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Author(s):  
Alejandro Hernández-Soto ◽  
Jason Pérez-Chávez ◽  
Rebeca Fait-Zuñiga ◽  
Randall Rojas-Vásquez ◽  
Andres Gatica-Arias ◽  
...  

The development of gamma rays mutant rice lines would be a solution for introducing variability in already farmer using varieties. In vitro gamma (60Co) mutagenesis reduces chimeras and allows a faster selection of desired traits but requires laboratory process optimization. The objective of the present work was the in vitro establishment of a recalcitrant rice embryogenic calli, the determination of its sensitivity to gamma radiation (Co-60), sequencing MATK and Rubisco for identification purposes, as well as generation optimization. The radiosensitivity of embryogenic calli resulted in an LD50 of 110Gy, while the 20% lethal dose was 64Gy. All sequenced genes matched perfectly with already reported MATK and Rubisco O. sativa genes with a clear SNP that identifies the local variety related to the southeast Asia Region. Callus induction improves with an MS with 2mg/L 2,4D, and the regeneration was achieved with an MS medium with 3mg/L BAP and 0,5mg/L NAA. The optimized radiation condition was 60Gy with an 83% regeneration in a semisolid medium, allowing a balance between mutation and regeneration. When increased to 80Gy, the regeneration rate falls to 29%. An immersion system (RITA®) of either 60 or 120 seconds every 8hours allowed a systematic and homogeneous total regeneration of the recalcitrant line, in contrast with the semisolid medium that resulted in positive but irregular regeneration. Other well-known recalcitrant cultivars, CR1821, CR1113 also had an improving regeneration in the immersion system, demonstrating its potential use for recalcitrant materials. To our knowledge, this is the first report on using an immersion system to allow regeneration of gamma-ray mutants from recalcitrant rice materials.


2021 ◽  
Vol 12 ◽  
Author(s):  
Sakari Välimäki ◽  
Teresa Hazubska-Przybył ◽  
Ewelina Ratajczak ◽  
Mikko Tikkinen ◽  
Saila Varis ◽  
...  

Somatic embryogenesis is being piloted for the commercial production of genetically improved Norway spruce (Picea abies L. Karst) forest regeneration material in Finland. The main challenge to making the process commercially relevant is the dependence on time-consuming and highly skilled manual labor. Automation and scaling up are needed to improve cost-effectiveness. Moving from the proliferation of embryogenic tissue on semisolid media to suspension cultures could improve process scalability. In a series of four experiments (overall, with 20 cell lines, 4–9 per experiment), the suitability of proliferation in suspension culture for Norway spruce somatic embryogenesis was evaluated based on the growth rate, indicators of stress conditions, good-quality cotyledonary embryo yield, and embling survival in a greenhouse. The proliferation rate in suspension was found equal to on semisolid media, but with a remarkable genotypic variation. Embryogenic tissue matured directly without pre-treatments from suspension onto semisolid media produced lower numbers of good-quality embryos than tissue matured from semisolid media. Rinsing the suspension-grown tissue with hormone-free liquid media before maturation improved embryo yield, bringing it closer to that of semisolid-grown tissue. Decreasing 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid concentrations in suspension proliferation media to 0.5 or 0.1 times those in semisolid media did not affect tissue growth and did not improve embryo production. The hydrogen peroxide (H2O2) content and guaiacol peroxidase activity were elevated in suspension cultures compared with semisolid medium, which had the same plant growth regulator content. In one experiment out of four, the greenhouse survival of germinants was lower when proliferation was carried out in full strength suspension than on semisolid media; in other experiments the survival rates were equal.


Author(s):  
Saikat Gantait ◽  
Manisha Mahanta

Abstract Background Gerbera jamesonii Bolus ex Hooker f. (African daisy) is listed among the top five most important ornamental plants in the global floricultural industry. To satisfy its demand, the floriculture industry relies on reproducible and effective propagation protocol while retaining the genetic uniformity of G. jamesonii. The present study, for the first time, reports the potential of picloram for enhanced induction of organogenic calli from leaves of G. jamesonii and its high-frequency indirect regeneration. Results The fastest induction of calli with maximum fresh and dry weight was recorded in the Murashige and Skoog (MS) semisolid medium supplemented with 1 mg/l picloram. In addition, callus induction was observed in 2,4-dichlorophenoxy acetic acid- and α-napthaleneaceticacid-supplemented media but with delayed response and reduced fresh and dry weight. The proliferated calli were transferred to shoot induction media containing MS salt and 0.5–1 mg/l N6-benzylaminopurine, kinetin, or thidiazuron. A mean number of ~6 shoots per callus were developed after 5 days of culture in the MS medium supplemented with 1 mg/l kinetin, with a mean length of 5.2 cm. Successful rooting of shoots was achieved in the MS medium fortified with 1.5 mg/l indole-3-acetic acid, wherein the earliest root initiation (~5 days), as well as the maximum number (~9) and length (~4.8 cm) of roots, were recorded. Complete plantlets were primarily acclimatized in sand before being transferred to a mixed substrate (of soil, sand, tea leaf waste, and cow urine) that secured >90% survival and further growth of the plantlets. Eventually, clonal fidelity of the in vitro regenerants assessed via inter-simple sequence repeats (ISSR) primers exhibited a monomorphic banding patterns that suggested genetic integrity within the plantlets as well as with their mother plant. Conclusions The results of the present study should be of interest for commercial propagation and mutagenesis- as well as genetic transformation-related research.


2021 ◽  
Author(s):  
Lourdes Delgado-Aceves ◽  
Liberato Portillo ◽  
Raquel Folgado ◽  
Felipe de Jesús Romo-Paz ◽  
Maria Teresa Gonzalez-Arnao

Abstract More than 50% of Agave species are endemic to Mexico. Among them, Agave peacockii is listed within the list of threatened species that require special protection. In this work, we aimed at developing new supplementary strategies to achieve micropropagation and perform cryopreservation of in vitro-grown shoot-tips of A. peacockii. For multiplication, the addition of two cytokinins, 6-benzylaminopurine (26.6 μM) and kinetin (27.84 μM) to MS semisolid medium significantly favoured the morphogenetic response and produced the highest shoot generation (87.00±17.18) after 60 d of culture. This interaction was more effective than using the same growth regulators separately. Propagated and rooted plantlets were successfully acclimated with 100% survival and a normal morphological development during greenhouse performance. For cryopreservation, an optimized protocol following droplet-vitrification approach allowed obtaining 98% and 96% regrowth before and after cryopreservation, respectively. Shoot-tips were excised of in vitro-propagated plants, subjected to preculture on MS semisolid medium with 0.3 M sucrose for 1d, loaded in solution with 0.4 M sucrose and 1.6 M glycerol for 20 min, exposed to vitrification solution PVS2 for 15 min, and then, immersed in liquid nitrogen in droplets of PVS2 placed on aluminium foil strips. The vegetative growth of cryo-derived plants and of the in vitro propagated plants was compared under greenhouse culture conditions. No significant differences were detected in most assessed characteristics after 120 d of acclimatization. The results presented here constitute new viable biotechnological approaches for the in vitro propagation and long-term conservation of endangered Agave germplasm.


Forests ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1408
Author(s):  
Diego Gago ◽  
Saladina Vilavert ◽  
María Ángeles Bernal ◽  
Conchi Sánchez ◽  
Anxela Aldrey ◽  
...  

The effect of sucrose concentration on the micropropagation of axillary shoots of willow was investigated. The following factors were examined: the culture system (semisolid medium in glass jars versus liquid medium in temporary immersion bioreactors), the type of explant (apical and basal sections), the frequency of immersion, and CO2 enrichment. Shoots and leaf growth were significantly higher in RITA® bioreactors than in the jars for all the sucrose treatments. Apical or basal sections of willow cultured in bioreactors under high light intensity (150 µmol m−2 s−1) and ventilated six times a day with CO2-enriched air were successfully proliferated without sucrose, whereas shoots cultured in jars did not proliferate well if sucrose concentration was 0.5% or lower. More roots were formed when sucrose was added to the medium. Shoots cultured in bioreactors were successfully acclimatized irrespective of the sucrose treatment and the root biomass when transferred to ex vitro conditions. This is the first report of photoautotrophic willow micropropagation, our results confirm the importance of proper gaseous exchange to attain autotrophy during in vitro propagation.


2021 ◽  
Vol 9 (09) ◽  
pp. 303-309
Author(s):  
Sosa-Rubio Edgar Enrique ◽  
◽  
Herrera-Cool Gilbert Jose ◽  
Zavaleta-Cordova Maria Del Carmen ◽  
◽  
...  

The objective of this study was to evaluate the biofertilizers effect in Panicum maximaum (cv. Mombaza) and Brachiaria brizantha tropical grasses production. Microorganisms were obtained in rhizosphere of plants. To establish an effective symbiosis with native strains of Azospirillum, Azotobacter and mycorrhizal fungi, experiments were carried out in greenhouse and field. The biofertilizers used in greenhouse were combined (CC), semisolid medium Nitrogen free with malate as nitrogen source (NFB), Azotobacter (azot) and Azospirillum (Azos). For mycorrhizal fungi, 6 treatments were used: T1-control, T2-fertilized, T3-brown spore, T4-honey spore, T4-black spore and T5-commercial spore. The microorganism used in field were those that showed effectivity in greenhouse. The treatments in field were T1: control, T2: inorganic fertilizer, T3: Azospirillum + Azotobacter, T4: mycorrhizal and T5: commercial biofertilizer. The variables evaluated were dry weight (DW), radicular weight (RW), radicular volume (RV), stem diameter (SD) and total height (TH). Results for B. brizantha indicate differences (P≤0.05). Application of Azospirillum + Azotobacter (T3) favored the development of the height of the plant and the diameter of the stem. The commercial biofertilizer (T5) increased the production of dry matter with 0.99 kg/m2. In respect with P. maximum (cv. Mombaza) grass, they were not detected significative differences (P≥0.05) between treatments, however, the biological results showed that inorganic fertilizer (T2) increased the dry matter production with 1.34 kg / m2 in comparison with Azospirillum + Azotobacter (T3) that showed 0.72 kg / m2.


2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Tuomas Kaprio ◽  
Alexander M. Lindström ◽  
Tiina Rasila ◽  
Olga Saavalainen ◽  
Ines Beilmann-Lehtonen ◽  
...  

Abstract Background Colon cancer (CC) is one of the most commonly diagnosed malignancies worldwide. Several biomarkers have been suggested for improved prognostic evaluation, but few have been implemented in clinical practice. There is a need for biomarkers that predict the tumor behavior in CC and allow stratification of patients that would benefit from adjuvant therapy. We recently identified and functionally characterized a previously unknown protein that we called ASTROPRINCIN (APCN) due to its abundance in astrocytes. APCN, also annotated as FAM171A1, is found in trophoblasts of early placenta. We demonstrated that high expression levels of APCN in cancer cells induced motility and ability of invasive growth in semisolid medium. Methods We screened by immunohistochemistry a tissue microarray material from the tumors of 429 CC patients with clinical follow-up in a test series and 255 CC patients in a validation series. Results We showed that low or absent APCN expression correlates with a favorable prognosis while high APCN expression was a sign of an adverse outcome. Cox uni- and multivariable analysis revealed that elevated tumor expression of APCN constitutes a robust marker of poor prognosis independent of stage, grade, patient’s age, or gender. Conclusion Our findings demonstrate that APCN is a novel independent prognostic marker in CC and could potentially select patients for more intense postoperative adjuvant treatment and follow-up.


2021 ◽  
Vol 3 (1) ◽  
pp. 32-40
Author(s):  
Ismi Isti'anah ◽  
Nisa Mubarik Rachmania ◽  
Aris Tjahjoleksono

Oil palm plantations have a good prospect in Indonesia. One of the efforts to improve the productivity of oil palm plantation is the application of bacteria as biological fertilizer. The research was conducted to characterize and apply the nitrogen-fixing and indole-3-acetic acid producing bacteria in oil palm seedlings. The bacteria was isolated from soil samples which taken from Taman Nasional Bukit Dua Belas (TNBD) Jambi. Nitrogen free bromthymol blue (NFB) is used as media for nitrogen-fixing bacterial isolation. Selected isolate named A13 had an ability to form white pellicle on the surface of the semisolid medium, increased the pH, and changed the color of medium from green to blue Isolate A13 was identified as Gram-negative bacteria and had a rods shape. Analysis of 16S rRNA gene sequence showed that isolate A13 had a similarity with Pseudochrobactrum assacharolyticum. Hypersensitivity assay on tobacco leaves showed that isolate A13 was not a pathogen. During 48 hours of incubation, isolate A13 produced a maximum of IAA at the 24th hour of incubation. Isolate A13 produced 0.675 ppm of ethylene/hour in Acetylene Reduction Assay and 69,839 ppm of IAA in HPLC methods. This was the first report on nitrogen fixation and IAA production by Pseudochrobactrum assacharolyticum and its application in the soil of oil palm seedlings. Application of isolate A13 in oil palm seedling increased significantly the number of lateral roots, stem diameter, and height of plants


2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Xiangchun Ruan ◽  
Xiaoling Deng ◽  
Meiling Tan ◽  
Chengbo Yu ◽  
Meishi Zhang ◽  
...  

Abstract Background Avian pathogenic Escherichia coli (APEC) strains cause infectious diseases in poultry. Resveratrol is extracted from Polygonum cuspidatum, Cassia tora Linn and Vitis vinifera, and displays good antimicrobial activity. The present study aimed to investigate the antibiofilm effect of resveratrol on APEC in vitro. The minimum inhibitory concentration (MIC) of resveratrol and the antibiotic florfenicol toward APEC were detected using the broth microdilution method. Then, the effect of resveratrol on swimming and swarming motility was investigated using a semisolid medium culture method. Subsequently, the minimum biofilm inhibitory concentration (MBIC) and the biofilm eradication rate were evaluated using crystal violet staining. Finally, the antibiofilm activity of resveratrol was observed using scanning electron microscopy (SEM). Meanwhile, the effects of florfenicol combined with resveratrol against biofilm formation by APEC were evaluated using optical microscopy (OM) and a confocal laser scanning microscopy (CLSM). Results The MICs of resveratrol and florfenicol toward APEC were 128 μg/mL and 64 μg/mL, respectively. The swimming and swarming motility abilities of APEC were inhibited in a resveratrol dose-dependent manner. Furthermore, resveratrol showed a significant inhibitory activity against APEC biofilm formation at concentrations above 1 μg/mL (p < 0.01). Meanwhile, the inhibitory effect of resveratrol at 32 μg/mL on biofilm formation was observed using SEM. The APEC biofilm was eradicated at 32 μg/mL of resveratrol combined with 64 μg/mL of florfenicol, which was observed using CLSM and OM. Florfenicol had a slight eradication effect of biofilm formation, whereas resveratrol had a strong biofilm eradication effect toward APEC. Conclusion Resveratrol displayed good antibiofilm activity against APEC in vitro, including inhibition of swimming and swarming motility, biofilm formation, and could eradicate the biofilm.


2021 ◽  
Vol 22 (2) ◽  
Author(s):  
Claudia Marcela Lopez Diaz ◽  
Isidro Elías Suárez Padrón ◽  
Alicia Humanez Alvarez

To evaluate the micropropagation response of arrow cane, Gynerium sagittatum (Aubl.), plants using a double-phase medium in the multiplication stage, explants consisting of stem sections with axillary meristems from cultivars Criolla, Criolla 1, and Martinera were established in vitro in a semisolid medium. Then, they were multiplied using a double-phase medium supplied at several Benzylaminopurine (BAP) concentrations (0.0, 0.5, 1.0, 1.5, and 2.0 mg/L), followed by rooting in a culture medium supplied at several Naphthaleneacetic acid (NAA) concentrations (0.0, 0.5, 1.0, 1.5, and 20 mg/L). Both multiplied unrooted and rooted microshoots were transferred ex vitro. Treatments were distributed with a completely randomized design; data were analyzed with an ANOVA and means separated with Tukey’s test. Explants from Criolla and Martinera cultured with 0.5 mg/L BAP resulted in higher multiplication rates. All microshoots transferred to the rooting medium rooted, although NAA significantly increased the number of roots and reduced root length. Plants from all three cultivars, in vitro rooted or unrooted transferred to ex vitro conditions, showed 100 % survival and adaptation. For Criolla and Martinera, 0.5 mg/L BAP statistically increased shoot multiplication rates and NAA increased adventitious root formation and reduced root length. Plants of all cultivars survived and adapted 100 % to ex vitro conditions.


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