In Vitro Cellular & Developmental Biology - Plant最新文献

A protocol for in vitro induction of autoallododecaploid Echinochloa crus-galli with colchicine treatment was established. Colchicine concentration and exposure time had significant effects on the callus survival rate, shoot differentiation rate, and chromosome doubling rate. With the increase of colchicine concentration and the extension of treatment time, the callus survival rate and shoot differentiation rate both decreased. Treatment with 0.05% colchicine for 48 hr was the most effective condition for polyploidization, yielding 42.99% dodecaploids. The authenticity of the induced dodecaploid was confirmed by nuclear DNA content, chromosome number, and stomatal and morphological characteristics. Dodecaploid plants showed the obvious “giant” effects of polyploid organs, as well as significantly reduced seed set rate. The concentrations of calcium, iron, most free amino acids, and total free amino acids in dodecaploid plants were significantly higher than those in hexaploid plants. The results showed that the chromosome of allohexaploid E. crus-galli could be redoubled. The dodecaploid E. crus-galli could still grow normally and showed advantages associated with polyploidization, such as enlarged organs and increased nutrient concentrations. The study enriched the germplasm resources of barnyard grass and laid a foundation for the utilization and theoretical research of E. crus-galli in the future.

Plants exposed to harsh external environments such as cold and drought display stunted growth and delayed development. WRKY proteins can bind to the cis-acting element W-box to activate or repress transcription of downstream target genes to regulate responses, including different abiotic and biotic stresses. In this experiment, Malus baccata (L.) Borkh was used to isolate the MbWRKY65 gene to study its cold and drought resistance functions. The outcomes demonstrated that MbWRKY65 is most similar to the apple MdWRKY65 protein. Its location in the nucleus is shown by subcellular localization data, which is in line with its role as a transcription factor. MbWRKY65 was expressed richer in roots and older leaves in response to cold and drought treatments. The transgenic tomato showed considerably reduced contents of malondialdehyde (MDA), hydrogen peroxide (H₂O₂), superoxide anion radical (O₂⁻), and relative conductivity during cold and drought stress in contrast to wild-type (WT) and unloaded lines (UL), but greater contents of proline and chlorophyll, as well as higher activity of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD). Stress-related genes (LeCBF1, LeABI3, LeABF4, LeCBF3, LeNCED1, and LeDREB1) in transgenic tomato plants were positively regulated under conditions of cold and drought, and in contrast to the WT and UL lines, the transgenic tomato showed a considerably greater expression level. In conclusion, MbWRKY65 actively contributes to plant tolerance to cold and drought.

Saposhnikovia divaricata (Turcz. ex Ledeb.) Schischk is a medicinal plant with a broad spectrum of pharmacological activities, which are attributed to the presence of various bioactive compounds, including chromones. The uncontrolled harvesting of this plant has resulted in the depletion of its natural resources. The ability of S. divaricata to regenerate in vitro was studied. Aseptic culture was established by sterilizing seeds and germinating them, both with and without the pericarp. Adventitious shoot formation through direct morphogenesis was achieved on Murashige and Skoog medium in the presence of different cytokinins: benzylaminopurine, meta-topolin, and thidiazuron, at concentrations of 0.1 µM, 0.5 µM, and 1.0 µM. It has been determined that the most preferable explants were the cotyledonary nodes of aseptic seedlings, as they enable the rapid production of 1.4 ± 0.5 to 10.2 ± 2.0 microshoots per explant, depending on the type and concentration of the cytokinin used. Successful regeneration also occurred from the first true leaves of seedlings in vitro. Microshoots were formed directly from the explant tissues, with their quantity ranging from 1.7 ± 0.7 to 4.4 ± 0.9. The highest number of microshoots was induced by thidiazuron, but after cultivation with meta-topolin, the regenerated plants showed the highest frequency of rooting, ranging from 66 to 97%. Somatic embryoids were obtained when the first true leaves were cultivated with 2,4-dichlorophenoxyacetic acid (10.0 µM). Morphogenesis occurred via the indirect pathway (through the callus formation stage), accompanied by the formation of up to 24.2 ± 6.1 somatic embryoids, with a rooting frequency reaching 96%. These studies provide a preliminary basis for developing a protocol for the clonal micropropagation of S. divaricata.

Campanula leblebicii, discovered by Yıldırım, is found only in the village of Ovacık in the Kemalpaşa district of İzmir province, and it is one of the valuable endemic species for Turkey flora. Petiole and leaf explants were cultured on full-strength Murashige and Skoog (MS) medium containing 30.0 g L⁻¹ sucrose; 4.0 g L⁻¹ Gelrite; 0.3 mg L⁻¹ 1-naphthaleneacetic acid (NAA); 0, 0.5, 1.0, 2.0, or 3.0 mg L⁻¹ zeatin (ZEA); and 0, 0.5, 1.0, 2.0, or 3.0 mg L⁻¹ 6-benzyladenine (BA) for callus initiation. To obtain shoot formation, callus was transferred to full-strength MS medium containing 0.3 mg L⁻¹ NAA; 0, 0.5, 1.0, 2.0, or 3.0 mg L⁻¹ gibberellic acid (GA₃); and 0, 0.5, 1.0, 2.0, 3.0 mg L⁻¹ BA. Efficient callus (93.33%) and shoot formation (73.33) were observed from petiole explants cultured on MS medium containing 0.3 mg L⁻¹ NAA and 1.0 mg L⁻¹ BA. Rooting was obtained spontaneously in all shoot initiation medium, but efficient rooting occurred on MS medium supplemented with 0.3 mg L⁻¹ NAA and 1.0 mg L⁻¹ BA. Plants were acclimatized to the pots including sterile peat and perlite (1:1, v/v), and, in total, 12 plants were acclimatized from one genotype.

An efficient in vitro propagation protocol for a medicinal plant, Persicaria glabra (Willd.) M. Gomez, has been established. Nodal explants were cultured in vitro on agar-gelled (0.8%) Murashige and Skoog (MS) medium containing different concentrations of cytokinins, namely N⁶-benzyladenine (BA), 6-(γ,γ-dimethylallylamino)purine (2-iP), or kinetin (KIN). BA (2.5 µM) was found to be the most effective cytokinin for axillary bud proliferation. A growth retardant ancymidol (ANC) was combined with 2.5 µM BA to enhance the multiple shoot induction and ensure the overall health of micro shoots. The nodal explants taken from micro shoots developed in 2.5 µM BA were subcultured on agar-gelled MS medium supplemented with different concentrations of ANC along with 2.5 µM BA. Among the different concentrations tested, 4.0 µM ANC combined with 2.5 µM BA produced a maximum number of multiple shoots (13 shoots per explant). A progressive decline in the shoot height was observed with increasing concentrations of ANC and significantly reduced shoot length (0.5 cm) at a higher concentration (12.0 µM ANC). Reduced leaf area and more chlorophyll were noticed in ANC-treated plants than in BA-alone-treated plants. This facilitated better growth and development of robust micro shoots. Well-developed micro shoots were excised from 4.0 µM ANC along with 2.5 µM BA and subjected to ex vitro rooting experiments using different concentrations of auxins, including α-naphthaleneacetic acid (NAA) or indole-3-butyric acid (IBA). The maximum number of roots (46.33 roots per shoot) and root length (14.53 cm) were observed in 0.5 mM IBA with 89.11% of the response. The rooted plants were successfully acclimatized in field conditions with 100% survival. The genetic fidelity analysis of in vitro–raised plants was tested using inter simple sequence repeat (ISSR) markers, and they showed genetic monomorphism with the mother plant. The present study suggests that adding ANC and BA would enhance the multiple shoots induction and high rate of acclimatization of P. glabra cultures. The addition of ANC resulted in a 2.6-fold increase in shoot number and avoided undesirable elongation of shoots. Healthy shoots, thus, produced in ANC combined with BA-added medium doubled the survival of plants during acclimatization over a BA-alone medium and can be used in many other plant species that have the problem of developing shoots of an unhealthy nature coupled with abnormal internodal elongations.

Triploid lacquer trees have a higher yield of raw lacquer and greater stress resistance; however, it is challenging to develop efficient and rapid seedling culture techniques because of the influence of metabolites with raw lacquer. In this study, the stem segments of a triploid lacquer tree were used to investigate the effects of explant sterilization treatment, different combinations of exogenous hormone concentrations on the induction of axillary buds, seedling development, and rooting, as well as transplanting substrate ratios. The results showed that the optimal combination of disinfection treatment for triploid Toxicodendron vernicifluum (Stokes) F. A. Barkl explants involves 75% alcohol immersion for 10 s, followed by 2.0% benzalkonium chloride treatment for 1 min and 0.1% HgCl₂ treatment for 4 min. The suitable medium for axillary bud induction was Murashige and Skoog (MS) containing 0.1 mg•L⁻¹ zeatin (ZT) and 0.1 mg•L⁻¹ 6-benzylaminopurine (6-BA), resulting in a high induction rate of 95.0%. Use of 1/2 MS supplemented with 0.5 mg•L⁻¹ 6-BA and 0.3 mg•L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) was the most effective for axillary bud proliferation. The combination of 1/2 MS containing 0.1 mg•L⁻¹ 6-BA, 0.5 mg•L⁻¹ 2,4-D, 0.5 mg•L⁻¹ indole-3-butyric acid (IBA), and 0.2% activated carbon (AC) was an optimum for hardening-off. With woody plant medium (WPM) supplemented with 0.5 mg•L⁻¹ naphthaleneacetic acid (NAA) and 0.5 mg•L⁻¹ 2,4-D and 0.5 g•L⁻¹ AC as a rooting medium, the rooting rate was 67.8%. Also, the appropriate transplanting substrate ratio for triploid Toxicodendron vernicifluum (Stokes) F. A. Barkl was grass charcoal:laterite:humus = 1:1:1. Collectively, the results provided the technical assurance for seedling scale multiplication and propagation, facilitating its application in production.

The Indian lotus (Nelumbo nucifera Gaertn.) is a popular ornamental plant and a source of traditional herbal medicine. Its various parts are widely used in the pharmaceutical, cosmeceutical, and nutraceutical industries. The recent study aimed to develop a high-frequency in vitro regeneration system in Indian lotus. This study utilizes the pink lotus cultivar from the Botanical Garden of CSIR-NBRI Lucknow. The study was successfully achieved through direct and indirect methods using different plant growth regulators (PGRs). A direct regeneration system was established using explants shoot apical meristem and plumule cultured on SIM supplemented with 17 combinations and concentrations of BAP and NAA. Both explants produced the highest number of shoots with a combination of 4.44 μM BAP and 0.55 μM NAA. The highest number of shoots per explant 25 ± 1.0 was developed from the shoot apical meristem, while the plumule explant developed 16.3 ± 0.5 shoots per explant. Thereafter, the plantlets were transferred to LRIM, which contained 17 combinations and concentrations of NAA or IBA and BAP. The maximum number of roots, per explant 23.6 ± 0.5, was developed from shoot apical meristem using 2.22 μM NAA and 0.54 μM BAP. The highest number of roots, per explant 23 ± 1.0, was developed from the plumule using 4.44 μM IBA. Indirect somatic embryogenesis has been established through callus culture. The leaf segments were cultured onto a callus induction medium supplemented with ten combinations of 2,4-D and BAP. The high-frequency callus formation 24.33 ± 0.5 was obtained with a 5.0 μM 2,4-D and 1.0 μM BAP combination. All developmental stages at the proembryo, globular, heart, torpedo, and mature embryos were formed on concentrations of 2,4-D and BAP. After inducing shoot and root growth, well-developed plantlets were transferred to the greenhouse, resulting in a success rate of 18.47%.

In this study, the aim was to try to increase the anthocyanin-producing in callus and to investigate the ultrastructure of the developing callus cells of leaf, apical meristem, and node explants of Vaccinium arctostaphylos L. To generate callus, sterilized explants were seeded in Woody Plant Medium (WPM) containing different concentrations of indoleacetic acid or zeatin. The most calluses containing anthocyanin were obtained from the apical meristem explant cultured on medium containing 2.0 mg L⁻¹ indoleacetic acid. The medium containing 2.0 mg L⁻¹ indoleacetic acid was modified with different sucrose concentrations and were prepared to increase anthocyanin production and callus biomass. The best callus production was obtained with apical meristem explants cultured on medium containing 50.0 g L⁻¹ sucrose and was observed as a pink color callus when examined under a fluorescence microscope with 525-nm green and 610-nm red wavelengths. The callus had autofluorescent. The cell ultrastructures of the pink- and yellow-colored callus growing in medium containing 50.0 g L⁻¹ sucrose were examined using a transmission electron microscope (TEM) after the preparation process. Cells that developed from yellow callus and were called type 1 had an elliptical or long, angular shape. Type 1 cells had a large vacuole. There were electron-dense vesicles around the vacuoles attached to the inner surface of the tonoplast, and anthocyanic vacuolar inclusion–like inclusions were seen in the vacuole. The cytoplasm and organelles were trapped between the vacuole and the cell wall. It was observed that cells developed from pink color callus (called type 2) often had an elliptical shape. Similar to type 1 cells, it has a large vacuole; its cytoplasm is trapped between the cell wall and the vacuole with very few intercellular spaces. Some of the vesicles had an anthocyanic vacuolar inclusion appearance within the vacuole. A macroautophagy-like fusion was placed between the cytoplasmic vesicles and the vacuole. These observations showed that the anthocyanin-producing cells of the V. arctostaphylos L. transported anthocyanins to the vacuole using the vesicular transport method. The current research is the first study to fill the gap in the literature, so it is extremely important as a source for future studies.

This study investigates the influence of various parameters on the efficiency of cultivating and preserving Lemna aequinoctialis duckweed samples in vitro, with the aim of minimizing time, labor, and storage expenses. The method addresses the issue of limited space and the risk of sample loss by storing multiple duplicates (more than one tube or flask per sample) in a space-efficient and cost-effective container. Due to the practical implications and capacity to decrease operating expenses, our results are helpful for large-scale implementation. Using a single frond resulted in a longer preservation effect compared to sample densities of three and five fronds. Initial samples can be cultured in nutrient solution with or without sugar, stored in tap water or nutrient solution, kept at a stable temperature of 25 ± 2°C or room temperature. The test tubes with duckweed were not covered, or covered by black paper up to the solution surface or higher. The outcomes demonstrated that all three duckweed samples (HNP_005, HNP_026, and HNP_031), whether grown in SSM or SFM media, were best preserved if the tubes were covered by black paper higher than the solution surface (CH) and can be kept at room temperature for at least 9 mo.

Cryopreservation for sugarcane (Saccharum spp. hybrids) germplasm conservation is well established. Virus elimination using droplet-vitrification (D-V) and cryo- or osmo-therapy has only been recently reported for sugarcane mosaic virus (SCMV). In this study, exposing large (3 mm) in vitro shoot tips of cultivars N12, N19, N58, and NCo376 infected with sugarcane yellow leaf virus (SCYLV) and NCo376 co-infected with SCMV and SCYLV were tested for virus elimination using both of the above-mentioned techniques. Cryo-therapy involved the exposure of infected in vitro shoot tips to the D-V protocol followed by recording recovery and virus-free shoot tips 16 wk after treatment. Osmo-therapy, consisting of the same treatment as cryo-therapy without immersion in liquid nitrogen (LN), was included for comparative purposes. Cryo-therapy resulted in 100% of the recovered shoots being SCYLV-free in cultivars N19, N58, and NCo376 and 83% in N12 when compared with untreated material. Osmo-therapy showed 58% (N12), 91% (N19 and N58), and 100% (NCo376) of shoots being clear of SCYLV when compared with untreated in vitro control plants (0 to 8%). Both techniques reduced the regrowth levels of treated shoot tips (22 to 57% recovery) when compared with untreated controls (92 to 97%). A novel finding of the study was that NCo376 co-infected with SCMV and SCYLV showed 100% virus-free recovered shoots after cryo-therapy and 92 to 100% of healthy shoots after osmo-therapy, compared with controls, which had 17 to 42% virus-free shoots. Plants from all cultivars that were re-tested 4 mo after hardening maintained their virus-free status. The described techniques for virus eradication offer a promising solution for the provision of clean vegetative planting propagules and safer germplasm exchange.