Plant Cell, Tissue and Organ Culture最新文献

Abstract

Handroanthus chrysanthus Jacq. S. O. Grose and Tabebuia rosea (Bertol.) Bertero ex A.DC are two forest species that grow in the coastal region of Ecuador and are threatened with extinction. A protocol for the mass multiplication of these species was developed using in vitro culture techniques. The cultures were initiated from seeds, and the effect of two culture media: Woody Plant Medium (WPM) and Murashige-Skoog (MS), two concentrations of NaClO (0.5% and 1%), and two disinfection times (3 and 5 min) was studied. During multiplication, the effect of three concentrations of two cytokinins: 6-benzylaminopurine (6-BAP) 4.4, 5.5, or 6.6 μM; kinetin 4.6, 5.75, or 6.9 μM) on the number of shoots, their length, and diameter was analyzed. This phase of the experiment was carried out in two successive multiplications. For rooting, two concentrations of indole-3-butyric acid (IBA) (2.45 and 4.9 μM) were tested, and the number of roots formed and their length were determined. It was demonstrated that the WPM medium is the most suitable for the in vitro culture of both species and that disinfection time and NaClO concentration affect each species differently. For the multiplication of H. chrysanthus, the most suitable cytokinin was 6-BAP 6.6 μM; T. rosea performed better in the absence of cytokinins. IBA 2.45 μM produced the best results for the rooting of H. chrysanthus, while for T. rosea, IBA 4.9 μM was the most suitable. The acclimatized plants showed a high survival rate, demonstrating the feasibility of using this methodology for the accelerated propagation of these endangered species.

Key message

In this research, the culture medium requirements and conditions for the micropropagation of H. chrysanthus and T. rosea were fine-tuned. This technique can be implemented to obtain plants for use in reforestation.

Doubled haploids have the great potential to enhance both breeding efficiency and genetic research in African marigold (Tagetes erecta L.). In order to obtain basic information about doubled haploids in African marigold, in the present study, we investigated the morphological characters in doubled haploids generated through anther culture. Screening of anther derived regenerants revealed that out of the 72 plants, 6 plants (8.33%) were found to be haploids (2n = x = 12), 66 plants (91.66%) were found to be diploids (2n = 2x = 24) similar to its donor parent. Six doubled haploid lines hence homozygous material were obtained by in vitro anther culture from African marigold genotype Af/R/L-1 which were subsequently characterised for various qualitative and quantitative traits. In terms of results, a single doubled haploid line was not superior for every attribute, and distinct doubled haploid lines were superior for different qualities associated to growth and flowering. Different growth rates and genetic makeup could be the cause of this.

Eurycoma longifolia Jack. is a commercially valuable medicinal plant with clinically proven anti-cancer and aphrodisiac properties. To ensure the sustainability of the production of E. longifolia products on a commercial scale, hairy roots (HR) were engineered. In this study, we used light-emitting diodes (LEDs) as elicitation agents to enhance the synthesis of three (3) anticancer compounds (eurycomanone, 9-hydroxycanthin-6-one and 9-methoxycanthin-6-one). HR cultures were cultured for 12 weeks under four different LED treatments, including blue light (100%), red light (100%) and a combination of blue and red light (60%: 40%). In addition, a white LED was used as a control. The effects of the treatments on growth, synthesis and anti-cancer properties were determined. The results show a significant difference (p < 0.05) between the treatments. The combination of blue and red LED produced the highest dried biomass of 0.316, 0.391 and 0.459 g/50mL at weeks 6, 8 and 10, respectively, which is 2.2, 1.7 and 1.5 times that of the white LED. In addition, the red LED produced the highest level of eurycomanone at the 8th and 12th week of culture, the combination of blue and red LED produced the highest level of 9-hydroxycanthin-6-one at the 8th and 12th week of culture, and 9-methoxycanthin-6-one at the 4th and 8th week of culture. The MTT assay showed significant activity of the crude extracts from all treatments against MCF-7 cancer cells. These results indicate that LED excitation is a promising technique for the production of anticancer agents from HR cultures of E. longifolia.

This review explores recent advances in the biotechnology of Lamprocapnos spectabilis (L.) Fukuhara (commonly known as bleeding heart), a valuable ornamental-medicinal perennial. The article covers in vitro morphogenesis, cryopreservation techniques, and methods for inducing variability. The establishment of in vitro cultures utilized Murashige and Skoog medium enriched with various auxins, cytokinins, gold nanoparticles, and plant extracts, under both fluorescent and wide-spectrum LED lighting. Axillary bud activation and indirect somatic embryogenesis were more efficient, particularly in the presence of kinetin and picloram, respectively, compared to adventitious shoot regeneration. Significant cultivar differences were observed, with ‘Valentine’ being the easiest and ‘White Gold’ the most challenging to culture in vitro. To mitigate stress caused by classical growth regulators, alternative substances such as nanoparticles and natural extracts were used. Gold nanoparticles enhanced shoot proliferation and plantlet quality, while coconut and rice extracts improved survival rates during acclimatization. Enhanced metabolite production was achieved using exogenous auxins and gold nanoparticles. Guaiacol peroxidase was identified as a sensitive oxidative stress marker, with glutathione reductase being the most stable under stress. Cryogenic techniques incorporating explant encapsulation, i.e. encapsulation-vitrification, showed high effectiveness and genetic stability of plants, with nanomaterials boosting effectiveness. Coconut extract also enhanced post-thaw shoot proliferation, while sesame extract served as a natural retardant for slow-growth cultures. Mutagenic effectiveness ranked as microwaves < nanoparticles < X-rays. Comprehensive genetic variability insights were provided by integrating multiple SPAR marker systems. This review underscores the promising biotechnological advancements for L. spectabilis, emphasizing the potential of in vitro techniques, innovative cryopreservation methods, and the application of nanoparticles and plant extracts to enhance micropropagation, genetic variability, and metabolite production, thereby contributing to the conservation and commercial sustainability of this valuable ornamental-medicinal perennial.

Although Ecuador is one of the richest places in the world in terms of biodiversity of species belonging to the Orchidaceae family, some of its species are endangered. The main factors that are threatening orchid species include destruction of their habitat, inadequate management of resources, environmental contamination, and overcollection of specimens. Each orchid capsule contains thousands of seeds; however, only 2–3% germinate under natural conditions. The limited germination is attributed to factors such as the lack of seed endosperm and the need for symbiotic relationships with mycorrhizae. The in vitro orchid culture may be a strategy to achieve their efficient propagation and thus contribute to their conservation. This study reports protocols for in vitro seed germination in four species of Ecuadorian orchids: two epiphytic species, Epidendrum jamiesonis and Oncidium pentadactylon, and two terrestrials, Pleurothallis pulchella and Elleanthus capitatus. A germination percentage higher than 30% was observed in all species, which led to successful seedling development. For Epidendrum jamiesonis, effective elongation and acclimatization stages are also reported. The plants obtained from the in vitro asymbiotic culture described here could promote conservation programs and serve as a reference for the culture of other orchid species.

Haploid induction is of great importance in the breeding of cross-pollinated crops such as maize, and it also expedites the development of new varieties in a shorter period in self-pollinated crops, i.e. soybean. The success rate of traditional techniques is almost negligible at less than 1%, and their applicability is dependent on external factors in soybean. There is a lack of standardized and reproducible systems, which makes it challenging to adopt the existing systems for haploid plant production. Therefore, there is a high demand for implementing innovative approaches for this crop due to the limitations of conventional methods. The BABY BOOM (BBM) gene, which generated haploidy at high rates (> 80%) when expressed in pearl millet or through ectopic expression in the egg cells of rice, maize, and tobacco, is a novel example with promising potential. In this study, we used the egg cell-specific promoter DD45, which was cloned from Arabidopsis, to ectopically express the native soybean BBM1 (GmBBM1) gene in soybean to observe the response to haploidy induction. Initially, the clone pDD45:GmBBM1 was successfully constructed and confirmed by PCR and Sanger sequencing. The construct was subsequently transformed into soybean via a half-seed approach. The expression of GmBBM1 in both flowers and leaves increased in the T₀ transgenic soybean lines. The T₀ plants and their seeds showed developmental abnormalities described by early senescence and flowering; however, the T₁ plants exhibited normal growth characteristics. The ploidy levels of the T₁ and T₂ plants were determined by flow cytometry and chromosome counting. The flow cytometry histograms revealed haploidy in the T₁ generation; however, further chromosome counting in T₂ plants revealed changes in chromosome number, and aneuploidy, which may be due to spontaneous doubling. This system is especially important in legume crops, as no reports exist on the application of the BBM1 system in soybean. Our study will provide valuable insights for future research and advancing soybean breeding with haploid induction.

Pinus massoniana is a critical afforestation and ecological tree species in China. However, the continued existence of this pine is severely threatened by pine wilt disease. Somatic embryogenesis serves as a highly efficient clonal propagation approach. Although significant progress has been made in somatic embryogensis research on P. massoniana, resulting in the successful regeneration of plants, the limited embryogenic potential of improved cell lines and loss of embryogenic properties resulting from prolonged proliferation have posed obstacles to the industrialization of SE production. In this study, we investigated the effect of phytosulfokine on embryo development of cell lines from P. massoniana which lead to a cascade of physicochemical changes. Eight embryogenic cell lines of P. massoniana were used to observe phenotype and cytological changes. Physiological factors and the contents of nutrients and endogenous hormones were measured before and after phytosulfokine addition. We found that PSK promoted a change in the embryogenic mass of P. massoniana, leading to their development from pro-embryogenic mass (PEM)I to PEMII or PEMIII stages of pro-embryos. In addition, PSK accumulated soluble sugar, protein, and starch, and maintained redox homeostasis during cell line proliferation by reducing H₂O₂ levels. Our findings increase our understanding of how PSK affects somatic embryogensis in P. massoniana, thereby providing a valuable tool for establishing efficient somatic embryogensis systems in conifer species.

Eggplant (Solanum melongena L.) is one of the essential vegetables worldwide, and cultivated genotypes of eggplant suffer from numerous abiotic and biotic stresses. A reproducible and efficient plant regeneration system is crucial for applying molecular breeding methods to overcome the difficulties of conventional breeding programs to improve eggplant germplasm, such as genetic transformation and genome editing techniques. The three explant types, cotyledon, hypocotyl, and leaf, were obtained from two different cultivars, Pusa purple long (PL) and Pusa green round (GR) of eggplant. Three explants were cultivated on media augmented with a variety of cytokinins, including BAP, mT, and ZEA, in different concentrations. The media supplemented with mT at 8.28 µM generated the highest number of shoots, which showed the optimum regeneration efficiency for all three explants in two eggplant genotypes. The cotyledon explants generated the optimum number of shoot buds on the medium amended with low concentrations of BAP (2.22 µM), KIN (2.32 µM), and ZEA (4.56 µM), and mT at 8.28 µM. The mT (8.28 µM) and BAP at 2.22 µM combinations produced 25.8 and 18.3 shoots in PL and GR genotypes, respectively. The addition of various concentrations of IAA (1.43 to 5.71 µM), IBA (1.23 to 4.92 µM), and NAA (1.34 to 5.37 µM) in combination with mT (8.28 µM) were evaluated to find out their role on the induction and proliferation of numerous shoot buds from cotyledon explants of two cultivars of eggplant. The medium augmented with mT (8.28 µM) and IAA (2.85 µM) produced 26.4 shoots and 17.8 shoots in cotyledon explants of PL and GR cultivars, respectively. The optimum rooting efficiency of shoots was recorded on the medium containing the IAA (5.71 µM) and produced complete plantlets. The plantlets showed 100% similarity with their mother plants.

BBX transcription factors have a transcriptional regulatory role in response to light, circadian cues, and brassinosteroid-light crosstalk signaling. However, the functions of BBX in soybean resistance to seed deterioration have not been shown. In our previous study, a soybean gene GmSBH1 and a HSE cis-element of GmSBH1 promoter were found in response to high temperature and humidity (HTH) stress. GmCOL4 was a candidate protein, which bound to HSE cis-element. In the present study, GmCOL4 was isolated and characterized. Subcellular localization and transcriptional activation assays showed that GmCOL4 was a nuclear protein with transcriptional activation function. The BBOX2 domain was found to play an obvious role in transcriptional activation activity of GmCOL4. Furthermore, GmCOL4 interacted with GmZTL1 was confirmed in vivo and in vitro. GmCOL4 and GmZTL1 presented different expression patterns among diverse soybean tissues and were synergistically involved in response to HTH stress in developing seeds. Overexpression of GmCOL4 and GmZTL1 could alter tobacco phenotypes and enhance developing seed tolerance to seed deterioration under HTH stress. Based on these results, a regulation network was conjectured, GmCOL4 interacts with GmZTL1 to co-regulate the GmSBH1 via directly binding to the HSE cis-element, thereby enhancing the soybean resistance to seed deterioration under HTH stress and affecting leaf development.

Soybean (Glycine max (L.) Merrill) contains isoflavones, and in particular genistein, which have clinically proven roles. As a result, this bioactive compound is greatly valued in the pharmaceutical industry. Hence, this study was aimed to develop an adventitious root culture system for the production of soy isoflavones (genistein) using hypocotyl explants. In solid culture system, auxin (IAA, 0–57.0 µM; NAA, 0–53.7 µM; and IBA, 0–49.2 µM), media strength (¼x, ½x, ¾x, 1x, and 2x) and in the liquid culture system, IBA (0–49.2 µM), media strength (¼x, ½x, ¾x, 1x, and 2x), and sugars (glucose, fructose, and sucrose) were optimized. After 30 days of culture, IBA at 39.3 µM demonstrated the maximum response in root parameters, with the 1x solid MS medium showing improved root parameters compared to other medium strengths. For liquid system culture optimization, full strength MS medium supplemented with 39.3 µM IBA showed the highest root biomass in liquid medium. Among sucrose concentrations, the highest accumulation of root biomass was observed at 3% (3.87 g flask ^− 1) followed by 4% (3.65 g flask ^− 1), however, 4% sucrose proved favorable for genistein synthesis (9.34 mg g^− 1 DW) compared to 3% (6.29 mg g^− 1 DW). Additionally, the maximum levels of phenolic and flavonoid content were observed at 4% sucrose, correlating with higher antioxidant activities in DPPH and FRAP assays. Gene expression analysis of isoflavone biosynthetic genes revealed higher levels of expression at 4% sucrose compared to the control. These results underscore the crucial role of determining the optimum culture conditions and the effect of sucrose in enhancing root biomass and genistein content in soybean adventitious root cultures.