation pattern of linoleic acid levels fit a model involving segregating alleles at two loci. In contrast, segregation for linolenic acid content fits a three-locus additive model. In this study, microspore culture technology provided a rapid method of defining F1 gametic segregation for inheritance analyses.

Embryogenesis following cryopreservation in isolated microspores of rapeseed (Brassica napus L.).

A simple procedure is described for cryopreservation of isolated microspores of rapeseed in liquid nitrogen without loss of embryogenic capacity (i.e. embryogenes is can still be induced following freezing). Microspores frozen in Lichter's (1982) medium with 13% sucrose produced ca. 10% of the embryos yielded by an unfrozen control. Microspores frozen in Lichter's medium with 13% sucrose, and supplemented with 0.5 M glycerol and 0.5 M DMSO produced no embryos. Regeneration of embryos obtained from frozen microspores yielded 88% diploid and 12% haploid plants, while embryos from unfrozen controls produced 7% diploids and 93% haploids. The potential to increase the efficiency of the rapeseed haploidy system using cryopreservation is discussed in light of these results.

The characterization of herbicide tolerant plants in Brassica napus L. after in vitro selection of microspores and protoplasts.

Brassica napus L.(cv Topas) plants tolerant to chlorsulfuron (CS) were isolated after selection experiments utilizing microspores and haploid protoplasts. The first microspore-derived plant (M-37,) was CS tolerant, haploid and sterile. Normal plant morphology and fertility was restored after colchicine doubling. A CS tolerant plant was also selected from protoplasts (P-26) isolated from microspore-derived embryo tissue and grown on medium containing CS. P-26 was aneuploid, CS tolerant and had very low fertility. The two selected lines produced selfed progeny which were tolerant to from 10-100 times the CS levels of the corresponding Topas plants. Microspores and protoplasts derived from the selfed plants were also CS tolerant. The segregation pattern for CS tolerance from reciprocally crossed progeny of M-37 and Topas was consistent with a semi-dominant nuclear mode of inheritance. Biochemical analysis of the two mutants indicated that the microspore-derived mutant and F1 crosses contained an altered acetohydroxyacid synthase (AHAS) enzyme, while the AHAS activity of the protoplast mutant was similar to Topas. Selfed seed from the M-37 plants have provided tolerance to CS in both greenhouse and field tests. S1 plants from a second microspore selected mutant (M-42) have tolerated 30 g/ha of CS in greenhouse tests. The two single-celled selection systems are discussed and the microspore selection system highlighted as a new method for in vitro selection.

The combination of polima cms and cytoplasmic triazine resistance in Brassica napus.

Protoplast fusion was used to combine cytoplasmic triazine resistance (ctr) and Polima type cytoplasmic male sterility (cms) in Brassica napus. The cybrids produced constitute the major biological input required for the production of commercial single-cross hybrid rapeseed bearing cytoplasmic triazine resistance. The results also indicate that Polima cms is associated with the mitochondrial genome.

Protoplast culture and plant regeneration from Brassica carinata Braun.

Protoplasts isolated from hypocotyls of three-day-old seedlings of Brassica carinata (Braun) cv R-2128 were cultured in a modified Nitsch and Nitsch liquid medium containing 13% sucrose, 0.4% Ficoll, 0.25 mg/l BA, 0.5 mg/l NAA and 0.5 mg/l 2,4-D. The density of medium caused the protoplasts and the developing microcalli to float on the surface of the liquid medium whereas all debris and lysed cells sank to the bottom of the culture plate. After 4-6 weeks developing microcalli were approximately 0.5 mm in diameter and were transferred onto MS medium containing 3% sucrose, 0.4% agarose, 200 mg/l casein hydrolysate, 5 mg/l BA and 0.5 mg/l NAA, pH 5.7. Approximately 20% of the calli transferred to this medium produced plantlets.

Efficient isolation of microspores and the production of microspore-derived embryos from Brassica napus.

Mechanical isolation of Brassica napus microspores from whole buds with a micro-blender enabled the rapid isolation of large numbers of microspores free from tissue and cellular debris. The procedure resulted in savings of time and labor and could be used independent of the size and number of buds to be examined. Between 700 and 1000 embryos per bud were obtained. Compared to anther removal there was no difference in embryo yields or quality. Microspore isolation under cool conditions and overnight incubation prior to plating improved the frequency of embryogenesis. Over 75% of the embryos developed into normal torpedo-stage structures if the medium was replenished during culture and the embryos placed on a gyratory shaker. Over 80% of the torpedo-shaped embryos would ultimately develop into plants. The implications of these techniques to genetic and physiological studies are discussed.

A simple culture method for Brassica hypototyl protoplasts.

Hypocotyl protoplasts from oil rape, Brassica napus L. cv. Isuzu were cultured in the dark at 25°C in a modified Nitsch and Nitsch medium containing 13% sucrose, 5 g/l Ficoll, 0.5 mg/l BAP, 1 mg/l NAA and 0.5 mg/l 2-4 D. Protoplasts floated on the surface of the medium and developed into microcolonies 0.5 mm in diameter in 4-6 weeks. The microcolonies also remained on the surface of the medium. Transfer to MS medium supplemented with 200 mg/l casein hydrolysate, 5mg/l BAP, 0.5 mg/l NAA and solidified with 0.6% agarose induced shoot regeneration in 3-4 weeks.

Restriction patterns reveal origins of chloroplast genomes in Brassica amphiploids.

Chloroplast (ct) DNA from the three elementary Brassica species (B. nigra (L.) Koch, B. oleracea L. and B. campestris L.) and the three amphiploid Brassica species (B. carinata A. Br., B. napus L. and B. juncea (L.) Czern.) was digested with fifteen restriction endonucleases. In all species restriction sites for enzymes with GC-rich recognition sequences were less frequent and not as variable as for those with AT-rich sequences. Comparisons between species revealed two distinct groups of ct DNA fragment patterns: complex one, composed of B. oleracea, B. napus, B. campestris and B. juncea and complex two, composed of B. nigra and B. carinata. The patterns of B. carinata were virtually identical to those of B. nigra and those of B. juncea were virtually identical to those of B. campestris indicating not only where the ct genomes of B. carinata and B. juncea originated, but also how little these genomes have been altered since the origin of these amphiploids. Ct DNA in B. napus shows more homology with that of B. oleracea than with that of B. campestris, but the ct genome of this amphiploid has diverged more from that of its putative parent than have those of the other two amphiploids.