Plant Biotechnology: Applications in In Vitro Plant Conservation and Micropropagation

1. Introduction

Micropropagation and conservation using in vitro methods are one of the most important technologies used to maintain the gene pool and genetic diversity. Nature contains a large variety of wild relatives and progenitors of today’s cultivated plant species; these are rich plant genetic resources for vital agricultural uses, but also for rare and endangered plants, which often have medicinal, decorative, forage, and other properties [1]. In addition, rare plants are important components of vegetation in a particular region. Their disappearance can lead to the destruction of key parts of the biological flora of plant communities [2]. Tremendous growth in the global population and human-mediated exploitation of natural resources has exerted significant pressure on diversity, and has led to the genetic erosion of important germplasms from their natural habitats [3].

Continuous efforts have been made in advancing micropropagation methods; for example, the inclusion of silicon nanomaterials under in vitro conditions has improved plant growth and reduced the regeneration of undesirable structural disorders in Cyperus rotundus L. [4]. Furthermore, a recent special topic collection has reported advances in in vitro method-based conservation of plant genetic resources and has recommended the importance of preserving genetic diversity to meet the challenges of crop performance for food for future generations [5]. Important crops like rice have been investigated, and a potential genetic resource, Oryza officinalis Wall, was improved using in vitro methods for reproductive pollen development to overcome its poor crossability and possibly introduce important traits into cultivated rice [6]. Advances in in vitro techniques may help overcome several challenges and contribute significantly to preserving and maintaining precious natural genetic resources for future crop resilience and food security [7].

The present Special Issue, “Plant Biotechnology: Applications in In Vitro Plant Conservation and Micropropagation”, aimed to present the most recent and innovative studies, tools, approaches, and techniques that have been successful in the in vitro conservation of rare plant species. Specifically, it includes protocols for callusogenesis in rare medicinal plants, the use of biotechnological methods to obtain secondary metabolites from rare and endangered plant species, rare plant seed germination, and population genetics research for the conservation of rare plant species. This Special Issue successfully presented eleven recent contributions to a broad audience, focusing on the role of in vitro plant conservation and micropropagation to advance the preservation of plant genetic resources.

2. Contributions and Overview of Published Studies

The present Special Issue featured eleven contributions, by authors from Chile, Russia, Saudi Arabia, Korea, India, Italy, France, Spain, Australia, Hungary, China and Portugal. The first contribution discusses in vitro propagation of Peumus boldus Molina, an endemic woody species of Chile’s sclerophyllous forests, using a temporary immersion system. The high antioxidant content in leaves and bark, low level of germination in natural habitats, and anthropogenic activities have contributed to the decline of its population in natural habitats. Boldo shoots were used to optimize in vitro propagation, and resulted in a high proliferation rate through temporary immersion, followed by ex situ rooting, with 77–83% of plantlets subsequently acclimatized. This method is potentially effective for the large-scale multiplication of this endemic and threatened species, aiding in the restoration of an important species of Chilean sclerophyllous forests.

The second contribution focuses on another winter-hardy species of the genus Actinidia Lindl., Actinidia kolomikta (Maxim) Maxim, whose fruits are a rich source of bioactive compounds. This study involved different iron chelates (FeEDTA and FeEDDHA) and cytokinins (6-benzylaminopurine, meta-Topolin, and 2-izopentyladenine) in the Quoirin and Lepoivre medium for in vitro culture methods, investigating the different morphological parameters. Observations revealed that the morphogenesis of A. kolomikta depends on the applied growth regulators, which also influence the adaptation process of plants. The developed protocol is suitable for regenerating multiple shoots for the increased micropropagation of promising A. kolomikta cultivars.

The third contribution reports the improved acclimatization of micropropagated Al-Taif Rose (Rosa damascena f. trigintipetala (Dieck) R. Keller) plantlets by using the Arbuscular Mycorrhizal Fungi Rhizophagus fasciculatus as an inoculant to study their growth, vigor, and physiological performance ex situ. A higher net CO₂ assimilation, stomatal conductance, transpiration rate, and biomass production were recorded, while proline content was lower upon inoculation in comparison to the control. The study established a link between in vitro micropropagated rose plants and the ex situ inoculation of Arbuscular mycorrhiza for the improved performance of this important ornamental and medicinal plant species.

The fourth contribution explored somatic embryogenesis to produce the Iris pallida Lam. plants, which have rhizomes rich in ketone compounds and a violet-smelling orris essence. These rhizomes are in high demand in the Italian perfume industry and so this plant has economic impacts for native producers. A combination of slow-growth storage (SGS) and the encapsulation of somatic embryos was performed to scale up synthetic seed production, which showed potential for germplasm conservation and also in the micropropagation cycle of orris.

The fifth contribution tested Coronilla viminalis Salisb., an endangered fodder legume from the Canary Islands and Northwestern Africa, which is adapted to drought, and for which conservation is essential. Intense loss of viability and low germination levels were addressed by using plant hormone-rich medium under aseptic conditions, as well as by using nodal segments in different medium combinations. The results showed superior germination, improved root development, and high levels of acclimatization of plantlets to facilitate the conservation of this endangered species.

The sixth contribution reported a clonal propagation method of a tropane-rich Duboisia species (Duboisia myoporoides, Duboisia leichhradtii and Duboisia hopwoodii) native to Australia and which has a high commercial importance. The method showed better performance with genotype dependency among different species for different supplemented media, particularly in different patterns of meristem induction, multiplication, and rooting percentage. The developed method potentially addresses large-scale clonal propagation for industrial supply and may also be useful for further research such as in vitro breeding and genetic modification experiments.

The seventh contribution studied the ultrastructural signs of senescence of compact Glycine max callus cells using electron microscopy to investigate their photosynthesizing potential, which is poorly known. The results demonstrated that green Glycine max callus tissue is composed of cells containing granal chloroplasts, which are structurally distinct from the mesophyll chloroplasts of leaves. The senescent features of these cells opened up discussion on whether the appearance of senescence in compact callus tissues is unavoidable or whether it may be repaired by optimizing cultivation conditions, thus improving the callus tissue’s quality and health. The findings of the study may be useful in the development of plant tissue culture methods that require the consideration of the contribution of photosynthesis to the CO₂ balance in closed culture vessels.

The eighth contribution developed an in vitro induction of callus and plant regeneration from nodal explants of Centratherum punctatum Cass., which has high medicinal and industrial importance for pharmaceutical purposes. Further, the phytochemicals from the callus, leaf, and roots from regenerated shoots were extracted in methanol and analyzed by gas chromatography. Quantification by GC-MS analysis showed the rich sources of diverse biocompounds in regenerated callus, leaves, and roots, and highlighted that the in vitro-regenerated plant or plant parts can provide a continuous source of raw materials for the utilization of secondary metabolites by mitigating overexploitation risks. Such chemical profiling is pivotal for extending future research into therapeutic uses.

Contribution nine reported an effective method for axillary shoot proliferation in Al-Taif rose using stem nodes, an important cultivar for the rose oil industry. The findings of the present study have demonstrated that the application of GA₃ promotes the emergence of buds; BAP exhibited superior efficiency for shoot proliferation, while dark incubation favored shoot proliferation as compared to light incubation. The developed method may improve micropropagation and the in vitro production of the Al-Taif rose to ensure uninterrupted supply for high commercial demand.

The tenth contribution established an efficient micropropagation method for the blueberry cultivar ‘ZY09’, which has a high growing demand for large-scale plantation. The application of (NH₄)₂SO₄ instead of NH₄NO₃ in woody plant medium facilitated the proliferation of microshoots. The optimal combination of plant growth regulators for the in vitro proliferation of blueberry microshoots was indole-3-butyric acid (0.1 mg·L⁻¹), thidiazuron (0.0005 mg·L⁻¹), and zeatin (1 mg·L⁻¹). Perlite was the most suitable substrate for ex vitro rooting. The observations may serve as a reference for optimizing other culture media to provide valuable technical support and theoretical guidance for large-scale and rapid production of micropropagated plantlets of the southern highbush blueberry cultivar.

The last and eleventh contribution reviewed and presented an overview of recent advances in blueberry (Vaccinium spp.) in vitro culture. The fruits are rich sources of anthocyanins and antioxidant compounds, which make it very demanding for dietary supplements and health ingredients. This review presented an updated overview of the most promising methods and techniques for the micropropagation of Vaccinium, thereby contributing to the ongoing development of the blueberry production industry and derivative products.

3. Conclusions and Future Perspectives

The present Special Issue focused on applications of in vitro plant conservation and micropropagation as a significant tool to address multiple issues in the micropropagation of important plant species. Several woody tree species were also discussed, where the methods can be utilized for the conservation of these important species in their natural habitats. Other reports highlighted the improvements in medicinal and industrially important plant species by inducing the callus, shoots, roots, and complete plants. Regenerated plants and parts thereof can be an important source when extracting biocompounds for medicinal and pharmaceutical uses. Overall, the Special Issue presented significant efforts to address multiple issues related to important plant species.