scholarly journals Agrobacterium tumefaciens-Mediated Genetic Transformation of the Ect-endomycorrhizal Fungus Terfezia boudieri

Genes ◽  
2020 ◽  
Vol 11 (11) ◽  
pp. 1293
Author(s):  
Lakkakula Satish ◽  
Madhu Kamle ◽  
Guy Keren ◽  
Chandrashekhar D. Patil ◽  
Galit Yehezkel ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Optical Density ◽  
Fluorescent Protein ◽  
Basic Knowledge ◽  
Selection Marker ◽  
Transformation System ◽  
Intensive Farming ◽  
Terfezia Boudieri ◽  
Desert Truffles

Mycorrhizal desert truffles such as Terfezia boudieri, Tirmania nivea, and Terfezia claveryi, form mycorrhizal associations with plants of the Cistaceae family. These valued truffles are still collected from the wild and not cultivated under intensive farming due to the lack of basic knowledge about their biology at all levels. Recently, several genomes of desert truffles have been decoded, enabling researchers to attempt genetic manipulations to enable cultivation. To execute such manipulations, the development of molecular tools for genes transformation into truffles is needed. We developed an Agrobacterium tumefaciens-mediated genetic transformation system in T. boudieri. This system was optimized for the developmental stage of the mycelia explants, bacterial optical density, infection and co-cultivation durations, and concentrations of the selection antibiotics. The pFPL-Rh plasmid harboring hph gene conferring hygromycin resistance as a selection marker and the red fluorescent protein gene were used as visual reporters. The optimal conditions were incubation with 200 μM of acetosyringone, attaining a bacterial optical density of 0.3 OD600; transfer time of 45 min; and co-cultivation for 3 days. This is the first report on a transformation system for T. boudieri, and the proposed protocol can be adapted for the transformation of other important desert truffles as well as ectomycorrhizal species.

scholarly journals Agrobacterium tumefaciens-Mediated Transformation for Investigation of Somatic Recombination in the Fungal Pathogen Armillaria mellea

2010 ◽  
Vol 76 (24) ◽  
pp. 7990-7996 ◽  
Author(s):  
Kendra Baumgartner ◽  
Phillip Fujiyoshi ◽  
Gary D. Foster ◽  
Andy M. Bailey
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Natural Populations ◽  
Reproductive Mode ◽  
Mitochondrial Haplotype ◽  
Transformation System ◽  
Serial Transfer ◽  
Haploid Nucleus ◽  
Somatic Recombination ◽  
Genetic Transformation System

ABSTRACT Armillaria root disease is one of the most damaging timber and fruit tree diseases in the world. Despite its economic importance, many basic questions about the biology of the causal fungi, Armillaria spp., are unanswered. For example, Armillaria undergoes matings between diploid and haploid mycelia, which can result in a recombinant diploid without meiosis. Evidence of such somatic recombination in natural populations suggests that this reproductive mode may affect the pathogen's ecology. Investigations of the mechanisms and adaptive consequences of somatic recombination are, however, hampered by the lack of a method to reliably synthesize somatic recombinants. Here we report the first genetic transformation system for the genus Armillaria. We transformed A. mellea with selective markers for use in diploid-haploid matings to reliably synthesize somatic recombinants. This was accomplished with Agrobacterium tumefaciens carrying pBGgHg, which carries the hygromycin phosphotransferase gene (hph). hph was integrated into transformants, as evidenced by serial transfer to selective media, PCR, reverse transcription-PCR (RT-PCR), and Southern hybridization. Nuclear and mitochondrial markers were developed to genotype synthesized mycelia. In matings between a wild-type diploid and hygromycin-resistant haploids (transgenic), we identified recombinant, hygromycin-resistant diploids and, additionally, hygromycin-resistant triploids, all with the mitochondrial haplotype of the haploid partner. Our approach created no mycelium in which the haploid nucleus was replaced by the diploid nucleus, the typical outcome of diploid-haploid matings in Armillaria. This genetic transformation system, in combination with new markers to track chromosomal and cytoplasmic inheritance in A. mellea, will advance research aimed at characterizing the significance of somatic recombination in the ecology of this important fungus.

scholarly journals Agrobacterium-mediated genetic transformation of Chinese chestnut (Castanea mollissima Blume)

2019 ◽  
Vol 140 (1) ◽  
pp. 95-103 ◽  
Author(s):  
Zhi-lin Sun ◽  
Xiao Li ◽  
Wan Zhou ◽  
Jun-di Yan ◽  
Yue-rong Gao ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Economic Value ◽  
Target Material ◽  
Normal Growth ◽  
Transformation Method ◽  
Transformation System ◽  
Embryogenic Calli ◽  
Chinese Chestnut ◽  
Castanea Mollissima

Abstract Chinese chestnut (Castanea mollissima) is an important germplasm resource for the breeding of Castanea species worldwide with vital ecological and economic value. Biotechnology overcomes the limitations of traditional breeding and accelerates germplasm improvement. However, a genetic transformation system for Chinese chestnut has not yet been established. In this study, a stable and efficient Agrobacterium-mediated genetic transformation method for Chinese chestnut is described. Embryogenic calli of C. mollissima cv. ‘Yanshanhongli’ were used as the target material. The sensitivity of embryogenic calli to kanamycin was determined, whereby the proliferation of non-transformed calli was completely inhibited at 180 mg/L. Antibiotic inhibition results for Chinese chestnut embryogenic calli showed that 50 mg/L cefotaxime and 500 μM timentin completely inhibited the growth of Agrobacterium tumefaciens but did not affect the normal growth of Chinese chestnut embryogenic calli. When embryogenic calli were co-cultured for 2 days with Agrobacterium tumefaciens strain AGL1 harboring the PBI121-EGFP plasmid, an embryogenic callus transformation efficiency of 4.55% was obtained, and two transgenic chimera were acquired. This Agrobacterium-mediated transformation system for Chinese chestnut provides a fundamental platform for genetic improvement of core germplasm and for further verification of gene function.

scholarly journals A simple and rapid protocol for the genetic transformation of Ensete ventricosum

Plant Methods ◽  
2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Jonathan Matheka ◽  
Jaindra Nath Tripathi ◽  
Ibsa Merga ◽  
Endale Gebre ◽  
Leena Tripathi
Keyword(s):  
Disease Resistance ◽  
Plant Regeneration ◽  
Genetic Transformation ◽  
Marker Gene ◽  
Selection Marker ◽  
Tropical Species ◽  
Transformation System ◽  
Regeneration System ◽  
Ensete Ventricosum ◽  
Multiple Buds

Abstract Enset (Ensete ventricosum), also known as Ethiopian banana, is a food security crop for more than 20 million people in Ethiopia. As conventional breeding of enset is very challenging, genetic engineering is an alternative option to introduce important traits such as enhanced disease resistance and nutritional value. Genetic transformation and subsequent regeneration of transgenic enset has never been reported mainly due to challenges in developing transformation protocols for this tropical species. Agrobacterium-mediated transformation could be a practical tool for the genetic improvement of enset. However, the efficiency of the transformation system depends on several parameters such as plant regeneration, genotype, explant, selection agent and Agrobacterium strains. As a first step towards the development of transgenic enset, a simple and rapid plant regeneration system was developed using multiple buds as explants. Induction and proliferation of multiple buds from shoot tip explants was achieved on Murashige and Skoog (MS) medium supplemented with 5 and 10 mg/l of 6-benzylaminopurine (BAP), respectively. Shoots were regenerated from multiple buds on MS media containing 2 mg/l BAP and 0.2% activated charcoal. Based on the optimized regeneration protocol, an Agrobacterium-mediated transformation method was developed using multiple buds as explants and the binary plasmid pCAMBIA2300-GFP containing the green florescent protein (gfp) reporter gene and neomycin phosphotransferase II (nptII) selection marker gene. Transgenic plantlets were obtained within 4 months at a frequency of about 1.25%. The transgenic lines were validated by PCR analysis using primers specific to the nptII gene. To obtain uniformly transformed plantlets, chimerism was diluted by subculturing and regenerating the transgenic shoots on a selective medium containing kanamycin (150 mg/l) for five cycles. The uniformity of the transgenic plants was confirmed by Southern blot hybridization and RT-PCR analyses on different tissues such as leaf, pseudostem and root of same transgenic plant. In the present study, we report a simple Agrobacterium-mediated transformation system for generating transgenic events of enset. To the best of our knowledge, this is the first report on the stable transformation and regeneration of transgenic events of enset. The transformation system established in this study can be used for the generation of transgenic enset with important traits such as disease resistance.

Genetic transformation of Ginkgo biloba by Agrobacterium tumefaciens

2000 ◽  
Vol 108 (4) ◽  
pp. 413-419
Author(s):  
Patricia Dupré ◽  
Jerôme Lacoux ◽  
Godfrey Neutelings ◽  
Dominique Mattar-Laurain ◽  
Marc-André Fliniaux ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Genetic Transformation ◽  
Ginkgo Biloba
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