scholarly journals Factors Affecting the Regeneration, via Organogenesis, and the Selection of Transgenic Calli in the Peach Rootstock Hansen 536 (Prunus persica × Prunus amygdalus) to Express an RNAi Construct against PPV Virus

Plants ◽  
2019 ◽  
Vol 8 (6) ◽  
pp. 178 ◽  
Author(s):  
Sabbadini ◽  
Ricci ◽  
Limera ◽  
Baldoni ◽  
Capriotti ◽  
...  
Keyword(s):  
Shoot Regeneration ◽  
Prunus Persica ◽  
Fluorescent Protein ◽  
In Vitro Regeneration ◽  
Naphthaleneacetic Acid ◽  
Prunus Amygdalus ◽  
Indirect Organogenesis ◽  
Egfp Gene ◽  
Rnai Construct

Prunus spp. is one of the most recalcitrant fruit tree species in terms of in vitro regeneration and transformation, mostly when mature tissues are used as explants. The present study describes the in vitro regeneration via indirect organogenesis, and Agrobacterium tumefaciens-mediated transformation of the peach rootstock Hansen 536 (Prunus persica × Prunus amygdalus) through the use of meristematic bulks (MBs) as starting explants. Efficient adventitious shoot regeneration was obtained when Hansen 536 MBs were cultured on an optimized medium consisting of modified McCown Woody Plant medium (WPM) enriched with 4.4 M 6-Benzyladenine (BA), 0.1 M 1-Naphthaleneacetic acid (NAA) and 6.0 g L−1 plant agar S1000 (B&V). MB slices were used later as starting explants for Agrobacterium-mediated transformation to introduce an RNAi construct “ihp35S-PPV194” against PPV virus. Transgenic events were identified by both green fluorescent protein (GFP) screening and kanamycin selection at different concentrations (0, 17 or 42 M). GFP-fluorescent proliferating callus lines were selected and confirmed to stably express the ihp35S-PPV194::eGFP gene construct by molecular analysis. Although shoot regeneration from these transgenic calli has not been obtained yet, this represents one of the few examples of successful attempts in peach genetic transformation from somatic tissues, and also serves as a useful in vitro system for future gene functional analysis in peach.

scholarly journals In Vitro Shoot Regeneration and Polyploid Induction of Rhododendron ‘Fragrantissimum Improved’

HortScience ◽  
2010 ◽  
Vol 45 (5) ◽  
pp. 801-804 ◽  
Author(s):  
Cary J. Hebert ◽  
Darren H. Touchell ◽  
Thomas G. Ranney ◽  
Anthony V. LeBude
Keyword(s):  
Shoot Regeneration ◽  
In Vitro Regeneration ◽  
Naphthaleneacetic Acid ◽  
Regenerative Capacity ◽  
Leaf Segments ◽  
Ornamental Traits ◽  
Hybrid Protocols ◽  
Mitotic Inhibitor ◽  
In Vitro Shoot Regeneration

Rhododendron L.‘Fragrantissimum Improved’ is an attractive cultivar with showy, fragrant flowers but has limited potential for breeding because it is a sterile wide hybrid. Protocols for in vitro regeneration and polyploid induction were developed for this cultivar as a means to potentially restore fertility and enhance ornamental traits. Combinations of thidiazuron (TDZ) at 0, 5, 10, 15, or 20 μM and 1-naphthaleneacetic acid (NAA) at 0, 2.5, 5, or 10 μM were used to induce shoot regeneration from leaves. Shoot regeneration was optimized (68% of leaf segments produced shoots) using 8.8 μM TDZ and 10 μM NAA. To induce polyploidy, regenerative callus was treated with 7.5, 15, 30, 60, or 90 μM of the mitotic inhibitor oryzalin for 1, 3, 5, 7, or 14 d in various combinations. Oryzalin significantly affected survival and shoot regenerative capacity. A percentage of homogenous, tetraploid shoots was recovered from treatments of 30 μM oryzalin for 1 (13%) or 3 (13%) days and 7.5 μM oryzalin for 7 (20%) or 14 (7%) days.

scholarly journals Application of Artificial Neural Network for Modeling and Studying In Vitro Genotype-Independent Shoot Regeneration in Wheat

2020 ◽  
Vol 10 (15) ◽  
pp. 5370 ◽  
Author(s):  
Mohsen Hesami ◽  
Jorge A. Condori-Apfata ◽  
Maria Valderrama Valencia ◽  
Mohsen Mohammadi
Keyword(s):  
Neural Network ◽  
Shoot Regeneration ◽  
In Vitro Regeneration ◽  
Generalized Regression Neural Network ◽  
Naphthaleneacetic Acid ◽  
Regeneration Frequency ◽  
Artificial Neural ◽  
2,4 Dichlorophenoxyacetic Acid ◽  
In Vitro Shoot Regeneration

Optimizing in vitro shoot regeneration conditions in wheat is one of the important steps in successful micropropagation and gene transformation. Various factors such as genotypes, explants, and phytohormones affect in vitro regeneration of wheat, hindering the ability to tailor genotype-independent protocols. Novel computational approaches such as artificial neural networks (ANNs) can facilitate modeling and predicting outcomes of tissue culture experiments and thereby reduce large experimental treatments and combinations. In this study, generalized regression neural network (GRNN) were used to model and forecast in vitro shoot regeneration outcomes of wheat on the basis of 10 factors including genotypes, explants, and different concentrations of 6-benzylaminopurine (BAP), kinetin (Kin), 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1-naphthaleneacetic acid (NAA), zeatin, and CuSO4. In addition, GRNN was linked to a genetic algorithm (GA) to identify an optimized solution for maximum shoot regeneration. Results indicated that GRNN could accurately predict the shoot regeneration frequency in the validation set with a coefficient determination of 0.78. Sensitivity analysis demonstrated that shoot regeneration frequency was more sensitive to variables in the order of 2,4-D > explant > genotype < zeatin < NAA. Results of this study suggest that GRNN-GA can be used as a tool, besides experimental approaches, to develop and optimize in vitro genotype-independent regeneration protocols.

scholarly journals Response of Different Explants for Callus Induction in Cucumber

2021 ◽  
Vol 2 (5) ◽  
pp. 71-75
Author(s):  
Hasina Sultana ◽  
Lutfun Nahar ◽  
M. Mofazzal Hossain ◽  
Totan Kumar Ghosh ◽  
Md. Sanaullah Biswas
Keyword(s):  
Shoot Regeneration ◽  
Callus Induction ◽  
In Vitro Regeneration ◽  
Leaf Disc ◽  
Dry Weight ◽  
Dichlorophenoxyacetic Acid ◽  
Naphthaleneacetic Acid ◽  
Regenerate Shoot ◽  
2,4 Dichlorophenoxyacetic Acid

In vitro regeneration of cucumber is relatively difficult for genetic improvement. In this regard, different concentrations of growth regulators and three types of explants (cotyledon, hypocotyl and leaf disc) were investigated for their efficiency on callus induction potential. Among different explants explored for callus induction with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), leaf disc responded earlier (4.67 days) and showed higher percentage of callus induction (91.50%) with 2 mg/l 2,4-D supplemented Murashige and Skoog (MS) media. The same concentration of 2,4-D resulted in the maximum callus fresh (0.56 g) and dry weight (0.39 g) from leaf disc explant. Then the callus was transferred to untreated, 2.0 mg/l BAP + 0.2 mg/l NAA + 1.0 mg/l Kn, 2.0 mg/l BAP + 1.0 mg/l NAA + 1.0 mg/l Kn and 2.0 mg/l BAP + 1.5 mg/l NAA + 1.0 mg/l Kn fortified MS medium. After transferring the callus of different explants to shoot regeneration media containing different concentrations of 6-benzylaminopurine (BAP), 1-naphthaleneacetic acid (NAA) and Kinetin (Kn), only cotyledon callus started to regenerate shoot. The combination of BAP (2 mg/l) + NAA (0.2 mg/l) + Kn (1 mg/l) showed highest shoot regeneration percentage (67.77%) and the maximum number of shoots (5.12) per explant were recorded in the treatment combination of 2 mg/l BAP + 0.2 mg/l NAA + 1 mg/l Kn. These results provided a basis for the optimization of the callus induction protocol of cucumber for genetic transformation.

scholarly journals Indirect Organogenesis and Plant Regeneration in Common Sage (Salvia officinalis L.): An Important Medicinal Plant of Iran

2017 ◽  
Vol 11 (5) ◽  
pp. 22 ◽  
Author(s):  
Samira Jafari ◽  
Mohammad Hossein Daneshvar ◽  
Mohammadreza Salehi Salmi ◽  
Amin Lotfi Jalal-Abadi
Keyword(s):  
In Vitro Regeneration ◽  
Salvia Officinalis ◽  
Naphthaleneacetic Acid ◽  
Regeneration System ◽  
Indirect Organogenesis ◽  
Salvia Officinalis L ◽  
Further Development ◽  
Important Medicinal Plant ◽  
Internode Explants

Salvia species are an important resource for medicinal industry. This research was conducted to develop an indirect organogenesis regeneration protocol for Salvia officinalis L. via which callus was obtained from leaf and internode explants, among these explants internode explant gave best result on MS medium supplemented with 0.5 mg/l 6-Benzylaminopurine (BAP), 2.0 mg/l 1-Naphthaleneacetic acid (NAA). The maximum percentage (70%) of regeneration was obtained with 0.5 mg thidiazuron (TDZ) from internode explants. Shootlets were highly rooted on MS/2 medium added with 1.0 mg/l indole-3-butyric acid (IBA). In vitro rooted seedlings were successfully acclimatized. This in vitro regeneration system will facilitate further development of reliable procedures for this genus.

Intensification de la régénération du pois (Pisum sativum L.), par le thidiazuron, via la formation de structures caulinaires organogènes

1997 ◽  
Vol 75 (3) ◽  
pp. 492-500 ◽  
Author(s):  
Delphine Popiers ◽  
Frédéric Flandre ◽  
Brigitte S. Sangwan-Norreel
Keyword(s):  
Pisum Sativum ◽  
In Vitro Regeneration ◽  
Naphthaleneacetic Acid ◽  
Cotyledonary Nodes ◽  
Pisum Sativum L ◽  
Embryo Axes ◽  
Initial Medium ◽  
Second Wave ◽  
Mature Seeds

In vitro regeneration of pea (Pisum sativum L.), a regeneration recalcitrant legume, was optimised using thidiazuron. Buds were initiated from the meristems of the cotyledonary nodes of embryo axes, isolated from mature seeds, and subcultured on Murashige and Skoog medium supplemented with 13.3 μM 6-benzylaminopurine, 16.1 μM α-naphthaleneacetic acid, and 0.2 μM 2,3,5-triiodobenzoic acid. Proliferation of buds was preceded by the formation of white nodular-like protrusions. These structures were cut transversally in fine slices and subcultured on the same medium or in presence of thidiazuron that produces a second wave of secondary budding. The best results (90–110 buds per expiant) were obtained with 10 μM thidiazuron. The capacity of regeneration was genotype independent and reproducible. Buds elongated on the initial medium, then formed roots in presence of 5.37 μM α-naphthaleneacetic acid. and developed into viable plants. Key words: Pisum sativum L., regeneration, meristems, embryo axes, thidiazuron.

scholarly journals Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

scholarly journals In vitro shoot regeneration of boldo from leaf explants

2010 ◽  
Vol 40 (10) ◽  
pp. 2210-2213
Author(s):  
Monalize Salete Mota ◽  
Juliana de Magalhães Bandeira ◽  
Eugenia Jacira Bolacel Braga ◽  
Valmor João Bianchi ◽  
José Antonio Peters
Keyword(s):  
Shoot Regeneration ◽  
Leaf Explants ◽  
Shoot Development ◽  
High Potential ◽  
Naphthaleneacetic Acid ◽  
Regeneration System ◽  
Bud Induction ◽  
Molecular Studies ◽  
In Vitro Shoot Regeneration

A shoot regeneration system for Plectranthus neochilus was studied from leaf explants. Leaves developed under in vitro conditions were cultured on Wood Plant Medium supplemented with 0.2mg dm-3 α-naphthaleneacetic acid (NAA) and different 6-benzilaminopurine (BAP) or thidiazuron (TDZ) concentrations (0, 1.5, 3.0, 4.5 and 6.0mg dm-3). An increase in percentage of responsive explants (85.3%) and in the number of shoots developed per explant (3.2) was observed when the explants were treated with 5.3 and 4.7mg dm-3 BAP, respectively. The leaf explants cultured on media supplemented with TDZ became vitreous and did not form buds. The regeneration system used is efficient for boldo bud induction and shoot development, showing high potential for advanced cellular and molecular studies.

scholarly journals In vitro regeneration and polyploidy induction in Pelargonium × hortorum L. H. Bailey

2009 ◽  
Vol 57 (5) ◽  
pp. 305-312
Author(s):  
Hana Vejsadová ◽  
Petra Kuchtová-Jadrná
Keyword(s):  
Flow Cytometry ◽  
Plant Growth ◽  
Shoot Regeneration ◽  
In Vitro Regeneration ◽  
Infiltration Method

The objective of this work was to induce in vitro shoot regeneration as influenced by plant growth re­gu­la­tors and ascertain an effective method of polyploidy induction using colchicine and oryzalin in two diploid cultivars ‘Gizela’ and ‘Black Velvet Scarlet F1’ of Pelargonium × hortorum L. H. Bailey. In both cultivars, benzyladenine (BA) significantly improved shoot regeneration compared to zeatin. The infiltration and the dip methods of explant treatment were used for polyploidy induction. Regenerants were analyzed using the flow cytometry (FCM). In ‘Gizela’ and ‘Black Velvet Scarlet F1’, 10 tetraploids on the level of 2n = 4x were found by the infiltration method. The tetraploidy was determined by the dip method in 4 shoots of ‘Gizela’ and 11 tetraploids were detected in ‘Black Velvet Scarlet F1’.

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