In Vitro Cellular & Developmental Biology - Plant最新文献

Byblis, a small genus of carnivorous plants predominantly found in Australia, is characterized by its passive trapping mechanism and unique floral features. The chemical composition of Byblis, including identified phenylethanoid glycosides, particularly acteoside, highlights its pharmacological potential with various biological activities. In vitro culture techniques have been established for propagation, with micropropagation protocols developed for different Byblis species. However, information on genetic transformation, vital for trait modification and enhanced pharmacological interest, remains limited. This study focuses on optimizing micropropagation, adventitious regeneration, and genetic transformation methods for Byblis liniflora. Adventitious regeneration rates were highest in medium with reduced Murashige and Skoog salts (MS/10) and sucrose (3 gL⁻¹) concentrations. Zeatin supplementation (1 mgL⁻¹) further improved regeneration rates and bud development with 100% of regenerated root explants and 8.8 shoots per explant. Liquid MB3 medium supplemented with indole-3-acetic acid (IAA) 5 mgL⁻¹ facilitated efficient rooting and acclimatization. The establishment of an efficient Rhizobium-mediated genetic transformation method yielded transgenic plants expressing green fluorescent protein (GFP). Molecular analysis confirmed transgene integration, marking the first successful genetic transformation in the Byblis genus. These advancements pave the way for exploring gene function and enhancing pharmacological properties, thereby broadening our understanding and utilization of carnivorous plants like Byblis.

An efficient and reproducible in vitro regeneration protocol was developed for elite cotton cultivar KC3 by using plant growth regulators (PGRs) in combination with seaweed polysaccharide (SP) extracts. The existence of polysaccharide in seaweed extract was confirmed by Fourier transform infrared spectroscopy (FT-IR) and carbon-13 (¹³C) nuclear magnetic resonance (NMR) spectroscopy analysis. The extracted SP extract efficacy was tested for in vitro plant regeneration. The maximum callus frequency (89.4%) was obtained from hypocotyl explant in the Murashige and Skoog (MS) medium supplemented with 4.0% glucose, 1.5 mg L⁻¹ thidiazuron (TDZ), 0.6 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), and 30.0 mg L⁻¹ SP. Remarkably, PGR- and SP-fortified medium inhibits the phenolic excretion from the explants. The well-developed yellow green friable texture of callus was transferred to shoot initiation medium. MS medium fortified with 4.0% glucose, 2.0 mg L⁻¹ 6-(γ,γ-dimethylallylamino) purine (2iP), 1.0 mg L⁻¹ kinetin (KIN), 1.0 mg L⁻¹ 6-benzylaminopurine (BA), and 40.0 mg L⁻¹ SP has shown maximum response (85.2%), and it produced 9.5 shoots per callus. The elongated shoots were cultured on root induction medium which consists of Murashige and Skoog (MS) salts with indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1-naphthaleneacetic acid (NAA), and SP. The results revealed that the maximum number of roots (12.9 per shoot) with 8.6 cm in length was achieved on MS medium supplemented with 0.6 mg L⁻¹ IBA, combined with 30.0 mg L⁻¹ SP. Therefore, modified MS medium with natural bio-stimulant has more potential and is more reliable for in vitro regeneration of plants by neutralizing the effects of phenolic compounds secreted by the explants.

Cardiac glycosides (CGs) are well known for treating congestive heart failure, and several CGs like digoxin, digitoxin, and ouabain are marketed as drugs. In the present study, we have biosynthesized two CGs (CGCL520/227 and CGCL534/209) and elicited them up to 537- and 357-fold respectively in response to methyl jasmonate (MJ) treatment. For identification of the key enzyme involved in its biosynthesis, a comparative transcriptome sequencing of control and MJ elicited (75.0 mg L⁻¹ for 3 d) callus culture was done. A total of 17,898 transcripts were expressed across all samples. Annotated unigenes were functionally categorized based on gene ontology. A total of 7625 unigenes were significantly matched in the KEGG database involved in 151 different plant metabolism pathways. Upon digital expression analysis, 2924 MJ-responsive transcripts were identified, and among them 166 were unique for MJ-treated samples. A majority of upregulated transcripts were categorized under hydrolase activity, oxido-reductase activity, metabolic processes, and carbohydrate metabolic process. Based on their role in terpenoid, steroid, and cardenolide pathways, 295 putative unigenes representing 24 gene families involved in CG biosynthesis were identified. Expression analysis revealed that 12 transcripts involved in steroid and cardenolide biosynthetic pathways were upregulated in response to MJ. The highest expression was recorded for squalene monooxygenase (SMO) with 43-fold upregulation, followed by sterol delta7 reductase (DWF5) with 22.2-fold. C-5 sterol desaturase (STE1), 4-diphosphocytidyl-2-C-methyl-D-erythritolkinase/4diphosphocytidyl-2C-methyl-D-erythritol synthase (CMK), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), acetyl-CoA C-acetyltransferase (AACT), mono-oxygenases (MO), and progesterone 5β-reductase (PBR) showed high and significant expressions of 16.4-, 16.1-, 14.8-, 14.7-, 13.4-, and 11.3-fold, respectively. This study not only identifies MJ-responsive CGs and related transcripts involved in CG biosynthesis, but also provides scope for the development of biotechnological process for biosynthesis and enrichment of targeted CGs using identified rate-limiting key enzymes.

Chinese artichoke (Stachys sieboldii [Miq.]) is a popular healthcare food owing to its high contents of stachyose. Viral infections have caused the severe degeneration of germplasm in this plant species. An efficient virus elimination and micropropagation system is required to produce Chinese artichoke plants that are free of viruses. A protocol for virus elimination in Chinese artichoke was established by comparing the potential for growth of the terminal buds using shoot apices as starting materials, in media with or without plant growth regulators (PGRs) and various concentrations of sucrose. Murashige and Skoog (MS) medium without any PGR performed better than that supplemented with PGRs as indicated by a higher rate of survival of shoot apices, higher plant height, more root numbers and adventitious buds, and increased biomass (P < 0.05). An experiment to further optimize the sucrose concentration demonstrated that MS medium with 5.0% sucrose (w/v) efficiently induced the growth of both roots and shoots, thus, achieving the efficient micropropagation of Chinese artichoke within 4 wk. Compared with the mother plants in which viruses had not been eliminated, the virus-free plants had significantly higher numbers of tubers, increased yield, and a higher content of stachyose (P < 0.05). Higher contents of endogenous hormones were detected in Chinese artichoke, which may explain the efficient micropropagation without the use of exogenous PGRs. This simple protocol enabled the production of virus-free Chinese artichoke to enhance the yield of tubers and high content of stachyose.

Lilium rosthornii Diels, commonly known as the Nanchuan lily in China, is a unique wild lily species with potential medicinal or ornamental value. The development and utilization are mainly restricted to its highly efficient regeneration and the scarcity of new germplasm. In this study, an efficient plant regeneration method for Nanchuan lily was established based on embryogenic callus induction, adventitious bud sprouting, and shoot proliferation and rooting. The results showed that Murashige and Skoog (MS) media supplemented with 0.5 mg L⁻¹ picloram (PIC) and 1.0 mg L⁻¹ 1-naphthaleneacetic acid (NAA) were suitable for embryogenic callus induction using outer bulblet scales (78.89% rate), and MS supplemented with 0.5 mg L⁻¹ 6-benzylaminopurine (6-BA), 1.0 mg L⁻¹ NAA, and 75.0 mg L⁻¹ ascorbic acid in darkness was optimal for embryogenic callus proliferation (2.62 coefficient). Moreover, callus inoculation in MS medium supplemented with 1.0 mg L⁻¹ thidiazuron (TDZ) and 0.1 mg L⁻¹ NAA significantly promoted shoot induction (3.16 coefficient) under relatively low light conditions. The shoot proliferation and rooting of MS plants treated with 0.1 mg L⁻¹ NAA and 0.5 mg L⁻¹ TDZ were greatest under light conditions at 93.33% and 98.89%, respectively. In addition, bulblet growth was induced by treatment with different combinations of colchicine at different concentrations for different durations. Focusing on the bulblet centre, 100% morphological changes were achieved for in vitro induction using 0.15% colchicine for 72 h of treatment. Compared with the control, three stable mutagenic plants with greater guard cells and reduced stomatal density were generated and were identified as chimaeras by flow cytometry analysis. These results provide support for improving tissue reproduction and breeding autotetraploid germplasm in Nanchuan lily.

Paspalum notatum Flüggé is a subtropical grass native from the Americas and one of the main components of the subtropical South American grasslands. Although P. notatum exhibits a high forage productivity and persistence, some limitations are observed in the species, such as forage quality due to high lignin content. The development of efficient tissue culture and plant transformation protocols in cultivars for forage production are necessary to embrace novel genetic improvement through transgenesis and genome editing. Efficient regeneration and transformation systems for many plant species can be hampered by genotype dependency, which can only be determined through extensive laboratory testing and evaluation. In this study, various factors extracted from protocols have been evaluated to establish efficient somatic embryogenesis, plant regeneration, and genetic transformation in apomictic initial variety INIA Sepé. Optimal somatic embryogenesis and regeneration from explant sources, such as mature seeds and meristematic tissue derived from in vitro propagated tillers, ranged between 79 and 88%. Microprojectile bombardment of two plasmids encoding a constitutively regulated reporter gene and nptII selectable marker expression constructs showed an average transformation efficiency of 10.3% from meristematic tissue as an explant source. Furthermore, the constitutive promoters, 35S and ZmUbi, were tested in GFP transient expression assays to evaluate optimal cassettes for transgene(s) expression. Translation of a wheat codon–optimized Cas9 sequence using Cas9:2A:tuGFP fusion protein was also tested in protoplasts. The information generated on promoter activity and Cas9 translation, and the establishment of an efficient transformation system, provided useful tools for future work in genetic engineering and genome editing in P. notatum cv. INIA Sepé.

Epipactis flava Seidenf., a Thai rheophytic orchid, is endangered due to habitat destruction and climate change. In vitro conservation serves as an effective technique for the ex situ preservation of orchid diversity. This study investigated the in vitro storage of E. flava seedlings under slow growth conditions using Murashige and Skoog (MS) medium supplemented with either sorbitol or mannitol individually at concentrations of 1.0%, 2.0%, 3.0%, and 4.0% (w/v) for 24 wk without subculture. The storage medium added with 3.0% (w/v) sucrose was used as the control. After 24 wk of storage, the storage medium containing 2.0% mannitol provided the highest survival rate (97.6%) while the highest leaf formation (70.8%) was achieved with 3.0% sucrose. The addition of 1.0 to 2.0% sorbitol, 1.0% mannitol, and 3.0 to 4.0% mannitol showed the highest shoot bud formation (100%). At the end of 24 wk, the plantlets were transferred to fresh medium for an 8-wk growth recovery. The plantlets derived from the storage medium with 3.0% mannitol exhibited the highest survival rate (73.5%). Conversely, the highest number of shoot buds (9.9 shoot buds per plantlet) was found on plantlets derived from storage medium with 1.0% sorbitol while the highest number of shoots (4.8 shoots per plantlet) was achieved in plantlets derived from 1.0% mannitol. Consequently, these results suggested that the storage medium with 3.0% mannitol is optimal for conserving plantlets for up to 24 wk, and all plantlets could successfully produce shoot buds and shoots during in vitro recovery. Hence, this strategy has potential in the conservation and utilization of this endangered species.

The GROWTH-REGULATING FACTOR (GRF) and its INTERACTING FACTOR (GIF) have been shown to stimulate regeneration of transgenic plants with studies reporting increased transformation efficiency in multiple species, including wheat, beet, and citrus. The present work evaluated the effects of overexpressing GRF4-GIF1 and GRF5 on the regeneration of transgenic plants in cassava (Manihot esculenta Crantz). Effects of GRF4-GIF1 and GRF5 sequences derived from Vitis vinifera and Arabidopsis thaliana were assessed by cloning expression cassettes under control of strong constitutive promoters. Friable embryogenic callus from cassava varieties 60444 and NASE 13 were transformed with Agrobacterium tumefaciens strains LBA4404 and LBA4404 THY-, and multiple independent transgenic plant lines recovered. Expression of the morphogenic genes did not enhance transformation efficiency, nor efficiency or timing of somatic embryo regeneration or whole plant recovery above the green fluorescent protein (GFP) control. Organogenesis experiments were carried out to observe effects of transgenic expression of these genes on morphogenesis from petiole, leaf-petiole, and stem explants. Results differed between the two genotypes evaluated. Expression of Vitis vinifera GRF4-GIF1 was effective for stimulation of rapid organogenesis and shoot regeneration at 30 to 37% from leaf-petiole explants of cultivar 60444. In contrast, Arabidopsis thaliana GRF5 was superior in stimulating shoot regeneration in cultivar NASE 13 with 40 to 50% of leaf-petiole explants regenerating shoots. In both cultivars, caulogenesis occurred rapidly within 3 to 4 wk culture on medium containing the cytokinin meta-topolin. Effects of overexpression of these morphogenic genes at the whole plant level were accessed by establishing plants in the greenhouse. GRF4-GIF1 overexpression resulted in significantly shorter plants with increased leaf size and total leaf area while AtGRF5 stimulated above average storage root weight.

Withania somnifera (Ashwagandha) is a valuable therapeutic plant used in many Ayurvedic drugs marketed for health restoration. However, its commercial production faces challenges due to poor seed viability, low germination rates, and the absence of standardized propagation methods. Therefore, this investigation aimed to establish a fast and highly efficient method for direct shoot regeneration in W. somnifera by using the apical meristem perforation technique, which is a novel approach. Initially, rapid seed germination was standardized by soaking the seeds in distilled water for varying durations (0 to 48 h). The highest rates of shoot initiation (90.40 ± 0.51%) and mean number of direct shoot regenerations (16.20 ± 0.50 per explant) were achieved on MS basal medium fortified with meta-topolin (mT) at 3.0 mg L⁻¹. For optimal root development, shoots (3 to 4 cm) were cultured on half-strength Murashige and Skoog (MS) medium with 3.0 mg L⁻¹ indole-3-butyric acid (IBA). Subsequently, well-rooted seedlings (90% survival rate) were acclimatized under glasshouse conditions in pots filled with a mixture of sandy loam and vermicompost (4:1). Anatomical examination of leaves and stems from micropropagated and acclimatized glasshouse plants revealed well-developed cuticles, differentiated mesophylls, and vascular bundles. Furthermore, the accumulations of withaferin A (1.26 ± 0.12 mg g⁻¹), withanone (1.39 ± 0.18 mg g⁻¹), and withanoside V (0.11 ± 0.04 mg g⁻¹) in greenhouse-acclimated plant leaves were 1.88, 2.28, and 2.30 times higher, respectively, than in tissue culture samples (0.52 ± 0.07 mg g⁻¹, 0.73 ± 0.02 mg g⁻¹, and 0.05 ± 0.02 mg g⁻¹). In conclusion, the described procedure offered an effective and straightforward method for large-scale production of W. somnifera plants for commercial metabolites.

Juglans mandshurica is an important component of Korean pine mixed and secondary forests in Northeast China and is also a precious timber and economic species. In this study, plant regeneration of J. mandshurica has been established using stems with bud and mature zygotic embryos as explants. The results showed that placing the stems with buds in low-temperature conditions (0 to 4°C) and in darkness for 5 d inhibited the browning of the explants and promoted bud sprouting; Na₂S₂O₃ also inhibited the browning of mature zygotic embryos significantly. The suitable basal media and hormones for bud sprouting and zygotic embryo germination were (1) WPM medium (woody plant medium) containing 2.0 mg·L⁻¹ 6-benzylaminopurine (6-BA) and 0.01 mg·L⁻¹ 3-indolebutyric acid (IBA) and (2) DKW medium (Driver and Kuniyuki medium) containing 2.5 mg·L⁻¹ 6-BA and 0.1 mg·L⁻¹ IBA. The suitable basal medium and hormone for rooting of shoots from buds was 1/2 Murashige and Skoog (MS) medium (the macro-elements were half-strength of MS medium) supplemented with 1.0 mg·L⁻¹ IBA, and the suitable basal medium and hormone for rooting of seedlings derived from zygotic embryo was 1/2 MS medium containing 1.0 mg·L⁻¹ IBA, and DKW medium with 2.5 mg·L⁻¹ 6-BA, 0.1 mg·L⁻¹ IBA, 0.2 mg·L⁻¹ gibberellin A3 (GA₃), and 0.2 g·L⁻¹ activated carbon (AC). About 95% of the rooted plantlets survived after acclimation and transplantation. These results provided a foundation for the scale propagation to meet the demands of forestry production of the species.