scholarly journals Recalcitrance of Cannabis sativa to de novo regeneration; a multi-genotype replication study

2020 ◽  
Author(s):  
Adrian S. Monthony ◽  
Sean T. Kyne ◽  
Christopher M. Grainger ◽  
A. Maxwell P. Jones
Keyword(s):  
Cannabis Sativa ◽  
De Novo ◽  
Leaf Explants ◽  
Plant Biotechnology ◽  
Replication Study ◽  
Induction Medium ◽  
Replication Studies ◽  
Somatic Tissues ◽  
Research Programmes ◽  
Shoot Induction Medium

1AbstractCannabis sativa is relatively recalcitrant to regeneration from somatic tissues, but several reports have been published demonstrating a response. Most reports show low levels of regeneration from somatic tissues, but a landmark publication by Lata et al. in 2010 reported regeneration from leaf explants with a 96% response rate, producing an average of 12.3 shoots per explant in a single, high-THC genotype. Despite the importance regeneration plays in plant biotechnology this protocol has not been used in subsequent papers in the decade since it was published, raising the concern that it is not reproducible. Many researchers are looking to build research programmes in this growing field, and it is important that the reproducibility and robustness of single-genotype C. sativa regeneration protocols undergo multi-lab validations to ensure they are reproducible across the species. Replication studies in this burgeoning field will help research groups avoid lost time and resources which arise from pursuing protocols that are not reproducible. Here we test the replicability of this protocol across 10 drug-type C. sativa genotypes. This protocol successfully induced callus in all 10 genotypes. Callus size and appearance substantially differed among cultivars, with the most responsive genotype producing 6-fold more callus than the least responsive genotype. However, the most successful shoot induction medium developed in the 2010 paper failed to induce regeneration in any of the cultivars tested, resulting in the eventual necrosis of the calli. Based on this replication study, it is evident that the existing regeneration protocol is not robust and could not be replicated in any of the 10 genotypes tested.

scholarly journals Recalcitrance of Cannabis sativa to de novo regeneration; a multi-genotype replication study

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0235525
Author(s):  
Adrian S. Monthony ◽  
Sean T. Kyne ◽  
Christopher M. Grainger ◽  
Andrew Maxwell P. Jones
Keyword(s):  
Cannabis Sativa ◽  
De Novo ◽  
Shoot Organogenesis ◽  
Genotypic Variation ◽  
Leaf Explants ◽  
Plant Biotechnology ◽  
Replication Study ◽  
Induction Medium ◽  
Drug Type ◽  
Shoot Induction Medium

Cannabis sativa is relatively recalcitrant to de novo regeneration, but several studies have reported shoot organogenesis or somatic embryogenesis from non-meristematic tissues. Most report infrequent regeneration rates from these tissues, but a landmark publication from 2010 achieved regeneration from leaf explants with a 96% response rate, producing an average of 12.3 shoots per explant in a single drug-type accession. Despite the importance regeneration plays in plant biotechnology and the renewed interest in this crop the aforementioned protocol has not been used in subsequent papers in the decade since it was published, raising concerns over its reproducibility. Here we attempted to replicate this important Cannabis regeneration study and expand the original scope of the study by testing it across 10 drug-type C. sativa genotypes to assess genotypic variation. In our study, callus was induced in all 10 genotypes but callus growth and appearance substantially differed among cultivars, with the most responsive genotype producing 6-fold more callus than the least responsive. The shoot induction medium failed to induce shoot organogenesis in any of the 10 cultivars tested, instead resulting in necrosis of the calli. The findings of this replication study raise concerns about the replicability of existing methods. However, some details of the protocol could not be replicated due to missing details in the original paper and regulatory issues, which could have impacted the outcome. These results highlight the importance of using multiple genotypes in such studies and providing detailed methods to facilitate replication.

Development of an Agrobacterium-mediated Transformation System for `Beurre Bosc' Pear

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 461d-461
Author(s):  
Richard L. Bell ◽  
Ralph Scorza ◽  
Chinnathambi Srinivasan
Keyword(s):  
Shoot Proliferation ◽  
Induction Medium ◽  
Shoot Induction ◽  
Transformation System ◽  
Gus Histochemical Assay ◽  
Young Leaves ◽  
Leaf Tissues ◽  
Shoot Induction Medium ◽  
Basal Salts

An efficient regeneration/transformation system was developed for `Beurre Bosc' pear. Young leaves were harvested from in vitro shoots proliferated on a medium containing MS basal salts and 5 BAP, 0.5 μM IBA, and 0.6M3. Shoot regeneration was optimized using a modification of the medium of Chevreau and Leblay (1993). Explants were cultured on shoot induction medium contained 10 μM TDZ and 1 μM IBA for 4 weeks in the dark, and then transfered to a similar, but auxinless, regeneration medium until shoots developed, usually after an additional 4 to 8 weeks. Leaf tissues were transformed by co-cultivation for 3 days with Agrobacterium tumefaciens EHA101 carrying a pGA482 plasmid containing NPTII, GUS, and rolC genes, followed by cultivation on SIM containing 300 mg/L timentin. Putative transgenic plants were selected on shoot induction medium containing 80mg/L kanamycin, and multiplied on shoot proliferation medium. Four clones were confirmed as transgenic using the GUS histochemical assay and Southern blots for the NPTII and rolC genes. Plants of each clone have been rooted and successfully transfered to the greenhouse for further analysis of gene expression.

scholarly journals Estimation of a term structure model of carbon prices through state spaces methods: a pitch

2020 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Thomas William Aspinall ◽  
Adrian Gepp ◽  
Geoff Harris ◽  
Simone Kelly ◽  
Colette Southam ◽  
...  
Keyword(s):  
Term Structure ◽  
Replication Study ◽  
The European Union ◽  
Research Project ◽  
Research Teams ◽  
Content Type ◽  
Term Structure Model ◽  
Replication Studies ◽  
Core Elements ◽  
Trading Scheme

Purpose The pitching research template (PRT) is designed to help pitchers identify the core elements that form the framework of any research project. This paper aims to provide a brief commentary on an application of the PRT to pitch an environmental finance research topic with a personal reflection on the pitch exercise discussed. Design/methodology/approach This paper applies the PRT developed by Faff (2015, 2019) to a research project on estimating the strength of carbon pricing signals under the European Union Emissions Trading Scheme. Findings The PRT is found to be a valuable tool to refine broad ideas into impactful and novel research contributions. The PRT is recommended for use by all academics regardless of field and particularly PhD students to structure and communicate their research ideas. The PRT is found to be particularly well suited to pitch replication studies, as it effectively summarizes both the “idea” and proposed “twist” of a replication study. Originality/value This letter is a reflection on a research teams experience with applying the PRT to pitch a replication study at the 2020 Accounting and Finance Association of Australia and New Zealand event. This event focused on replicable research and was a unique opportunity for research teams to pitch their replication research ideas.

scholarly journals EMBRIOGENESIS SOMATIK ANGGREK BULAN Phalaenopsis amabilis (L.) Bl: STRUKTUR DAN POLA PERKEMBANGAN

2007 ◽  
Vol 13 (1) ◽  
pp. 33-38
Author(s):  
Edy Setiti Wida Utami ◽  
Issirep Soemardi ◽  
Taryono Taryono ◽  
Endang Semiarti
Keyword(s):  
Somatic Embryo ◽  
Somatic Embryos ◽  
Zygotic Embryo ◽  
Leaf Explants ◽  
Gellan Gum ◽  
Development Pattern ◽  
Induction Medium ◽  
Embryo Germination ◽  
Maturation Medium ◽  
One Year

Research of the structure and development pattern of somatic embryos from callus of leaf explants moon orchid Phalaenopsis amabilis (L) Bl had been done. One year old of plantlets were used as explants sources. Basal leaf of these explants were cultured in Somatic Embryo Induction Medium (SEIM) e.i.: NP(New Phalaenopsis) medium added with 2 mg/L NAA, 1 mg/L BA, 10 g/L sucrose, and 2 g/L gellan gum. Then somatic embryos were transferred to EMM (Embryo Maturation Medium) e.i. NP medium added with 1 mg/L NAA, 1 mg/L BA, 10 g/L sucrose, and 2 g/L gellan gum. Finally, mature somatic embryo were transferred to NP medium without plant growth regulator as Embryo Germination Medium (EGM). The origin of somatic embryos initially from single cell at the pheriphery of embryogenic callus. These cells then devided in mitotic repeatedly formed globular proembryo, elongation embryo, and completed embryo. The structure and development pattern of somatic embryos as the same as with zygotic embryo.

scholarly journals Molecular Basis for Natural Vegetative Propagation via Regeneration in North American Lake Cress, Rorippa aquatica (Brassicaceae)

2019 ◽  
Vol 61 (2) ◽  
pp. 353-369 ◽  
Author(s):  
Rumi Amano ◽  
Hokuto Nakayama ◽  
Risa Momoi ◽  
Emi Omata ◽  
Shizuka Gunji ◽  
...  
Keyword(s):  
Shoot Regeneration ◽  
Vegetative Propagation ◽  
Molecular Basis ◽  
Auxin Transport ◽  
Vascular Tissue ◽  
De Novo ◽  
Leaf Explants ◽  
Experimental Models ◽  
Polar Auxin Transport ◽  
Proximal Side

Abstract Some plant species have a striking capacity for regeneration in nature, including regeneration of the entire individual from explants. However, due to the lack of suitable experimental models, the regulatory mechanisms of spontaneous whole plant regeneration are mostly unknown. In this study, we established a novel model system to study these mechanisms using an amphibious plant within Brassicaceae, Rorippa aquatica, which naturally undergoes vegetative propagation via regeneration from leaf fragments. Morphological and anatomical observation showed that both de novo root and shoot organogenesis occurred from the proximal side of the cut edge transversely with leaf vascular tissue. Time-series RNA-seq analysis revealed that auxin and cytokinin responses were activated after leaf amputation and that regeneration-related genes were upregulated mainly on the proximal side of the leaf explants. Accordingly, we found that both auxin and cytokinin accumulated on the proximal side. Application of a polar auxin transport inhibitor retarded root and shoot regeneration, suggesting that the enhancement of auxin responses caused by polar auxin transport enhanced de novo organogenesis at the proximal wound site. Exogenous phytohormone and inhibitor applications further demonstrated that, in R. aquatica, both auxin and gibberellin are required for root regeneration, whereas cytokinin is important for shoot regeneration. Our results provide a molecular basis for vegetative propagation via de novo organogenesis.

scholarly journals YUCCA-Mediated Biosynthesis of the Auxin IAA Is Required during the Somatic Embryogenic Induction Process in Coffea canephora

2020 ◽  
Vol 21 (13) ◽  
pp. 4751
Author(s):  
Miguel A. Uc-Chuc ◽  
Cleyre Pérez-Hernández ◽  
Rosa M. Galaz-Ávalos ◽  
Ligia Brito-Argaez ◽  
Víctor Aguilar-Hernández ◽  
...  
Keyword(s):  
De Novo ◽  
Leaf Explants ◽  
Coffea Canephora ◽  
Naphthaleneacetic Acid ◽  
Induction Process ◽  
Exogenous Addition ◽  
Pre Treatment ◽  
Process Of Se ◽  
Somatic Embryogenic ◽  
Indole 3 Acetic Acid

Despite the existence of considerable research on somatic embryogenesis (SE), the molecular mechanism that regulates the biosynthesis of auxins during the SE induction process remains unknown. Indole-3-acetic acid (IAA) is an auxin that is synthesized in plants through five pathways. The biosynthetic pathway most frequently used in this synthesis is the conversion of tryptophan to indol-3-pyruvic acid (IPA) by tryptophan aminotransferase of Arabidopsis (TAA) followed by the conversion of IPA to IAA by enzymes encoded by YUCCA (YUC) genes of the flavin monooxygenase family; however, it is unclear whether YUC-mediated IAA biosynthesis is involved in SE induction. In this study, we report that the increase of IAA observed during SE pre-treatment (plants in MS medium supplemented with 1-naphthaleneacetic acid (NAA) 0.54 µM and kinetin (Kin) 2.32 µM for 14 days) was due to its de novo biosynthesis. By qRT-PCR, we demonstrated that YUC gene expression was consistent with the free IAA signal found in the explants during the induction of SE. In addition, the use of yucasin to inhibit the activity of YUC enzymes reduced the signal of free IAA in the leaf explants and dramatically decreased the induction of SE. The exogenous addition of IAA restored the SE process in explants treated with yucasin. Our findings suggest that the biosynthesis and localization of IAA play an essential role during the induction process of SE in Coffea canephora.

scholarly journals Silicon Promotes Adventitious Shoot Regeneration and Enhances Salinity Tolerance ofAjuga multifloraBunge by Altering Activity of Antioxidant Enzyme

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Iyyakkannu Sivanesan ◽  
Byoung Ryong Jeong
Keyword(s):  
Salinity Tolerance ◽  
Shoot Regeneration ◽  
Microscopic Analysis ◽  
Adventitious Shoot ◽  
Induction Medium ◽  
Shoot Induction ◽  
Electron Microscopic ◽  
Sod Activity ◽  
Scanning Electron Microscopic Analysis ◽  
Shoot Induction Medium

We investigated the effect of Si concentration on shoot regeneration and salinity tolerance ofAjuga multiflora. Addition of Si to the shoot induction medium significantly increased the frequency of shoot induction. The average number of shoots regenerated per explant decreased on the medium containing NaCl alone, while there was less decrease when the shoot induction medium was supplemented with both NaCl and Si. The shoot induction percentage increased linearly with increasing concentration of Si in the NaCl containing medium. Addition of Si to the shoot induction medium significantly increased SOD, POD, APX, and CAT activity in regenerated shoot buds as compared with the control. The inclusion of Si to the NaCl containing medium significantly increased the SOD activity in leaves and roots, while it decreased POD, APX, and CAT activity in both organs. Scanning electron microscopic analysis showed that there are no distinct differences in the structure of stomata between the control and Si-treated plants. However, NaCl treatment significantly affected the structure and number of stomata as compared to the control. Wavelength dispersive X-ray analysis confirmed the high Si deposition in trichomes of plants grown in the Si containing medium but not in plants grown in the medium without Si.

scholarly journals Establishment of a Rapid and Efficient Micropropagation System for Succulent Plant Haworthia turgida Haw.

HortScience ◽  
2017 ◽  
Vol 52 (9) ◽  
pp. 1278-1282 ◽  
Author(s):  
Boling Liu ◽  
Hongzhou Fang ◽  
Chaorong Meng ◽  
Ming Chen ◽  
Qingdong Chai ◽  
...  
Keyword(s):  
Callus Induction ◽  
Adventitious Shoots ◽  
Germplasm Conservation ◽  
Leaf Explants ◽  
Adventitious Shoot ◽  
Induction Medium ◽  
Naphthaleneacetic Acid ◽  
Root Induction ◽  
Ms Medium ◽  
Succulent Plant

In the present study, the effect of plant growth regulators (PGRs) on callus regeneration, adventitious shoot differentiation, and root formation of Haworthia turgida Haw. was investigated. The greatest callus induction percentage (95.6%) was achieved with leaf explants inoculated on Murashige and Skoog (MS) medium with 1.0 mg·L−1 6-benzyladenine (BA) and 0.1 mg·L−1 1-naphthaleneacetic acid (NAA), and this callus induction medium supplemented with 2.5 mg·L−1 thidiazuron (TDZ) was optimal for callus proliferation. The maximum number of shoots (25.7) was obtained when the callus was cultured on MS medium supplemented with 1.0 mg·L−1 BA and 0.2 mg·L−1 2,4-dichlorophenoxyacetic acid (2,4-D). The highest number of roots per shoot (6.2) and highest rooting frequency (82.0%) were obtained when adventitious shoots were inoculated on MS medium with 0.05 mg·L−1 NAA. Regenerated plantlets were transferred to a mixture of vermiculite and soil and acclimated in a greenhouse. The survival rate of the transplanted plantlets was about 91.6%. The rate of ex vitro rooting was 83.3%, indicating that this technique is effective for root induction in H. turgida. This study has established a rapid and efficient micropropagation system that can be beneficial for commercial cultivation and germplasm conservation of H. turgida.

Control of de novo root regeneration efficiency by developmental status of Arabidopsis leaf explants

2019 ◽  
Vol 46 (3) ◽  
pp. 133-140 ◽  
Author(s):  
Jing Pan ◽  
Fei Zhao ◽  
Guifang Zhang ◽  
Yu Pan ◽  
Lijun Sun ◽  
...  
Keyword(s):  
De Novo ◽  
Leaf Explants ◽  
Developmental Status ◽  
Root Regeneration ◽  
Regeneration Efficiency
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