Choice Of Explants Research Articles

Improving transformation and regeneration efficiency in medicinal plants: Insights from other recalcitrant species.

Medicinal plants are integral to traditional medicine systems world-wide, being pivotal for human health. Harvesting plant material from natural environments, however, has led to species scarcity, prompting action to develop cultivation solutions that also aid conservation efforts. Biotechnological tools, specifically plant tissue culture and genetic transformation, offer solutions for sustainable, large-scale production and enhanced yield of valuable biomolecules. While these techniques are instrumental to the development of the medicinal plant industry, the challenge of inherent regeneration recalcitrance in some species to in vitro cultivation hampers these efforts. This review examines the strategies for overcoming recalcitrance in medicinal plants using a holistic approach, emphasising the meticulous choice of explants, e.g. embryonic/meristematic tissues; plant growth regulators, e.g. synthetic cytokinins; and use of novel regeneration-enabling methods to deliver morphogenic genes e.g. GRF/GIF chimeras and nanoparticles, which have been shown to contribute to overcoming recalcitrance barriers in agriculture crops. Furthermore, it highlights the benefit of cost-effective genomic technologies that enable precise genome editing and the value of integrating data-driven models to address genotype-specific challenges in medicinal plant research. These advances mark a progressive step towards a future where medicinal plant cultivation is not only more efficient and predictable but also inherently sustainable, ensuring the continued availability and exploitation of these important plants for current and future generations.

Choice of explant, its size and method of its placement in a test tube to increase the productivity of clonal micropropagation of Fioletoviy ranniy variety

The paper is devoted to the optimization of one of the factors influencing the success of micropropagation – the choice of the optimal part of vine to take explant, size of the explant and the method of its exposure in a test tube. The studies have confirmed that the place, where micro-cutting was taken, its size and its orientation during planting can affect the speed of growth processes and the survival rate of plants. The experiment was put on the Purple Early grapevine variety. The part of vine for taking micro-cutting, best size of explant and the method of its orientation in the test tube are established.

Cannabis sativa: From Therapeutic Uses to Micropropagation and Beyond.

The development of a protocol for the large-scale production of Cannabis and its variants with little to no somaclonal variation or disease for pharmaceutical and for other industrial use has been an emerging area of research. A limited number of protocols have been developed around the world, obtained through a detailed literature search using web-based database searches, e.g., Scopus, Web of Science (WoS) and Google Scholar. This article reviews the advances made in relation to Cannabis tissue culture and micropropagation, such as explant choice and decontamination of explants, direct and indirect organogenesis, rooting, acclimatisation and a few aspects of genetic engineering. Since Cannabis micropropagation systems are fairly new fields, combinations of plant growth regulator experiments are needed to gain insight into the development of direct and indirect organogenesis protocols that are able to undergo the acclimation stage and maintain healthy plants desirable to the Cannabis industry. A post-culture analysis of Cannabis phytochemistry after the acclimatisation stage is lacking in a majority of the reviewed studies, and for in vitro propagation protocols to be accepted by the pharmaceutical industries, phytochemical and possibly pharmacological research need to be undertaken in order to ascertain the integrity of the generated plant material. It is rather difficult to obtain industrially acceptable micropropagation regimes as recalcitrance to the regeneration of in vitro cultured plants remains a major concern and this impedes progress in the application of genetic modification technologies and gene editing tools to be used routinely for the improvement of Cannabis genotypes that are used in various industries globally. In the future, with more reliable plant tissue culture-based propagation that generates true-to-type plants that have known genetic and metabolomic integrity, the use of genetic engineering systems including “omics” technologies such as next-generation sequencing and fast-evolving gene editing tools could be implemented to speed up the identification of novel genes and mechanisms involved in the biosynthesis of Cannabis phytochemicals for large-scale production.

CRISPR/Cas9 gene editing in legume crops: Opportunities and challenges

AbstractLegumes are an excellent source of proteins and health‐promoting phytochemicals. Recognizing their importance in human nutrition and sustainable agricultural production, significant efforts are currently being made to accelerate genetic gain related to yield, stress resilience, and nutritional quality. Recent increases in genomic resources for multiple legume crops have laid a solid foundation for application of transformative breeding technologies such as genomic selection and genome editing for crop improvement. In this review, we focus on the recent plant‐specific advances in CRISPR/Cas9‐based gene editing technology and discuss the challenges and opportunities to harnessing this innovative technology for targeted improvement of traits in legume crops. Gene‐editing methods have been successfully established for soybean, cowpea, chickpea, and model legumes such as Medicago truncatula and Lotus japonicus. However, the recalcitrance of other legumes to in vitro gene transfer and regeneration has posed a serious challenge to application of gene editing. We discuss various modifications to in vitro culture methods, in terms of the choice of explant, media composition, and DNA delivery and gene‐editing detection methods that can potentially improve the rate of transformation and regeneration of whole plant in legume crops. Although gene‐editing technology can bring enormous benefits to legume breeding, regulatory hurdles are a cause for serious concern. We compare the regulatory environments existing in the European Union and the United States of America. A favorable regulatory framework and public acceptance are important factors in realizing CRISPR's potential benefits to global food security.

Obtaining micro cuttings of potatoes by clonal micropropagation

At present, high-quality healthy planting material of potato tubers cannot be obtained without the use of clonal micropropagation technology, with the help of which, through micro-cuttings, it is possible to recover from viral, viroid, bacterial and fungal infections and mass propagation of economically valuable varieties. The wrong choice of explants and ineffective sterilization are the most common causes of suppression of the growth of microplants, bacterial infections, necrosis of shoot tips, habituation and growth of tissues, lack of rooting at the stage of rhizogenesis and successful acclimatization at the stage of adaptation to non-sterile conditions, etc.

In Vitro Plant Tissue Culture: Means for Production of Passiflora Species

Passiflora genus includes over 600 species native to tropical and subtropical areas of America, appreciated for the production of fruit and medicinal value. Their ornamental potential is especially appreciated in North America and in Europe. With the expansion of the flower trade and the use of secondary metabolites in the pharmaceutical industry, a need for the constant monopolization of new technologies and alternative in vitro techniques that allow to obtain a uniform, high quality material free of pests and diseases occurs. Passiflora’s tissue cultures began to be studied in 1966, raising more and more interest of researchers worldwide. Depending on the source and type of the explant, plant growth regulators, and the used genotype, direct and indirect organogenesis are the main regeneration pathways for Passiflora. The latest approaches regarding the choice of explant and its source, the plant material surface sterilization and the specific requirements of each micropropagation stage are presented within our review. To this genus, the reduced gas exchange of in vitro growing of seedlings has been shown as the main cause of lack of success. In this regard, for regeneration and obtained improvements in morphogenesis, different protocols have been developed by using inhibitors of ethylene. In recent years, studies suggest that via somatic embryogenesis, starting from mature and immature zygotic embryos, regenerated plants that have maintained their mother plant ploidy can be successfully obtained. This confirms the callus cultures as main path to obtain in vitro regenerated Passiflora plants.

The Cryobiotechnology of Oaks: An Integration of Approaches for the Long-Term Ex Situ Conservation of Quercus Species

Conventional dry seed storage is unlikely for about one third of all tree species (and nearly half of evergreen rain forest trees) as they probably produce desiccation sensitive (recalcitrant) seeds. Consequently, international ex situ conservation targets for threatened trees will be difficult to achieve without innovation, especially in cryobiotechnology. We assessed progress in the development of various cryobiotechnology approaches for the preservation of oaks (Quercus), which are keystone species of functioning landscapes, important to the bioeconomy and under increasing threats from the spread of pests and diseases under a changing climate. Various tissues of oaks can be used for banking, from pollen grains to embryo axes. Pollen from five oak species have been shown to be highly desiccation tolerant, making dry pollen storage at low temperatures (including in liquid nitrogen) a valuable technology to support conservation and breeding programs. Somatic embryo (SE) technology and/or shoot tip in vitro technology is available for 39 species, and SE cryopreservation is routinely performed on three commercial species and shoot tips cryopreservation successful in two more species. Seed embryonic axes are the preferred explants for oak ex situ conservation, with tissue survival and regeneration of plants after cryopreservation recorded for 14 and seven species respectively; although differential responses between the shoot and root meristems in the axes are known. Dormant bud preservation seems promising, but is under-researched. Overall, these results indicate the possibility of establishing an integrated platform for the ex situ conservation of oak species based on cryobiotechnology. Challenges of explant choice, optimization of methodologies and large-scale application do remain. However, multiple approaches for the cryopreservation of oak genetic resources are available and implementation programmes should not be delayed, particularly in the centres of species diversity.

Somatic embryogenesis in Larix: the state of art and perspectives.

Clonal propagation of conifers using somatic embryogenesis is essential for the selection of tree species, and for the implementation of afforestation and reforestation. In combination with cryopreservation, somatic embryogenesis creates the basis for the development of economically valuable lines of clones and elite genotypes. The industrial use of such genetically verified clone lines in forestry can significantly increase forest productivity compared to any conventional methods for improving tree crops that are available. Larch is considered as one of the main conifer candidates for large-scale reforestation, not only due to the vastness of its habitat, but also due to the unique quality of its wood, rapid growth and high ecological plasticity. However, the vast majority of larch species are characterized by uneven yields and extremely low seed quality. In this regard, obtaining planting material for reforestation from larch seeds on seed plantations is not advisable, but can be successfully implemented in afforestation programs using somatic embryogenesis technologies. Research on the somatic embryogenesis of larch has been conducted for over 30 years, which allowed considerable experience in this field to be accumulated. To date, the conditions for the initiation and maintenance of embryogenic cultures, as well as for the formation and development of somatic embryos have been determined. Significant progress has been made in the study of both the factors affecting these processes and the molecular mechanisms that underlie the various stages of embryogenesis. Nevertheless, despite the successes achieved, knowledge available today on the somatic embryogenesis of representatives of the genus Larix is still not enough to develop technologies for producing valuable plant-breeding material in vitro. This review analyzes the current state of research on the problem of somatic embryogenesis of representatives of the genus Larix. Particular attention is paid to the choice of explants for somatic embryogenesis, the composition of the media for cultivation, the dependence of the potential of somatic embryogenesis on the duration of cultivation, and the genetic control of somatic embryogenesis.

RESPUESTA MORFOGENÉTICA DE DOS ESPECIES DE AGAVE REGENERADAS IN VITRO

<p><strong>Background</strong>: Agaves in Mexico are plants with great economic, gastronomic and cultural value. Despite the multiple forms of reproduction of plants belonging to the genus <em>Agave</em>, several of these species are threatened due to their slow growth, low percentage of seed viability, destruction of its habitat, excessive looting, interruption of its ripening cycle due to the use of their different organs,however, it has been posible to establish biotechnological techniques for its propagation, conservation and genetic improvement. <strong>Objective</strong>: At the present work were selected a representative species of Agave, one of them for pulque production (<em>A. salmiana</em>) (As) and the other one in the production of mezcal (<em>Agave marmorata</em>) (Am) with the aim to establish an efficient micropropagation protocol optimizing the concentrations of the citokinin benzylaminopurine (BA) and the auxin 2,4-Dichlorophenoxyacetic acid (2,4-D), on the <em>in vitro </em>morphogenesis of both species.<strong> Methodology: </strong>Two <em>in vitro </em>culture systems were conducted in three explants (zygotic embryonic axis (E1), <em>in vitro </em>meristematic zone (E2) and <em>ex vitro</em> meristematic zone (E3)). <strong>Results</strong>: Shoots regeneration was achieved in all tested experiments; the highest number of shoots in As was 23.8 for explant using the E2 of directly regenerated <em>in vitro</em> germinated plants using a concentration of 10 mg L<sup>-1</sup> of BA, while in Am 24.7 shoots/explant were obtained with 5 mg L<sup>-1</sup> of BA using the zygotic embryonic axis who throughing a callus phase. <strong>Implications</strong>: The results obtained help to understand the importance of developing specific micropropagation protocols for each species, as well as contribuiting in the masive multiplications of <em>A. salmiana</em> and <em>A. marmorata</em> by understanding the effect of cytokinin BA and auxin 2,4-D and their combination in the formation of shoots by direct and indirect organogenesis. This provides a conservation alternative and an opportunity to startgenetic improvment programs. <strong>Conclusion</strong>: The results of this research suggest that the choice of explant, multiplication system, species and plant growth regulators are the key to obtain a greater number of shoots with high <em>ex vitro</em> survival rates.</p>

Regeneration of shoots via direct somatic embryogenesis from the leaf surface of Scaevola taccada (Gaertn.) roxb. – a climate resilient species of coastal areas

Scaevola taccada (Gaertn.) Roxb. (family Goodeniaceae) is a large littoral shrub of great economic and ecological importance. Plantlets were regenerated from the in vitro raised leaf explants via direct somatic embryogenesis in this study. Nodes were cultured on Murashige and Skoog's (MS) medium augmented with 1.5 mg L −1 6-benzylaminopurine (BAP) and the in vitro raised leaves used as secondary explants to induce somatic embryogenesis directly from the leaf surface on the MS medium supplemented with two kinds of cytokinins, namely BAP and N6-furfuryladenine (Kinetin). The highest percentage of explants (92%) responded with globular somatic embryos after 4 weeks of incubation on 2.0 mg L −1 BAP. The anatomical observations of somatic embryos (SE) revealed that continuous cell divisions lead to the formation of proembryos and followed by globular somatic embryos. The bipolar structures with the shoot apical meristems formation observed after 4 weeks of cultures. Light microscopic evaluations showed that direct somatic embryogenesis from leaf explants were nonsynchronous. Culturing of four weeks old somatic embryos (SE) on MS medium fortified with 0.25 mg L −1 BAP and 0.15 mg L −1 IAA induced substantial changes in formation of multiple shoots. It was observed that full strength MS medium retarded the germination frequency of SE, and tip browning and vitrified shoots were detected. The reduced concentrations of ammonium nitrate salt (half) in the MS medium increased number of healthy shoots (36 shoots per culture vessel). Similarly, increased quantity of calcium chloride (CaCl2) in the medium (1.5 times) alleviated tip browning of the shoots. The shoots were successfully rooted in vitro on half strength MS medium + 2.0 mg L −1 IBA with 12.6 roots per shoot. The rooted shoots were hardened in the soilrite® in the greenhouse and finally transferred to the field. After 3 months, 100% survival rate was recorded under in vivo conditions. The developed protocol could be used for genetic transformation systems in S. taccada as leaves might be the better choice of explants.

Choice of Explant for Plant Genetic Transformation.

Particle bombardment or biolistic transformation is an efficient, versatile method. This method does not need any vector for the gene transfer and is not dependent on the cell type, species, and genotype. The success of any transformation technique depends on the starting experimental materials or the explants. Here, we describe the factors that have influenced the choice of explants in biolistic transformation. Many general factors in the selection of explants in the development of transgenic plants are presented here. Therefore, this chapter provides extensive guidelines regarding the choice of explants for researchers working on various plant genetic transformation techniques.

Embryogenic Calli Explants and Silicon Carbide Whisker-Mediated Transformation of Cotton (Gossypium hirsutum L.).

Rapid growth in the genetic transformation of plants is the outcome of versatile transformation methods, explant nature, and media regimes. Modern biotechnologists have now a toolkit embraced with different plant transformation methods to generate specific and targeted genetic variation for performance improvement of crop plants. Genetic information are created by proper custom synthesis/amplification of DNA sequences from natural sources, modification during gene cloning, and choice of regulatory sequences and delivered to plants via different plant transformation techniques. Cotton is known by different names like king of fiber crops, white gold, etc., due to its socioeconomic involvement in society livelihood. So cotton is the host of several transgenes delivered for the purpose of trait development of improvement outcompeting its wild counterparts. At present most of the cotton adopted by farmers is biotech and contributes significantly in meeting farmers and industry demands. It is the versatile nature of cotton that it has been subjected to different genetic transformation methods to provide the breeders with an opportunity to develop alien traits or improve the endogenous gene performance that are very difficult or impossible to develop through conventional breeding methods. Landmark achievements were achieved by expanding explant choice such as calli as explants as it reduces the extent of labor, time, and effort and thereby becoming cost-effective cotton transformation. Cotton calli becomes differentiated into embryogenic and non-embryogenic which requires regular screening, keeping in view texture, color, and growth behavior. Here we describe the calli features which are peculiar in nature when used as explants in a novel physical way of cotton transformation with different genes by using embryogenic calli as continuous source of explants. The dawn of genome editing has opened another horizon in transformation research and development enhancing the scope of cotton transformation.

Improvement of regeneration in pepper: a recalcitrant species

Organogenesis is influenced by factors like genotype, type of explant, culture medium components, and incubation conditions. The influence of ethylene, which can be produced in the culture process, can also be a limiting factor in recalcitrant species like pepper. In this work, bud induction was achieved from cotyledons and hypocotyls—from eight pepper cultivars—on Murashige and Skoog (MS) medium supplemented with 22.2 μM 6-benzyladenine (6BA) and 5.71 μM indole-3-acetic acid (IAA), in media with or without silver nitrate (SN) (58.86 μM), a suppressor of ethylene action. In the SN-supplemented medium, the frequencies of explants with buds and with callus formation were lower in both kinds of explant, but higher numbers of developed shoots were isolated from explants cultured on SN. Bud elongation was better in medium with gibberellic acid (GA3) (2.88 μM) than in medium free of growth regulators or supplemented with 1-aminocyclopropane-1-carboxylic acid (ACC) at 34.5 μM. However, isolation of shoots was difficult and few plants were recovered. The effect of adding SN following bud induction (at 7 d) and that of dark incubation (the first 7 d of culture) was also assessed in order to improve the previous results. When SN was added after bud induction, similar percentages of bud induction were found for cotyledons (average frequency 89.37% without SN and 94.37% with SN) whereas they doubled in hypocotyls (50% without SN and 87.7% with SN). In addition, in both kinds of explant, the number of developed plants able to be transferred to soil (developed and rooted) was greatly increased by SN. Dark incubation does not seem to improve organogenesis in pepper, and hypocotyl explants clearly represent a better explant choice—with respect to cotyledonary explants—for the pepper cultivars assayed.

Doubled Haploid Transgenic Wheat Lines by Microspore Transformation.

Microspores are preferred explant choice for genetic transformation, as their use shortens the duration of obtaining homozygous transformants. All established gene-delivery methods of particle bombardment, electroporation, and cocultivation with Agrobacterium tumefaciens were optimized on androgenic microspores or derived tissues. In the biolistic gene delivery method 35-40days old haploid microspore embryoids were used for genetic transformation, whereas freshly isolated androgenic microspores were used for genetic transformation in the electroporation and Agrobacterium cocultivation-based methods. The genetic transformation methods of biolistic gene-delivery and electroporation gave rise to the chimeric plants, whereas the method involving cocultivation with Agrobacterium yielded homozygous transformants. These methods were tested on a large number of cultivars belonging to different market classes of wheat, and found to be fairly independent of the explant genotype. Other benefits of using microspores or derived tissues for transformation are: (1) a few explant donors are required to obtain desired transformants and (2) the time required for obtaining homozygous transformants is about 8months in case of spring wheat genotypes and about a year in case of winter wheat genotypes.

Can explant choice help resolve recalcitrance problems in in vitro propagation, a problem still acute especially for adult conifers?

Key message There are tissues distributed throughout the plant that have a higher morphogenic capacity than others in the plant, and these could perhaps solve recalcitrance problems.

Auxin Transport Inhibitor 2,3,5 Triiodobenzoic Acid Induced Direct Shoot Morphogenesis in Millingtonia hortensis L.f.

Tissue culture studies have shown that in vitro response of explants is largely dependent on media composition, auxincytokinin ratio and the choice of explants. Pierik defines the phenomenon wherein the position of explants in the mother plant influences its subsequent in vitro growth and development as topophysis. The plant hormone auxin is assumed to provide positional information for patterning during development. It is precise determination of auxins distributed across tissues and how it is sensed in space and time are uncertain. Interaction between several auxin response factors (ARFs), transcriptional activators and repressors in a complex network, control auxin-regulated gene expression.

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.

Tissue culture of Muscari species: present achievements and future perspectives

Species of the Muscari genus (Asparagaceae) are highly specialized ornamental plants, but some of their natural populations are under threat. Biotechnology, including in vitro tissue culture, allows for clonal material of important germplasm Muscari to be produced in volume. This review focuses on the achievements that have been attained in the in vitro tissue culture of members of this genus and evaluates the effects that biotic (genotype, explant choice) and abiotic factors (physical and chemical conditions in vitro) have on regeneration. Current applications and future perspectives of tissue-cultured Muscari species as a tool for large-scale clonal propagation as well as germplasm storage and genetic improvement of germplasm via other biotechnological methods are also discussed.

Somatic Embryogenesis and Plant Regeneration from Zygotic Embryo in <i>Carica papaya</i> L., cv. Red-Lady

Somatic embryogenesis creates a number of opportunities to facilitate large-scale propagation, synthetic seed production, genetic improvement through somaclonal variation, in vitro mutagenesis, protoplast fusion and genetic transformation. Induction of somatic embryogenesis from the vegetative parts of papaya plant was met with low success rates and a slow process of regeneration. Success depends on the choice of explants, the species being used and on various methods of embryogenesis will study. The most suitable explant for somatic embryogenesis is a large portion of either meristematic tissue or cell that retain an ability to express totipotency. Various investigations were made on somatic embryo induction of Carica spp. such as ovules of immature fruits, zygotic embryos from immature fruits, axillary buds, peduncles of immature fruits etc. In this study, somatic embryo induction from zygotic embryo of an immature fruit was examined. Various concentrations of 2,4-D and two levels of sucrose concentrations with or without addition of glutamine were assessed to determine the best response for induction of somatic embryogenesis from immature zygotic embryo of immature fruits in papaya cv. Red-lady (Carica papaya L.). The results showed that MS medium supplemented with 5.0 mg/l 2,4-D + 400 mg/l glutamine + 60 g/l sucrose promoted the formation of highest (70%) percentage of somatic embryo and also the average maximum number (35.80±4.40) of somatic embryo per explant. The highest (44%) percentage of somatic embryo germination into complete plantlets were obtained on MS medium devoid of plant growth regulators. The embryo developed only shoots of 22% and only roots of 10% instead of complete plantlet formation in this medium of MS0. The average highest (4.25 ± 0.32 cm) shoot length per germinated somatic embryo derived complete plantlets were also achieved on MS0 medium. The maximum (90%) percentage of root induction and average highest (7.50 ± 0.75) number of root per somatic embryo derived shoot and also the average maximum (5.20 ± 0.40 cm) root length were obtained on MS medium without plant growth regulators. The survival rate of somatic embryo derived plantlets in the field was about 10% to 50% depending on management practices.

Palm (Arecaceae) somatic embryogenesis

Tissue culture comprises several techniques for the mass propagation of selected genotypes. In the case of palms, the most promising and efficient micropropagation technique is somatic embryogenesis. Ever since somatic embryogenesis was first induced in oil palm several decades ago, 19 palm species have been induced to develop somatic embryos from several types of explants. Optimization of somatic embryo multiplication protocols has been the central focus of research, but embryo maturation and conversion remains poorly-studied in many palm species. This review summarizes the advances made in both the study and optimization of palm somatic embryogenesis (SE) protocols. Explant choice and inoculation method, media components, culture method, histology of early somatic embryogenesis, gene expression, biochemical changes, embryo maturation and conversion, and acclimatization of regenerated plantlets are presented and critically discussed.