Agrobacterium tumefaciens mediated genetic transformation of Ocimum gratissimum: A medicinally important crop

A protocol for genetic transformation mediated by Agrobacterium tumefaciens and production of transgenic Dendrobium lasianthera has been developed for the first time. The 8-week-old protocorm explants were used as target of transformation with Agrobacterium tumefaciens strain LBA4404 carrying plasmid pG35SKNAT1. Several parameters such as infection period, Agrobacterium density, concentration of acetosyringone, and co-cultivation period were evaluated for the transformation efficiency. The data were analyzed using one-way analysis of variance (ANOVA) and Duncan's Multiple Range Test (DMRT) with p < 0.05. Subsequently, KNAT1 gene expression was confirmed by polymerase chain reaction (PCR) analysis. The highest efficiency of transformation (70%) obtained from protocorm explants infected with Agrobacterium culture was at the OD 600 concentration of 0.6 for 30 min, and co-cultivated with acetosyringone 100 µM for 5 days. The results of confirmation by PCR analysis show that the KNAT1 gene has been integrated and expressed in the genome of Dendrobium lasianthera transgenic.

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“…The bacterial density in suspension may influence efficiency of transformation [23], [24], [25]. The transformation efficiency described in (Fig.…”

Section: Discussionmentioning

confidence: 99%

Agrobacterium tumefaciens-mediated transformation of Dendrobium lasianthera J.J.Sm: An important medicinal orchid

Utami

Hariyanto

Manuhara

2018

Journal of Genetic Engineering and Biotechnology

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“…Agrobacterium cell concentration and infection time are also important factors influencing the genetic transformation efficiency (Khan et al, 2015). Lower Agrobacterium density or shorter infection time cannot guarantee sufficient gene transfer, while higher Agrobacterium concentration or longer infection time could harm the plant cells and result in browning and even death of the explants.…”

Section: Discussionmentioning

confidence: 99%

Agrobacterium tumefaciens mediated transformation of chicory (Cichorium intybus L.) with Mytilus galloprovincialis foot protein type five (MGFP-5)

Lv¹,

Zhang²,

Gao³

et al. 2019

PAK.J.BOT.

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Mytilus galloprovincialis foot protein type 5 (Mgfp-5) can be used as medical adhesive or in underwater environments due to its significant adhesive ability and biodegradable properties. To obtain sufficient Mgfp-5 for further study, genetic engineering has been abundantly used, while plant genetic engineering was rarely applied. This study reports an efficient protocol for the direct regeneration of chicory (Cichorium intybus L.), with a regeneration frequency of 79.18% in the case of inoculation on MS+1.5 mg/L 6-BA+0.2 mg/L NAA medium. The plant expression vector pRI101-Mgfp of the Mgfp-5 gene was transformed into chicory via Agrobacterium mediation. After plant selection and regeneration, regenerated plants with resistance were successfully obtained. The efficient transformation system of the Agrobacterium-mediated genetic transformation consists of leaf explants immersed in Agrobacterium at O.D.(600) of 0.6 for 10 min after being pre-cultured for 3 d, followed by co-culture for 3 d. Then, these explants were transferred to medium containing 50 mg/L kanamycin 1 selection power. As proved via PCR, RT-PCR, and Western blot analysis, the Mgfp-5 gene was integrated into the chicory genome, accompanied by normal transcript and expression, as well as stable genetic transformation and inheritance. These results underpin future research toward providing a feasible method for the production of Mgfp-5 protein.

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“…Our study revealed that treatment of bacterial suspension with Tween 20 at 0.01% increased the susceptibility of explants to Agrobacterium infection, resulting in a striking elevation of transient gus expression (62%). Tween 20 has been successfully used to enhance the transformation efficiency of plants by many researchers (Kim et al, 2009;Paul et al, 2012;Khan et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

Direct shoot organogenesis and Agrobacterium tumefaciens mediated transformation of Solanum trilobatum L.

Sakthivel¹,

Jayashre²,

Largia³

et al. 2016

Turk J Biol

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Solanum trilobatum L. is one of the most valued medicinal plants of Indian Ayurveda and Siddha medicine. An efficient protocol for direct shoot organogenesis and Agrobacterium tumefaciens mediated transformation using in vitro derived leaf explants of S. trilobatum was established for the first time. High frequency direct shoot regeneration (89%) was achieved in MS medium containing 2 mg/L 6-benzylaminopurine and 0.25 mg/L indole-3-acetic acid. The Agrobacterium strain EHA 105 harboring the binary vector pCAMBIA 1301, which contained hygromycin phosphotransferase (hpt) as a selectable marker gene and β-glucuronidase (gus) as a reporter gene, was used for the transformation. The factors that influence the transformation frequency were optimized as follows: age of explants (40 days old), acetosyringone concentration (150 µM), duration of preculture (2 days), bacterial optical density (O.D. 0.4), Tween 20 concentration (0.01%), infection time (15 min), pH of cocultivation medium (5.4), and cocultivation duration (3 days). The putative transformants were screened on selection medium containing 15 mg/L hygromycin. The transgenic plants were confirmed by histochemical gus analysis, PCR, and RT-PCR analysis. A stable transformation efficiency of 64% was achieved under optimized transformation conditions. Thus, the protocol can be used for genetic improvement of this plant in the future.

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Agrobacterium tumefaciens mediated genetic transformation of Ocimum gratissimum: A medicinally important crop

Cited by 32 publications

References 31 publications

Agrobacterium tumefaciens-mediated transformation of Dendrobium lasianthera J.J.Sm: An important medicinal orchid

Agrobacterium tumefaciens-mediated transformation of Dendrobium lasianthera J.J.Sm: An important medicinal orchid

Agrobacterium tumefaciens mediated transformation of chicory (Cichorium intybus L.) with Mytilus galloprovincialis foot protein type five (MGFP-5)

Direct shoot organogenesis and Agrobacterium tumefaciens mediated transformation of Solanum trilobatum L.

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