Biotechnology of ornamental plants: when beauty joins science—preface from the editors

Biotechnological tools have a central place in the ornamental crops, where they have been used for their micropropagation as also for the generation of genetic novelties by mutagenesis, gene transfer, or through the manipulation of ploidy levels for their subsequent exploitation both in breeding and commercially. The articles in this Special Issue of Plant Cell Tissue and Organ Culture focus on several of these biotechnological approaches developed in a range of ornamental species and crops.

This special issue starts with a review by Malakar et al. (https://doi.org/10.1007/s11240-022-02300-w) on the uses of biotechnology tools in Heliconia, commonly known as ‘False-Bird-of-Paradise’. The 250–300 tropical Heliconia species are increasingly popular ornamental cut flowers due to their unusual elegance and attractive colours. A season-free supply of cut spikes is crucial to keep pace with market demand and here micropropagation can come to the rescue. It is recognized that developing novel Heliconia hybrids faces various phenological and physiological barriers, including a reduced production of viable pollen, self-incompatibility and a lack of bird-mediated pollination. Thus, embryo rescue, mutagenesis and polyploidisation have been explored to overcome these barriers. All these biotechnological approaches have been surveyed in this comprehensive review.

Adenium obesum, desert rose, is a member of the Apocynaceae which is threatened with extinction. Araújo et al. (https://doi.org/10.1007/s11240-021-02184-2) examined the effect of varying wavelengths from light-emitting diodes (LEDs) (white LED—6400  K—λ = 525 nm; blue LED—λ = 430 nm; green LED—λ = 520 nm; orange LED—λ = 595 nm; purple LED—λ = 440 nm “2 blue LEDs + 1 red LED”; and red LED—λ = 670 nm) and control—absence of LED, combined with Murashige and Skoog (MS) or MRA (developed by the authors) media on the in vitro germination of seeds and initial growth of A. obesum seedlings, and on the production of photosynthetic pigments and soluble sugars. The highest germination and lowest mortality rates occurred under red LED light combined with MRA medium or purple LED combined with MS medium, while the best initial growth was observed under red LED irrespective of culture media. The pigment contents were highest under white light with MS medium or green light with MRA medium.

Light quality was also one factor studied by Farrokhzad et al. (https://doi.org/10.1007/s11240-022-02272-x) who examined the effects of different wavelengths of LED and fluorescent light used for culture of the orchid Phalaenopsis amabilis, measuring shoot proliferation and photomorphogenesis response. For this purpose, the endogenous levels of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), trans-Zeatin (ZEA), Kinetin (KIN), abscisic acid (ABA), gibberellic acid (GA₃), melatonin (MEL), as well as the DNA methylation patterns were measured. The best shoot proliferation was observed under blue + green irradiation, while fresh and dry weight were increased under white LEDs but were not significantly different from fluorescent and blue + red. ZEA content was higher in fluorescent, white and red light while KIN content was higher in fluorescent and blue + green treatments. IAA and IBA contents were slightly higher under red + blue and red light. The auxin/cytokinin ratio was highest under green and red + blue and lowest under fluorescent, white and red + green. ABA was not significantly affected by the light wavelength. GA₃ was increased by red + blue and green + blue light, and decreased by the fluorescent, white, red + green, and blue light. MEL content was higher in blue and red and lower in red + green. The highest percentage of unmethylated sites was found under fluorescent and red + blue light, followed by white LEDs. Optimum branching was associated with higher KIN content, total cytokinins and GA₃ under blue + green. However, transfer to the full light spectrum was required for optimized biomass and normal growth.

With its bright and colourful blooms, New Guinea Impatiens (Impatiens hawkeri W. Bull) has a high ornamental appeal and has been the object of several biotechnology studies in the past. However, de novo regeneration is rare among them, and Zhang et al. (https://doi.org/10.1007/s11240-022-02282-9) developed efficient direct shoot organogenesis from hypocotyls with partial cotyledons of two cultivars of New Guinea Impatiens. They showed the impact on in vitro morphogenesis of the explant sterilization method, basal medium, AgNO₃, sucrose and plant growth regulators contents in the medium. Interestingly, hyperhydricity has been problematic with Impatiens tissue cultures, and the authors found that this could be alleviated by reducing sucrose (to 20 g/L) or through the addition of AgNO₃ (1 mg/L).

In a study with Gladiolus hybridus, Isah et al. (https://doi.org/10.1007/s11240-022-02309-1) examined the promoting impact of copper sulphate-hydrate (CuSO₄·5H₂O, 0–15 μM), silver nitrate (AgNO₃, 0–60 μM) and their combinations in the medium on callus growth and morphogenesis. Thus, when used alone, AgNO₃ was more efficient than any of the CuSO₄·5H₂O treatments, with maximum effect at 40 μM, but treatments combining high levels of these two metal micronutrients provoked a larger promotion of morphogenesis which was associated with essential physiological biochemical changes.

Askari and Visser (https://doi.org/10.1007/s11240-022-02328-y) compared scale explants of different sizes regarding their in vitro regeneration competence of adventitious bulblets in the genus Lilium. Thus, large explants (6 × 18 mm) gave significantly more bulblet growth than small ones (6 × 6 mm) and bulblets grew better following their transfer to fresh medium. In addition, they showed that leaving a small piece of the original scale attached to such bulblets increased their subsequent growth. Interestingly, the authors found that the position of the explant in the scale also affected bulblets growth, being optimum in explants cut from the middle basal half of a scale as opposed to the edge of the apical half of the scale. On the other hand, during bulblet regeneration, the content of polysaccharides in the explant markedly decreased while the total soluble sugars increased. There also were many more starch granules in cells of the basal explants compared with apical scale explants. Finally, the number of vascular bundles increased during culture in all scale explants irrespective of their position.

Tree peony (Paeonia sect. Moutan) is an economically important woody plant of interest both for its medicinal properties and its ornamental and oil values but is difficult to breed and propagate conventionally. Thus, there is a need to develop reliable biotechnology tools, as undertaken in two independent reports by Xu et al. (https://doi.org/10.1007/s11240-021-02216-x). First, the authors developed a protocol for in vitro regeneration of P. ostii ‘Feng Dan’ from cotyledon-derived embryogenic callus induced on modified MS medium with N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) and α-naphthylacetic acid (NAA), subsequently transferred to modified woody plant medium with CPPU and thidiazuron (TDZ) for optimal induction of meristematic nodules and leaf cluster differentiation. A combination of 6-benzyladenine (BA) + gibberellin (GA₃) supported the best shoot elongation, rooting and survival rates. Second, they studied the histology of induction of meristematic nodules and of shoot organogenesis (https://doi.org/10.1007/s11240-021-02208-x). Using a dissecting microscope, light microscope, scanning electron microscopy (SEM) and transmission electron microscopy, they revealed the complex developmental sequence, dynamic changes of extracellular matrix and ultrastructural characteristics during the morphogenesis of meristematic nodules in tree peony ‘Feng Dan’.

Capparis decidua (Forssk.) Edgew. is another well-known ornamental shrub, originating from arid and semi-arid regions. Ram et al. (https://doi.org/10.1007/s11240-022-02234-3) gave an example of technology transfer from lab to land for commercial propagation of this species using mature nodal explants, and they studied the synergism of thidiazuron (TDZ) with auxins and cytokinins, coupled with ex vitro rooting and acclimatization. The differentiation of axillary shoots was best on MS medium with TDZ plus NAA, their initial multiplication was optimal on MS medium with TDZ, BA and NAA, while shoot subculturing was optimal on MS medium with TDZ, BA, KIN and an ammonium sulphate supplement. These microshoots were rooted in vitro (on ¼ strength MS medium with IBA and activated charcoal) or ex vitro (with a pulse treatment of IBA and β-naphthoxyacetic acid) where the percentage of plant survival was significantly higher. Most micropropagated plants of this important ornamental and horticultural shrub survived in the field and fruits could be harvested twice a year.

Bamboos are an important worldwide non-timber forest product with rising interest due to their environmentally friendly applications. The giant bamboo Dendrocalamus asper is a large-sized ornamental bamboo with sweet edible shoots and culms that are useful for structural purposes. Sanches Ornellas et al. (https://doi.org/10.1007/s11240-022-02311-7) reported a method for the induction of somatic embryogenesis from callus obtained from young spikelets collected pre-anthesis and cultured on MS medium with 2,4-D combined with 2iP or KIN. They also examined whether the effect of explant cutting in half and its orientation on the medium improved callus induction. Thus, 2,4-D was essential for callus induction, and its combination with 2iP or KIN favoured the formation of somatic embryos that matured on a medium with a reduced 2,4-D level and germinated on growth regulator-free medium.

Despite the worldwide commercial importance of Rosa species, its regeneration via somatic embryogenesis remains problematic. Cai et al. (https://doi.org/10.1007/s11240-022-02299-0) tried to improve the induction rate of somatic embryos in callus derived from the leaflets and petioles of Rosa chinensis cv. ‘Old Blush’. They found that while leaves yielded embryogenic calluses, petioles did not. Thus, white and reddish-brown translucent calluses were obtained on media with 2,4-D and embryos proliferated on a medium with KIN. Normally constituted somatic embryos with two cotyledons exhibited the fastest germination and highest survival rates.

Nasri et al. (https://doi.org/10.1007/s11240-021-02163-7) generated more than 2000 mutants with new inflorescence colours and shapes in four chrysanthemum cultivars, by treating leaf discs with various concentrations of ethyl methanesulfonate (EMS) and characterized them phenotypically and with molecular (inter simple sequence repeat, ISSR and inter-retrotransposon-amplified polymorphism, IRAP) markers. Of these, 58 mutants including 28 leaf and 32 flower mutants were retained for analyses of phenotype and molecular variation. Phenotypic differences observed included leaf and inflorescence variability in terms not only of size, shape and colour but also of plant height and days to flowering. ISSR and IRAP primers enabled a classification of the mutants based on cultivar and to a certain extent based on the EMS concentrations used, confirming their usefulness for discrimination of real variants. This allowed for earlier selection and a reduction of the mutant population size.

When new genotypes of outstanding ornamental value are created, there is a need to quickly multiply them, and Nowakowska et al. (https://doi.org/10.1007/s11240-021-02206-z) evaluated the effect of meta-Topolin (mT), zeatin (ZEA), 6-benzyladenine (BA), thidiazuron (TDZ), 2-isopentenyladenine (2iP), or the combination of 2iP + ZEA on proliferation of shoots of Rhododendron ‘Kazimierz Odnowiciel’, a relatively new cultivar with large, beautiful flowers and high frost tolerance. The concentrations of chlorophyll, carotenoids and soluble sugars as well as catalase activity were highest on the medium containing ZEA + 2iP but was also coupled with genetic changes as assessed using ISSR markers. A polymorphism below 4% relative to the mother plant was recorded for microshoots established on ZEA.

In an appealing study, Manokari et al. (https://doi.org/10.1007/s11240-022-02315-3) examined for the first time the histochemical development of cell wall composition in velamentous roots of in vitro propagated plantlets of the grey orchid, Vanda tessellata. These roots are crucial for the epiphytic adaptation of plants, as they resist desiccation, contribute to plant anchorage and permit efficient absorption of water and nutrients. The authors analysed kinetically lignin, cellulose, pectin and suberin during various micropropagation stages, revealing the interaction between developmental stages and cell wall composition of velamentous tissues, exodermis, endodermis and vascular bundles. Thus, velamen layers and xylem vessels were rich in lignin, cellulose and polysaccharides, while the exodermis and endodermis were rich in pectin and suberin. Ex vitro, the highest amount of lignin, cellulose and pectin accumulated in the roots, while suberin amount was highest in velamen tissues, xylem vessels, exodermis and endodermis. The results suggest a major involvement of the deposition of cell wall materials in roots as the key to adaptation during hardening ex vitro.

One of the main issues of concern in the regeneration of plants in vitro is the cytogenetic stability of the regenerants and, while in ornamentals, the production of ploidy mosaics may be of commercial interest, there is still the need to understand the mechanisms behind it. Thus, Fritsche et al. (https://doi.org/10.1007/s11240-022-02238-z) examined the occurrence of endopolyploidy in orchid protocorm-like bodies (PLBs), a common morphogenetic pathway for orchid micropropagation. In order to assess the effect of endopolyploidy in explants on the cytogenetic stability of regenerated plantlets, the authors used Epidendrum fulgens as the model and flow cytometry as the method to analyse endopolyploidy in pollinia, petals, labella, leaf bases, leaf tips, root tips and protocorm bases and apices, before their subsequent use as explants for PLB induction and plant regeneration. Even with contrasting ploidy patterns in tissue samples, endopolyploidy was detected in all explants but was highest in protocorm bases and root tips, and lowest in petals and protocorm apices. Pollinia failed to produce PLB or callus, while plantlet regeneration was easier from PLBs induced from protocorm bases than from leaves and roots. Doubled ploidy levels recorded in PLB-regenerated plantlets from protocorm bases and leaf bases were not directly associated with the proportion of endopolyploid cells or cycle value of those explants.

Kulus et al. (https://doi.org/10.1007/s11240-022-02236-1) studied the effect of gold nanoparticles (AuNPs), microwaves and X-rays as mutagens on the in vitro development, physiological activity, acclimatization efficiency and genetic integrity of bleeding heart (Lamprocapnos spectabilis) ‘Valentine’. This herbaceous species is valued in landscape architecture, gardens and floristry for its decorative leaves and long-viable heart-shaped red, pink and white flowers, produced in spring. The profiles of primary and secondary metabolites and the enzymatic activity in the produced plants were studied, and the usefulness of various genetic markers in mutant detection was compared. Also, the genome size of L. spectabilis was estimated for the first time. The addition of AuNPs medium had a positive impact on the in vitro development and multiplication of plants, while plants subjected to the longest microwave irradiation (3 × 9 s) and the non-treated control had the lowest acclimatization efficiency. Mutagen effects depended on the agent type and dose, but all of them significantly affected the activity and profile of most enzymes and phytochemicals studied. Mutations could be detected by DAMD (Directed Amplification of Minisatellite DNA), RAPD and SCoT (Start Codon Targeted Polymorphism) markers, but not by ISSR. Phenotype variation in leaf shape was found in four plants.

Buddleja lindleyana Fort. is a garden ornamental also commonly used as a medicinal plant. To broaden its germplasm resources, Yan et al. (https://doi.org/10.1007/s11240-022-02245-0) treated seeds of B. lindleyana with colchicine and found that it had a dose–response inhibitory effect on seed germination and seedling height. Colchicine treatment yielded 30 mutant plants (22 diploids, 3 tetraploids and 5 chimaeras, as identified by flow cytometry) with morphological variations (leaf malformation, leaf colour macular, early leaf bud germination, uneven leaf surface and leaf hyperplasia, and even yellow-leaf). Among them, tetraploids were mainly from colchicine at the highest concentration (3.0%) and a seed soaking time of 48 h. The length and width of guard cells and stomata significantly differed between diploids and tetraploids, as also in terms of leaf width and leaf shape index differences were detected, while there were no significant leaf length differences among diploid, tetraploid and chimera.

Nakano and Hoshino (https://doi.org/10.1007/s11240-022-02267-8) developed a colchicine-mediated polyploidisation protocol for Haemanthus albiflos Jacq. (Amaryllidaceae) that permitted the simultaneous recovery of tetraploid and octoploid plants from treated diploid calluses derived from immature embryos. Colchicine at 0.2% (w/v) was tested for 24, 48 and 72 h with best responses for the 48 h treatment. The ploidy level of plants was assessed by flow cytometry and chromosome counting. This is the first report on the production of octoploid plants in the Amaryllidaceae.

Chen et al. (https://doi.org/10.1007/s11240-022-02283-8) developed a microspore embryogenesis and chromosome doubling strategy in ornamental kale (Brassica oleracea var. acephala) where they examined for the first time in kale the use of L-ascorbic acid sodium salt (VcNa) to enhance the embryogenesis and regeneration responses from isolated microspores. Overall, VcNa at pH 5.8 increased embryogenesis, the optimum concentration being of 4 μM VcNa. For chromosome doubling, the roots of haploid plants were dipped in colchicine solution or colchicine was added to solid MS medium. Optimum chromosome doubling of haploids (~ 50%) was achieved by dipping their roots in 750 mg/L colchicine solution for 4–6 h and 1000 mg/L colchicine solution for 2 h. Twenty independent doubled haploid lines were analysed and found to exhibit excellent ornamental traits, in terms of colour of inner (dark pink, pink, light pink, light pink, white) and outer leaves (dark or light green), plant type (high, medium and dwarf), plant habit (compact plants of better ornamental and application value for cut flowers, or loose plants) and period of colour change.

In a study with Cerastigma willmottianum Stapf (Plumbaginaceae), a highly decorative native Chinese plant used in green areas and landscapes, Shi et al. (https://doi.org/10.1007/s11240-022-02277-6) improved the ornamental traits through the polyploidisation of wild plants by treating the apical buds in vitro with colchicine, trifluralin or pendimethalin. Of these, trifluralin appeared best both in terms of plant survival and of the polyploidization rate, with the latter assessed after 5–8 successive rounds of selection (purification generations) by morphological observations, stomatal measurements, analysis of their DNA content and chromosome counting. Compared to diploids, tetraploids were more compact and had larger flowers and heart-shaped leaves, coupled with a greater net photosynthetic rate and higher CO₂ assimilation efficiency, thereby adding to the improved ornamental value a high carbon sequestration ability.

Su et al. (https://doi.org/10.1007/s11240-022-02262-z) devised an efficient cryopreservation procedure for Chrysanthemum cv. Chuju. This consisted of the culture of adventitious shoot clusters on MS medium with 100 µM melatonin (at 4 °C for 4 days), followed by preculture of excised shoot tips on MS medium with 0.4 M sucrose (4 days, at 4 °C in dark), prior to their loading in 60% plant vitrification solution 2 (PVS2) for 20 min at 0 °C and then by immersion in 100% PVS2 solution for 60 min. After a 1 h incubation in liquid nitrogen (LN), they were rapidly rewarmed (2 min at 39 °C), and liquid MS medium with 1.2 M sucrose was used as the unloading solution. Following post-culture in recovery medium for shoot regeneration, two thirds of the cryopreserved shoot tips survived and regenerated. Interestingly, biochemical and histological analyses showed that exogenous melatonin improved the survival rate by reducing oxidative stress and maintaining the morphological structure of shoot tip cells. ISSR analyses did not reveal any polymorphic bands in regenerants recovered from the cryopreserved shoot tips.

Aimed at improving survival post-cryopreservation, Liu et al. (https://doi.org/10.1007/s11240-022-02261-0) studied the protective function of ApY₂SK₂ dehydrin against oxidative stress in embryogenic callus of Agapanthus praecox. Dehydrins (DHNs) are members of the late embryogenesis-abundant (Lea) protein family with a central role in seed protection against dehydration and adaptation of plants to abiotic stresses including oxidative stress, critical in cryopreservation. The authors genetically transformed ApY₂SK₂ type DHN into embryogenic callus (EC) of Agapanthus praecox by overexpression (OE) and RNA interference (RNAi) techniques to evaluate the in vivo oxidative stress protective effect of DHNs during cryopreservation. Then, cell viability had opposite trends between OE and RNAi cell lines, as the cell relative death ratio of ApY₂SK₂-OE EC was significantly decreased, and in ApY₂SK₂-RNAi cells, it was significantly increased after cryopreservation. In parallel, during cryopreservation, overexpression of ApY₂SK₂ increased non-enzymatic antioxidant contents, antioxidant enzyme activities and up-regulated CAT, POD and GPX expression. Thus, ApY₂SK₂ can affect ROS metabolism, alleviate excessive H₂O₂ and OH· generation, activate the antioxidant system, improve cellular REDOX balance and reduce membrane lipid peroxidation damage of plant cells during cryopreservation.

Polat et al. (https://doi.org/10.1007/s11240-022-02305-5) studied the effects of chitosan oligomers (CHI) with acetylation degrees (DA) of 10% and 20% on in vitro seed germination and organ development in the weed Ageratum houstonianum Mill., and examined photosynthetic pigment production, lignin content and lipid peroxidation analyses. Chitosan decreased seed germination and delayed seedling growth, but CHI-20DA was best for shoot production and CHI-10DA improved shoot elongation. At a high concentration, CHI-20DA favoured photosynthetic pigment production and induced higher lipid peroxidation than CHI-10DA, but the lowest CHI-20DA level gave the highest lignin content. While leaf traits were not significantly affected by chitosan, the highest CHI-20DA treatment significantly enhanced rhizogenesis although with a dramatic reduction of root length. Hence, high concentrations of CHI-10DA or low concentrations of CHI-20DA might be used instead of synthetic growth regulators in A. houstonianum production provided attention is paid to avoid nutritional deficiency resulting from an altered shoot and root growth, due to an excessive use of high-acetylated chitosan.

Lotz et al. (https://doi.org/10.1007/s11240-021-02223-y) developed a method for genetic transformation of California poppy (Eschscholzia californica) in the Ranunculales that is an early branching lineage of all dicots and hence highly informative phylogenetically, which is widely used as a model to study the conservation of flower developmental control genes. The authors reported a strategy for the efficient and stable Agrobacterium tumefaciens-mediated genetic transformation, somatic embryo induction and regeneration, as well as a rapid method for protoplast isolation and transformation of E. californica. Thus, gene functions can be studied in both the single-cell and full plant contexts towards a modification of alkaloid biosynthetic pathways.

The manuscripts included in this special issue reflect well the proportion of publications in recent years on the several existing biotechnological tools for ornamental species and crops. While micropropagation has been most often developed and optimized, it is closely followed by polyploidization, which holds great promise for the generation of plants with improved ornamental traits such as modification in the plant growth habit, denser foliage or larger flowers with thicker petals. Also of interest are the opportunities offered by several techniques and tools for a better sustainability in plant production for ornamentals but also more generally, such as the use of energy-saving LED light sources for culture chamber and greenhouse cultivation, as well as the possibility to decrease the quantity of fertilizers and pesticides required for production by exploiting transformed, modified, edited and/or mutagenized novel genotypes. The ornamental sector is characterized by the rapid turnover of varieties, with novel phenotypes being required much more often than in other groups of crops. Main targets are a wider array of traits that must be quickly marketable and, ideally, provide a rapid return on investment for breeders. Such genetic novelties frequently show innovative flower colours, a modified plant growth and architecture, a different flowering season, new flower shapes and sizes with improved fragrance and better vase life. For some species, various traits affecting seed production (e.g. male sterility, absence of self-incompatibility), tolerance to abiotic stress, pests and diseases are also of paramount importance. It can be anticipated that genetically manipulated ornamental genotypes, especially genome-edited plants, will soon gain an increasing market share, by providing novel specifications that will better adapt to a changing market and also new genotypes with increased capacity to produce various secondary metabolites of pharmaceutical or cosmetic interest.