Plant Regeneration Research Articles

Somatic embryogenesis and plant regeneration from suspension cultures of Sapindus trifoliatus

Somatic embryogenesis and plant regeneration from suspension cultures of Sapindus trifoliatus

Genome editing of a recalcitrant wine grape genotype by lipofectamine-mediated delivery of CRISPR/Cas9 ribonucleoproteins to protoplasts.

The main bottleneck in the application of biotechnological breeding methods to woody species is due to the in vitro regeneration recalcitrance shown by several genotypes. On the other side, woody species, especially grapevine (Vitis vinifera L.), use most of the pesticides and other expensive inputs in agriculture, making the development of efficient approaches of genetic improvement absolutely urgent. Genome editing is an extremely promising technique particularly for wine grape genotypes, as it allows to modify the desired gene in a single step, preserving all the quality traits selected and appreciated in elite varieties. A genome editing and regeneration protocol for the production of transgene-free grapevine plants, exploiting the lipofectamine-mediated direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to target the phytoene desaturase gene, is reported. We focused on Nebbiolo (V. vinifera), an extremely in vitro recalcitrant wine genotypeused to produce outstanding wines, such as Barolo and Barbaresco. The use of the PEG-mediated editing method available in literature and employed for highly embryogenic grapevine genotypes did not allow the proper embryo development in the recalcitrant Nebbiolo. Lipofectamines, on the contrary, did not have a negative impact on protoplast viability and plant regeneration, leading to the obtainment of fully developed edited plants after about 5 months from the transfection. Our work represents one of the first examples of lipofectamine use for delivering editing reagents in plant protoplasts. The important result achieved for the wine grape genotype breeding could be extended to other important wine grape varieties and recalcitrant woody species.

Comparative transcriptome and functional analyses provide insights into the key factors regulating shoot regeneration in highbush blueberry.

Establishing an efficient plant regeneration system is a crucial prerequisite for genetic engineering technology in plants. However, the regeneration rate exhibits considerable variability among genotypes, and the key factors underlying shoot regeneration capacity remain largely elusive. Blueberry leaf explants cultured on a medium rich in cytokinins exhibit direct shoot organogenesis without prominent callus formation, which holds promise for expediting genetic transformation while minimizing somatic mutations during culture. The objective of this study is to unravel the molecular and genetic determinants that govern cultivar-specific shoot regeneration potential in highbush blueberry (Vaccinium corymbosum L.). We conducted comparative transcriptome analysis using two highbush blueberry genotypes: 'Blue Muffin' ('BM') displaying a high regeneration rate (>80%) and 'O'Neal' ('ON') exhibiting a low regeneration rate (<10%). The findings revealed differential expression of numerous auxin-related genes; notably, 'BM' exhibited higher expression of auxin signaling genes compared to 'ON'. Among blueberry orthologs of transcription factors involved in meristem formation in Arabidopsis, expression of VcENHANCER OF SHOOT REGENERATION (VcESR), VcWUSCHEL (VcWUS), and VcCUP-SHAPED COTYLEDON 2.1 were significantly higher in 'BM' relative to 'ON'. Exogenous application of auxin promoted regeneration, as well as VcESR and VcWUS expression, whereas inhibition of auxin biosynthesis yielded the opposite effects. Overexpression of VcESR in 'BM' promoted shoot regeneration under phytohormone-free conditions by activating the expression of cytokinin- and auxin-related genes. These findings provide new insights into the molecular mechanisms underlying blueberry regeneration and have practical implications for enhancing plant regeneration and transformation techniques.

Development of indirect somatic embryogenesis and plant regeneration system with immature embryos of the cultivated traditional Chinese medicinal herb Polygonatum cyrtonema

The dried rhizome of Polygonatum cyrtonema represents a highly valued traditional Chinese medicine. With a growing demand, the imperative arises for the establishment of an efficient tissue culture system for large-scale propagation. Here, we successfully developed a tissue culture system capable of yielding P. cyrtonema seedlings through somatic embryogenesis using immature embryos as explants within a 120-day timeframe. An optimum callus induction rate was achieved through a combination of 1.0 mg/L 6-BA and 0.5 mg/L 2,4-D in MS medium, resulting in the formation of numerous highly active callus. Subsequent transfer of primary callus to a medium containing 1.0 mg/L 6-BA and 1.0 mg/L 2,4-D in MS medium for 30 days yielded induction rates of 97.78%, 89.62%, 73.33%, and 66.67% for globular, heart, torpedo, and cotyledonary embryos, respectively. The highest rate of somatic embryo normal germination (73.33%) was observed in the MS+6-BA 2.0 mg/L+NAA 1.0 mg/L medium. Subsequent transfer of germinated somatic embryos to MS+IAA 0.5 mg/L+IBA 1.0 mg/L medium for 20 days resulted in robust tissue culture seedlings, with a survival rate of 93.33% when transplanted into a seedling substrate consisting of soil: perlite (3:1, v/v). This study not only marks the inaugural instance of somatic embryogenesis in Polygonatum species but also paves a foundation for the advent of breeding traditional Chinese medicinal herb P. cyrtonema via recent advanced biotechnological strategies.

A Walk on the Wild Side: Genome Editing of Tuber-Bearing Solanum bulbocastanum.

Solanum bulbocastanum is a wild diploid tuber-bearing plant. We here demonstrate transgene-free genome editing of S. bulbocastanum protoplasts and regeneration of gene-edited plants. We use ribonucleoproteins, consisting of Cas9 and sgRNA, assembled in vitro, to target a gene belonging to the nitrate and peptide transporter family. Four different sgRNAs were designed and we observed efficiency in gene-editing in the protoplast pool between 8.5% and 12.4%. Twenty-one plants were re-generated from microcalli developed from individual protoplasts. In three of the plants we found that the target gene had been edited. Two of the edited plants had deletion mutations introduced into both alleles, whereas one only had a mutation in one of the alleles. Our work demonstrates that protocols for the transformation of Solanum tuberosum can be optimized to be applied to a wild Solanum species.

N6-benzyladenine (BAP)-Based Seed Preconditioning Enhances the Shoot Regeneration of Seedling-Derived Explants for Subsequent Indirect Gene Transfer in Soybeans (Glycine max [L.] Merrill.)

This study evaluated the effects of N6-benzyladenine (BAP) seed preconditioning and seedling-derived explants on in vitro plant regeneration potential in soybeans (Glycine max [L.] Merrill.). The findings showed that seed preconditioning with 2.55 mg/L BAP prior to germination significantly influenced seedling establishment and the development of shoots, shoot elongation, and rooting on MS media supplemented with BAP and TDZ, compared to the negative (MS-NC) and positive (MS-NP) controls. The results also showed significant differences based on the genotypes, with Dundee recording 91.0% germination over a minimum of 5 days, compared to 74.2% with Peking, followed by 87.5% and 80.0% overall shoot induction frequency in these genotypes, respectively. Regenerated shoots were successfully elongated on MS medium supplemented with 0.5 mg/L BAP plus 0.6 mg/L GA3 and rooted on hormone-free medium, for 3‒4 weeks, and then hardened in the acclimatization growth room under elevated light levels. Overall, this study revealed that BAP preconditioning of seeds enhances the frequency of bud initiation and shoot proliferation, mostly in whole-seedling and cotyledonary node explants subcultured on MS-E and MS-A media supplemented with BAP in combination with TDZ.

Autonomous differentiation of transgenic cells requiring no external hormone application: the endogenous gene expression and phytohormone behaviors.

The ectopic overexpression of developmental regulator (DR) genes has been reported to improve the transformation in recalcitrant plant species because of the promotion of cellular differentiation during cell culture processes. In other words, the external plant growth regulator (PGR) application during the tissue and cell culture process is still required in cases utilizing DR genes for plant regeneration. Here, the effect of Arabidopsis BABY BOOM (BBM) and WUSCHEL (WUS) on the differentiation of tobacco transgenic cells was examined. We found that the SRDX fusion to WUS, when co-expressed with the BBM-VP16 fusion gene, significantly influenced the induction of autonomous differentiation under PGR-free culture conditions, with similar effects in some other plant species. Furthermore, to understand the endogenous background underlying cell differentiation toward regeneration, phytohormone and RNA-seq analyses were performed using tobacco leaf explants in which transgenic cells were autonomously differentiating. The levels of active auxins, cytokinins, abscisic acid, and inactive gibberellins increased as cell differentiation proceeded toward organogenesis. Gene Ontology terms related to phytohormones and organogenesis were identified as differentially expressed genes, in addition to those related to polysaccharide and nitrate metabolism. The qRT-PCR four selected genes as DEGs supported the RNA-seq data. This differentiation induction system and the reported phytohormone and transcript profiles provide a foundation for the development of PGR-free tissue cultures of various plant species, facilitating future biotechnological breeding.

Potential effects of brown howler monkey extinction on dispersal services in fragmented forests void of large dispersers.

The dispersal of large-seeded species strongly depends on medium-sized and large frugivores, such as primates, which are highly susceptible to population declines. In the Atlantic Forest, brown howler monkeys Alouatta guariba are medium-sized folivorous-frugivorous species that are likely to occur in small to large fragments where the largest frugivores are extinct. However, populations of this primate have been suffering from forest fragmentation, habitat loss, hunting, and the direct and indirect effects of yellow fever outbreaks, which increase the importance of understanding their role as seed dispersers and the impacts of their potential loss. The richness and abundance of large-seeded species might also be reduced in smaller fragments, which could directly affect the magnitude of the potential impact of disperser extinction on plant recruitment. Here, we tested the following mutually exclusive predictions on the effect of fragment size on plant richness and relative density of medium- and large-seeded species consumed by brown howler monkeys in fragments smaller than 1500ha: the number and the relative density of plant species potentially affected by the local extinction of these monkeys will be (1) directly related to forest fragment size, or (2) not related to forest fragment size. Plant richness and the relative density of large- and medium-sized seed species consumed by brown howler monkeys did not vary with fragment size, corroborating our second prediction. Thus, the local extinction of brown howler monkeys would have a similar potentially negative impact on plant regeneration for the range of tested fragment sizes. We discuss the limitations of our results and suggest other lines of enquiry for the refinement of our conclusions.

Establishment of somatic embryogenesis regeneration system and transcriptome analysis of early somatic embryogenesis in Litchi chinensis

Litchi chinensis Sonn. is an important economic fruit tree in tropical and subtropical regions. Regrettably, the efficiency of plant regeneration via somatic embryogenesis in litchi is typically low due to the poor conversion of embryos to plants. The purpose of this study was to establish a regeneration system via somatic embryogenesis from immature embryos explants in ‘Heiye’ cultivar of litchi. Our results demonstrated that MS medium supplemented with 2.0mg L−1 2,4-D was optimal for callus induction. For somatic embryo (SE) induction, MS medium containing 0.5 g L−1 activated charcoal (AC) was the most effective, while the use of zeatin (ZT) and thidiazuron (TDZ) resulted in abnormal somatic embryos. The rooting and regeneration rate of 2.15% and 17.5%, respectively, were achieved using MS medium supplemented with 0.5 g L−1 AC. Furthermore, transcriptome analysis was performed on embryogenic callus (EC), globular embryo (GE), and heart embryo (HE) to explore the molecular mechanisms of early somatic embryogenesis. 2,587 common DEGs between EC_vs_GE and EC_vs_HE were identified, and the expression patterns of these common DEGs were separated into twelve major clusters. GO annotation and KEGG pathway analysis revealed that these common DEGs were implicated in plant hormone signal transduction, auxin-activated signaling pathway, and other biological processes. Additionally, differentially expression transcription factors were identified, and the function of LcBBM2 which is specifically highly expressed during early somatic embryogenesis was verified. Overexpression of LcBBM2 in tomato promotes callus and shoot formation. Therefore, this study can provide a theoretical basis and technical support for genetic breeding improvement of litchi.

Response surface methodology and artificial intelligence modeling for in vitro regeneration of Brazilian micro sword (Lilaeopsis brasiliensis)

In this study, response surface methodology (RSM) was used to optimize in vitro regeneration of the Brazilian micro sword (Lilaeopsis brasiliensis) aquatic plant, followed by data prediction and validation using machine learning algorithms. The basal salt, sucrose and Benzyaminopurine (BAP) concentrations were derived from Box-Behnken design of RSM. The response surface regression analysis revealed that 1.0 g/L MS + 0.1 mg/L BAP + 25 g/L sucrose was optimized for maximum regeneration (100%), shoot counts (63.2), and fresh weight (1.382 g). The RSM-based predicted scores were fairly similar to the actual scores, which were 100% regeneration, 63.39 shoot counts, and 1.44 g fresh weight. Pareto charts analysis illustrated the significance of MS for regeneration and fresh weight but remained insignificant. Conversely, MS × BAP was found to be the most crucial factor for the shoot counts, with MS coming in second and having a major influence. The analysis of the normal plot ascertained the negative impact of elevated MS concentration on shoot counts and enhanced shoot counts from the combination of MS × BAP. Results were further optimized by constructing contour and surface plots. The response optimizer tool demonstrated that maximum shoot counts of 63.26 and 1.454 g fresh weight can be taken from the combination of 1.0 g/L MS + 0.114 mg/L BAP + 23.94 g/L. Using three distinct performance criterias, the results of machine learning models showed that the multilayer perceptron (MLP) model performed better than the random forest (RF) model. Our findings suggest that the results may be utilized to optimize various input variables using RSM and verified via ML models.Key messageOptimization of in vitro whole plant regeneration of Brazilian sword wood using response surface methodologyData analysis through ANOVA, response surface regression anlaysis and machine learningGraphical presentation of data via Pareto charts, normal plots, contour plots and surface plots for optimizationBetter performance of ANN-based MLP model compared to decision tree based RF modelGraphical abstract

Identification and characterization of the WOX Gene Family revealed two WUS Clade Members associated with embryo development in Cunninghamia lanceolata

The WUSCHEL-related homeobox (WOX) gene family is vital for plant development and stress response. In this study, we conducted a comprehensive analysis of WOX genes in Cunninghamia lanceolata (C. lanceolata) and subsequently explored the potential roles of two ClWOX genes within the WUS clade. In total, six ClWOX genes were identified through a full-length transcriptome analysis. These genes, exhibiting conserved structural and functional motifs, were assigned to the ancient clade and Modern/WUS clade, respectively, through a phylogenetic analysis. Our expression analysis indicated that these ClWOX genes were highly expressed in the middle and late developmental stages of zygotic embryos in C. lanceolata. Moreover, only ClWOX5 and ClWOX6 within the Modern/WUS clade exhibited transcriptional activity, and their expressions were also induced in response to auxin and wounding. Overexpression of ClWOX5 and ClWOX6 in Arabidopsis caused a partially sterile phenotype, resulting in a very low seed setting rate. Transcriptomic analysis revealed that expressions of many embryo-defective (EMB) genes, phytohormone-related genes, and transcription factors (TFs) were dramatically altered in ClWOX5 and ClWOX6 transgenic plants, which suggested that ClWOX5 and ClWOX6 may play specific important roles in embryo development via complex gene networks. In addition, overexpression of ClWOX5 and ClWOX6 in leaf segments promoted shoot regeneration in tobacco, indicating that ClWOX5 and ClWOX6 can promote plant regeneration and could be used to improve genetic transformation. In conclusion, these results help to elucidate the function of the WOX gene and provide a valuable basis for future studies of the developmental regulation and applications of WOX genes in C. lanceolata.

High Efficiency In vitro Whole Plant Regeneration via Desiccated Callus in Oryza sativa cv. MTU1010

For most people across the planet, rice is also considered as most beneficial crops in terms of fulfilling every day's energy and nutritional needs. Worldwide food safety is seriously threatened since severe limitations on rice output caused by both abiotic and biotic variables. Even though rice features much more developed regeneration mechanism than other agricultural crops, majority of desi cultivars continue to stay as impervious to genetic alterations and regeneration. Therefore, among preliminary steps in the transgenic plant generation necessitates to improve tissue culture procedures to produce viable plants via embryogenic calli mediated regeneration. In this study, we reporting that enhanced rate of plant regeneration was observed from mature seed-derived embryogenic calli of the indica rice cultivar MTU1010 following partially desiccation procedure. We studied the effects of several plant growth regulators on the efficiency of inducing embryogenic callus and regeneration of whole plantlets. The MS-CIM medium with 2.0 mg-1 2,4, D showed the highest callus induction percentage. Maximum rate of shoot regeneration (92%) was observed from 48hr partially dehydrated calli, when enriched with high cytokinin (2 mg/l kinetin) and lower auxin (0.5 mg/l NAA) concentrations. Calli that had been moderately dehydrated showed significantly greater regeneration rates than undehydrated calli. Upon being transferred onto a rooting medium, the well-rooted plantlets were shifted to shaded glass house for hardening. Hence, this approach is rather simple to apply and can be employed in genetic modification studies alongside other biotechnological endeavours.

Клональное микроразмножение лесных ягодных растений рода Rubus

Cloudberry (Rubus chamaemorus L.) and arctic raspberry (Rubus arcticus L.) are highly nutritional and medicinal but lowyield berries, with some populations being on the verge of extinction. Micropropagation biotechnologies are cost-effective and may provide healthy and plentiful planting material for these valuable berries. Clonal micropropagation of cloudberry and arctic raspberry requires new methods adapted for Russian varieties. This research featured the effect of sterilizing agents on the survival rate of explants of R. chamaemorus and R. arcticus, as well as the effect of growth regulators in the nutrient medium on their organogenesis in vitro.&#x0D; Berries obtained from regenerant plants of R. chamaemorus (Arkhangelsk and Vologda varieties) and R. arcticus (Sofia and Galina varieties) underwent a biochemical analysis. Further research involved the effect of sterilizing agents and exposure time on the viability of explants, as well as the effect of the nutrient medium composition and the concentration of growth regulators on the development of microshoots and roots in vitro.&#x0D; In case of lateral buds, the highest survival rate of both types of explants (80–96%) belonged to the samples sterilized with 0.2% silver nitrate and 5% Lysoformin 3000 for 15 min. In case of etiolated shoots (79–100%), it was the samples treated with 0.2% silver nitrate, 0.01% Nika 2, and 5% Lysoformin 3000 for 10 min. The maximal total length of microshoots in R. chamaemorus (19.4–22.7 cm) was registered at 0.1 mg/L Cytodef in the Murashige and Skoog medium. The maximal total length of roots (46.0–56.6 cm) was obtained when the medium contained 0.5 mL/L indolylacetic acid. As for R. arcticus, the maximal total lengths of microshoots (22.4–22.8 cm) and roots (86.6–89.3 cm) occurred at the same concentrations of growth regulators on 1/2 Murashige and Skoog medium.&#x0D; In this research, Cytodef and indolylacetic acid applied in the process of clonal micropropagation of R. chamaemorus and R. arcticus made it possible to increase the yield of high-quality planting material for commercial plantations.

Plant regeneration capacity in seeds of three species of Miconia (Melastomataceae) may be related to endogenous polyamine profiles.

In plant tissue culture, differences in endogenous levels of species-specific plant growth regulators (PGRs) may explain differences in regenerative capacity. In the case of polyamines (PAs), their dynamics and distribution may vary between species, genotypes, tissues, and developmental pathways, such as sexual reproduction and apomixis. In this study, for the first time, we aimed to assess the impact of varying endogenous PAs levels in seeds from distinct reproductive modes in Miconia spp. (Melastomataceae), on their in vitro regenerative capacity. We quantified the free PAs endogenous content in seeds of Miconia australis (obligate apomictic), Miconia hyemalis (facultative apomictic), and Miconia sellowiana (sexual) and evaluated their in vitro regenerative potential in WPM culture medium supplemented with a combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). The morphogenic responses were characterized by light microscopy and scanning electron microscopy and discussed regarding the endogenous PAs profiles found. Seeds of M. sellowiana presented approximately eight times more putrescine than M. australis, which was associated with a higher percentage of regenerated calluses (76.67%) than M. australis (5.56%). On the other hand, spermine levels were significantly higher in M. australis. Spermine is indicated as an inhibitor of auxin-carrying gene expression, which may have contributed to its lower regenerative capacity under the tested conditions. These findings provide important insights into in vitro morphogenesis mechanisms in Miconia and highlight the significance of endogenous PA levels in plant regeneration. These discoveries can potentially optimize future regeneration protocols in Miconia, a plant group still underexplored in this area.

Interplay of biotic and abiotic factors shapes tree seedling growth and root-associated microbial communities

Root-associated microbes can alleviate plant abiotic stresses, thus potentially supporting adaptation to a changing climate or to novel environments during range expansion. While climate change is extending plant species fundamental niches northward, the distribution and colonization of mutualists (e.g., arbuscular mycorrhizal fungi) and pathogens may constrain plant growth and regeneration. Yet, the degree to which biotic and abiotic factors impact plant performance and associated microbial communities at the edge of their distribution remains unclear. Here, we use root microscopy, coupled with amplicon sequencing, to study bacterial, fungal, and mycorrhizal root-associated microbial communities from sugar maple seedlings distributed across two temperate-to-boreal elevational gradients in southern Québec, Canada. Our findings demonstrate that soil pH, soil Ca, and distance to sugar maple trees are key drivers of root-associated microbial communities, overshadowing the influence of elevation. Interestingly, changes in root fungal community composition mediate an indirect effect of soil pH on seedling growth, a pattern consistent at both sites. Overall, our findings highlight a complex role of biotic and abiotic factors in shaping tree-microbe interactions, which are in turn correlated with seedling growth. These findings have important ramifications for tree range expansion in response to shifting climatic niches.

Episodic deterioration of plant diversity in rich fens with or without in situ oil sands exploration disturbance

Peatlands with ground surfaces near the water table may be sensitive to disturbances that reduce this distance to water. Shrubby rich fens (SRF) in northern Alberta, Canada, are restricted to the wettest landscape positions and are characterized by brown mosses and stunted trees on weakly consolidated and saturated substrates. In situ oil sands exploration (OSE) practices have established thousands of drilling pads to explore for deeply buried bitumen deposits, with many located on peatlands. In the development of OSE pads on peatlands, the vegetation layer is removed, eliminating the functionally important hummock‐hollow surface topography. We examined the spontaneous regeneration of bryophytes and vascular plants and surface microtopography on SRF, 7 and again 14 years after pad abandonment. In both sampling years, drilling pad surfaces were closer to the water table (mean pad elevation 2012 = 3.3 cm, 2019 = 5.6 cm) and had minor topography compared to adjacent reference habitat. We found, however, over the 7 years, that for both drilling pads and reference habitat, bryophyte and vascular plant richness decreased, and most species declined in frequency. Furthermore, for pads and reference habitat, density of tree seedlings decreased, and expansion of hummock‐forming mosses was limited. Findings suggest that drilling pads and their natural counterparts on SRF may be prone to episodic reductions in plant diversity and ecosystem retrogression, likely resulting from prolonged surface saturation during summers with high rainfall. Because of their wet landscape positions and environmental vulnerability, SRF are likely the peatland habitats slowest to recover following OSE pad construction.

Organelle Ca2+/CAM1-SELTP confers somatic cell embryogenic competence acquisition and transformation in plant regeneration.

Somatic cell totipotency in plant regeneration represents the forefront of the compelling scientific puzzles and one of the most challenging problems in biology. How somatic embryogenic competence is achieved in regeneration remains elusive. Here, we discover uncharacterized organelle-based embryogenic differentiation processes of intracellular acquisition and intercellular transformation, and demonstrate the underlying regulatory system of somatic embryogenesis-associated lipid transfer protein (SELTP) and its interactor calmodulin1 (CAM1) in cotton as the pioneer crop for biotechnology application. The synergistic CAM1 and SELTP exhibit consistent dynamical amyloplast-plasmodesmata (PD) localization patterns but show opposite functional effects. CAM1 inhibits the effect of SELTP to regulate embryogenic differentiation for plant regeneration. It is noteworthy that callus grafting assay reflects intercellular trafficking of CAM1 through PD for embryogenic transformation. This work originally provides insight into the mechanisms responsible for embryogenic competence acquisition and transformation mediated by the Ca2+/CAM1-SELTP regulatory pathway, suggesting a principle for plant regeneration and cell/genetic engineering.

Direct Plant Regeneration from Nodal Explants of Grapefruit (Citrus paradisi Macfad.)

Aims: The present study was undertaken to develop a protocol for in vitro regeneration and multiplication of disease-free quality planting material of Grapefruit (Citrus paradisi Macfad.). Study Design: Completely Randomized Design (CRD) Place and Duration of Study: The study was conducted at the Plant Tissue Culture Laboratory, Department of Horticulture, University of Agricultural Sciences, Gandhi Krishi Vignana Kendra, Bengaluru, from 2020 to 21. Methodology: Nodal segments from the young shoots of field growing plants were used as explants to conduct the experiment. The explants were sterilized and placed on MS medium supplemented with different concentrations and combinations of growth regulators, namely BAP, Kinetin and Gibberellic acid. Results: Among the different combinations of growth regulators, the combination of BAP 1.0 mgL-1 and GA3 3.0 mgL-1 produced maximum number of shoots (7) from nodal segments Conclusion: The use of growth regulators such as BAP and GA3 is reliable for shoot regeneration even when the field explants are used.

A Review on Flower Bulb Micropropagation: Challenges and Opportunities

This comprehensive review scrutinizes tissue culture and micropropagation methodologies in geophytes, focusing on bulbous plants. The examination encompasses key stages, including somatic embryogenesis, bulb growth, dormancy breaking, and planting. Studies underscore the pivotal role of plant growth regulators (PGRs) in plant regeneration and bulb growth. Bioreactor systems for healthy plant regeneration, rooting methods, acclimatization strategies, and considerations for ex vitro survival are elucidated. The review also delves into somaclonal variation dynamics and acknowledges the burgeoning field of gene editing, particularly Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) studies, as a promising avenue for enhancing valuable compound content in geophytes. In addition to addressing challenges in flower bulb micropropagation, this review briefly highlights emerging opportunities, including the potential integration of artificial intelligence (AI) to optimize culture conditions, predict growth parameters, and enhance efficiency in bulb production. The conclusion emphasizes the necessity of a multifaceted approach integrating biochemistry, physiology, and molecular biology to address existing challenges and improve tissue culture protocols for diverse geophyte species. This review article also intends to highlight how tissue culture techniques could contribute to the development and valorization of flower bulbs in today’s scenario of the ornamental industry.

Structure and vulnerability of the ichthyochory network in wetland forests of Central Amazonia

AbstractThe interaction between fish and plants is vital for the dynamics of ecosystems since it influences plant distribution and regeneration patterns. In the floodable areas of the Amazon, these interactions are mainly structured by the flood pulse, which enables seed dispersal via water (hydrochory) and fish (ichthyochory), and which contributes to the formation of complex mutualistic networks. Our study evaluated the structure (modularity, nestedness, specialisation, and robustness) of the network of dispersal of seeds by frugivorous fish in a floodplain forest in Central Amazonia. We expect the binary network to have a nested pattern, while the weighted network is expected to have a non‐nested structure; that generalist frugivorous species should constitute the core of the network, while specialist frugivorous species will be peripheral; that the size of fish and seeds is related to specialisation, and that the robustness of the network weakens as generalist frugivorous fish are removed. We found 5012 intact seeds from 49 plant species in the digestive tracts of 11 species of frugivorous fish. A nested and modular pattern was found for the binary and weighted networks, albeit with a low degree of nestedness. Our network proved relatively robust when the frugivores were removed, whereby all the fish and plant species became peripheral. No relationship was found between the size of the fish and the seeds and the degree of specialisation. Considering the anthropogenic impacts that can cause modifications in seed dispersal networks, knowing the structure of mutualistic networks is fundamental in order to be able infer the vulnerability of the interactions as a result of changes in the ecosystem.