The global flower industry thrives on novelty. Genetic engineering is providing a valuable means of expanding the floriculture gene pool so promoting the generation of new commercial varieties. Commercialisation of genetically engineered flowers is currently confined to novel coloured carnations. However, further products are expected given the level of activity in the field. In general terms engineered traits are valuable to either the consumer or the producer. At present only consumer traits appear able to provide a return capable of supporting what is still a relatively expensive molecular breeding tool. The biosynthesis of floral pigments, particularly anthocyanins, has been elucidated in great detail in model flowers such as petunia. This knowledge is now being applied to an understanding of a wide range of other flowers and providing a means of targeting colour modification in these species. The engineering of novel traits in a given variety also rests on capabilities in plant transformation that are continuing to expand at a rapid rate. The expression of genes transferred across genera is not always predictable and so requires considerable trial and error to arrive at stable phenotype of commercial interest. Manipulation of metabolic pathways, often requiring introduction of multiple genes can also be problematic. This is a reflection of the complexity of interactions within and between cells at a gene and gene product level. An understanding of gene function is only an essential first step in engineering novel traits. The production of novel flower colour has been the first success story in floriculture genetic engineering. Other traits that have received attention include floral scent, floral and plant morphology, senescence of flowers both on the plant and post-harvest and disease resistance.

Genetic Engineering in Floriculture

This review provides an informative and broad overview of orchid biotechnology, addressing several important aspects such as molecular systematics, modern breeding, in vitro morphogenesis, protoplast culture, flowering control, flower color, somaclonal variation, orchid mycorrhiza, pathogen resistance, virus diagnosis and production of virus-free plants, functional genomics, genetic transformation, conservation biotechnology and pharmaceutical biotechnology. This resource will provide valuable insight to researchers who are involved in orchid biology and floriculture, using biotechnology to advance research objectives. Producing an improved orchid through biotechnology for industrial purposes or to serve as a model plant for pure and applied sciences is well within reach and many of the current techniques and systems are already employed at the commercial production level.

The Application of Biotechnology to Orchids

Paphiopedilum orchids are among the world’s most popular orchid due to their impressively beautiful flowers. Propagation of these orchid genera has been hampered by the naturally slow growth rate of the plant, which renders it very difficult to be propagated through conventional methods. In vitro culture techniques have provided a useful alternative technology for propagating this recalcitrant species. In this study, the propagation of P. rothschildianum was achieved through the in vitro formation of secondary protocorm-like bodies (PLBs) from the primary PLB that developed from stem-derived callus. The PLBs were cultured on half-strength MS medium supplemented with different concentrations (1.0, 2.0, 3.0, and 4.0 μM) of 6-benzyladenine (BA) and kinetin for the induction of secondary PLBs. The highest number of secondary PLBs formed was obtained on half-strength MS medium supplemented with 4.0 μM kinetin, with an average of 4.1 PLBs per explant after 8 weeks of culture. The secondary PLBs continued to proliferate further and formed 9.5–12.1 new PLBs per secondary PLB after being subcultured onto half-strength plant growth regulator-free MS medium supplemented with 60 g/L banana homogenate (BH). These tertiary PLBs were subcultured onto media containing different organic additives, such as BH, coconut water, potato homogenate, and tomato homogenate, for plantlet regeneration. Among the organic additives tested, the addition of 20% CW to half-strength MS medium resulted in the best average plantlet regeneration percentage from the PLBs, 67.9%, after 8 weeks of culture.

In vitro propagation of Paphiopedilum orchid through formation of protocorm-like bodies

A transformation procedure for phalaenopsis orchid established by using immature protocorms for Agrobacterium infection was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. Protocorms obtained after 21 days of culture on liquid New Dogashima medium were inoculated with Agrobacterium strain EHA101(pIG121Hm) harboring both β-glucuronidase (GUS) and hygromycin resistance genes. Subculture of the protocorms on acetosyringone-containing medium 2 days before Agrobacterium inoculation gave the highest transformation efficiencies (1.3–1.9%) based on the frequency of hygromycin-resistant plants produced. Surviving protocorms obtained 2 months after Agrobacterium infection on selection medium containing 20 mg l−1 hygromycin were cut transversely into two pieces before transferring to recovery medium without hygromycin. Protocorm-like bodies (PLBs) proliferated from pieces of protocorms during a 1-month culture on recovery medium followed by transfer to selection medium. Hygromycin-resistant phalaenopsis plants that regenerated after the re-selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by Southern blot analysis. A total of 88 transgenic plants, each derived from an independent protocorm, was obtained from ca. 12,500 mature seeds 6 months after infection with Agrobacterium. Due to the convenient protocol for Agrobacterium infection and rapid production of transgenic plants, the present procedure could be utilized to assess expression of transgenes under different genetic backgrounds, and for the molecular breeding of phalaenopsis.

Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination

Oncidium is an important ornamental plant but the study of its functional genomics is difficult. Erycina pusilla is a fast-growing Oncidiinae species. Several characteristics including low chromosome number, small genome size, short growth period, and its ability to complete its life cycle in vitro make E. pusilla a good model candidate and parent for hybridization for orchids. Although genetic information remains limited, systematic molecular analysis of its chloroplast genome might provide useful genetic information. By combining bacterial artificial chromosome (BAC) clones and next-generation sequencing (NGS), the chloroplast (cp) genome of E. pusilla was sequenced accurately, efficiently and economically. The cp genome of E. pusilla shares 89 and 84% similarity with Oncidium Gower Ramsey and Phalanopsis aphrodite, respectively. Comparing these 3 cp genomes, 5 regions have been identified as showing diversity. Using PCR analysis of 19 species belonging to the Epidendroideae subfamily, a conserved deletion was found in the rps15-trnN region of the Cymbidieae tribe. Because commercial Oncidium varieties in Taiwan are limited, identification of potential parents using molecular breeding method has become very important. To demonstrate the relationship between taxonomic position and hybrid compatibility of E. pusilla, 4 DNA regions of 36 tropically adapted Oncidiinae varieties have been analyzed. The results indicated that trnF-ndhJ and trnH-psbA were suitable for phylogenetic analysis. E. pusilla proved to be phylogenetically closer to Rodriguezia and Tolumnia than Oncidium, despite its similar floral appearance to Oncidium. These results indicate the hybrid compatibility of E. pusilla, its cp genome providing important information for Oncidium breeding.

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Complete Chloroplast Genome Sequence of an Orchid Model Plant Candidate: Erycina pusilla Apply in Tropical Oncidium Breeding

The present protocol was aimed at establishing a routine transformation procedure via Agrobacterium tumefaciens for an important Oncidium orchid cultivar. An expression vector containing hptII and gusA genes driven by the cauliflower mosaic virus (CaMV) 35S promoter was successfully introduced into the Oncidium orchid genome by A. tumefaciens. Protocorm-like bodies (PLBs) derived from protocorms, were the target explants for transformation. The transformation was performed through two stages of cocultivation, the first stage occurring on antibiotic-free medium for 3 days, and the subsequent stage on medium containing 100 mg/l timentin for 1 month. Among 1,000 inoculated PLBs, 108 putatively transformed PLBs were proliferated on 5 mg/l hygromycin selection medium. A total of 28 independent transgenic orchid plants were obtained, from which six transgenic lines that were positive for β-glucuronidase were randomly selected and confirmed by Southern, northern and western blot analyses. These results indicated that the foreign DNA was successfully integrated into the orchid genome and expressed transcriptionally and translationally in these orchid plants. The present transformation method reported is suitable for improving the Oncidium orchid through genetic engineering.

H.-H. Hsiao

Papers

Agrobacterium tumefaciens-mediated transformation of an Oncidium orchid