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Tiwari, P.; Sharma, A.; Bose, S.K.; Park, K. Breeding Approaches for Orchid Cultivation and Conservation. Encyclopedia. Available online: (accessed on 16 April 2024).
Tiwari P, Sharma A, Bose SK, Park K. Breeding Approaches for Orchid Cultivation and Conservation. Encyclopedia. Available at: Accessed April 16, 2024.
Tiwari, Pragya, Abhishek Sharma, Subir Kumar Bose, Kyeung-Il Park. "Breeding Approaches for Orchid Cultivation and Conservation" Encyclopedia, (accessed April 16, 2024).
Tiwari, P., Sharma, A., Bose, S.K., & Park, K. (2024, February 26). Breeding Approaches for Orchid Cultivation and Conservation. In Encyclopedia.
Tiwari, Pragya, et al. "Breeding Approaches for Orchid Cultivation and Conservation." Encyclopedia. Web. 26 February, 2024.
Breeding Approaches for Orchid Cultivation and Conservation

Orchid cultivation is gaining momentum attributed to the development of new orchid varieties with unique features of colors and appearance. To bridge this demand and supply gap, both traditional and molecular breeding approaches are employed with consistent efforts.


1. Classical Breeding Strategies

1.1. Crossbreeding

Orchid genera Phalaenopsis and Oncidium are widely grown orchids for commercial production. Orchids are both self-pollinated and cross-pollinated species. However, the self-pollinating species sometimes produce a smaller number of seeds than cross-pollinated flowers [1][2]. Crossbreeding or hybridization using both natural and artificial approaches splendidly integrates the excellent traits of two parents in their hybrid offspring. One of the oldest natural orchid hybrids, Phalaenopsis intermedia, which is a result of a cross between P. aphrodite and P. rosea, was described in 1853. Meanwhile, the first commercial artificial hybrid orchid Calanthe dominyi was developed as a cross between C. masuca and C. furcate [3][4]. The production of these commercial natural and hybrid orchids was achieved using crossbreeding approaches. However, several factors including hybrid combination fertility, targeted traits quality assessment, and superior hybrid offspring selection play an important role and must be considered [5]. The generation F1 derived from the cross between parents with targeted contrasting traits usually has large phenotypic differences from their parents. For example, the F1 generation derived from the parent having a large flower and short flowering time compared to the parent having a small flower and long flowering time, viz. Ionmesa Popcorn ‘Haruri’, produces flowers with distinct notable differences from its parents. However, hybrids sometimes bear problems associated with germination; for example, the occurrence of intraspecific < intrageneric < intergeneric degree of genetic relationship in hybrid Cymbidium seeds results in difficult seed germination and culturing [6]. Similar problems associated with the hybridization process, parent incompatibility, and post-fertilization embryo abortion failing the distinct hybridization have been reported [7]. Commercial orchid cultivation suffers from several associated breeding barriers such as the large and complex polyploid genome, slow growth, and long life cycle; consequently, it takes a long period to generate new cultivars using cultivation through the traditional breeding system. The low transformation efficiency makes the development of new varieties with desired traits a challenging task [8][9]. For example, the high commercial value orchid Phalaenopsis takes more than 2 years to switch from the vegetative to the reproductive phase [10]. Therefore, the applicability of these conditions remains to be studied in major commercial orchid species. Seed germination is one of the key aspects of the traditional breeding system, as it is directly associated with the efficient success of crossbreeding; therefore, in-depth studies are required for a deeper understanding of the seed germination mechanisms and plant developmental characteristics of an effective breeding system. Hence, a suitable cultivation approach is required for hybrid seeds developed from crossbreeding and stable growth of the hybrid population.

1.2. Selection Breeding

Selection breeding differentiates from crossbreeding where hybrid selection is based on natural variations in traits [11]. Selection breeding specifically concerns three important genetic parameters: genetic correlations between traits, trait heritability, and interactions between genotypes and the environment [1]. Like classical orchid breeding strategies of crossbreeding and selection breeding, biotechnological interventions have resulted in the origin of mutational breeding and molecular marker-assisted breeding approaches for orchids.

1.3. Mutation Breeding

The mutational breeding approach utilizes physical and chemical mutagens to improve individual traits and will shorten the breeding cycle in orchids. Mutational breeding has produced several orchids with improved traits, viz. aroma, higher medicinal content, enhanced shelf life, and stress resistance [4]. Polyploidization using colchicine and other mutagen treatments is one of the key approaches used for mutation in orchids such as Cymbidium, Dendrobium, Oncidium, and Phalaenopsis [12][13][14][15][16][17]. The higher levels of genomic heterozygosity can allow the enhanced mutation rate in a short time duration; however, the random and unpredictable nature of mutagenesis can take place throughout the genome which can lead to other physio-morphological problems in orchid mutants. Therefore, a lot of studies are still required to understand the basis of mutation breeding in orchids for the identification of suitable genotypes, explants, mutagen types, and their optimized dose concentration to produce mutant orchids with desired traits.

1.4. Molecular Marker-Assisted Breeding (MMAB)

The molecular marker-assisted breeding approach utilizes the accuracy of molecular biology tools and techniques for fast, accurate, and environmental-influence-free orchid breeding and natural and artificial hybrid selection [18]. The MMAB uses the most prevalent, versatile, and high-potential markers such as restriction fragment length polymerase (RFLP), amplified fragment length polymerase (AFLP), insertional simple sequence repeats (ISSR), and single nucleotide polymorphism (SNP) markers [1]. Most of these markers are widely used in modern orchid breeding and have achieved good results. Li et al. [19] developed a set of wide-range genic SSR markers in Cymbidium ensifolium to evaluate the genetic relationship and trait mapping in the orchid population. These SSR markers facilitate the identification of genetic relationships and have been successively used for the identification of root growth mechanisms and secondary metabolites-related gene identification, along with flower shape and color-related genes in Phalaenopsis [20][21]. Similarly, SNP markers were also utilized to construct integrated genetic maps of the Dendrobium genome along with the identification of several important QTL sites [22]. Although MMAB has played an important role as a modern orchid breeding approach, it mainly targets phylogenetics for determining genetic relationships between orchid species. Therefore, co-integrated approaches of traditional, and modern orchid breeding strategies and biotechnologies are required for improved orchid breeding.

2. In Vitro Orchid Propagation in Plant Tissue Culture

In vitro micropropagation provides a convenient and feasible approach for orchids, especially for orchid seeds that are difficult to germinate and grow in a natural environment [23]. Also, it provides a platform for biotechnologies for orchid improvements and genetic engineering. In vitro micropropagation represents a promising tool for the conservation of several threatened and endangered orchids and has been successfully applied in several species such as Paphiopedilum armeniacum, Bulbophyllum nipondhii, Paphiopedilum insigne, and Anoectochilus elatus [24][25][26][27].
In the last decades, plant tissue cultures have been instrumental in the rapid propagation and ex situ conservation of orchids, using different methods and explants including flower stalks, shoot tip nodes, stem bids, root tips, and rhizome segments [28]. The composition of culture media plays a major role in in vitro seed germination and micropropagation using different explants [29][30]. For example, Murashige and Skoog (MS) medium enhances germination in the orchid Geodorum densiflorum, whereas Knudson C medium [31] is more suitable for Paphiopedilum seeds compared to other orchid-specific culture media [32]. It was also observed that a low concentration of mineral salts in MS medium (viz. ½ MS or ¼ MS) promotes seed germination in some terrestrial orchids [33][34]. The constituents of culture media, especially plant growth regulators, auxins, and cytokinins, have a significant impact on the growth, germination, and development of orchid seeds and explants in vitro [35][36]. Along with the plant growth promoters, the addition of organic nutrient sources like coconut water and potato extract in culture media was also found to promote orchid in vitro seed germination [25][37].
The different orchid species show great variations in physiological and morphological features, requiring different conditions for growth in plant tissue culture. Different species respond differently to growth factors (depending on the genotype), and some are recalcitrant in in vitro conditions [38]. In terrestrial orchids, such as Paphiopedilum and Cypripedium, meristem explant survival and PLB induction are difficult. In addition, a method developed for a particular species may not work for another species. Therefore, for targeted genetic transformation, a plant regeneration protocol must be developed for a particular plant genotype [38], and the identification of variations in genotype must occur before plant transformation is necessary. The subsequent discovery of asymbiotic seed germination (for in vitro plant propagation) has been widely employed to develop orchid plants in vitro and direct somatic embryogenesis [39], totipotent callus induction, shoot-bud formation from different explants, PLB formation from cell suspension culture, and others, which have immensely contributed to the micropropagation and creation of transgenic hybrids [40].

3. Cryopreservation Techniques

Low temperature and dry storage-based preservation are key approaches to orchid germplasm conservation [41][42]. However, this low temperature and dry storage-based method is successful for 1 to 6 months of germplasm preservation but fails to provide high viability of germplasm under preservation for longer durations [43][44]. Cryopreservation is the best germplasm preservation approach as all the metabolic and physiological processes cease at the temperature −196 °C [45]. The pretreatment of orchid seeds and pollens germplasm through vitrification, desiccation, and encapsulation–dehydration methods of removing the cell water content before cryopreserving in liquid nitrogen are employed [46][47].

3.1. Vitrification

Sakai [48] introduced the technique of vitrification typically used for the longer preservation of immature and mature orchid seeds with higher than average water content. The vitrification method uses a high osmolarity vitrification solution containing glycerol, dimethyl sulfoxide, and ethyl glycol as cryoprotectants. The seeds for preservation are kept in this high osmolarity vitrification solution that reduces the intracellular water content of seeds and vitrified by penetration of these cryoprotectants through osmoregulation, thus reducing the freezing temperature and preventing cells from ice nucleation injuries [47][49]. Vitrification has helped in the conservation through cryopreservation of immature and high-water-content seeds of several orchid genera, viz. Bletilla, Cymbidium, Dendrobium, Encyclia, Phaius, and Vanda [45].

3.2. Desiccation

Desiccation-based cryopreservation is found to be more suitable for mature orchid seeds. The process of desiccation includes the slow drying of seeds under a controlled desiccation rate under constant relative humidity or drying with silica gel or with CaCl2.6H2O salt solution to reduce the water content of the seeds before preserving them in liquid nitrogen [45]. Several orchids, viz. Bletilla formosana, Caladenia flava, Dactylorhiza fuchsii, Diuris fragrantissima, Eulophia gonychlia, Gymnadenia conopsea, Orchis coriophora, Paphiopedilum rothschildianum, Pterostylis sanguinea, Thelymitra macrophylla, and Dendrobium candidum, were successively cryopreserved using the desiccation technique [45][50].

3.3. Encapsulation–Dehydration

Encapsulation–dehydration, a technique developed for artificial seed production, is the third method for cryopreservation [51]. The encapsulation–dehydration approach uses the in vitro cultured orchid plant tissues, seeds, and embryos, partially desiccated with silica beads or airflow of the laminar bench to reduce the cellular water content. These partially dried tissues are trapped and encapsulated in sodium alginate beads before keeping them in liquid nitrogen for cryopreservation [50]. The encapsulation–dehydration techniques are generally applied to a few orchids, viz. Cyrtopodium hatschbachii and Oncidium bifolium [52][53].

4. Genetic Engineering and Generation of Hybrids with ‘High-Value’ Traits

Genetic engineering in orchids confers ‘high-value’ traits to the hybrids with desired characteristics and has been attempted in the genera viz. Phalaenopsis, Cattleya, Cymbidium, Dendrobium, Oncidium, Paphiopedilum, and Vanda, with documented translational success. PLBs derived from shoot tip cultures are used as target material for transformation in Phalaenopsis sp., while protocorm has also been frequently used [54]. A relatively large number of transgenic plants can be generated by protocorm transformation due to the presence of thousands of seeds in one fruit of Phalaenopsis sp. However, to achieve a successful outcome, it is indispensable to improve the transformation efficiency for efficient orchid breeding. Furthermore, the genetic engineering procedures in orchids are applied through either particle bombardment or Agrobacterium-mediated transformation to achieve the delivery of the desired gene. The initial genetic transformation studies were limited to the biolistic-mediated transformation [55][56]. The very first successful Agrobacterium-mediated genetic transformation was achieved in the orchid genera Phalaenopsis expressing the GUS gene construct [57]. The Agrobacterium-mediated genetic transformation was found to be more efficient than the biolistic-mediated genetic transformation in terms of incorporation of low copy number genes at transcriptionally active chromosomal regions [58].
The last decade has documented genetically engineered orchids through Agrobacterium-mediated genetic transformation systems in Phalaenopsis, Dendrobium, Cymbidium, Oncidium, and Vanda [59][60][61][62][63]. The genetic engineering approaches in orchids are not only restricted to the overexpression of heterologous genes for the desired traits but also gene silencing to knock out genes in Dendrobium ‘Sonia’ and Oncidium hybrids [64]. Now, with the availability of the complete genomes of Dendrobium officinale and P. equestris, the genetic engineering approaches facilitate CRISPR/Cas9-mediated genome editing in different orchid species [65]. Liu et al. [64] delivered a phytoene synthase-RNAi construct into PLBs of Oncidium hybrid. The downregulation of PSY and geranyl synthase genes was observed in the transgenic orchid, and endogenous levels of abscisic acid and gibberellic acid were decreased in dwarf plants [64]. In another study, Agrobacterium-mediated insertion of KNAT1 (a key gene for bud apical meristem differentiation) and AtRKD4 (a key gene for embryonic differentiation) into the orchid genome induced organogenesis and bud development, thereby improving the yield of native and transgenic orchids.


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