Efficient Plant Regeneration Research Articles

Establishment of callus induction and plantlet regeneration systems of Peucedanum Praeruptorum dunn based on the tissue culture method

BackgroundPeucedanum praeruptorum Dunn has typical stacked umbels and medicinal value; however, the lack of an effective tissue culture system for P. praeruptorum has limited the large-scale propagation of its seedlings.ResultsWe systematically established an in vitro regeneration system for P. praeruptorum using young leaves and stems as explants. Tissue culture plantlets were successfully obtained within 123 and 90 d of somatic embryogenesis and organogenesis, respectively. Combined plant growth regulators (PGRs) were optimized to promote efficient plant regeneration at each stage of the culture process. Specifically, embryogenic callus induction was superior in Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 6-benzyladenine (BA) and 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). For somatic embryonic development, the highest differentiation rates were achieved using BA, 2,4-D, and 6-furfuryl aminopurine (6-KT). Induction of organogenesis resulted in the highest differentiation rates and proliferation coefficients of buds in MS medium supplemented with BA and α-naphthaleneacetic acid (NAA). Moreover, regeneration of P. praeruptorum seedlings was achieved by adjusting the BA and indole-3-butyric acid (IBA) concentrations in 1/2 MS medium.ConclusionOur results provide a technical system for the rapid propagation of P. praeruptorum, which can facilitate germplasm improvement, resource conservation, and further genetic transformation of Peucedanum species.

Crucial Factors Influencing the Efficiency of Androgenesis in Oat (Avena sativa L.) Through Anther and Microspore Cultures

Historically, traditional crossbreeding schemes have predominated in oat breeding. In vitro culture techniques seek to expedite the breeding process and enhance selection efficiency. Maximum yields are achieved from hybrid plants produced by crossing pure (homozygous) lines with the desired traits. Homozygous lines can be produced through conventional breeding methods, which are time-consuming and costly. Alternatively, the production of homozygous lines can be accelerated by producing doubled haploid (DH) plants derived from (haploid) male gametophytes or their microspores (androgenesis). This method condenses the various stages required for producing homozygous lines in a single generation, resulting in significant time and cost savings. These and other advantages render androgenic DHs the preferred choice in numerous important crops where any of the various in vitro experimental techniques (anthers culture or isolated microspores culture) are well-established. However, in the case of oat (Avena sativa L.), an efficient plant regeneration method remains not very effective compared to the most common cereals, possibly due to the known recalcitrance of this cereal to in vitro culture. This review presents the methods through anther and microspore cultures utilized in the production of oat DHs revealing the crucial factors influencing the efficiency of this method in oat (Avena sativa L.).

Efficient Plantlet Regeneration from Branches in Mangifera indica L.

Mango (Mangifera indica L.) is one of the most significant tropical and subtropical fruit species, with high ecological and economic value. However, research on the in vitro culture of mangoes is relatively weak, so establishing an efficient and stable mango plant regeneration system is of great significance. In this study, a preliminary mango regeneration system was established with Mangifera indica L. cv. Keitt from young branches as the starting explants. The results showed that the optimal plant growth regulator (PGR) formula for direct adventitious shoot induction on the branches was 1 mg/L 6-benzylaminopurine (6-BA) + 0.1 mg/L a-naphthaleneacetic acid (NAA), with an adventitious shoot induction rate of 73.63% and an average of 6.76 adventitious shoots. The optimal basal medium for adventitious shoot induction was wood plant medium (WPM), with an adventitious shoot induction rate of 63.87% and an average of 5.21 adventitious shoots. The optimal culture medium for adventitious shoot elongation was WPM + 1 mg/L 6-BA + 0.5 mg/L NAA, with an adventitious shoot elongation rate of 89.33% and an average length of 5.17 cm. The optimal formula for the induction of mango rooting was Douglas fir cotyledon revised medium (DCR) + 3 mg/L indole-3-butyric acid (IBA), with a maximum rooting rate of 66.13% and an average rooting quantity of 6.43. The genetic fidelity of the in vitro-regenerated plants was evaluated using inter-simple sequence repeat (ISSR) molecular markers. There was no difference between the in vitro-regenerated plants and the parent plant. This study provides an efficient and stable propagation system for Mangifera indica L., laying the foundation for its rapid propagation and genetic improvement.

β-glucans, SAM, and GSH fluctuations in barley anther tissue culture conditions affect regenerants’ DNA methylation and GPRE

BackgroundMicrospore embryogenesis is a process that produces doubled haploids in tissue culture environments and is widely used in cereal plants. The efficient production of green regenerants requires stresses that could be sensed at the level of glycolysis, followed by the Krebs cycle and electron transfer chain. The latter can be affected by Cu(II) ion concentration in the induction media acting as cofactors of biochemical reactions, indirectly influencing the production of glutathione (GSH) and S-adenosyl-L-methionine (SAM) and thereby affecting epigenetic mechanisms involving DNA methylation (demethylation—DM, de novo methylation—DNM). The conclusions mentioned were acquired from research on triticale regenerants, but there is no similar research on barley. In this way, the study looks at how DNM, DM, Cu(II), SAM, GSH, and β-glucan affect the ability of green plant regeneration efficiency (GPRE).ResultsThe experiment involved spring barley regenerants obtained through anther culture. Nine variants (trials) of induction media were created by adding copper (CuSO4: 0.1; 5; 10 µM) and silver salts (AgNO3: 0; 10; 60 µM), with varying incubation times for the anthers (21, 28, and 35 days). Changes in DNA methylation were estimated using the DArTseqMet molecular marker method, which also detects cytosine methylation.Phenotype variability in β-glucans, SAM and GSH induced by the nutrient treatments was assessed using tentative assignments based on the Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy. The effectiveness of green plant regeneration ranged from 0.1 to 2.91 plants per 100 plated anthers. The level of demethylation ranged from 7.61 to 32.29, while de novo methylation reached values ranging from 6.83 to 32.27. The paper demonstrates that the samples from specific in vitro conditions (trials) formed tight groups linked to the factors contributing to the two main components responsible for 55.05% of the variance (to the first component DNM, DM, to the second component GSH, β-glucans, Cu(II), GPRE).ConclusionsWe can conclude that in vitro tissue culture conditions affect biochemical levels, DNA methylation changes, and GPRE. Increasing Cu(II) concentration in the IM impacts the metabolism and DNA methylation, elevating GPRE. Thus, changing Cu(II) concentration in the IM is fair to expect to boost GPRE.

Meta-Topolin induced highly efficient plant regeneration from various explants of eggplant (Solanum melongena L.)

Meta-Topolin induced highly efficient plant regeneration from various explants of eggplant (Solanum melongena L.)

Developmental regulators in promoting genetic transformation efficiency in maize and other plants

Given global agricultural challenges such as population growth, climate change, and limitations on resources and the environment, as well as increasing diversity in breeding goals, relying on traditional breeding methods is inadequate to provide food security requirements and promote sustainable development. Genetic transformation technology has become an effective tool for performing functional genomics research and molecular breeding. In this study, we conducted an in-depth analysis of 1669 literary works to investigate the potential of developmental regulators (DRs) in enhancing the efficiency of plant genetic transformation, with a concentration on their use in maize. Through multi-omics data analysis, we identified 12 homologous DRs from various species that are potentially applicable to maize. We identified a total of 41 possible disease resistances (DRs) for maize genetic transformation. Further experimental verification of ZmWIND1, a novel regulator belonging to the ERF/AP2 transcription factor (TF) family, showed that it significantly improved the efficiency of plant regeneration and transformation efficiency in maize. Specifically, compared to the control group, the callus induction rates for the pG3GB411-ZmWIND1 vector increased to 60.22 % and 47.85 % in Xiang249 and Zheng58, respectively. Transformation efficiency increased to 37.5 % in Xiang249 and 16.56 % in Zheng58, both significantly surpassing the control group. These findings have the potential to broaden the range of transformable maize varieties and lines, as well as introduce new genetic transformation methods in agricultural biotechnology, underscoring the immense potential to enhance genetic transformation efficiency through systematic exploration and application of DRs in maize.

A simple and efficient non-tissue culture method for genetic transformation of grape immature zygotic embryos via VvBBM overexpression

The process of genetic transformation of grapevine has long been hampered by the inefficiency and complexity of tissue culture-based methods, severely limiting cultivar improvement and functional genome research. In this study, we aimed to develop a novel, non-tissue culture-based method for the genetic transformation of grape immature zygotic embryos without relying on tissue culture. This approach involves the overexpression of the Vitis vinifera BABY BOOM (VvBBM) gene, a key member of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor family, which is known to enhance plant regeneration and transformation efficiency. Using ‘Cabernet Sauvignon’ immature zygotic embryos, we achieved a transformation efficiency of 42.85 % under non-tissue culture conditions, significantly reducing the cycle duration to approximately two months compared to traditional genetic transformation methods. Phenotypic and molecular analyses of the transformed grapevine plants confirmed the integration and overexpression of the VvBBM gene, resulting in improved growth metrics, including increased plant height, root length, and total root number, in a genotype-independent manner. Our results highlight the potential of VvBBM overexpression to circumvent the limitations imposed by tissue culture dependence, providing a rapid, efficient, and scalable alternative to conventional methods. Our study not only provides a practical approach to overcoming the barriers to genetic transformation in grapevine, but also opens new avenues for the application of this technology in the breeding of other economically important crops.

Comparative Analyses of Green Plantlet Regeneration in Barley (Hordeum vulgare L.) Anther Culture

The efficient doubled haploid (DH) plant production methods play a key role in accelerating the breeding of new varieties and hybrids in cultivated plants. Consequently, DH plant production methods are continuously improving for barley (Hordeum vulgare L.) breeding and research programs. Two plant regeneration (FHGR and K4NB) and three rooting media (MSr, N6I and ½N6I + Ca) were compared with four F1 barley cross-combinations to clarify the effect of medium on the regeneration of green and albino plantlets and acclimatization. The plant regeneration efficiency was higher using K4NB medium (74.53 green plantlets/100 anthers and 30.85 albino/100 anthers) compared to FHGR (55.77 green plantlets/100anthers and 21.32 albino/100 anthers). The percentage of acclimatization was highest when the K4NB regeneration medium was combined with the MSr rooting medium. Altogether, 61.83% of the anther culture-derived plantlets of 8 cross-combinations acclimatized to the greenhouse conditions, and 1403 acclimatized plantlets were produced from the F1 cross-combinations. Haploid (22.52%), diploid (69.37%) and tetraploid (8.11%) plantlets were identified among the 111 tested green plantlets by flow cytometric analyses. The tetraploid lines can be explored to offer new scopes for future barley research and breeding directions. Nearly one thousand DH plants have been integrated into our barley breeding program.

Proteomic and Phosphoproteomic Analyses during Plant Regeneration Initiation in Cotton (Gossypium hirsutum L.).

Somatic embryogenesis (SE) is a biotechnological tool used to generate new individuals and is the preferred method for rapid plant regeneration. However, the molecular basis underlying somatic cell regeneration through SE is not yet fully understood, particularly regarding interactions between the proteome and post-translational modifications. Here, we performed association analysis of high-throughput proteomics and phosphoproteomics in three representative samples (non-embryogenic calli, NEC; primary embryogenic calli, PEC; globular embryos, GE) during the initiation of plant regeneration in cotton, a pioneer crop for genetic biotechnology applications. Our results showed that protein accumulation is positively regulated by phosphorylation during SE, as revealed by correlation analyses. Of the 1418 proteins that were differentially accumulated in the proteome and the 1106 phosphoproteins that were differentially regulated in the phosphoproteome, 115 proteins with 229 phosphorylation sites overlapped (co-differential). Furthermore, seven dynamic trajectory patterns of differentially accumulated proteins (DAPs) and the correlated differentially regulated phosphoproteins (DRPPs) pairs with enrichment features were observed. During the initiation of plant regeneration, functional enrichment analysis revealed that the overlapping proteins (DAPs-DRPPs) were considerably enriched in cellular nitrogen metabolism, spliceosome formation, and reproductive structure development. Moreover, 198 DRPPs (387 phosphorylation sites) were specifically regulated at the phosphorylation level and showed four patterns of stage-enriched phosphorylation susceptibility. Furthermore, enrichment annotation analysis revealed that these phosphoproteins were significantly enriched in endosomal transport and nucleus organization processes. During embryogenic differentiation, we identified five DAPs-DRPPs with significantly enriched characteristic patterns. These proteins may play essential roles in transcriptional regulation and signaling events that initiate plant regeneration through protein accumulation and/or phosphorylation modification. This study enriched the understanding of key proteins and their correlated phosphorylation patterns during plant regeneration, and also provided a reference for improving plant regeneration efficiency.

Efficient plant regeneration through callus culture in Hedychium spicatum Buch.Ham. Ex. D. Don using response surface methodology

Efficient plant regeneration through callus culture in Hedychium spicatum Buch.Ham. Ex. D. Don using response surface methodology

Effect of salt stress on plant regeneration efficiency in primed and non-primed seed's calli of rice (Oryza sativa L.) variety Swarna.

This study leads with the primed seeds of rice (var. Swarna) with distilled water (D.W.) and various concentrations of Mg(NO3)2 (0-8mM)/Kinetin (0-5ppm) alone or in combination with screen out the regeneration medium induced tolerance level of NaCl. To fulfill the objective, the primed and non-primed rice seeds were inoculated in MS medium supplemented with 30gL-1 maltose + 1gL-1 casein hydrolysate and 2mgL-1 of 2,4-D for callus induction and cultured up to 45days in two sets: one set for regeneration purpose in NaCl-induced regeneration medium and another set was used to study the physiological potentiality of the callus. The 45-day-old calli were transferred into regeneration medium MSR (MS medium for regeneration) (BAP: NAA: Kinetin = 4:1:1) containing NaCl with a concentration range of 0 to 300mM. The number of regenerating calli and shoot regeneration percentage, number of plantlets obtained from one callus, recovery of plantlets from each concentration of NaCl and proline estimation from the leaf of the regenerated plantlets were determined from one set obtained after 45days. The calli obtained from another set after 45days, the frequencies of total and embryogenic calli induction percentage, fresh and dry weights, proline content, nitrate reductase and superoxide dismutase activities were measured. The calli obtained from 2.5ppm kinetin + 4mMMg(NO3)2 primed seeds were showed best result as compared to the other treatments for the above-mentioned parameters in different concentrations of NaCl-induced medium and survive up to 200mM concentrations of NaCl.

Multiple cytokinin elevates indirect regeneration potential of indica rice mature embryo

The concept of producing high quality mature seed derived callus competent to genetic transformation followed by efficient full plant regeneration is not unique in rice. But the process is lacking in terms of regeneration efficiency, genotype dependency especially in indica rice. Further, most of the research has only been focused on elite indica cultivars and popular local varieties are neglected. Our extensive literature study also revealed that among all the factors responsible for higher rice regeneration efficiency, the combination, concentration, and type plant growth regulators (PGRs) are mostly responsible for indica rice genotypes. Hence, the present study is focused on standardization of popular local indica rice cultivars for callus induction and regeneration by tweaking the PGRs in culture media, according to their amenability. The plant regeneration potential was assessed on both traditional organogenesis frequency and morphology-based scoring matrix. This revealed the best suitable PGR concentration-combination for efficient organogenesis and the genotype Khitish to be most responsive for indirect regeneration (78.68 ± 1.50 %). The highly polymorphic SSR marker based clonal fidelity test also validated the absence of somaclonal variation among the regenerated plants. This study would be helpful in improving the regeneration potential in local indica rice varieties and genetic transformation studies.

Indirect plant regeneration in Dioscorea rotundata and Dioscorea alata yam genotypes using different explants

Yam (Dioscorea sp.) is an important staple crop for millions of smallholder farmers in West Africa. In this study, we aimed to develop an efficient indirect plant regeneration system for three important yam genotypes “Kukrupa,” “Mankrong Pona” and “Matches.” The potential of leaf primordia, petioles and axillary bud explants for callus induction on Murashige and Skoog (MS) medium supplemented with different concentrations (0, 3, 5, 7 and 9 mg/l) of 2,4-dicholorophenoxy acetic acid (2,4-D) or picloram were tested. Callus induction was influenced by auxin concentration as well as explant. The highest callus induction was achieved using leaf primordia (“Kukrupa” and “Mankrong Pona”) explants on 3 mg/l picloram or axillary buds (“Matches”) on 5 mg/l picloram. Transfer of callus to 6-benzylaminoapurine (BAP) amended medium resulted in the formation of at least two shoots per callus clump. Plantlets were obtained on basic yam medium supplemented with 0.2 mg/l 1-naphthalene acetic acid (NAA), 3 mg/l kinetin and 0.5 mg/l BAP after four weeks. Post acclimatization plantlet survival was high (75 − 82.5%). The indirect plant regeneration protocol reported through this study helps in the improvement of important yam genotypes through tissue culture-oriented genome editing, in vitro mutagenesis and also for its mass propagation.

Different phosphorus preferences among arbuscular and ectomycorrhizal trees in a subtropical forest

Phosphorus (P) is one of the most limiting elements for efficient plant growth and regeneration, coexisting plants are found to be able to partition soil P components through different types of mycorrhizal symbiosis. However, previous evidences were mostly based on artificial addition of different forms of P chemicals, there is still a lack of observational and experimental verifications on how tree species respond to soil P heterogeneity in natural soils. Herein, we conducted a field survey to investigate how spatial distribution of tree species with different types of mycorrhizal associations are related with soil P compositions. We found that arbuscular mycorrhizal (AM) trees tend to distribute in soils rich in inorganic P, while ectomycorrhizal (ECM) trees distribute in habitats with low inorganic P. We further established a shade-house experiment to evaluate how P availability of field soils affects seedling performance of AM and ECM trees. Biomass of AM seedlings was highest when grew in soil with the medium level of inorganic P, while ECM seedlings grown best in soil with the lowest inorganic P level. Seedling survival and mycorrhizal colonisation rates decreased, while specific root length and specific area of seedling roots increased, with increasing inorganic P concentrations. Using field soil with natural composition of P-forms and spatial heterogeneity, our findings confirmed the hypothesis that AM trees prefer inorganic P while ECM trees are able to utilise organic P in a more realistic setting, which regulated spatial distributions and promoted coexistence of subtropical tree species at local scale.

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.

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.

Overexpression of HbGRF4 or HbGRF4-HbGIF1 Chimera Improves the Efficiency of Somatic Embryogenesis in Hevea brasiliensis.

Transgenic technology is a crucial tool for gene functional analysis and targeted genetic modification in the para rubber tree (Hevea brasiliensis). However, low efficiency of plant regeneration via somatic embryogenesis remains a bottleneck of successful genetic transformation in H. brasiliensis. Enhancing expression of GROWTH-REGULATING FACTOR 4 (GRF4)-GRF-INTERACTING FACTOR 1 (GIF1) has been reported to significantly improve shoot and embryo regeneration in multiple crops. Here, we identified endogenous HbGRF4 and HbGIF1 from the rubber clone Reyan7-33-97, the expressions of which dramatically increased along with somatic embryo (SE) production. Intriguingly, overexpression of HbGRF4 or HbGRF4-HbGIF1 markedly enhanced the efficiency of embryogenesis in two H. brasiliensis callus lines with contrasting rates of SE production. Transcriptional profiling revealed that the genes involved in jasmonic acid response were up-regulated, whereas those in ethylene biosynthesis and response as well as the S-adenosylmethionine-dependent methyltransferase activity were down-regulated in HbGRF4- and HbGRF4-HbGIF1-overexpressing H. brasiliensis embryos. These findings open up a new avenue for improving SE production in rubber tree, and help to unravel the underlying mechanisms of HbGRF4-enhanced somatic embryogenesis.

Genetic Transformation of Potato without Antibiotic-Assisted Selection

The genetic engineering of plants often relies on the use of antibiotic or herbicide resistance genes for the initial selection of primary transgenic events. Nevertheless, the commercial release of genetically modified crops containing any marker gene encounters several challenges stemming from the lack of consumer acceptance. The development of strategies enabling the generation of marker-free transgenic plants presents an alternative to address public concerns regarding the safety of biotech crops. This study examined the capabilities of highly regenerative potato cultivars to develop transgenic plants without the presence of selective substances in their media. Internodal segments of in vitro potato plants were inoculated with the Agrobacterium strain AGL0 carrying plasmids, which contained the GFP or RFP gene driven by the CaMV 35S promoter to monitor the transformation process by observing in vivo green or red fluorescence. Despite the absence of selective pressure, inoculated explants demonstrated comparable or even higher transient expression compared to experiments based on antibiotic assistant selection. Consequently, under non-selective conditions, non-transgenic, chimeric, and fully fluorescent potato plantlets were concurrently developed. Among the five tested cultivars, the regeneration efficiency of non-chimeric transgenic plants varied from 0.9 (‘Chicago’) to 2.7 (#12-36-42) plants per 100 detached plantlets. Depending on the regenerative characteristics of potato varieties (early, intermediate, or late), a specific time interval can be determined when a blind collection of transgenic plantlets is more successful, streamlining the transformation procedure. The results indicate that the outlined procedure is simple and reproducible, consistently achieving the transformation efficiency of 7.3–12.0% (per 100 inoculated explants) in potato cultivars without selective pressure. The described transformation procedure holds the potential for obtaining cisgenic or intragenic potato plants with new valuable traits that do not carry marker genes.

An insight into tissue culture-induced variation origin shared between anther culture-derived triticale regenerants

BackgroundThe development of the plant in vitro techniques has brought about the variation identified in regenerants known as somaclonal or tissue culture-induced variation (TCIV). S-adenosyl-L-methionine (SAM), glutathione (GSH), low methylated pectins (LMP), and Cu(II) ions may be implicated in green plant regeneration efficiency (GPRE) and TCIV, according to studies in barley (Hordeum vulgare L.) and partially in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927). Using structural equation models (SEM), these metabolites have been connected to the metabolic pathways (Krebs and Yang cycles, glycolysis, transsulfuration), but not for triticale. Using metabolomic and (epi)genetic data, the study sought to develop a triticale regeneration efficiency statistical model. The culture’s induction medium was supplemented with various quantities of Cu(II) and Ag(I) ions for regeneration. The period of plant regeneration has also changed. The donor plant, anther-derived regenerants, and metAFLP were utilized to analyze TCIV concerning DNA in symmetric (CG, CHG) and asymmetric (CHH) sequence contexts. Attenuated Total Reflectance–Fourier Transfer Infrared (ATR-FTIR) spectroscopy was used to gather the metabolomic information on LMP, SAM, and GSH. To frame the data, a structural equation model was employed.ResultsAccording to metAFLP analysis, the average sequence change in the CHH context was 8.65%, and 0.58% was de novo methylation. Absorbances of FTIR spectra in regions specific for LMP, SAM, and GSH were used as variables values introduced to the SEM model. The average number of green regenerants per 100 plated anthers was 2.55.ConclusionsThe amounts of pectin demethylation, SAM, de novo methylation, and GSH are connected in the model to explain GPRE. By altering the concentration of Cu(II) ions in the medium, which influences the amount of pectin, triticale’s GPRE can be increased.

Efficient plant regeneration through direct shoot organogenesis and two-step rooting in Eucommia ulmoides Oliver.

Eucommia ulmoides Oliver (E. ulmoides Oliver), a multipurpose woody plant, holds great economic significance due to its expansive medicinal, food and industrial applications. The rapid advancement of E. ulmoides in various fields has resulted in the inadequacy of existing breeding methods to meet its growth and annual production demands. Consequently, there is an urgent need for innovative propagation strategies. This study introduces an optimized micropropagation protocol for E. ulmoides, facilitating direct shoot organogenesis from nodal segments with axillary buds. We systematically examined the impact of basal medium composition, plant growth regulators, photosynthetic photon flux density, and sucrose concentration on bud sprouting. Employing cuttings with axillary buds as propagation material, we achieved a shortened cultivation period of merely 4 weeks for bud elongation and proliferation, marking a substantial enhancement in propagation efficiency. Notably, the Driver Kuniyuki Walnut medium, supplemented with 20.0 g L-1 sucrose and 2.0 mg L-1 trans-zeatin, induced shoots sprouting with a 100% success rate and an average length of 5.18 cm per nodal segment, equating to a great bud propagation rate of approximately 500%. Furthermore, a light source with an intensity of 80 μmol m-2 s-1 was shown the most economical choice. To address the primary challenge of inducing roots in regenerated plants, we employed a refined two-step rooting technique. This method yielded the optimal rooting frequency of 93.02%, producing an average of 5.90 adventitious roots per plantlet, each with an average length of 2.77 cm. The micropropagation program developed in this work will be the cornerstone for the preservation of the germplasm of E. ulmoides and its long-term use in medicinal and industrial applications.