Breeding of ornamental orchids with focus on Phalaenopsis: current approaches, tools, and challenges for this century.

Ornamental orchid breeding programs have been conducted to develop commercially valuable cultivars with improved characteristics of commercial interest, such as size, flower color, pattern, shape, and resistance to pathogens. Conventional breeding, including sexual hybridization followed by selection of desirable characteristics in plants, has so far been the main method for ornamental breeding, but other techniques, including mutation induction by polyploidization and gamma irradiation, and biotechnological techniques, such as genetic transformation, have also been studied and used in ornamental breeding programs. Orchids are one of the most commercially important families in floriculture industry, having very particular reproductive biology characteristics and being a well-studied group of ornamentals in terms of genetic improvement. The present review focuses on the conventional and biotechnological techniques and approaches specially employed in breeding Phalaenopsis orchids, the genus with highest worldwide importance as an ornamental orchid, highlighting the main limitations and strengths of the approaches. Furthermore, new opportunities and future prospects for ornamental breeding in the CRISPR/Cas9 genome editing era are also discussed. We conclude that conventional hybridization remains the most used method to obtain new cultivars in orchids. However, the emergence of the first biotechnology-derived cultivars, as well as the new biotechnological tools available, such as CRISPR-Cas9, rekindled the full potential of biotechnology approaches and their importance for improve ornamental orchid breeding programs.

Light and Ethephon Overcoming Seed Dormancy in Friar's Crown (Melocactus zehntneri, Cactaceae), a Brazilian Cactus.

Seed germination in Melocactus and other cactus species is hampered by dormancy. However, most studies failed to achieve high seed-germination rates, suggesting a complex mechanism of dormancy in Cactaceae. Thus, the objective of this study was to evaluate whether factors such as light and phytoregulators overcome the dormancy in the seeds of the friar's crown cactus (Melocactus zehntneri). Two consecutive experimental sets were designed: one with seed germination under filter paper conditions and different wavelengths and Photosynthetically Photon Flux Densities (PPFDs); and one in vitro experiment using a culture medium to evaluate the influence of different phytoregulators, such as gibberellic acid (GA3), benzylaminopurine (BAP) and ethephon (ET), both in the germination of seeds of M. zehntneri. Seeds of M. zehntneri are positive photoblastic. Red light and gradual increases in PPFD resulted in the highest germination rates (60.8-61.7%) and germination speed index (4.4-4.5). In vitro seeding in culture media increased the germination percentage to 76% in control without phytoregulators. Ethephon showed a major effect in releasing the germination of dormant seeds of M. zehntneri, totaling 98% of seeds germinated under in vitro conditions, while GA3 and BAP showed minor or no effect on germination. The present study resulted in an efficient in vitro technique for germination and a better understanding of cacti seed dormancy.

In Vitro Polyploidization of Brassolaeliocattleya Hybrid Orchid.

The Cattleya (Orchidaceae-Laeliinae subtribe) intergeneric hybrids, such as Brassolaeliocattleya (Blc.), have great ornamental value, due to their compact-size, with large and high color diversity of flowers. Artificial induction of polyploidy brings agronomic, ornamental and genetic benefits to plants. Polyploidization efficiency depends on factors, such as the type of antimitotic, polyploidization method, concentrations, exposure times and type of explant. This study aimed to develop a protocol to polyploidize Blc. orchids, by testing two types of explants (seeds and protocorms), concentrations and exposure times to colchicine. The effects of colchicine on the in vitro development of explants were also investigated. The responses of explants to colchicine depended on the concentrations, exposure time and the interaction of these factors. Flow cytometric analysis evidenced high endopolyploidy and allowed the separation of polyploidized (4C, 8C and 16C peaks) from non-polyploidized (only 2C and 4C peaks) plants. The highest percentage of polyploid plants was regenerated from protocorms (16.4%) treated with colchicine instead of seeds (3.2%). Protocorms treated with colchicine at 500-750 µM for 18 h resulted in the best percentage of polyploidization. Additionally, in vitro natural polyploidization using protocorms was reported (11.5%). Cytological analyses allowed an estimation of the number of chromosomes of the parents (≡70), polyploidized (≡140) and non-polyploidized progeny (≡70).

Polyploidization in Orchids: From Cellular Changes to Breeding Applications.

Polyploidy occurs naturally in plants through cell division errors or can artificially be induced by antimitotic agents and has ecological effects on species adaptation, evolution, and development. In agriculture, polyploidy provides economically improved cultivars. Furthermore, the artificial induction of polyploids increases the frequency; thus, it accelerates obtaining polyploid plants used in breeding programs. This is the reason for its use in developing many crops of economic interest, as is the case of orchids in the flower market. Polyploidy in ornamental plants is mainly associated with flowers of larger size, fragrance, and more intense coloring when compared to naturally diploid plants. Currently, orchids represent the largest flower market worldwide; thus, breeding programs aim to obtain flowers with the larger size, durability, intense colors, and resistance to pathogens. Furthermore, orchid hybridization with polyploidy induction has been used to produce improved hybrid cultivars. Thus, the objective of this review was to compile information regarding the natural occurrence, importance, and methods of induction of polyploidy in orchids. The study also summarizes the significance of polyploids and techniques associated with artificially inducing polyploidy in different orchids of commercial relevance.

Somatic embryogenesis from flower tepals of Hippeastrum aiming regeneration of virus-free plants.

Hippeastrum hybridum is an important bulbous flower plant in world floriculture, which are propagated conventionally by the technique known as double or twin scales to obtain plants with clonal origin. However, this technique promotes the propagation of systemic diseases, particularly mosaic-inducing viruses. The aim of this paper was to evaluate the somatic embryogenesis (SE) from tepals as an alternative to provide a technique for SE induction and to obtaining virus-free plantlets of Hippeastrum from infected plants. The concentrations of 2,4-Dichlorofenoxiacetic Acid (2,4-D) and thidiazuron (TDZ) was evaluated in SE induction pathway. The monitoring of viruses during the assays with tepals was performed by Reverse Transcription-Polymerase Chain Reaction. SE induction was obtained, for the first time, in tepal segments from flower buds of Hippeastrum. The 2,4-D was the main factor for embryogenic callus induction, and TDZ increased the SE induction rate. However, conversion of somatic embryos into plantlets were only developed in free-2,4-D media, replaced by 1.0 mg L-1 6-Benziladenine. Out of five virus species monitored during the experiment, Cucumber mosaic virus was detected in tepals and Hippeastrum mosaic virus in leaves of donor plants. The SE-derived plantlets that germinated in vitro were acclimatized and tested negative for all viruses assayed.

An Overview of Orchid Protocorm-Like Bodies: Mass Propagation, Biotechnology, Molecular Aspects, and Breeding.

The process through induction, proliferation and regeneration of protocorm-like bodies (PLBs) is one of the most advantageous methods for mass propagation of orchids which applied to the world floricultural market. In addition, this method has been used as a tool to identify genes of interest associated with the production of PLBs, and also in breeding techniques that use biotechnology to produce new cultivars, such as to obtain transgenic plants. Most of the molecular studies developed have used model plants as species of Phalaenopsis, and interestingly, despite similarities to somatic embryogenesis, some molecular differences do not yet allow to characterize that PLB induction is in fact a type of somatic embryogenesis. Despite the importance of species for conservation and collection purposes, the flower market is supported by hybrid cultivars, usually polyploid, which makes more detailed molecular evaluations difficult. Studies on the effect of plant growth regulators on induction, proliferation, and regeneration of PLBs are the most numerous. However, studies of other factors and new technologies affecting PLB production such as the use of temporary immersion bioreactors and the use of lighting-emitting diodes have emerged as new tools for advancing the technique with increasing PLB production efficiency. In addition, recent studies on Phalaenopsis equestris genome sequencing have enabled more detailed molecular studies and the molecular characterization of plantlets obtained from this technique currently allow the technique to be evaluated in a more comprehensive way regarding its real applications and main limitations aiming at mass propagation, such as somaclonal variation.

Micropropagation in the Twenty-First Century.

Despite more than a century of research on effective biotechnological methods, micropropagation continues to be an important tool for the large-scale production of clonal plantlets of several important plant species that retain genetic fidelity and are pest-free. In some cases, micropropagation is the only technique that supports the maintenance and promotes the economic value of specific agricultural species. The micropropagation of plants solved many phytosanitary problems and allowed both the expansion and access to high-quality plants for growers from different countries and economic backgrounds, thereby effectively contributing to an agricultural expansion in this and the last century. The challenges for micropropagation in the twenty-first century include cost reduction, enhanced efficiency, developing new technologies, and combining micropropagation with other systems/propagation techniques such as microcuttings, hydroponics, and aeroponics. In this chapter, we discuss the actual uses of micropropagation in this century, its importance and limitations, and some possible techniques that can effectively increase its wider application by replacing certain conventional techniques and technologies.

Nonfood GM Crops in Tropical Regions: A Reasonable Way to Promote the Technology for Increased Agricultural Sustainability.

Modifying a plant genetically is the most remarkable technology developed for agriculture production; however, the use of genetically modified (GM) food crops has raised concerns regarding their impact on the human health and environment. Nevertheless, the nonfood GM crops, such as for biofuel and wood production, can be a solution to increase the yield and avoid deforestation promoted by the expansion of cultivable land. Thus, the biosafety regulatory framework of different countries can be revised and studied on a case-by-case basis, considering the rapid release of nonfood GM uses for the development of a more sustainable agriculture, particularly in tropical regions.

Methods for genetic transformation in Dendrobium.

KEY MESSAGE: The genetic transformation of Dendrobium orchids will allow for the introduction of novel colours, altered architecture and valuable traits such as abiotic and biotic stress tolerance. The orchid genus Dendrobium contains species that have both ornamental value and medicinal importance. There is thus interest in producing cultivars that have increased resistance to pests, novel horticultural characteristics such as novel flower colours, improved productivity, longer flower spikes, or longer post-harvest shelf-life. Tissue culture is used to establish clonal plants while in vitro flowering allows for the production of flowers or floral parts within a sterile environment, expanding the selection of explants that can be used for tissue culture or genetic transformation. The latter is potentially the most effective, rapid and practical way to introduce new agronomic traits into Dendrobium. Most (69.4 %) Dendrobium genetic transformation studies have used particle bombardment (biolistics) while 64 % have employed some form of Agrobacterium-mediated transformation. A singe study has explored ovary injection, but no studies exist on floral dip transformation. While most of these studies have involved the use of selector or reporter genes, there are now a handful of studies that have introduced genes for horticulturally important traits.

Advances in Dendrobium molecular research: Applications in genetic variation, identification and breeding.

Orchids of the genus Dendrobium are of great economic importance in global horticultural trade and in Asian traditional medicine. For both areas, research yielding solid information on taxonomy, phylogeny, and breeding of this genus are essential. Traditional morphological and cytological characterization are used in combination with molecular results in classification and identification. Markers may be useful when used alone but are not always reliable in identification. The number of species studied and identified by molecular markers is small at present. Conventional breeding methods are time-consuming and laborious. In the past two decades, promising advances have been made in taxonomy, phylogeny and breeding of Dendrobium species due to the intensive use of molecular markers. In this review, we focus on the main molecular techniques used in 121 published studies and discuss their importance and possibilities in speeding up the breeding of new cultivars and hybrids.

Asymbiotic in vitro seed propagation of Dendrobium.

The ability to germinate orchids from seeds in vitro presents a useful and viable method for the propagation of valuable germplasm, maintaining the genetic heterogeneity inherent in seeds. Given the ornamental and medicinal importance of many species within the genus Dendrobium, this review explores in vitro techniques for their asymbiotic seed germination. The influence of abiotic factors (such as temperature and light), methods of sterilization, composition of basal media, and supplementation with organic additives and plant growth regulators are discussed in context to achieve successful seed germination, protocorm formation, and further seedling growth and development. This review provides both a basis for the selection of optimal conditions, and a platform for the discovery of better ones, that would allow the development of new protocols and the exploration of new hypotheses for germination and conservation of Dendrobium seeds and seedlings.

Dendrobium micropropagation: a review.

Dendrobium is one of the largest and most important (ornamentally and medicinally) orchid genera. Tissue culture is now an established method for the effective propagation of members of this genus. This review provides a detailed overview of the Dendrobium micropropagation literature. Through a chronological analysis, aspects such as explant, basal medium, plant growth regulators, culture conditions and final organogenic outcome are chronicled in detail. This review will allow Dendrobium specialists to use the information that has been documented to establish, more efficiently, protocols for their own germplasm and to improve in vitro culture conditions based on the optimized parameters detailed in this review. Not only will this expand the use for mass propagation, but will also allow for the conservation of important germplasm. Information on the in vitro responses of Dendrobium for developing efficient protocols for breeding techniques based on tissue culture, such as polyploidization, somatic hybridization, isolation of mutants and somaclonal variants and for synthetic seed and bioreactor technology, or for genetic transformation, is discussed in this review. This is the first such review on this genus and represents half a decade of literature dedicated to Dendrobium micropropagation.

Symbiotic in vitro seed propagation of Dendrobium: fungal and bacterial partners and their influence on plant growth and development.

The genus Dendrobium is one of the largest genera of the Orchidaceae Juss. family, although some of its members are the most threatened today. The reason why many species face a vulnerable or endangered status is primarily because of anthropogenic interference in natural habitats and commercial overexploitation. The development and application of modern techniques and strategies directed towards in vitro propagation of orchids not only increases their number but also provides a viable means to conserve plants in an artificial environment, both in vitro and ex vitro, thus providing material for reintroduction. Dendrobium seed germination and propagation are challenging processes in vivo and in vitro, especially when the extreme specialization of these plants is considered: (1) their biotic relationships with pollinators and mycorrhizae; (2) adaptation to epiphytic or lithophytic life-styles; (3) fine-scale requirements for an optimal combination of nutrients, light, temperature, and pH. This review also aims to summarize the available data on symbiotic in vitro Dendrobium seed germination. The influence of abiotic factors as well as composition and amounts of different exogenous nutrient substances is examined. With a view to better understanding how to optimize and control in vitro symbiotic associations, a part of the review describes the strong biotic relations of Dendrobium with different associative microorganisms that form microbial communities with adult plants, and also influence symbiotic seed germination. The beneficial role of plant growth-promoting bacteria is also discussed.

In vitro conservation of Dendrobium germplasm.

Dendrobium is a large genus in the family Orchidaceae that exhibits vast diversity in floral characteristics, which is of considerable importance to orchid breeders, biotechnologists and collectors. Native species have high value as a result of their medicinal properties, while their hybrids are important as ornamental commodities, either as cut flowers or potted plants and are thus veritable industrial crops. Thus, preservation of Dendrobium germplasm is valuable for species conservation, breeding programs and the floriculture industry. Cryopreservation represents the only safe, efficient and cost-effective long-term storage option to facilitate the conservation of genetic resources of plant species. This review highlights 16 years of literature related to the preservation of Dendrobium germplasm and comprises the most comprehensive assessment of thorough studies performed to date, which shows reliable and reproducible results. Air-drying, encapsulation-dehydration, encapsulation-vitrification, vitrification and droplet-vitrification are the current cryopreservation methodologies that have been used to cryopreserve Dendrobium germplasm. Mature seeds, pollen, protoplasts, shoot primordia, protocorms and somatic embryos or protocorm-like bodies (PLBs) have been cryopreserved with different levels of success. Encapsulation-vitrification and encapsulation-dehydration are the most used protocol, while PLBs represent the main explant explored.