Plant Cell, Tissue and Organ Culture最新文献

Abstract

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 message

• Optimization of in vitro whole plant regeneration of Brazilian sword wood using response surface methodology

• Data analysis through ANOVA, response surface regression anlaysis and machine learning

• Graphical presentation of data via Pareto charts, normal plots, contour plots and surface plots for optimization

• Better performance of ANN-based MLP model compared to decision tree based RF model

Graphical abstract

Abstract

Plumbago zeylanica L., a wild shrub, is a vital natural source of plumbagin, a potent 1,4-naphthoquinone renowned for its anti-cancer properties, notably effective against breast, prostate, and ovarian cancers. Traditional plumbagin extraction, involving root uprooting and plant destruction, raises ecological concerns. The primary objective of this study is to enhance plumbagin production by incorporating the elicitation process into in vitro cultivation with regenerated plants that retain all of their intact organs. Seven different elicitors categorized into three distinct groups were employed to stimulate plumbagin content. Among the various elicitors used, this study marks the first application of biogenic silver nanoparticles (AgNPs) from Curcuma amada in stimulating plumbagin production in this plant. The maximum plumbagin content, recorded at 8.98 ± 0.24 mg/g dry weight basis, was found in the roots when elicited with AgNPs at a concentration of 15 mg/l. In addition to that, biotic elicitors (yeast extract, chitosan and casein hydrolysate) and heavy metals (lead, cobalt and nickel) also successfully elicit plumbagin in the root and aerial parts of the plants, quantified through High Performance Liquid Chromatography (HPLC). In our study, we found that certain elicitors induced root browning and tissue necrosis, as confirmed by propidium iodide (PI) staining. The most significant browning effects were observed with chitosan from biotic sources and lead from heavy metals, while no such effects were associated with AgNPs at any concentration. Utilizing intact, entire plants as the subjects for elicitation in our study is a valuable aspect. This approach closely replicates the natural process occurring in intact plants, enhancing the relevance of our findings to practical situations.

Clonal forestry is an important approach for intensive management as it involves vegetative propagation, which does not entail gene separation and recombination, thereby retaining the excellent traits of the parent trees. Exploring essential genes involved in rooting of cuttings seems urgent in Catalpa bungei as it is the capital method in vegetative propagation. In this study, we identified a homolog of the SHR gene involved in the development of adventitious roots (ARs) in C. bungei through multiple alignment, homologous cloning, qPCR detection, and transgenic techniques. The CbSHR gene encodes a protein of 445 amino acids, and the expression of the CbSHR gene reaches its peak at the callus differentiation stage (S4, about 30 days after cutting) during AR development. By overexpressing the CbSHR gene in tobacco, the number of roots and their length were increased compared with the wild-type line, thus identifying a pivotal homolog, the CbSHR gene in C. bungei, which promotes the initiation and elongation of ARs. This result provided a candidate gene for genetic improvement of cutting, and will also contribute to understanding the molecular mechanisms of AR development in C. bungei.

Abstract

The haploidy technique is a useful tool for quickly producing pure, fully homozygous lines. Artemisia annua L. is a medicinal plant that produces artemisinin, a widely used antimalarial drug. Because of its extremely small flowers (≤ 3.0 mm), this study conducted microscopic observations to determine the types of flowers of A. annua suitable for microspore embryogenesis, as well as their corresponding microspore development stages, and obtained embryoids (non-zygotic embryos) from isolated microspore cultures. The media for inducing embryoid production were based on Nitsch and Nitsch medium containing 13% or 17% sucrose and the following plant growth regulators: (1) a combination of naphthaleneacetic acid and 6-benzyladenine (MCA medium) and (2) a combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (MCAD medium). The results indicated that based on the proportions of uninucleate and binucleate microspores, flowers at the prebloom and early bloom stages contained sufficient late uninucleate to early binucleate microspores suitable for inducing embryogenesis. The production of microspore-derived embryogenic (MDE) structures was faster in MCA13 and MCA17 media than in MCAD13 and MCAD17 media. MCA13 and MCAD13 media induced the production of more callus-like structures than MCA17 and MCAD17 media. Thus, the addition of 2, 4-D to MCAD medium inhibited the growth of MDE structures. Globular embryoids emerged from the multicellular cluster.

Melia azedarach L. is an important multipurpose plant (ornamental, landscape, shade-tree, timber industry) with biopesticide and medicinal potential due to natural compounds, mainly limonoids that have insecticide and antimicrobial effect. Propagation of M. azedarach through conventional methods is difficult and azadirachtin production low, therefore in vitro tissue culture can constitute an effective alternative for stable, continuous and high-yield secondary metabolites production. For this purpose, in the present study, the effects of explant type (leaves, immature flowers), plant growth regulators [2,4-D or TDZ (0, 1, 2 mg/L), 2,4-D + TDZ (1 + 1, 1 + 2, 2 + 1, 2 + 2 mg/L)], UV-B radiation exposure time (0, 1, 2, 3, 4 h/day), and incubation period (2, 4 weeks) on producing azadirachtin and growth parameters (fresh weight, dry weight, growth index %) in vitro callus culture of M. azedarach were assessed. Results showed that leaf explants gave superior percentage for callus induction (100%) (4 weeks) and fresh weight (54.77 mg) (8 weeks) compared with immature flower explants (96.67%, 51.20 mg) under 2 mg/L 2,4-D + 2 mg/L TDZ. Leaf-derived calli exhibited significantly higher growth parameters and azadirachtin content than immature flower-derived calli under the same UV-B exposure time and incubation period in MS medium under 2 mg/L 2,4-D + 2 mg/L TDZ. The maximum increase in azadirachtin and growth parameters was achieved in leaf-derived callus by the highest UV-B exposure time of 4 h/day and the longest incubation period of 4 weeks (fresh weight: 1139.95 mg, dry weight: 115.35 mg, growth index: 279.98%, azadirachtin: 14.93 mg/g dry weight). The process of callus culture in association with UV-B irradiation as an elicitor can be a viable option for the production of azadirachtin in a large-scale bioreactor fulfilling the ever escalating industrial demand for plant-derived extracts. These results can further be manipulated as a sustainable method for the production of a natural and environmentally friendly pesticide (e.g. azadirachtin).

Abstract

Applying stress factors such as high temperatures during the different stages of somatic embryogenesis is important to uncover the molecular mechanisms of stress response and adaptation, and as a strategy to produce plants adapted to harsh environmental conditions derived from climate changes. In this sense, the present work aims to study the effect of high temperatures applied during the maturation of somatic embryogenesis in the ploidy stability, the amino acid and polyamine profiles of the somatic embryos obtained, and the morphological characteristics of the somatic plantlets. The results revealed that the maturation temperature did not affect the morphology of the resulting somatic plantlets, nor the ploidy and genome size of phenotypically normal somatic embryos, whose ploidy and DNA content levels were similar to those found in mature zygotic embryos. Nonetheless, a slight but significant reduction of the genome size of aberrant somatic embryos was observed. The maturation temperature changed the levels of glycine, arginine, lysine, and ornithine. These last three amino acids are precursors of the polyamines detected. Regarding this, putrescine levels were higher in somatic embryos from the highest maturation temperature (5 min pulse at 60 ºC), however, the amount of this polyamine in all samples was much lower than spermidine, spermine and cadaverine. In conclusion, the different temperatures applied did not lead to substantial changes in the ploidy level, endogenous PAs of the somatic embryos developed, or in the morphology of the somatic plantlets. Significant changes in the endogenous amino acids were observed, which may be linked to PAs metabolism and other metabolic pathways involved in stress response.

We established optimal conditions for the mass production of microtubers from suspension cultures of Pinellia ternata cells including the relative effects of regulators of plant growth, carbon sources, nitrogen sources, pH and inoculation amount on the growth of suspended cells and the induction of microtubers. Histological analysis was used to investigate the morphogenesis of microtubers and the quality of the microtuber-propagated tubers was evaluated. The results showed that the optimal culture conditions for inducing P. ternata microtubers from suspension culture included MS medium containing 1.5 mg·L⁻¹ of 6-BA, 1.5 mg·L⁻¹ of NAA, 10 g·L⁻¹ of sucrose and 0.2 g·L⁻¹ CH at pH 5.8; the best initial inoculation amount was determined to be 20 g·L⁻¹. After 60 days of culture under these conditions, the maximum yield of P. ternata cells was 7.54 g; furthermore, the embryonic callus generated microtubers via organogenesis at a frequency of 88.5%. The microtubers were planted directly into soil to permit growth, the seedling rate was approximately 50%. Microtuber-propagated tubers were of better quality, with a succinic acid content similar to that of field-cultivated tubers, but with the content of trigonelline and adenosine that were superior to those of cultivated tubers. Collectively, these data indicate that microtubers can be efficiently produced from suspension cultures and that microtuber-propagated tubers can be used for commercial use and field production in the P. ternata industry.

Somatic embryogenesis (SE) is a technique aimed at producing plant embryos in vitro and is considered a highly promising method for micropropagation. Here, we have established an efficient pathway for plant regeneration through somatic embryogenesis in S. chinensis. In the study, it was found that dark culture conditions significantly increased the rooting rate of plants through SE regeneration. Under dark conditions, radicle primordia were initiated during the globular embryo stage and developed from the heart-shaped to the torpedo-shaped embryo stages. The levels of IAA and ABA in somatic embryos subjected to the dark treatment were significantly lower (190.9 ng/g and 525.1 ng/g) from the globular to heart-shaped embryo stages compared to those in somatic embryos exposed to light (597.5 ng/g and 749.188 ng/g). Additionally, the concentrations of GA₃ and ZR were lower at all stages under light treatment. Transcriptome sequencing and bioinformatics analysis revealed that the pathways and processes predominantly enriched in differentially expressed genes in somatic embryos under dark conditions were plant hormone signaling, circadian rhythm and phenylpropanoid biosynthesis. QRT-PCR was employed to validate the expression of genes related to plant hormone signaling transduction. The results were consistent with the transcriptome sequencing results. This work has laid the foundation for applied research and could prove useful in future programs aimed at improving reluctant woody plants. Furthermore, the findings can potentially be extended to other species as well.

Virus-induced gene silencing (VIGS) is an effective reverse genetics method used to study the gene function by analysis of plants at phenotype and molecular levels. Here, the standardization of the protocol for VIGS through Agrobacterium-mediated transformation was successfully achieved. Stem cuttings with roots were established in Coleus forskohlii. Phytoene desaturase gene from Coleus forskohlii (CfPDS) was isolated and cloned in TRV2 vector to construct CfPDS:TRV2. CfPDS:TRV2 construct was transformed in Agrobacterium tumefaciens. Agro-infiltration was carried out using leaf infiltration, agrodrench and submerged methods. Submerged method gave prominent results as compared to others. The expression of CfPDS gene was significantly decreased in plants agro-infiltrated with CfPDS:TRV2 construct resulting in photobleached areas of the affected parts of leaves. CfPDS:TRV2 treated plants showed lower chlorophyll a concentration and production of reactive oxygen species (ROS) as compared to TRV1 + 2 treated control plants. This study resulted in the establishment of VIGS protocol for Coleus forskohlii that can be further used to engineer the genes involved in the secondary metabolite pathway.

Abstract

The current work specifies an effort to conserve the highly endangered plant Decalepis salicifolia by utilizing encapsulation technology for high plantlet recovery, short-term storage, and conservation. In order to determine the best culture conditions for maximizing the ability of synseeds to develop into complete plantlets, nodal segments (NS) were encapsulated in a sodium alginate (SA) matrix. The best solution for creating isodiametric beads was determined to be a 3% sodium alginate with 100 mM CaCl₂ solution. Regeneration of the encapsulated segment was reported directly or after storage at 4 °C for up to 8 weeks. The highest shoot regrowth frequency (90.8%) and highest microshoots per encapsulated nodal segment i.e. 4.90 were recorded when Murashige and Skoog (MS) basal medium was supplemented with 5.0 µM 6-benzyl adenine (BA) + 1.0 µM indole-3-butyric acid (IBA) and encapsulated nodal segment were inoculated onto the nutrient medium comprised of MS + 5.0 µM BA. Microshoots rooted effectively on MS medium augmented with indole-3-butyric acid IBA (2.5 µM). Plantlets achieved from preserved synthetic seeds were acclimatized and relocated in the natural condition successfully with an immortality rate of 87.1%. Subsequently, it was planted in garden soil and exhibited no morphological changes. Gas Chromatography and Mass Spectrometry (GC-MS) of leaf extract, obtained from the donor plant as well as in vitro derived encapsulated regenerated plantlets shows the presence of diverse chemical compounds of immense pharmacological properties and number of biochemical compounds are almost similar in both, which established the biochemical similarity between them. Genetic similarity between the donar plant and the synseed-derived plant was confirmed by the presence of monomorphic bands produced with the help of the start codon targeted (SCoT) marker.