Reverse breeding: a novel breeding approach based on engineered meiosis.

Reverse breeding (RB) is a novel plant breeding technique designed to directly produce parental lines for any heterozygous plant, one of the most sought after goals in plant breeding. RB generates perfectly complementing homozygous parental lines through engineered meiosis. The method is based on reducing genetic recombination in the selected heterozygote by eliminating meiotic crossing over. Male or female spores obtained from such plants contain combinations of non-recombinant parental chromosomes which can be cultured in vitro to generate homozygous doubled haploid plants (DHs). From these DHs, complementary parents can be selected and used to reconstitute the heterozygote in perpetuity. Since the fixation of unknown heterozygous genotypes is impossible in traditional plant breeding, RB could fundamentally change future plant breeding. In this review, we discuss various other applications of RB, including breeding per chromosome.

Low-coverage resequencing detects meiotic recombination pattern and features in tomato RILs.

Traditional plant breeding relies on meiotic recombination for mixing of parental alleles to create novel allele combinations. Detailed analysis of recombination patterns in model organisms shows that recombination is tightly regulated within the genome, but frequencies vary extensively along chromosomes. Despite being a model organism for fruit developmental studies, high-resolution recombination patterns are lacking in tomato. In this study, we developed a novel methodology to use low-coverage resequencing to identify genome-wide recombination patterns and applied this methodology on 60 tomato Recombinant Inbred Lines (RILs). Our methodology identifies polymorphic markers from the low-coverage resequencing population data and utilizes the same data to locate the recombination breakpoints in individuals by using a variable sliding window. We identified 1,445 recombination sites comprising 112 recombination prone regions enriched for AT-rich DNA motifs. Furthermore, the recombination prone regions in tomato preferably occurred in gene promoters over intergenic regions, an observation consistent with Arabidopsis thaliana, Zea mays and Mimulus guttatus. Overall, our cost effective method and findings enhance the understanding of meiotic recombination in tomato and suggest evolutionarily conserved recombination associated genomic features.

Plastid transformation in lettuce (Lactuca sativa L.) by polyethylene glycol treatment of protoplasts.

A detailed protocol for PEG-mediated plastid transformation of Lactuca sativa cv. Flora, using leaf protoplasts, is described. Successful plastid transformation using protoplasts requires a large number of viable cells, high plating densities, and an efficient regeneration system. Transformation was achieved using a vector that targets genes to the trnI/trnA intergenic region of the lettuce plastid genome. The aadA gene, encoding an adenylyltransferase enzyme that confers spectinomycin resistance, was used as a selectable marker. With the current method, the expected transformation frequency is 1-2 spectinomycin-resistant cell lines per 10(6) viable protoplasts. Fertile, diploid, homoplasmic, plastid-transformed lines were obtained. Transmission of the plastid-encoded transgene to the T1 generation was demonstrated.

Hybrid recreation by reverse breeding in Arabidopsis thaliana.

Hybrid crop varieties are traditionally produced by selecting and crossing parental lines to evaluate hybrid performance. Reverse breeding allows doing the opposite: selecting uncharacterized heterozygotes and generating parental lines from them. With these, the selected heterozygotes can be recreated as F1 hybrids, greatly increasing the number of hybrids that can be screened in breeding programs. Key to reverse breeding is the suppression of meiotic crossovers in a hybrid plant to ensure the transmission of nonrecombinant chromosomes to haploid gametes. These gametes are subsequently regenerated as doubled-haploid (DH) offspring. Each DH carries combinations of its parental chromosomes, and complementing pairs can be crossed to reconstitute the initial hybrid. Achiasmatic meiosis and haploid generation result in uncommon phenotypes among offspring owing to chromosome number variation. We describe how these features can be dealt with during a reverse-breeding experiment, which can be completed in six generations (∼1 year).

Reverse breeding in Arabidopsis thaliana generates homozygous parental lines from a heterozygous plant.

Traditionally, hybrid seeds are produced by crossing selected inbred lines. Here we provide a proof of concept for reverse breeding, a new approach that simplifies meiosis such that homozygous parental lines can be generated from a vigorous hybrid individual. We silenced DMC1, which encodes the meiotic recombination protein DISRUPTED MEIOTIC cDNA1, in hybrids of A. thaliana, so that non-recombined parental chromosomes segregate during meiosis. We then converted the resulting gametes into adult haploid plants, and subsequently into homozygous diploids, so that each contained half the genome of the original hybrid. From 36 homozygous lines, we selected 3 (out of 6) complementing parental pairs that allowed us to recreate the original hybrid by intercrossing. In addition, this approach resulted in a complete set of chromosome-substitution lines. Our method allows the selection of a single choice offspring from a segregating population and preservation of its heterozygous genotype by generating homozygous founder lines.

Stable plastid transformation in lettuce (Lactuca sativa L.).

Although plastid transformation in higher plants was first demonstrated in the early 1990s it is only recently that the technology is being extended to a broader range of species. To date, the production of fertile transplastomic plants has been reported for tobacco, tomato, petunia, soybean, cotton and Lesquerella fendleri (Brassicaceae). In this study we demonstrate a polyethylene glycol-mediated plastid transformation system for lettuce that generates fertile, homoplasmic, plastid-transformed lines. Transformation was achieved using a vector that targets genes to the trnA/trnI intergenic region of the lettuce plastid genome employing the aadA gene as a selectable marker against spectinomycin. Spectinomycin resistance and heterologous gene transcription were shown in T(1) plants derived from self-pollinated primary regenerants demonstrating transmission of the plastid-encoded transgene to the first seed generation. Crossing with male sterile wild-type lettuce showed that spectinomycin resistance was not transmitted via pollen. Constructs containing the gfp gene showed plastid-based expression of green fluorescent protein. The lettuce plastid could have potential both as a production and a delivery system for edible human therapeutic proteins.