Explant Type Research Articles

Optimizing in vitro micropropagation strategies in Santalum album L. through different explants

This study focuses on optimization of in vitro propagation protocols for Santalum album L. (Indian sandalwood) to address the increasing global demand and to aid in conservation aspects. This investigation evaluated various micropropagation parameters including explant types, basal media, disinfection methods and plant growth regulators (PGR). Murashige and Skoog medium consistently outperformed other media, with shoot tips showing the highest morphogenetic response (66.66%). Surface disinfection with 4% NaOCl for 10 min was the most effective and resulted in the explant survival rate of 86%. For shoot induction, 2 mg/L kinetin in MS medium resulted in the highest number of shoots (3.81) and longest shoots (5.39 cm) after 60 days. An effect was observed when PGR was combined with 5.0 mg/L kinetin + 2.0 mg/L BAP, corresponding to a shoot induction rate of 65.75%. Root induction was recorded at 14.79% after 10 days with the best treatment (MS + 1 mg/L IBA). Callus culture showed limited success as only one treatment (MS + 1 mg/L BAP + 25% coconut water) showed a regenerative response of 7.14%. These results provide a foundation for micropropagation of Sandal while highlighting areas that require further optimization, particularly in root induction and acclimation phases.

Optimizing In Vitro Propagation of Haworthia truncata Schönland Using Leaf, Root, and Inflorescence.

Haworthia truncata, a species native to South Africa, is characterized by its limited growth and scarcity, contributing to high production costs. Countries like China and Turkey are known for exporting Haworthia globally. Tissue culture offers an efficient method for mass-producing unique and beautiful species such as H. truncata. This study tested Murashige and Skoog (MS) basal media supplemented with various concentrations of IBA (0.05-1.5 mg/L), NAA (0.05-0.25 mg/L), and BA (0.25-1.5 mg/L) to promote shoot proliferation. MS medium without plant growth regulators (PGRs) was also tested as a control. Different explant types (leaf, root, and inflorescence) were analyzed for their potential in direct and indirect regeneration. Inflorescence explants showed the highest callus induction with 1.5 mg/L IBA, while optimal shoot proliferation occurred at 1 mg/L IBA. Callus induction was optimal for leaf explants with 0.05 mg/L NAA and 0.25 mg/L BA, and shoot proliferation was highest at 0.05 mg/L NAA and 1 mg/L BA. Root explants achieved maximum callus induction with 0.25 mg/L BA and 0.25 mg/L NAA, with the best shoot proliferation using 0.05 mg/L NAA and 1 mg/L BA. The highest rooting percentage of regenerated shoots was obtained on ½ MS medium with 1.5 mg/L IBA.

Induction of stigma-like structures in saffron (Crocus sativus L.): Exploring factors and metabolite analysis.

Saffron (Crocus sativus L.) has held significant cultural and medicinal value since the Greek-Minoan civilization. As a triploid spice with vegetative propagation from the Iridaceae family, the three-branch style of C. sativus flowers, known as saffron, constitutes the most economically valuable part of the plant, renowned for its diverse medicinal properties. This study explores the in vitro induction of stigma-like structures (SLSs) from various explants of the Ghaen ecotype flower. The study found that the optimal sampling time for the majority of explants was the third week of October. Ovary explants exhibiting a prolonged response to hormonal treatments for the production of SLSs. Furthermore, intact, and injury ovary explants were found to be the most effective explant types for inducing SLSs. The explants were cultured on MS, 1/2MS, LS and B5 basal media supplemented with various combinations and concentrations of plant growth regulators. The results indicated that the B5 medium, enriched with 5-10 mg/ L BAP and 5-10 mg/ L NAA was the most effective treatment for inducing SLSs in all types of explants. Quantitative and qualitative analyses of saffron compounds in SLSs indicated similarities with natural saffron, albeit at significant lower concentrations: crocin (up to 10.2 mg/g), picrocrocin (up to 4.8 mg/g), and safranal (up to 9.7 mg/g). The highest accumulation of the three studied secondary metabolites was observed in the SLSs of style (24.4 mg/g), stigma (28.3 mg/g), and ovary (21.4 mg/g) explants, respectively. This study introduces a comprehensive procedure for producing SLSs containing the three most important metabolites of saffron for the first time.

Poplar transformation with variable explant sources to maximize transformation efficiency

For decades, Agrobacterium tumefaciens-mediated plant transformation has played an integral role in advancing fundamental and applied plant biology. The recent omnipresent emergence of synthetic biology, which relies on plant transformation to manipulate plant DNA and gene expression for novel product biosynthesis, has further propelled basic as well as applied interests in plant transformation technologies. The strong demand for a faster design-build-test-learn cycle, the essence of synthetic biology, is, however, still ill-matched with the long-standing issues of high tissue culture recalcitrance and low transformation efficiency of a wide range of plant species especially food, fiber and energy crops. To maximize the utility of plant material and improve the transformation productivity per unit plant form, we studied the regeneration and transformation efficiency of different types of explants, including leaf, stem, petiole, and root from Populus, a woody perennial bioenergy crop. Our results show that root explants, in addition to the above-ground tissues, have considerable regeneration capacity and amenability to A. tumefaciens and, the resulting transformants have largely comparable morphology, reporter gene expression, and transcriptome profile, independent of the explant source tissue. Transcriptome analyses mapped to regeneration stages and transformation efficiencies further revealed the expression of the auxin and cytokinin signaling and various developmental pathway genes in leaf and root explants undergoing early organogenesis. We further report high-potential candidate genes that may potentially be associated with higher regeneration and transformation efficiency. Overall, our study shows that explants from above- and belowground organs of a Populus plant are suitable for genetic transformation and tissue culture regeneration, and together with the underlying transcriptome data open new routes to maximize plant explant utilization, stable transformation productivity, and plant transformation efficiency.

SOMATIC EMBRYOGENESIS IN SAINFOIN (Onobrychis viciifolia Scop.)

Sainfoin (Onobrychis viciifolia Scop.) is a perennial legume used for forage production. The aim of this study was to investigate the effects of different basal media (Murashige&Skoog-MS, Schenk& Hildebrant-SH, Gamborg-B5 or CHU-N6), explant types (cotyledon and hypocotyl), and growth regulators (kinetin and thidiazuron) on the induction of somatic embryogenesis in sainfoin. For this purpose, hypocotyl and cotyledon explants isolated from in vitro grown sterile seedlings were cultured in solid MS, SH, B5, or N6 basal media supplemented with 2 mg/L 2,4-dichlorophenoxyacetic acid, 0.2 mg/L kinetin or thidiazuron, and 1 g/L proline. The developing embryonic callus was then transferred to a solid MS medium containing 3% sucrose. As a result of the experiment, callus weights per explant varied between 248.3-534.7 mg, 408.7-609.1, 220.3-537 mg and 127.3-428.3 mg in MS, B5, N6, and SH basal media respectively, depending on explants and growth regulators. Average callus weights were higher in B5 and N6 basal media than in MS and SH media, TDZ-containing media than kinetin containing media and cotyledon explant than hypocotyl explant. The highest frequency of explants developing somatic embryos (mean 33.3%) was obtained from kinetin-containing MS or B5 basal media, while the highest embryo per explant (mean 2.6 somatic embryo per explant) was obtained from kinetin-containing MS medium. The somatic embryo number per hypocotyl explant was higher than that of cotyledon explants.

In vitro Propagation of Banana (Musa spp.) cv. Neypoovan through Male Bud Culture

Neypoovan is an important banana cultivar widely cultivated in South India. The demand for planting material of this cultivar is huge and tissue-cultured plantlets are not available owing to its inherent problems in the initial establishment of the culture and slow rate of multiplication by using shoot tips as explant. The present study investigated on direct regeneration and mass multiplication of the banana cultivar Neypoovan (AB) through In vitro male bud culture. The experiment which was conducted at the College of Agriculture, Vellayani explored both flower buds and floral hands as explants. The establishment media evaluated involved basal MS as control and 15 hormone treatments involving different levels of BA (2, 4 and 6 mgL−1), TDZ (0.2, 0.4 and 0.6 mgL−1), kinetin (0.5, 1.0 and 2.0 mgL−1), combination of BA (2, 4 and 6 mgL−1) with kinetin (1.0 mgL−1) and combination of BA (2, 4 and 6 mgL−1) with NAA (1.0 mgL−1). Floral hands in 4 mgL−1 BA + 1mgL−1 NAA achieved higher culture establishment percentage (100) and lesser number of days for establishment (26.78 days) when compared to whole buds. Standardization of shoot multiplication involved three levels of BA viz., 2, 4 and 6 mgL−1 in standard MS solid media. The treatment with 4 mgL−1 BA registered the least number of days to shoot initiation (96.45 days) and significantly higher number of shoots per culture (9.73). The study involved different rooting media viz., MS, MS + 250 mg charcoal, MS + 1mgL−1 NAA and MS + 0.5mgL−1 NAA. Days to root initiation was least in full MS (5.25 days) which also recorded the highest root length (10.23 cm). The present investigation highlighted the possibility of using floral hands as a promising explants type for direct regeneration of banana cv. Neypoovan.

Identification of quantitative trait loci for in vitro plant regeneration from leaf microexplants in cucumber (Cucumis sativus L.).

Plant regeneration in tissue cultures is crucial for the application of biotechnological methods to plant breeding. However, the genetic basis of in vitro plant regeneration is not fully understood. For cucumber, regeneration protocols from different types of explants have been reported, but thus far, the molecular basis of regeneration from cotyledon explants has only been studied. The aim of this work was to identify quantitative trait loci (QTLs) for in vitro plant regeneration from cucumber leaf microexplants. Plant regeneration was evaluated using a population of recombinant inbred lines (RILs) developed from a cross between line B10, characterized by high regeneration efficiency, and the low regeneration efficiency line Gy14. All RILs were scored for frequency of callus formation, organogenesis, and shoot regeneration. RILs with regeneration efficiencies higher than that of line B10 have been observed. QTLs for the frequency of organogenesis and shoot regeneration were identified. All the QTLs were mapped on cucumber chromosome 6, explaining 11.9 to 20% of the phenotypic variance. The major-effect QTL for organogenesis or6.1 was located on the upper arm of chromosome 6. The QTLs for shoot regeneration frequency, sr6.1A and sr6.1B, were located on the lower arm of chromosome 6. Analysis of the genomic region corresponding to these QTLs combined with gene expression profiling revealed that CsARF6 and CsWOX9 are gene candidates underlying these QTLs. This study is a step toward identifying the genes controlling the ability of cucumber plant regeneration from leaf explants.

The content of endogenous hormones in explants and calluses of <i>Lavandula angustifolia</i> Mill. at the initial stages of <i>in vitro</i> culture

The content of endogenous hormones (auxin IAA, cytokinines, ABA) in explants of various types (segments of leaf, bud, stem), primary calluses induced from them, as well as morphogenic and non-morphogenic calluses at the initial stages of in vitro culture by the immunoassay method was studied for the first time for Lavandula angustifolia Mill. The maximum value of hormone levels in such explants as segments of bud was shown. An increase in the content of hormones in primary calluses was revealed in comparison with similar characteristics in all types of explants. The higher level of the active form of cytokinin (trans-zeatin) and ABA, as well as the lower level of the inactive form of cytokinin (zeatin-nucleotide) and auxin IAA were identified in morphogenic callus compared with non-morphogenic callus. It is suggested that the content of endogenous hormones in explants and calluses of L. angustifolia is due to their histological status. The conclusion is made about the unified histophysiological mechanisms of callusogenesis and morphogenesis in vitro in the studied plant.

Effect of explant type and growth regulators on in vitro regeneration of apricot (Prunus armeniaca L.) Al-Amar rootstock

Apricot is a highly recalcitrant species for shoot regeneration in addition the plant regeneration capacity is strongly genotype – dependent. Thus, this study aimed to establish in vitro regeneration of the Egyptian Al-Amar apricot rootstock. Two explant types (Cotyledon and hypocotyl) were cultured on woody plant medium (WPM) supplemented with three concentrations of thidiazuron (TDZ) in combination with eight concentration of indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). The cotyledons showed direct somatic organogenesis as the explants formed buds directly on regeneration media. The highest percentage of cotyledons producing buds was 78.00% when cultured on WPM containing 13.62 µM TDZ and 2.46 µM IBA. In contrast hypocotyls expressed indirect somatic organogenesis, as the explants produced callus first before bud formation. The highest percentage was 96.87% in hypocotyls cultured on WPM fortified with 15.89 µM TDZ and 1.61 µM NAA. The shoot proliferation was achieved when buds from cotyledons and hypocotyls were cultured on Soot Regeneration Medium (SRM) supplemented with 8.87 µM 6-Benzylaminopurine (BAP) and 0.54 µM NAA. The highest shoot formation with an average of 6.4 shoots per explant was obtained from hypocotyls, while an average of 2.0 shoots per explants was achieved from cotyledons. The regenerated shoots were rooted on WPM including 9.80 µM IBA. The results revealed that the regeneration of Al-Amar rootstock was more successful through hypocotyls and could therefore facilitate its genetic manipulation.

In Vitro Callus Culture of Salvia Officinalis L. and the Effect of Some Amino Acids on Rosmarinic Acid Accumulation

This study investigated the in vitro callus formation ability of various explants obtained from the Salvia officinalis (common sage) and the production of rosmarinic acid in the resulting calli. Four types of explants, including young leaves (1-1.5 cm), nodes, and stem pieces, were cultured on MS medium containing 0.8 mg/l 2.4-D, 0.5 mg/l NAA, and 2.0 mg/l BAP to induce callus formation. The highest callus formation (100%) was achieved from young leaf explants. After 4 weeks, the formed calli were transferred to an MS medium containing 0.1 mg/l NAA+1.0 mg/l BAP, and in vitro rosmarinic acid production was enhanced by adding L-tyrosine (10 mg/l) and L-phenylalanine (10 mg/l) to the nutrient medium. The calli were cultured in these media for 1 and 2 months, and the production of rosmarinic acid in sage calli was analyzed using HPLC. The addition of amino acids to the medium significantly increased rosmarinic acid production in the calli. Furthermore, the results varied significantly with different amino acids and callus culture durations. Tyrosine was observed to be more effective in increasing rosmarinic acid production in sage calli. Moreover, calli cultured for 2 months in both tyrosine- and phenylalanine-supplemented media exhibited higher rosmarinic acid production compared to the control group cultured for only 1 month.

Effects of Thin Cell Layer Technique and Nutrient Media Contents of Regeneration and Browning Characteristics of the Laurus nobilis L. Explants

The aim of this study is in order to investigate the effects of explant source, explant type and MS media composition containing different concentrations of sucrose, activated carbon and Coconut milk for regeneration and browning of Laurus nobilis L. using thin cell layer (TCL) culture system. A higher rate of callus (57.15%) and shoot (2%) regeneration and a lower rate of blackening were determined in transversely cut stem TCL explants compared to leaf explants. While 1.33% callus regeneration was achieved in leaf explants; shoot regeneration could not be achieved. While more callus regeneration (35.17%) was found in explants taken from the field, more shoot regeneration (1.5%) and lower rate of browning were obtained in explants taken from in vitro. In the sugar trials, the highest callus regeneration (40.83%) was defined in MS medium containing 30 g/L sucrose supplemented with 1 mg/L BAP, and the highest shoot regeneration (2.5%) was determined in MS medium containing 45 g/L and 60 g/L sucrose supplemented with 1 mg/L BAP. When explant type, explant source and nutrient media composition are considered together; the highest callus regeneration (100%) was obtained in field-sourced stem TCL explants cultured in medium containing 25 mL/L coconut milk and 1 mg/L BAP. The highest shoot regeneration (6.6%) was determined in in vitro stem TCL explants cultured in MS media containing 30, 45, 60 g/L sucrose and 1 mg/L BAP. The lowest percentage of browning (50%) was obtained from in vitro stem TCL explants cultured in MS medium containing 2 g/L activated carbon and 1 mg/L BAP.

The Effect of Explant Type and Growth Regulators on Callus Induction in Passiflora quadrangularis

The Effect of Explant Type and Growth Regulators on Callus Induction in Passiflora quadrangularis

In Vitro Plant Regeneration of Sulla coronaria from Floral Explants as a Biotechnological Tool for Plant Breeding

Sulla coronaria L. Medik., a biennial forage legume typical of the Mediterranean basin, plays a significant role in foraging systems due to its high nutritional value, ability to increase ruminant live weight, and potential to reduce methane emissions. However, its allogamous nature complicates genetic improvement and the development of new varieties with desired traits. Given these challenges, this study aims to develop, for the first time, a successful protocol for the in vitro meristematic shoot regeneration of S. coronaria. The experiment utilizes four different flower explants (anther with filament, ovary, petals, and whole immature flower) collected from twenty distinct S. coronaria biotypes with three plant growth regulator (PGR) combinations and under both light and dark conditions. In terms of the regeneration response, the key factors appear to be the combinations of PGRs and the type of explant used. The interactions between all the factors do not seem to be significant.

OBTAINING ASEPTIC CULTURE IN VITRO OF RHODIOLA SEMENOWII L. FROM VARIOUS TYPES OF EXPLANTS

In vitro cultivation of callus cells allows for the stable production of secondary metabolites, being an alternative to the use of medicinal plant biomass from natural populations. Standardization of cultivation protocols and technological processes for the synthesis of secondary metabolites in vitro conditions determines significant interest in the use of tissue culture methods (PTC – Plant Tissue Culture). The aim of the study was to obtain an aseptic culture of Rhodiola semenowii in vitro from various types of explants, as well as the effect of the hormonal composition of the nutrient medium on the induction of callusogenesis in the culture of isolated organs of R. semenowii. As a result of the research, the optimal mode of obtaining R. semenovii aseptic cultures from various explants was determined. It has been shown that the use of 70% EtOH in combination with 0.01% HgCl2 or 20% NaClO makes it possible to obtain aseptic cultures from any type of R. semenovii explants. It has been established that the type of explant is R. semenovii influenced both the frequency of callus formation and the beginning of its formation. The least optimal types of R. semenovii explants are stem and rhizome segments, the frequency of callusogenesis varied from 5.8% to 75.6%. The induction of callusogenesis also depended on the hormonal composition of the nutrient medium: the highest percentage of callus formation in all types of explants was on media containing 0.5mg L−1 thidiazuron (TDZ) + 1.0 mg L−1 1-naphthaleneacetic acid (NAA) and 0.5mg L−12,4D + 2.0 mg L−1 6-Benzylaminopurine (BAP).

Establishment of Agrobacterium-Mediated Transient Transformation System in Desert Legume Eremosparton songoricum (Litv.) Vass.

Eremosparton songoricum (Litv.) Vass. is a desert legume exhibiting extreme drought tolerance and the ability to withstand various harsh environments, making it a good candidate for investigating stress tolerance mechanisms and exploring valuable stress-resistant genes. However, the absence of a genetic transformation system for E. songoricum poses significant limitations for functionally validating these stress-resistant genes in situ. In this study, we developed an Agrobacterium-mediated transient transformation system for E. songoricum utilizing the β-glucuronidase (GUS) gene as a reporter. We investigated three types of explants (seedlings, assimilated branches and callus) and the effects of different Agrobacterium strains, seedling ages, OD600 values, acetosyringone (AS) concentrations, sucrose concentrations and infection times on the transformation efficiency. The results reveal that the optimal transformation system was infecting one-month-old regenerating assimilated branches with the Agrobacterium strain C58C1. The infection solution comprised 1/2 MS medium with 3% sucrose and 200 μM AS at an OD600 of 0.8, infection for 3 h and then followed by 2 days of dark cultivation, which achieving a maximum transformation rate of 97%. The maximum transformation rates of the seedlings and calluses were 57.17% and 39.51%, respectively. Moreover, we successfully utilized the assimilated branch transient transformation system to confirm the role of the previously reported transcription factor EsDREB2B in E. songoricum. The overexpression of EsDREB2B enhanced drought tolerance by increasing the plant's reactive oxygen species (ROS) scavenging capacity in situ. This study established the first transient transformation system for a desert legume woody plant, E. songoricum. This efficient system can be readily applied to investigate gene functions in E. songoricum. It will expedite the exploration of genetic resources and stress tolerance mechanisms in this species, offering valuable insights and serving as a reference for the transformation of other desert plants and woody legumes.

Improved Protocol for Efficient Agrobacterium-Mediated Transient Gene Expression in Medicago sativa L.

Medicago sativa L. (Alfalfa) is a globally recognized forage legume that has recently gained attention for its high protein content, making it suitable for both human and animal consumption. However, due to its perennial nature and autotetraploid genetics, conventional plant breeding requires a longer timeframe compared to other crops. Therefore, genetic engineering offers a faster route for trait modification and improvement. Here, we describe a protocol for achieving efficient transient gene expression in alfalfa through genetic transformation with the Agrobacterium tumefaciens pCAMBIA1304 vector. This vector contains the reporter genes β-glucuronidase (GUS) and green fluorescent protein (GFP), along with a selectable hygromycin B phosphotransferase gene, all driven by the CaMV 35s promoter. Various transformation parameters-such as different explant types, leaf ages, leaf sizes, wounding types, bacterial concentrations (OD600nm), tissue preculture periods, infection periods, co-cultivation periods, and different concentrations of acetosyringone, silver nitrate, and calcium chloride-were optimized using 3-week-old in vitro-grown plantlets. Results were attained from data based on the semi-quantitative observation of the percentage and number of GUS spots on different days of agro-infection in alfalfa explants. The highest percentage of GUS positivity (76.2%) was observed in 3-week-old, scalpel-wounded, segmented alfalfa leaf explants after 3 days of agro-infection at a bacterial concentration of 0.6, with 2 days of preculture, 30 min of co-cultivation, and the addition of 150 µM acetosyringone, 4 mM calcium chloride, and 75 µM silver nitrate. The transient expression of genes of interest was confirmed via histochemical GUS and GFP assays. The results based on transient reporter gene expression suggest that various factors influence T-DNA delivery in the Agrobacterium-mediated transformation of alfalfa. The improved protocol can be used in stable transformation techniques for alfalfa.

Implementation of biotechnological techniques for the propagation and introduction of Trichosanthes kirilowii Maxim

Trichosanthes kirilowii Maxim. is classified among the 50 most important medicinal plants used in traditional Asian medicine. This species is endemic and reproduction from seeds in the conditions of Central Europe is inefficient. The present study focused on the implementation of effective propagation of T. kirilowii, including standardisation of seed germination conditions, reproduction of the plant based on micropropagation, and introduction to field conditions. Four types of explants were tested on six types of media in various variants of culture conditions. Additionally, anatomical and histological analyses of specimens obtained through the in vitro cultivation were carried out, with particular emphasis on the site of initiation of aboveground and underground shoots. Microscopic analyses showed that the T. kirilowii shoots and roots obtained in the in vitro culture formed in the process of organogenesis via direct regeneration. Even though the callus did not participate in the regeneration of the entire plant, the micropropagation protocol proposed in the study was efficient: 6–8 shoots were obtained from 1 explant. The entire micropropagation process from the explant to an adult individual growing in the ground lasted 122 days. The protocol for T. kirilowii micropropagation in vitro cultures provides a fast, efficient, and robust approach to propagation of this species. The biotechnological applications in the reproduction of T. kirilowii will help not only to increase the number of daughter plants but also to multiply individuals of the appropriate sex, which will increase the probability of production of fruits in this dioecious species.

EXPRESIÓN EMBRIOGÉNICA DIRECTA EN Agave cupreata

<p><strong>Background:</strong> <em>Agave cupreata</em> is a specie that can only reproduce by seeds and could take up to 15 years until flowering, placing it at risk of extinction due to its agroindustrial exploitation to produce mezcal. <strong>Objective:</strong> To evaluate the type of explant, the vitamin complex and 2,4-D concentrations on the direct embryogenic response. <strong>Methodology:</strong> Explants of leaf, root and stem sections were evaluated. Also, the vitamins MS (Murashige and Skoog, 1962) and L2 (Phillips and Collins, 1979), five concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), (0.0, 0.1, 0.3, 0.6 and 0.9 mg L<sup>-1</sup>) and six concentrations of indole-3-acetic acid (IAA), (0.0, 0.1, 0.5, 1.0, 2.0 and 3.0 mg L<sup>-1</sup>) were assayed for the expression of somatic embryos directly. <strong>Results: </strong>The leaf explant, the vitamin L2 complex, and the concentration of 0.9 mg L<sup>-1</sup> of 2,4-D and 0.5 mg L<sup>-1</sup> of IAA turned out to be the most efficient for the formation of pro-embryogenic masses and the expression of direct somatic embryos in the globular state. <strong>Implications:</strong> These results lay the groundwork for the future regeneration of <em>A. cupreata</em> plants via direct somatic embryogenesis, potentially facilitating their conservation and mass propagation. This contributes to the sustainability of mezcal; boosts the local economy, preserves biodiversity, and ensures the continued production of this endangered species. <strong>Conclusions:</strong> For the first time, the successful expression of somatic embryos directly from <em>A. cupreata</em> explants was evidenced.</p>

12209 Validation Of A Novel Placenta Explant Protocol For Studying Circadian Rhythms In The Mouse Placenta In Early, Mid And Late Pregnancy

Abstract Disclosure: J.S. Lee: None. A.M. Yaw: None. H.M. Hoffmann: None. Introduction: The placenta serves as an interface facilitating the exchange of essential nutrients and oxygen between the mother and fetus. Preeclampsia, a complication of pregnancy driven by placental dysfunction, is one of the leading causes of maternal, fetal, and neonatal morbidity and mortality. While deregulation of the genes driving circadian rhythm has been associated with preeclampsia, the role of circadian rhythms in the placenta largely remains unknown. To address this gap of knowledge, our goal was to establish and validate an organotypic ex vivo preparation to study placenta circadian rhythms at gestation day (GD) 10, GD14, and GD18 in the mouse. Methods: To establish an organotypic placenta culture GD10, GD14, and GD18 placental explants, we sectioned the placenta into 350μm sections using a vibratome. The explants were prepared from the validated circadian Per2::Luciferase reporter mouse, allowing automated circadian rhythms recording of the explants in a LumiCycle. We compared explant success rate and rhythm quality in 2x2mm section vs whole slice explants at GD14. Using hematoxylin and eosin (H&E) staining we validated the explants to be decidua which is strictly of maternal origin, labyrinth which is strictly of fetal origin, and the junctional zone which consists of binucleated cells fused of maternal and fetal cells. Results: Through H&E staining, we confirmed the decidua is enriched with neutrophils, the junctional zone contains spongiotrophoblasts and glycogen trophoblasts, and the labyrinth has red blood cells. Both explant types were enriched with >80% of cells from the targeted region. We next established a protocol to record Per2::Luciferase rhythms for <6 days. The fragility of the GD10 placentas required the whole explant protocol, whereas the GD18 2x2mm sectioned explants produced strong rhythms with a high success rate. At GD14, the whole explants resulted in stronger rhythms with higher amplitude and higher goodness of fit compared to the 2x2mm explants. Conclusion: We found that depending on GD recording success differs. Specifically, the whole placental explant provides a more robust and reliable model for studying circadian rhythms in GD10 and GD14, whereas 2x2mm explants are successful at GD18. This novel placental preparation protocol will be valuable in studying disease- and drug-induced changes to circadian rhythms in a placenta layer-specific manner in the mouse. Presentation: 6/1/2024

Importance of Media Composition and Explant Type in Cannabis sativa Tissue Culture.

Producing uniform Cannabis sativa (Cannabis) for medicinal/recreational flower production through sexual propagation has been problematic, leading to dominance of clonal propagation from "mother plants" in the cannabinoid industry, which also faces significant limitations. Cannabis tissue culture (TC) methods have been developed to overcome these challenges, but the long-term health and maintenance of Cannabis explants in TC have been largely overlooked in previous studies. The current study focused on the development of an efficient and optimized micropropagation protocol covering the entire process, with a specific focus on the health and performance in the multiplication stage. Multiplication media were formulated hormone-free to avoid longer-term vitrification issues, resulting in single-main-shoot cultures rather than multiple-shoot cultures. This instigated the use of stage II explant types different from the standard shoot tips previously used for multiple shoot cultures. Multiplication media were further improved from the basal salt composition via nitrogen and calcium additives. The optimized protocol was used on eight diverse Cannabis cultivars to test its applicability across various genetic backgrounds. Results indicated that the protocol was effective for conservation purposes across all cultivars and achieved good long-term multiplication rates for some but not all. The outcomes of this study mark a significant stride towards an efficient Cannabis TC methodology ready for more comprehensive industrial applications.