Hygromycin promotes somatic embryogenesis in spinach

Milojević, Jelena; Tubić, Ljiljana; Nolić, Vladimir; Mitić, Nevena; Ćalić﻿﻿﻿, Dušica; Vinterhalter, Branka; Zdravković-Korać, Snežana

doi:10.1007/s11240-012-0117-x

Cited by 12 publications

(8 citation statements)

References 27 publications

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“…This discrepancy might be due to the different genotypes used in both studies. It has been reported that the concentrations of hygromycin used for transformation vary greatly depending on genotype, type of explants and even explant size (Milojević et al, 2012). Since 10 mg L −1 hygromycin was proved to be lethal to the wild type crambe, we thus used this concentration as the highest limit in the subsequent transformation experiments.…”

Section: Resultsmentioning

confidence: 99%

“…To overcome this problem, delayed selection or stepwise selection methods have been studied in some plant species. The delayed selection means delaying transfer of explants to selection media after Agrobacterium co-culture, while the stepwise selection means that applying a lower concentration of selective agent first, followed by a gradual increase in concentration to a certain level (Visser et al, 1989; Bhuiyan et al, 2011; Milojević et al, 2012). It has been supposed that the initially low selective pressure allows the recovery of transformed cells, while the high selective pressure in subsequent transfers will eliminate untransformed cells and allow only the transformants to grow.…”

Section: Resultsmentioning

confidence: 99%

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Efficient selection and evaluation of transgenic lines of Crambe abyssinica

Li¹,

Fan²,

Gruber³

et al. 2013

Front. Plant Sci.

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Crambe abyssinica is a dedicated oilseed crop suitable for production of industrial feedstocks. Genetic modification of crambe has progressed substantially in the last few years, but the transformation efficiency needs to be further improved. Meanwhile, developing a reliable molecular system including Southern blot and qRT-PCR analyses is desired for effectively evaluating transgenic lines and gene expression levels of both endogenous and transgenes. In this study, we have developed an efficient transformation protocol with hygromycin as the selective agent for crambe transformation. In the regeneration test, addition of hygromycin at concentration of 5 mg L−1 resulted in 18% of shoot regeneration using crambe hypocotyls as explants, while no regeneration occurred when the hygromycin concentration reached 10 mg L−1. Based on this result, the hygromycin concentration up to 10 mg L−1 was used in the subsequent transformations. The results showed that the transformation efficiency under constant low selection pressure (H3-H3) was similar to that under higher selection pressure first, followed by transfer to lower selection pressure (H10-H3). The PCR, Southern blot and fatty acid composition analyses confirmed the integration of transgenes in the crambe genome. We have also optimized the Southern and qRT-PCR methods for future studies on crambe or related species. For Southern blot analysis on crambe, more than 50 μg DNA is required for a clear band. The choice of enzymes for DNA digestion was not rigid for confirmation of the T-DNA integration, while for determining the copy number of transgenes, suitable enzymes should be chosen. Increasing the enzyme concentration could improve the digestion and 20 μl enzyme was recomended for a complete digestion of up to 80 μg crambe DNA. For qRT-PCR analysis, around 20 days after flowering was observed to be the suitable sampling time for expresseion analysis of genes invovled in the seed oil biosynthesis.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Efficient selection and evaluation of transgenic lines of Crambe abyssinica

Li¹,

Fan²,

Gruber³

et al. 2013

Front. Plant Sci.

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“…This can be more toxic and kills sensitive cells faster. As a result, it has been widely used as a resistance gene for plant transformation in different species, such as for cassava (Zhang and Puonti-Kaerlas, 2000), castor (Sujatha andSailaja, 2005), dendrobium (Suwanaketchanatit et al, 2007), grape (Fan et al, 2008), rapeseed (Liu et al, 2011) and spinach (Milojević et al, 2012). The hygromycin antibiotic has also been successfully used as a selection agent in oil palm transformation, but no study has been reported describing the production of stable transformation plants.…”

Section: A R T I C L E I N P R E S Smentioning

confidence: 99%

Genetic Transformation of Oil Palmbased on Selection With Hygromycin

Bahariah¹

2020

JOPR

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“…Although the genotype has generally shown a significant effect, regeneration has been achieved either through organogenesis or embryogenesis depending mainly on the explant type and auxin/cytokinin ratio: cotyledon explants and low ratios facilitate the former, whereas root explants and high ratios favor the latter. In addition, low temperature (14°C), photoperiod, light intensity, and GA 3 content had substantial effects on shoot regeneration ( Geekiyanage et al, 2006 ; Chin et al, 2009 ; Nguyen et al, 2013b ), while low levels of hygromycin (0.5 mg l -1 ) have been found to promote SE ( Milojević et al, 2012 ). An optimized regeneration protocol has been recently published ( Nguyen et al, 2013a ).…”

Section: Regeneration and Transformationmentioning

confidence: 99%

Genetic Transformation and Genomic Resources for Next-Generation Precise Genome Engineering in Vegetable Crops

2017

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In the frame of modern agriculture facing the predicted increase of population and general environmental changes, the securement of high quality food remains a major challenge to deal with. Vegetable crops include a large number of species, characterized by multiple geographical origins, large genetic variability and diverse reproductive features. Due to their nutritional value, they have an important place in human diet. In recent years, many crop genomes have been sequenced permitting the identification of genes and superior alleles associated with desirable traits. Furthermore, innovative biotechnological approaches allow to take a step forward towards the development of new improved cultivars harboring precise genome modifications. Sequence-based knowledge coupled with advanced biotechnologies is supporting the widespread application of new plant breeding techniques to enhance the success in modification and transfer of useful alleles into target varieties. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system, zinc-finger nucleases, and transcription activator-like effector nucleases represent the main methods available for plant genome engineering through targeted modifications. Such technologies, however, require efficient transformation protocols as well as extensive genomic resources and accurate knowledge before they can be efficiently exploited in practical breeding programs. In this review, we revise the state of the art in relation to availability of such scientific and technological resources in various groups of vegetables, describe genome editing results obtained so far and discuss the implications for future applications.

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Hygromycin promotes somatic embryogenesis in spinach

Cited by 12 publications

References 27 publications

Efficient selection and evaluation of transgenic lines of Crambe abyssinica

Efficient selection and evaluation of transgenic lines of Crambe abyssinica

Genetic Transformation of Oil Palmbased on Selection With Hygromycin

Genetic Transformation and Genomic Resources for Next-Generation Precise Genome Engineering in Vegetable Crops

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