In Vitro Cellular & Developmental Biology - Animal

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ISSN / EISSN : 1071-2690 / 1543-706X
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Jingjing Hou, Hao Chen, Yuzhen Fang, Ying Zhu, Bing Han, Chuanqing Sun,
In Vitro Cellular & Developmental Biology - Animal pp 1-10; doi:10.1007/s11627-021-10202-3

The rice (Oryza sativa L. ssp. indica) cultivar “93-11” is a reference genotype used in many studies; however, its recalcitrance during transformation and regeneration greatly limits functional genomics and breeding research. In this study, we developed an efficient Agrobacterium tumefaciens–mediated transformation system for “93-11”, based on the phosphor mannose isomerase (PMI) positive selection system. Calli of “93-11” were transformed with the Agrobacterium strain EHA105 harboring a binary vector, containing the PMI gene and an RNAi sequence targeting TILLER ANGLE CONTROLLING 1 (TAC1). We also developed a method for removing Agrobacterium from the callus following co-cultivation and determined the optimal conditions for PMI selection and callus differentiation. Compared with the hygromycin phosphotransferase (HPT) selection system, newly generated calli were recovered at higher rates on the PMI selection medium, with “93-11” transformation frequency reaching 7.50%. Notably, the down-regulation of TAC1 in the “93-11” genetic background led to a more compact plant architecture. Overall, this work presents a genetic transformation system suitable for the reference indica variety “93-11” using a non-antibiotic selectable agent. This advance will facilitate functional genomic research and the improvement of agronomic traits for indica varieties recalcitrant to transformation.
, Hassan Salehi
In Vitro Cellular & Developmental Biology - Animal pp 1-14; doi:10.1007/s11627-021-10193-1

While the antimicrobial activity of silver nanoparticles (AgNPs) is well established, the phytostimulatory and/or phytotoxic influences of AgNPs in closed tissue culture vessels is more controversial, to some extent. This is because numerous research papers have been published that ultimately conclude apparently contradictory results. In this paper, the physiological responses which AgNPs induce are studied in both tobacco micro-shoots and tobacco seedlings via the utilization of a number of morphological, biochemical, and molecular approaches. This report provides direct evidence that AgNPs positively regulate growth response in tobacco via a curtailing of ethylene production and an inhibition of the general ethylene signaling pathway. Tobacco seedlings exposed to media supplemented with high concentrations of AgNPs (150 μg mL−1) were shown to exhibit a hypersensitivity response. However, when compared to the control, tobacco seedlings exposed to sub-lethal concentrations of AgNPs (50 μg mL−1) displayed increased leaf fresh weight and plant height, as well as a 6-fold increase to root length. Inductively coupled plasma mass spectrometry (ICP-MS) confirmed the presence of silver ions (Ag+) in AgNP-treated tobacco leaves, and given that silver is known to be a potent inhibitor of ethylene action, this result led us to question whether AgNPs might be inhibiting ethylene receptor expression. Subsequent qRT-PCR testing in tobacco tissue treated with 50 μg mL−1 AgNP revealed a downregulation of ETR1, ERS1, and CTR1, which are all key ethylene signaling genes, as well as a downregulation of an important downstream ethylene-synthesizing gene, ACS2. This clearly established that the increase to root length in response to AgNPs might be occurring via an AgNP-dependent suppression of ethylene signaling genes. Further, gas chromatography analysis confirmed that tobacco planted on media supplemented with sub-lethal concentrations of AgNPs exhibited a reduction of gaseous ethylene production in closed in vitro vessels. Plants exposed to a higher concentration of AgNPs in their media did not show any additional inhibitory effects, with regard to ethylene production. Nonetheless, ultimately, the collective data implies that AgNPs inhibit various aspects of the ethylene signaling pathway, in addition to inhibiting the production of ethylene itself, and both of these inhibitions occur in a dose-dependent manner.
Shou-Wei Tian, Si-Nian Xing,
In Vitro Cellular & Developmental Biology - Animal pp 1-11; doi:10.1007/s11627-021-10187-z

Vegetables are an important component of the agriculture production systems and an important part of a healthy diet. Traditional breeding methods are difficult to meet the growing demands for yield and quality of vegetable crops; therefore, new approaches are required. CRISPR/Cas9 gene-editing technology has the advantages of short time period, simple and quick operation, and high editing efficiency. It has become an important tool for plant science and molecular breeding and has been widely used in the creation and genetic improvement of vegetable germplasm resources. This article mainly reviews the application progress and challenges of CRISPR/Cas9 in vegetable improvement, to provide a new perspective for vegetable breeding.
In Vitro Cellular & Developmental Biology - Animal pp 1-10; doi:10.1007/s11627-021-10186-0

Gentiana scabra is an important medicinal herb that is used to treat inflammation, tuberculosis, rheumatism, cholecystitis, and hepatitis. The primary bioactive components in G. scabra are secoiridoids derived from the roots and rhizomes of the plant. In this study, we sought to establish a means of reliably producing these secoiridoids through G. scabra in vitro adventitious root culture. To that end, we studied the effect of phytohormones, light condition, explant type, and medium composition on adventitious root induction and secoiridoid production. Maximal induction frequency (100%) was ultimately achieved when using leaf explants cultured on Murashige and Skoog (MS) medium containing 3.0 mg L−1 NAA and 0.25 mg L−1 TDZ with a 16/8-h photoperiod, yielding 53.20 ± 3.15 roots per explant. We explored root growth curves over an 8-wk period in MS liquid medium prepared as above, and observed maximal fresh weight (FW) and dry weight (DW) values of 26.94 ± 0.38 g per flask and 1.79 ± 0.032 g per flask, respectively, in 4-wk-old cultures. The accumulation of secoiridoids (gentiopicroside, swertiamarin, and sweroside) in these adventitious root cultures was assessed via high-performance liquid chromatography (HPLC), revealing maximal gentiopicroside (25.59 ± 0.65 mg g−1 DW), swertiamarin (1.61 ± 0.04 mg g−1 DW), and sweroside (4.42 ± 0.11 mg g−1 DW) levels after 4 wk. Together, our results reveal that G. scabra adventitious root cultures represent a viable approach to the in vitro production of secoiridoids. This is the first study to our knowledge to have explored the isolation of secoiridoids following the in vitro growth of G. scabra adventitious roots. These data may represent an important step toward the development of large-scale adventitious root cultures for commercial secoiridoids production.
, T. D. Silva
In Vitro Cellular & Developmental Biology - Animal pp 1-13; doi:10.1007/s11627-021-10177-1

This study investigated developmental changes in cold stressed microspores of Indica rice variety At 303. After 3 d at 10°C, approximately 76% of microspores were in the late uni-nucleate stage. Even after 5 d, 49% of viable cells remained in the late uni-nucleate stage without advancing to the bi-nucleate stage. In comparison, microspores undergoing normal gametogenesis in planta progressed rapidly from uni-nucleate to bi-nucleate stage eventually forming tri-nucleate pollen during this period. Thus, cold stress prevented normal microspore development and retained cells in the late uni-nucleate stage which is the most favorable stage for in vitro induction of sporophytic development in rice. Uni-nucleate microspores subjected to cold stress showed a characteristic pattern consisting of several minute vacuoles surrounding a centrally positioned nucleus, which can be interpreted as an early indicator of sporophytic determination in Indica rice microspores. During in vitro culture phase, freshly plated yellow anthers became brown. After 4 wk in culture, 51% of the anthers had discolored. Significantly, all yellow anthers contained only non-viable cells whereas 10% of the brown anthers had few viable cells. Some microspores in brown anthers underwent division on callus induction medium. The first division was symmetrical and occurred after 2 wk. The second division occurred after 4 wk and resulted in four-celled structures. Anther-derived callus was either compact or friable. Histo-differentiation occurred mostly from compact callus. Cell clusters, each delimited by a protoderm, were observed in histological sections of callus grown for 2 to 4 wk on regeneration medium. Within a cellular unit, two heterogeneous cell populations were arranged in concentric rings with larger cells in the center and smaller cells towards the periphery. However, an apical-basal polarity that is present in embryo-like structures was not observed. Therefore, it may be surmised that in Indica rice, regeneration from anther-derived callus takes place not by the formation of somatic embryos but by direct organogenesis.
Xiuyu Li, Qi Guo, Chao Han, Juan Li, Li Dong, Sen Cao, Zijie Zhang, Yuhan Sun, Ruohan Wang,
In Vitro Cellular & Developmental Biology - Animal pp 1-8; doi:10.1007/s11627-021-10192-2

We explored multiple experimental treatments for regenerating tissue-cultured shoots of tetraploid Robinia pseudoacacia L., and assessed the most suitable combination of treatments for the development of a highly efficient regeneration system. Leaflets of tissue-cultured shoots were used as explants to study the effects of four different treatments on leaf regeneration capacity. Treatments included leaflet position on donor leaves, light conditions during culturing, the orientation of leaves on the growth medium, and the number of cut wounds per leaf. After determining an appropriate stem explant culture period, we assessed the influence of four treatments on regenerative capacity. The 35-d-old leaf explants produced the largest amount of callus tissue, while the highest average callus growth index was observed on the leaves cultured for an additional 30 d. Each leaf produced an average of five adventitious buds. The greatest numbers of buds and callus tissues were observed in the leaves collected from the uppermost position on the shoot, treated with six cut wounds per leaf, and grown under illuminated conditions.
In Vitro Cellular & Developmental Biology - Animal pp 1-15; doi:10.1007/s11627-021-10200-5

In vitro micropropagation is an important technique for coffee multiplication performed by somatic embryogenesis, which can be indirect, with the formation of calluses, or direct, when embryos are formed directly from explant cells. In the present study, we characterized the ontogenesis of somatic embryos regenerated via indirect and direct somatic embryogenesis in leaf explants from Coffea arabica ‘Mundo Novo’ and assessed the development of these embryos in leaf explants from adult plants maintained ex situ and in vitro. Anatomical analyses showed that leaf explants present structural differences depending on their origin. In the direct pathway, tissue develops more rapidly in explants from in vitro plants than in those from ex situ explants. In both methods, the formation of a pro-embryogenic mass was found to be essential for embryo formation. In the indirect pathway, calluses from ex situ material presented elongated cells and a loose external appearance. Conversely, calluses from in vitro material presented large regions of meristematic cells with a single large nucleus and dense cytoplasm. We propose that indirect somatic embryogenesis in in vitro–grown explants may accelerate genetic breeding in coffee, and it represents the most efficient condition for somatic embryogenesis, producing embryos in less time than the required by ex situ plants (indirect: 260 d in ex situ conditions, 62 d in vitro; direct: 270 d ex situ, 86 d in vitro).
Deguo Han, Jiaxin Han, Tianlong Xu, , Chunya Yao, Tiemei Li, Xiaohan Sun, Xinhui Wang,
In Vitro Cellular & Developmental Biology - Animal pp 1-11; doi:10.1007/s11627-021-10199-9

Abiotic stress, such as drought, salt, and cold, affects normal plant growth. Ethylene response factors (ERFs) play an important role in the responses of plants to different stresses. An ERF gene was cloned from Malus baccata (L.) Borkh and named as MbERF12. A subcellular localization study proved that MbERF12 was a nucleus-localized protein. The expression level of MbERF12 was higher in stems and roots, which was markedly affected by high salinity, low temperature, and ethephon treatments. When MbERF12 was transformed into Arabidopsis thaliana, it obviously improved salt and low-temperature tolerance in transformed plants. Overexpression of MbERF12 in transformed A. thaliana also led to higher levels of peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) and higher contents of ethylene, chlorophyll, and proline, while the content of malondialdehyde (MDA) was lower, especially when being dealt with low-temperature and high-salinity stresses. MbERF12 plays a key role in the response to salt and cold stresses in Arabidopsis by improving the scavenging ability for reactive oxygen species (ROS) through ethylene signal transduction. We proposed that MbERF12 may function as a positive regulator for abiotic stress responses and can be considered as a potential gene for improvement of cold and high-salinity tolerance in plants.
Qin Wang, Rong Fu, Caihua Kong, Kena Liu, Huaxin Si,
In Vitro Cellular & Developmental Biology - Animal, Volume 57, pp 610-619; doi:10.1007/s11626-021-00586-2

This study investigates the protective effect of small peptides from Periplaneta americana (SPPA) on hydrogen peroxide (H2O2)–induced apoptosis of ovarian granular cells. H2O2 was applied to human ovarian granular cells (KGN cell strains). Cell viability was tested by cell counting Kit-8 (CCK-8). Cell apoptosis was tested by flow cytometry, and a cell apoptosis model was established. The model cells were treated with SPPA, and the cell survival rate was monitored using the CCK-8 method. The oxidative stress state of cells was examined using SOD, ROS, MDA, and NO kits. The protein expression levels of SIRT1, p53, and the apoptosis-related gene Caspase3 were measured using Western Blot methodology. Relative to the control group, cell viability declined significantly after the H2O2 treatment only (P < 0.01), while the apoptosis rate increased significantly (P < 0.01). The activity of SOD was weakened significantly (P < 0.01), while the cell levels of ROS, MDA, and NO increased dramatically (P < 0.01). Cell viability dramatically recovered (P < 0.01), and the SOD activity is hugely increased (P < 0.01) after SPPA treatment. In contrast, contents of ROS, MDA, and NO decreased sharply (P < 0.01), and significant dose-response relationships are characterized. Moreover, the H2O2 treatment group showed significantly downregulated expression of SIRT1 (P < 0.01) but significantly upregulated expressions of p53 and Caspase3 (P < 0.01) compared to the control group. Following the SPPA treatment of apoptosis cells, expression of SIRT1 increased significantly, while expressions of p53 and Caspase3 declined significantly (P < 0.01). This study suggests that SPPA inhibits H2O2-induced human KGN cell apoptosis through antioxidation, and the SIRT1/p53 signal pathway mediates the antioxidation.
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