Wallace D. Beversdorf

High frequency embryogenesis through isolated microspore culture in Brassica napus L. and B. carinata Braun

Abstract Isolated microspores from six cultivars of Brassica napus and one of B. carinata were cultured in modified Nitsch and Nitsch (NN) medium supplemented with 13% (W/V) sucrose, 0.05 mg/l benzyladenine (BA) and 1.00 mg/l nahpthaleneacetic acid (NAA). Embryogenic responses were observed at cultured temperatures ranging from 22 to 32°C. For most genotypes tested, the highest frequency of embryos occurred at 30°C and 7–54 embryos per anther (approx. 17 000 microspores per anther) developed. Although incubation at 30°C produced the highest frequency of embryos, lower culture temperatures induced better quality embryos. A split temperature culture regime of incubation at 32°C for 3 days followed by incubation at 25°C resulted in both high embryo yields and a high percentage of normal embryos. Plantlet development from microspore-derived embryos appeared to be influenced by both genotype and medium.

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

SummaryProtoplast 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.

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

SummaryThe 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.

Cytoplasmic male sterility in rapeseed (Brassica napus L.) : 1. Restriction patterns of chloroplast and mitochondrial DNA.

SummaryRestriction patterns of chloroplast (cp) and mitochondrial (mt) DNA in Brassica napus rapeseed reveal the alloplasmic nature of cytoplasmic male sterility in this crop. Both the Shiga and Bronowski systems probably exploit cytoplasmic diversity in B. napus cultivars arising from introgression of cytoplasm from the other rapeseed species, B. campestris. Nuclear genes specific to these systems do not cause sterility in maintainers (Bronowski and Isuzu-natane) because they have a campestris cytoplasm, but give rise to sterility in napus cytoplasms. In the course of hybridization to napus cultivars a line with the triazine resistant cytoplasm (a campestris cytoplasm) has undergone an alteration in the mt genome rendering its restriction pattern more similar than previously to that of napus. The alteration may be an inversion between 7.2 and 3.4 kb in length.

Enhanced plant regeneration from microspore-derived embryos of Brassica napus by chilling, partial desiccation and age selection

Germination was readily induced in recalcitrant microspore-derived embryos of Brassica napus ‘Topas’ when they were exposed to a period of chilling (9–12 days at 4°C) or partial desiccation (rapid or slow air drying) prior to germination. In general, embryos thirty-five days old had the highest germination rates as compared to younger or older ones. Populations of embryos were induced to germinate at a rate of over 90% under specific temperature, desiccation and age conditions. Comparisons to an embryogenic B. napus winter line, F346, are made.

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.

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 β-glucuronidase (GUS) and NPT II genes. Both the transient gene expression of β-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 β-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 β-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.

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

SummaryMicrospore-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.

Linear mitochondrial plasmid inBrassica has terminal protein

SummaryA linear 11.3 kb plasmid occurs in preparations of mitochondrial DNA from certainBrassica species and cultivars. Evidence that protein is associated with the ends of the plasmid was obtained in this study. Plasmid DNA ran as a distinct band during electrophoresis only if it had been treated with proteinase K. The hybridization patterns of clones from different regions of the plasmid to restriction digests of the protein-DNA complex revealed that the protein is associated with the end fragments of the plasmid. Exonuclease treatments indicated that the 5′ ends of the plasmid DNA molecule are blocked but that the 3′ ends are free.

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 Lichters (1982) medium with 13% sucrose produced ca. 10% of the embryos yielded by an unfrozen control. Microspores frozen in Lichters 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.