Jean Carlos Cardoso

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.

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.

In vitro flowering of Dendrobium

Dendrobium plants are the most frequently used orchids to study the process of in vitro flowering. Among the various environmental cues and plant growth regulators employed thus far, cytokinins have shown the most prominent effects on the transition from vegetative to reproductive stage. A clear correlation between flowering establishment and enhancement of endogenous cytokinins has been observed. However, the experimental utilization of a large amount of different Dendrobium species and hybrids, combined with a large diversity of variable media types and media compositions make interpretation of flowering control in the in vitro environment difficult. The combination of a specific model Dendrobium in vitro regeneration system or protocol with a simple, but well defined culture medium, could contributed, to some degree, to reaching a clearer consensus about the physiological and molecular mechanisms controlling the Dendrobium flowering phase change.

Levantamento de espécies da família Orchidaceae em Águas de Sta. Bárbara (SP) e seu cultivo

The biodiversity of the Orchidaceae family species presenting potential ornamental, medical and nutritional values was studied. Aguas de Santa Barbara (Brazil) was chosen due to the presence of gallery forests along the rivers Pardo and Novo, due to the occurrence of areas covered with Savannah vegetation and also due to the risks of environmental degradation by the presence of intense tourist and industrial activities in the area. Mapping and evaluation of the species with potential for ornamental purposes were done with the objective of developing techniques for their cultivation. Farms and areas of preservation with gallery forest, as well as some areas covered with Savannah vegetation located in Rio Novo and Rio Pardo counties were visited and data on the average temperature and relative air humidity were collected. Predominant species found in the area belong to the genus Brassavola, Catassetum, Cattleya, Eulophidium, Epidendrum, Ionopsis, Microlaelia, Oncidium, Pleurothallis, Polystachya and Rodriguezia, indicating the large biodiversity present in the area. Most of these species are micro orchids with no ornamental value. However due to the presence of great genetic variability they could be used for breeding purposes.

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.

Adventitious shoot induction from leaf segments in Anthurium andreanum is affected by age of explant, leaf orientation and plant growth regulator

Anthurium is one of the main potted plants on the flower market, and micropropagation is the most used technique for clonal and large scale propagation of this species. Calli, adventitious shoots and regenerated plantlets were obtained from leaf segments of Anthurium andraeanum. The influence of genotype, explant orientation, plant growth regulator, and age of donor plants were tested. We obtained regenerated plants in three (‘Red One’, ‘Red Dark’, and ‘Snow White’) out of four different cultivars. The ‘White Beauty’, however, was considered to be recalcitrant under these conditions. Therefore, the second study was conducted only with this recalcitrant cultivar, and the use of juvenile rather than adult leaf explants enhanced plantlet regeneration at the frequency of 0.3-2.8 plantlets per leaf segment. The use of 1.0 mg·L−1 6-benzylaminopurine (BAP) and no addition of 2,4-D in the culture medium resulted in direct plantlet regeneration of Anthurium. Plantlets obtained from ‘Red One’ and ‘White Beauty’ were successfully micropropagated and acclimatized. In ex vitro phases, leaf-derived plants of these two genotypes maintained their most important horticultural and ornamental characteristics.

Gibberellic acid and water regime in the flowering induction of Brassocattleya and Cattleya hybrid orchids

The influence of gibberellic acid (GA 3 ) and water regime was evaluated in the flowering induction and quality of two orchid hybrids belonging to the genera Cattleya (C.) and Brassocattleya (Bc.). The experiment was carried out in the Biotechnology and Orchid Culture Sector of Shunji Nishimura Technology Foundation, Pompeia, Sao Paulo State, Brazil. Five GA 3 concentrations (0, 125, 250, 500 and 1,000 mg L -1 ) were tested through four consecutive leaf applications in adult plants that had already flowered at least once, besides two water conditions (one and four irrigations per week). Applications were performed in October and November for Bc. Marcella Koss and in January and February for C. Irene Holguin. Flowering could not be induced in the latter by gibberellic acid. In Bc. Marcella Koss, the application of 250 mg L -1 GA 3 combined with decreased irrigation frequency induced flowering in around 83% plants. By using the same GA 3 concentration but frequent irrigation, only 17% plants were induced to flower. The number and size of flowers increased after application of higher GA 3 concentrations. This work allowed developing a commercial technique with the use of gibberellic acid (GA 3 ) to induce flowering in Bc. Marcella Koss hybrid orchid.

Tissue disinfection for preparation of Dendrobium in vitro culture

Abstract Establishing an aseptic in vitro culture for Dendrobium, or for any plant in fact, is the most important step towards developing an effective in vitro tissue culture including micropropagation protocol. Success in initial aseptic culture will contribute to the successful production of in vitro cultures that may involve the initiation or formation of callus and/or protocorm-like bodies (PLBs), the induction, regeneration or multiplication of shoots, and the preparation and proliferation of plantlets suitable for acclimatization. The initiation of an aseptic culture is closely related to the appropriate selection of an explant source and its preparation, including its (in vivo) pre-treatment if necessary and subsequent disinfection procedures. Care in the choice of explant and the application of an appropriate disinfection protocol can successfully reduce, or eliminate, contamination in in vitro cultures while reducing the negative impact on plant tissues and plantlet regeneration. Many unique aseptic culture procedures for Dendrobium genus have been reported in the literature, very often specific to particular tissues or genotypes, and this review not only highlights the details of such protocols, but also provides practical advice for novice – and even seasoned – orchidologists who wish to research Dendrobium in vitro, although it is cautioned that there is currently no universal aseptic culture procedure that can be applied to all conditions, all explants or all genotypes.

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.