Cryopreservation and assessment of genetic fidelity Acorus calamus Linn., an endangered medicinal plant.

BACKGROUND: Acorus calamus Linn. is a medicinally valuable monocot plant belonging to the family Acoraceae. Over-exploitation and unscientific approach towards harvesting to fulfill an ever-increasing demand have placed it in the endangered list of species. OBJECTIVE: To develop vitrification-based cryopreservation protocols for A. calamus shoot tips, using conventional vitrification and V cryo-plate. MATERIALS AND METHODS: Shoot tips (2 mm in size) were cryopreserved with the above techniques by optimizing various parameters such as preculture duration, sucrose concentration in the preculture medium, and PVS2 dehydration time. Regenerated plantlets obtained post-cryopreservation were evaluated by random amplified polymorphic DNA (RAPD) to test their genetic fidelity. RESULTS: The highest regrowth of 88.3% after PVS2 exposure of 60 min was achieved with V cryo-plate as compared to 75% after 90 min of PVS2 exposure using conventional vitrification. After cryopreservation, shoot tips developed into complete plantlets in 28 days on regrowth medium (0.5 mg/L BAP, 0.3 mg/L GA3, and 0.3 mg/L ascorbic acid). RAPD analysis revealed 100% monomorphism in all cryo-storage derived regenerants and in vitro donor (120-days-old) plants. CONCLUSION: Shoot tips of A. calamus that were cryopreserved had 88.3% regrowth using V cryo-plate technique and the regerants retained genetic fidelity. https://doi.org/10.54680/fr24210110412.

The activation of Arabidopsis axillary buds involves a switch from slow to rapid committed outgrowth regulated by auxin and strigolactone.

Arabidopsis thaliana (Arabidopsis) shoot architecture is largely determined by the pattern of axillary buds that grow into lateral branches, the regulation of which requires integrating both local and systemic signals. Nodal explants - stem explants each bearing one leaf and its associated axillary bud - are a simplified system to understand the regulation of bud activation. To explore signal integration in bud activation, we characterised the growth dynamics of buds in nodal explants in key mutants and under different treatments. We observed that isolated axillary buds activate in two genetically and physiologically separable phases: a slow-growing lag phase, followed by a switch to rapid outgrowth. Modifying BRANCHED1 expression or the properties of the auxin transport network, including via strigolactone application, changed the length of the lag phase. While most interventions affected only the length of the lag phase, strigolactone treatment and a second bud also affected the rapid growth phase. Our results are consistent with the hypothesis that the slow-growing lag phase corresponds to the time during which buds establish canalised auxin transport out of the bud, after which they enter a rapid growth phase. Our work also hints at a role for auxin transport in influencing the maximum growth rate of branches.

Comprehensive study on in vitro propagation of some imported peach rootstocks: in vitro explant surface sterilization and bud proliferation.

The present study was conducted in the Laboratory of Tissue Culture, Horticulture Department, Faculty of Agriculture, Damietta University, Egypt. The objective of this study was to establish a micropropagation protocol suitable for three imported peach rootstocks: Okinawa (P. persica), Nemared (P. persica × P. davidiana) × P. persica), and Garnem (P. dulcis × P. persica) in vitro. The results showed that soaking the explants in sodium hypochlorite (NaOCl) at 20% for 15 min produced the highest responsiveness (82.81%), survival (96.61%), with the lowest mortality (3.14%) and contamination (0.24%). Explants of the Garnem genotype had the best response (89.12%), survival (90.62%), lowest mortality (0.00%), and highest contamination (9.37%) when compared to the other genotypes. In comparison with axillary buds, the shoot tip displayed the highest responsiveness, survival, and death (100, 87.40, and 12.59%, respectively), as well as the least significant contamination (0.00%). Additionally, the percentages of responsive, survived, dead, and contaminated explants at the various collection dates varied significantly. The 6-benzylaminopurine (BAP) concentrations used (3 to 5.0 mg/L) demonstrated similar behavior in terms of in vitro proliferation, with rates of 3.77 to 6.11, 4.33 to 8.88, and 3.33 to 7.44 shoot numbers per explant for the Okinawa, Nemared, and Garnem peach rootstocks, respectively, indicating that the number of shoot proliferations is genotype-dependent. Additionally, using 5.0 mg/L BAP in combination with 0.2 mg/L IBA significantly increased average shoot proliferation (96.29%), number of shoots per explant (7.48), and average leaf number/explant (16.33) compared to the other treatments. Based on these results, adventitious bud development was enhanced during in vitro multiplication of the Okinawa, Nemared, and Garnem peach rootstocks by the synergistic interaction of indole-butyric acid (IBA) and 6-benzylaminopurine (BAP).

Unraveling the molecular mechanisms governing axillary meristem initiation in plants.

MAIN CONCLUSION: Axillary meristems (AMs) located in the leaf axils determine the number of shoots or tillers eventually formed, thus contributing significantly to the plant architecture and crop yields. The study of AM initiation is unavoidable and beneficial for crop productivity. Shoot branching is an undoubted determinant of plant architecture and thus greatly impacts crop yield due to the panicle-bearing traits of tillers. The emergence of the AM is essential for the incipient bud formation, and then the bud is dormant or outgrowth immediately to form a branch or tiller. While numerous reviews have focused on plant branching and tillering development networks, fewer specifically address AM initiation and its regulatory mechanisms. This review synthesizes the significant advancements in the genetic and hormonal factors governing AM initiation, with a primary focus on studies conducted in Arabidopsis (Arabidopsis thaliana L.) and rice (Oryza sativa L.). In particular, by elaborating on critical genes like LATERAL SUPPRESSOR (LAS), which specifically regulates AM initiation and the networks in which they are involved, we attempt to unify the cascades through which they are positioned. We concentrate on clarifying the precise mutual regulation between shoot apical meristem (SAM) and AM-related factors. Additionally, we examine challenges in elucidating AM formation mechanisms alongside opportunities provided by emerging omics approaches to identify AM-specific genes. By expanding our comprehension of the genetic and hormonal regulation of AM development, we can develop strategies to optimize crop production and address global food challenges effectively.

Dissecting the Roles of the Cytokinin Signaling Network: The Case of De Novo Shoot Apical Meristem Formation.

Numerous biotechnological applications require a fast and efficient clonal propagation of whole plants under controlled laboratory conditions. For most plant species, the de novo regeneration of shoots from the cuttings of various plant organs can be obtained on nutrient media supplemented with plant hormones, auxin and cytokinin. While auxin is needed during the early stages of the process that include the establishment of pluripotent primordia and the subsequent acquisition of organogenic competence, cytokinin-supplemented media are required to induce these primordia to differentiate into developing shoots. The perception of cytokinin through the receptor ARABIDOPSIS HISTIDINE KINASE4 (AHK4) is crucial for the activation of the two main regulators of the establishment and maintenance of shoot apical meristems (SAMs): SHOOTMERISTEMLESS (STM) and the WUSCHEL-CLAVATA3 (WUS-CLV3) regulatory circuit. In this review, we summarize the current knowledge of the roles of the cytokinin signaling cascade in the perception and transduction of signals that are crucial for the de novo establishment of SAMs and lead to the desired biotechnological output-adventitious shoot multiplication. We highlight the functional differences between individual members of the multigene families involved in cytokinin signal transduction, and demonstrate how complex genetic regulation can be achieved through functional specialization of individual gene family members.

The Shoot Apical Meristem: An Evolutionary Molding of Higher Plants.

The shoot apical meristem (SAM) gives rise to the aerial structure of plants by producing lateral organs and other meristems. The SAM is responsible for plant developmental patterns, thus determining plant morphology and, consequently, many agronomic traits such as the number and size of fruits and flowers and kernel yield. Our current understanding of SAM morphology and regulation is based on studies conducted mainly on some angiosperms, including economically important crops such as maize (Zea mays) and rice (Oryza sativa), and the model species Arabidopsis (Arabidopsis thaliana). However, studies in other plant species from the gymnosperms are scant, making difficult comparative analyses that help us understand SAM regulation in diverse plant species. This limitation prevents deciphering the mechanisms by which evolution gave rise to the multiple plant structures within the plant kingdom and determines the conserved mechanisms involved in SAM maintenance and operation. This review aims to integrate and analyze the current knowledge of SAM evolution by combining the morphological and molecular information recently reported from the plant kingdom.

In vitro propagation of Indonesian stevia (Stevia rebaudiana) genotype using axenic nodal segments.

OBJECTIVE: The high industrial demand for Stevia cultivation (Stevia rebaudiana) has increased due to its high stevioside content derived from the leaves. However, the low germination rate makes the cultivation of the plant become the main obstacle. Therefore, an efficient cultivation technique is required. This present work aims to analyze the effect of five combinations of Kinetin (Kin) and benzyladenine (BA) on stevia micropropagation using nodal segment explants. RESULTS: The micropropagation of stevia was performed using Murashige and Skoog (MS) medium supplemented with BA and Kin. We analyzed different organogenesis and callogenesis responses. In addition, the number of shoots and root formed during in vitro culture were also observed. Our results demonstrated that all treatments with Kin, both alone and in combination with BA, resulted in the development of callus on all nodal segment explants. Explants treated in MS with 1 mg L-1 BA exhibited the best average of shoot number (36.27). In contrast, the treatment without PGR resulted in the best root formation (2.6). The overall results suggested that different combination of BA and Kin resulted in distinct organogenesis responses, where 1 mg L-1 of BA was potentially used for boosting the number of shoots in micropropagation of stevia accession Mini.

In vitro regeneration of triploid from mature endosperm culture of Passiflora edulis "Mantianxing".

The regeneration of shoots from endosperm tissue is a highly effective method to obtain triploid plants. In this study, we elucidated the establishment of an in vitro regeneration system from endosperm culture for the production of Passiflora edulis "Mantianxing." The highest callus induction rate (83.33%) was obtained on the media supplemented with 1.0 mg/L TDZ. Meanwhile, the MS medium containing 1.0 mg/L 6-BA and 0.4 mg/L IBA gave the optimum 75% shoot bud induction. Chromosome analysis revealed that the chromosomal count of P. edulis "Mantianxing" regenerated from endosperm tissues was 27 (2n = 3x = 27), which indicated that shoots regenerated from endosperm tissues were triploids. Triploid P. edulis had more drought resistance than diploid plants. Our study provided a method for breeding of passion fruit by means of a stable and reproducible regeneration system from endosperm culture, leading to the generation of triploid plants.

Spatial IMA1 regulation restricts root iron acquisition on MAMP perception.

Iron is critical during host-microorganism interactions1-4. Restriction of available iron by the host during infection is an important defence strategy, described as nutritional immunity5. However, this poses a conundrum for externally facing, absorptive tissues such as the gut epithelium or the plant root epidermis that generate environments that favour iron bioavailability. For example, plant roots acquire iron mostly from the soil and, when iron deficient, increase iron availability through mechanisms that include rhizosphere acidification and secretion of iron chelators6-9. Yet, the elevated iron bioavailability would also be beneficial for the growth of bacteria that threaten plant health. Here we report that microorganism-associated molecular patterns such as flagellin lead to suppression of root iron acquisition through a localized degradation of the systemic iron-deficiency signalling peptide Iron Man 1 (IMA1) in Arabidopsis thaliana. This response is also elicited when bacteria enter root tissues, but not when they dwell on the outer root surface. IMA1 itself has a role in modulating immunity in root and shoot, affecting the levels of root colonization and the resistance to a bacterial foliar pathogen. Our findings reveal an adaptive molecular mechanism of nutritional immunity that affects iron bioavailability and uptake, as well as immune responses.

Enhancing cryopreservation survival in Petunia × Calibrachoa 'Light Yellow' callus: Insights into material characteristics and genetic integrity.

Petunia × Calibrachoa 'Light Yellow' (× Petchoa 'Light Yellow') is a kind of perennial herbaceous flower obtained through intergeneric hybridization of Petunia and Calibrachoa with high ornamental value and wide application, facing challenges in seed acquisition. Expanding propagation through tissue culture is an economically efficient means. Hence, establishing an effective procedure for the storage of callus is essential for × Petchoa 'Light Yellow'. Cryopreservation is an effective method for the in vitro propagation and long-term preservation of × Petchoa 'Light Yellow' germplasms. For formulating the optimization of the vitrification procedure, first, an orthogonal experimental design was employed to pinpoint critical steps in the vitrification protocol (pre-culture, osmoprotection, dehydration, and dilution) for Petunia × Calibrachoa callus tissues and then five additional factors (pre-culture, osmoprotection I and II, dehydration, and dilution) were optimized to further reduce the sample water content and enhance cell viability levels. The vitrification procedure was described as follows: callus tissues were precultured in MS solid medium with 0.3 M sucrose for 5 d, incubated with osmoprotection solution I and II for 15 min at 25 °C, respectively, cryoprotected with PVS2 for 30 min at 0 °C, and rapidly immersed in liquid nitrogen. Cryopreserved callus tissues were then diluted in MS liquid medium with 1.2 M sucrose for 20 min at 25 °C and recovered on MS solid medium with 0.5 mg/L 6-BA and 0.1 mg/L NAA, and sucrose. The cell viability measured by TTC staining was approximately 16 %-18 % after 72 h-recovery. Following 45 days, the relative survival of callus reached up to 49.48 %. Furthermore, EST-SSR analysis showed no significant difference in the genetic stability of cryopreserved callus compared to the control. Based on the cryopreservation of × Petchoa 'Light Yellow' callus, we further evaluated the response of callus water contents to the osmotic stress in the optimized and original protocols (CK) for a higher cryopreservation survival. A comparative analysis of water content demonstrated that the procedure of gradual and gentle dehydration significantly improved water content and cell survival. Ultrastructural changes between cryopreserved and non-cryopreserved callus were examined and high vacuolation emerged as a key determinant, indicating its substantial impact on the low survival of cryopreserved cells, which should help us to understand the effectiveness of osmotic protectants in dehydration.

Tipping the balance: The dynamics of stem cell maintenance and stress responses in plant meristems.

Plant meristems contain pools of dividing stem cells that produce new organs for plant growth and development. Environmental factors, including biotic and abiotic stresses and nutrient availability, affect meristem activity and thus the architecture of roots and shoots; understanding how meristems react to changing environmental conditions will shed light on how plants optimize nutrient acquisition and acclimate to different environmental conditions. This review highlights recent exciting advances in this field, mainly in Arabidopsis. We discuss the signaling pathways, genetic regulators, and molecular mechanisms involved in the response of plant meristems to environmental and nutrient cues, and compare the similarities and differences of stress responses between the shoot and root apical meristems.

Visualizing plant intracellular inorganic orthophosphate distribution.

Intracellular inorganic orthophosphate (Pi) distribution and homeostasis profoundly affect plant growth and development. However, its distribution patterns remain elusive owing to the lack of efficient cellular Pi imaging methods. Here we develop a rapid colorimetric Pi imaging method, inorganic orthophosphate staining assay (IOSA), that can semi-quantitatively image intracellular Pi with high resolution. We used IOSA to reveal the alteration of cellular Pi distribution caused by Pi starvation or mutations that alter Pi homeostasis in two model plants, rice and Arabidopsis, and found that xylem parenchyma cells and basal node sieve tube element cells play a critical role in Pi homeostasis in rice. We also used IOSA to screen for mutants altered in cellular Pi homeostasis. From this, we have identified a novel cellular Pi distribution regulator, HPA1/PHO1;1, specifically expressed in the companion and xylem parenchyma cells regulating phloem Pi translocation from the leaf tip to the leaf base in rice. Taken together, IOSA provides a powerful method for visualizing cellular Pi distribution and facilitates the analysis of Pi signalling and homeostasis from the level of the cell to the whole plant.

Direct Organogenesis of Citrus Cultivars from Shoot Tip Nodal Segments.

BACKGROUND: Citrus cultivar improvement via conventional breeding strategies is impeded by factors related to its reproductive biology. The orange is a hybrid between pomelo (Citrus maxima) and mandarin (Citrus reticulata). Among various orange cultivars, Valencia oranges have a bit of bitter tang mixed in with their sweetness, as Navel oranges are, the most widely cultivated citrus species, quite sweeter, and also don't contain any seeds. Tangelo mandarin orange cultivar is a hybrid of C. reticulata × C. maxima or × C. paradisi. OBJECTIVE: The present study was undertaken to optimize the hormonal composition of the media with regard to plant growth regulators for in vitro propagation of sweet orange cultivars from nodal segment explants. METHODS: The nodal segment explants were collected from three citrus cultivars, Washington Navel, Valencia and Tangelo. Murashige and Skoog (MS) medium supplemented with sucrose and different concentrations of growth regulators was used for shoot proliferation and root induction, and the optimum medium composition was assessed. The patent for Citrus Tissue Culture was obtained from the Office of Research Affairs, Haramaya University. RESULTS: The results indicate that the highest shoot response was recorded for Washington's navel with maximum shoot proliferation rate (99.75%), shoot number per explant (1.76), shoot length (10.70 cm), leaf number per explants (3.54) after three weeks of culture. In all experiments, no growth was observed for the basal MS medium. Phytohormone combinations of IAA (1.2 mg/L) and kinetin (2.0 mg/L) were found to be the best for shoot proliferation. Among the cultivars, there were significant differences for the highest rooting rate (81.255), root number (2.22), and root length (2.95 cm) variables for Washington Navel. The lowest rooting rate (48.45%), root number (1.47) and root length (2.26 cm) were observed for Valencia. The highest rooting rate (84.90%), root number per microshoot (2.22) and root length (3.05 cm) was on MS medium supplemented with 1.5 mg/L NAA. CONCLUSION: A comparison of different concentrations of IAA and NAA on root induction of microshoots from nodal segments of citrus cultivars demonstrated NAA was a more effective hormone than IAA.

Far-red light inhibits lateral bud growth mainly through enhancing apical dominance independently of strigolactone synthesis in tomato.

The ratio of red light to far-red light (R:FR) is perceived by light receptors and consequently regulates plant architecture. Regulation of shoot branching by R:FR ratio involves plant hormones. However, the roles of strigolactone (SL), the key shoot branching hormone and the interplay of different hormones in the light regulation of shoot branching in tomato (Solanum lycopersicum) are elusive. Here, we found that defects in SL synthesis genes CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and CCD8 in tomato resulted in more lateral bud growth but failed to reverse the FR inhibition of lateral bud growth, which was associated with increased auxin synthesis and decreased synthesis of cytokinin (CK) and brassinosteroid (BR). Treatment of auxin also inhibited shoot branching in ccd mutants. However, CK released the FR inhibition of lateral bud growth in ccd mutants, concomitant with the upregulation of BR synthesis genes. Furthermore, plants that overexpressed BR synthesis gene showed more lateral bud growth and the shoot branching was less sensitive to the low R:FR ratio. The results indicate that SL synthesis is dispensable for light regulation of shoot branching in tomato. Auxin mediates the response to R:FR ratio to regulate shoot branching by suppressing CK and BR synthesis.

Multiple feedbacks on self-organized morphogenesis during plant regeneration.

Decades of research have primarily emphasized genetic blueprint as the driving force behind plant regeneration. The flow of information from genetics, which manifests as biochemical properties, including hormones, has been extensively implicated in plant regeneration. However, recent advancements have unveiled additional intrinsic modules within this information flow. Here, we explore the three core modules of plant regeneration: biochemical properties, mechanical forces acting on cells, and cell geometry. We debate their roles and interactions during morphogenesis, emphasizing the potential for multiple feedbacks between these core modules to drive pattern formation during regeneration. We propose that de novo organ regeneration is a self-organized event driven by multidirectional information flow between these core modules.

A gene regulatory network critical for axillary bud dormancy directly controlled by Arabidopsis BRANCHED1.

The Arabidopsis thaliana transcription factor BRANCHED1 (BRC1) plays a pivotal role in the control of shoot branching as it integrates environmental and endogenous signals that influence axillary bud growth. Despite its remarkable activity as a growth inhibitor, the mechanisms by which BRC1 promotes bud dormancy are largely unknown. We determined the genome-wide BRC1 binding sites in vivo and combined these with transcriptomic data and gene co-expression analyses to identify bona fide BRC1 direct targets. Next, we integrated multi-omics data to infer the BRC1 gene regulatory network (GRN) and used graph theory techniques to find network motifs that control the GRN dynamics. We generated an open online tool to interrogate this network. A group of BRC1 target genes encoding transcription factors (BTFs) orchestrate this intricate transcriptional network enriched in abscisic acid-related components. Promoter::ß-GLUCURONIDASE transgenic lines confirmed that BTFs are expressed in axillary buds. Transient co-expression assays and studies in planta using mutant lines validated the role of BTFs in modulating the GRN and promoting bud dormancy. This knowledge provides access to the developmental mechanisms that regulate shoot branching and helps identify candidate genes to use as tools to adapt plant architecture and crop production to ever-changing environmental conditions.

Detailing Early Shoot Growth Arrest in Kro-0 x BG-5 Hybrids of Arabidopsis thaliana.

Shoot growth directly impacts plant productivity. Plants adjust their shoot growth in response to varying environments to maximize resource capture and stress resilience. While several factors controlling shoot growth are known, the complexity of the regulation and the input of the environment are not fully understood. We have investigated shoot growth repression induced by low ambient temperatures in hybrids of Arabidopsis thaliana Kro-0 and BG-5 accessions. To continue our previous studies, we confirmed that the Kro-0 allele of DYNAMIN-RELATED PROTEIN 3B causes stunted shoot growth in the BG-5 background. We also found that shoot growth repression was most pronounced near the apex at a lower temperature and that the cells in the hybrid stem failed to elongate correctly. Furthermore, we observed that shoot growth repression in hybrids depended on light availability. Global gene expression analysis indicated the involvement of hormones, especially strigolactone, associated with the dwarf phenotype. Altogether, this study enhances our knowledge on the genetic, physiological and environmental factors associated with shoot growth regulation.

The Growth-Inhibitory Effect of Increased Planting Density Can Be Reduced by Abscisic Acid-Degrading Bacteria.

High-density planting can increase crop productivity per unit area of cultivated land. However, the application of this technology is limited by the inhibition of plant growth in the presence of neighbors, which is not only due to their competition for resources but is also caused by growth regulators. Specifically, the abscisic acid (ABA) accumulated in plants under increased density of planting has been shown to inhibit their growth. The goal of the present study was to test the hypothesis that bacteria capable of degrading ABA can reduce the growth inhibitory effect of competition among plants by reducing concentration of this hormone in plants and their environment. Lettuce plants were grown both individually and three per pot; the rhizosphere was inoculated with a strain of Pseudomonas plecoglossicida 2.4-D capable of degrading ABA. Plant growth was recorded in parallel with immunoassaying ABA concentration in the pots and plants. The presence of neighbors indeed inhibited the growth of non-inoculated lettuce plants. Bacterial inoculation positively affected the growth of grouped plants, reducing the negative effects of competition. The bacteria-induced increase in the mass of competing plants was greater than that in the single ones. ABA concentration was increased by the presence of neighbors both in soil and plant shoots associated with the inhibition of plant growth, but accumulation of this hormone as well as inhibition of the growth of grouped plants was prevented by bacteria. The results confirm the role of ABA in the response of plants to the presence of competitors as well as the possibility of reducing the negative effect of competition on plant productivity with the help of bacteria capable of degrading this hormone.