Plant Cell, Tissue and Organ Culture最新文献

Use of meta-Topolin (mT) has significantly affected tissue culture results in several plant species. In this study, a fast and efficient micropropagation protocol for Withania somnifera was standardized. Also, a comparison was made between the effect of using M.S. media supplemented with mT and M.S. media supplemented with different concentrations of B.A.P. + AdSO₄ on the in vitro regeneration of W. somnifera. M.S. media supplemented with I.B.A. was used to obtain a healthy and advantageous root system. The resulting plantlets were analyzed for their total chlorophyll and protein content. The comparison revealed that 2.5 mg/l mT supplemented M.S. media showed better results with 88.9 ± 0.42% bud breaking percentage, a shoot multiplication rate of 22.4 ± 4.16 shootlets per explants, total chlorophyll content of 0.887 ± 0.004 mg/g and protein content of 25.67 ± 0.25%. Healthy adventitious root systems were observed in regenerated shootlets inoculated in M.S. media supplemented with 2.0 mg/l I.B.A. During hardening, 80% of plants treated initially with mT survived. Whereas treatment with M.S. media supplemented with 1.0 mg/l B.A.P. + 50 mg/l AdSO₄ resulted in bud breaking percentage of 61.1 ± 0.09%, a shoot multiplication rate of 15.8 ± 7.81 shootlets per explant, total chlorophyll content of 0.7194 ± 0.0055 mg/g and protein content of 23.33 ± 0.25%. During hardening, 78% of plantlets treated initially with B.A.P. + AdSO₄ survived. Therefore, the study concludes that mT may be used as an alternative source of cytokinin for obtaining healthy plantlets with a higher rate of plant regeneration in W. somnifera.

Stylosanthes (stylo) species are commercially important forage and pasture legumes in tropical and subtropical regions, but their vulnerability to chilling and frost remains a challenge. The REVEILLE (RVE) proteins, known as circadian clock components, have been implicated in abiotic stress responses in several plant species. Using RNA-seq analysis, we identified SgRVE1 as a cold-responsive gene in stylos. Further quantitative Real-time PCR analysis confirmed that SgRVE1 was rapidly induced by low temperature in different tissues, including roots, leaves, stems, and flowers. Multiple sequence alignment revealed that SgRVE1 shares a SHAQKFF-class MYB domain with the CCA1 clade of the RVE family. Phylogenetic analysis demonstrated that SgRVE1 clusters within the CCA1 clade and is closely related to AtRVE1. SgRVE1 also shows high sequence identities with three Arachis RVE1 orthologs, ranging from 95 to 96%. Overexpressing SgRVE1 in Arabidopsis thaliana activated AtCBF1,2,3 gene transcription, promoted osmoprotectant accumulation and enhanced cold tolerance. A yeast one-hybrid assay suggests that SgRVE1 binds to two Evening Element (EE) motifs, EE-like (EEL: AATATCT) and Short EE (SEE: AAATATCT), which has been identified in AtCBF1,2,3 promoter sequences. This study provides the first evidence of a RVE gene in stylos and reveals its role in the transcriptional regulation of CBF genes and the cold-responsive mechanism.

Vanilla is a high-value tropical orchid cultivated for its aromatic fruit capsules that are used in foods, perfumes, and industrial products. Vanilla planifolia (Jacks ex. Andrews) is the most important commercially grown species, but its production is constrained by poor yield, variable quality, low genetic diversity and limited horticultural advancements. A closely related species, Vanilla pompona Schiede, characteristics which could be useful in breeding improved varieties: large fruit, potent aroma, and resistance to Fusarium oxysporum f. sp. vanilla. Here we describe tissue culture-based regeneration and Agrobacterium-mediated stable transformation systems for V. pompona. Vegetatively propagated tissue was used to test the efficacy of hygromycin and phosphinothricin selection and to assess the efficiency of three Agrobacterium tumefaciens strains (EHA105, AGL1.1, GV3101) in transformation protocols. Results revealed that for V. pompona, kanamycin is not an effective selection marker, whereas hygromycin and phosphinothricin can be used for screening transformants. AGL1.1 provided the highest transformation efficiency (37%) as compared to strains EHA105 (11%) and GV3101 (4%). Additionally, we incorporated the use of firefly luciferase as a visual reporter of transformation and were able to demonstrate that it provides more robust assessment than that of green fluorescent protein. Finally, we report a novel quantitative imaging method to assess the growth responses of V. pompona protocorm-like bodies in response to selection that could be useful to other plant transformation and selection efforts.

The large-leaved Homalomena (LLH, Homalomena pendula) represents an endangered medicinal plant species within Vietnam, primarily attributed to its recognized tonic properties. Despite its imminent threat of extinction within Vietnamese ecosystems, the development of a robust protocol for molecular species identification and large-scale propagation of LLH remains absent. Consequently, we present the first conservation endeavor for LLH based on plant micropropagation techniques, with plant materials validated through anatomical observations and DNA barcoding (rbcL). Our investigation yielded five rbcL sequences specific to LLH, serving as the current best barcode for LLH identification and thereby facilitating forthcoming taxonomic endeavors. Optimization of in vitro culture conditions revealed that the Murashige and Skoog (MS) medium supplemented with 2 mg/L 6-benzylaminopurine, 0.5 mg/L α-naphthaleneacetic acid, and 60 g/L mashed potato, alongside the incorporation of 0.5 mg/L indole-3-butyric acid to the basal MS medium, yielded optimal outcomes for shoot proliferation and root development, respectively. After successful micropropagation, acclimatization of rooted plantlets to a substrate comprising soil, coconut coir, and rice husk (in a 1:1:1 ratio) culminated in a 100% survival rate among the plants.

Plant cell cultures are precious tools for investigating the response of plants to altered gravity at the cellular level. In the present study, the effects of clinorotation on the growth and cell cycle progression of cultured Nicotiana tabacum cells were investigated. Exposure to 2D-clinostat for 12 h increased the percentage of the cells in the G1 phase from 80 to 83.2%, while significantly reduced the percentage of those cells at the G2/M transition, compared to their corresponding control cells. When the duration of exposure was extended, the rate of cells transition to the M phase increased, ultimately promoted the exponential growth phase after 168 h. During the first 24 h of clinorotation, a significant rise in the levels of simple sugars within the cells was observed. The ferric-reducing antioxidant power (FRAP) of tobacco cells exhibited a downward trajectory that continued until 48 h. This research showed the influence of clinorotation on plant cells dependent on the exposure duration. The cells exhibited signs of stress after a short exposure, possibly due to high levels of soluble sugars that could impede cell advancement in the G1 phase by negatively affecting radical scavenging capacity (RSC). Upon extending the exposure duration to 168 h, the cells were adapted to the altered gravity conditions and improved their growth, probably due to a rise in auxin and gibberellin production. The results suggest cultured cells are a viable candidate, for examining plants in long-term space missions.

Graphical abstract

Cibotium barometz (L.) J. Sm., a fern, holds significant value in traditional Chinese medicine. This research aimed to establish a rapid tissue breeding technology for Cibotium barometz (L.) and provide a theoretical basis for its artificial breeding and industrial planting. The optimum conditions for spores germination were selected, including both the best sterilization time and the ideal spore germination medium. Subsequently, the optimal medium for proliferation and differentiation were chosen, and the optimal concentration of plant hormones was screened to determine the optimal medium. The rooting experiment was performed in two single-factor experiments by using differentiated sporophytes as materials. These experiments showed that the optimal sterilization time was 11 min and the best medium for germination was 1/8MS + 0.02 mg/L IAA. MS medium was suitable for proliferation, while WPM medium was suitable for sporophyte differentiation. The best medium for growth was MS + 1.0 mg/L IAA + 0.5 mg/L 6-BA. In the absence of sucrose, the seedlings barely grew and failed to differentiate into sporophytes. They showed optimal growth and differentiation at a sucrose concentration of 20 g/L. The plant growth regulators plant growth regulators used, including IAA, NAA, 6-BA, and GA₃, showed more effective differentiation, wherease KT had a slightly weaker effect. The rooting rate reached the maximum when the concentrations of ABT and IBA were at 1.5 mg•L^− 1 and IAA was at 1.0 mg•L^− 1. The IBA had a significant effect on the average and the longest root length, while the three PGRs had no obvious effect on the average or maximum diameter. Plant tissue culture seedlings still have good genetic stability after subsequent generation. In this study, the optimal cultivation conditions for rapid breeding technology in tissue culture, covering stages from germination and growth to differentiation and rooting, were obtained. Moreover, sporophytes were successfully transplanted to the external environment. This study provided a theoretical reference for the industrial planting of C. barometz.

Hedychium spicatum (Family-Zingiberaceae), commonly known as spiked ginger lily, is widely known for its medicinal properties and high market demand. The species is harvested mainly from the wild to meet the raw material requirements for the pharmaceutical and cosmaceutical industries; therefore, it needs urgent attention for its conservation and mass production. The present study developed an efficient in vitro propagation protocol for large-scale species production. The central composite design-response surface methodology (CCD-RSM) was designed to optimize the plant growth regulators (PGRs) concentration for maximum callus production, shoot regeneration, and rooting. The seed was used as explants in Murashige and Skoog (MS) medium with different concentrations of naphthalene acetic acid (NAA; 2.5-5.0 µM) in combination with thidiazuron (TDZ; 2.5-5.0 µM) for callus induction. TDZ (4.76 µM) with NAA (2.84 µM) showed maximum callus induction (100%) after 6 weeks of incubation. Callus pieces were transferred to MS medium with different concentrations of TDZ, NAA, and Indole-3-butyric acid (IBA) for shoot regeneration. The highest regeneration frequency (100%) was observed on MS medium enriched with TDZ (2.72 µM) and NAA (3.5 µM) that showed a maximum number of shoots/explants (16.34 no.). Regenerated shoots were rooted better (average number of roots/shoot − 11.75) on MS medium with NAA (2.11 µM) and IBA (1.17 µM) in combination. After the subsequent acclimatization and hardening process in the greenhouse, the plants were planted in the experimental field, and their survival rate was 89% after 2 years of establishment. The protocol established in the present study has prospects to meet the challenges of quality planting material for large-scale cultivation and raw material sources for industrial utilization.

Coconut palm is of great economic importance and contributes to income generation, employment, nutrition and food security in the commercial sector. Malaysia, which is among the top 10 coconut-producing countries in the world, plays a notable role, while Indonesia is the largest producer. The Matag coconut, a hybrid of the Malayan Yellow Dwarf and Tagnanan Tall varieties, is characterised by its excellent nut quality and higher yield of 25,000 to 30,000 nuts per hectare annually. Despite its fame, the Matag coconut has faced problems, particularly disease susceptibility, which has been accelerated by globalisation and has led to the extinction of many crops. Somatic embryogenesis, an in vitro breeding technique, is a promising avenue for coconut regeneration and micropropagation. However, the genotype dependence and high heterozygosity of coconut lead to variability among the progeny, which limits the effectiveness of this method. The conventional use of fluorescent lamps for in vitro propagation, which are customised to the needs of the plants, has disadvantages such as the generation of unnecessary wavelengths and high energy consumption. The integration of the thin cell layer (TCL) method into tissue culture, particularly via the organogenesis route, is proving to be a transformative technique for coconut propagation. This review highlights the potential enhancement of this novel approach through the use of red and blue light-emitting diodes (LEDs). The use of these LEDs aims to optimise the propagation and acclimatisation of the ‘Matag’ coconut and offers a viable solution for both research and commercial applications.

Viola odorata plant extracts are extensively used in indigenous medicine to treat respiratory diseases. V. odorata’s limited availability in nature, due to geographical conditions and adulteration, makes continuous supply of high-quality plant raw material challenging. This results in low-quality plant extracts that can have inconsistent and sometimes adverse effects when used in medicine. As an effective solution, in vitro plant biomass cultivation conserves plant diversity, ensuring high-quality raw materials with uniform quality and quantity. Toward this, the current study focuses on establishing bioprocess strategy to achieve maximum. V. odorata biomass productivity up to bioreactor level. In this context, in vitro culture conditions were rationally optimised, focusing upon both statistical optimization and one-factor-at-a-time (OFAT) approach, for improved biomass productivity. Inoculum density, macronutrient concentration, shaking speed, temperature, pH and light intensity were systematically optimized to achieve the maximum biomass. This led to increased biomass production from 10.2 ± 0.8 g DW L^− 1 to 21.7 ± 0.8 g DW L^− 1 within 12 days of the batch cultivation period. Optimized culture conditions were implemented in three different types of bioreactors and cultivation in a stirred tank reactor produced ∼ 19.7 g DW L^− 1 of maximum biomass within a 12 day batch cultivation period. Further, the metabolite profile of the bioreactor cultivated biomass (alcoholic extract) was compared with that of the natural plant biomass using gas chromatography-mass spectrometry-based metabolomics. The antibacterial activity of the plant extract was tested against three bacteria responsible for respiratory tract infections, and their antioxidant activity was compared with that of Vitamin E, wherein the results demonstrated comparable activity. The novelty of this study is in the fact that the V. odorata plant cells were cultivated in bioreactors for the first time for potential medicinal applications. It provides an alternative method for sustainable production of plant biomass, thereby conserving the natural plant sources.

Graphical Abstract

The use of lids with microporous membranes can enhance in vitro plant growth by promoting gas exchange, including ethylene exchange. We aimed to evaluate the influence of the microporous membrane, explant type, and culture medium on the in vitro development of Dalbergia nigra shoots, as well as on the protein profile, polyamines (PAs), ethylene and CO₂ levels. A total of 465 proteins were identified, of which 148 were differentially accumulated proteins, being 73 proteins up- and 75 down-accumulated in shoots grown in sealed-lid culture flasks containing microporous membranes compared to those grown without. The use of microporous membranes plays an important role in the accumulation of proteins related to photosynthetic activity process, such as ruBisCO_large domain-containing protein; ribulose bisphosphate carboxylase small chain; ribulose bisphosphate carboxylase small chain protein; and chlorophyll a-b binding protein, chloroplastic. Conversely, the decreased accumulation of proteins associated with amino acid synthesis linked to ethylene biosynthesis, such as aspartate aminotransferase, in shoots grown with microporous membranes was related to the lower level of ethylene than in shoots grown without membranes. The microporous membrane promoted an increase in total free PAs and putrescine contents in shoots, while higher CO₂ levels were detected in shoots grown without a microporous membrane. This is the first study showing the effects of promoting gas exchange on shoot development in D. nigra, in addition to exploring the interaction of protein accumulation with ethylene. This research can improve the understanding of propagation systems in D. nigra, an endangered species.