Transformation of alfalfa chloroplasts and expression of green fluorescent protein in a forage crop

Wei, Zhengyi; Liu, Yanzhi; Lin, Chung-Hung; Wang, Yunpeng; Cai, Qinan; Dong, Yingshan; Xing, Shaochen

doi:10.1007/s10529-011-0709-2

Cited by 35 publications

(18 citation statements)

References 34 publications

(31 reference statements)

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“…Operon transformation mediated by the IL-60 system presents several advantages over plastid transformation, as described by other authors (Elghabi et al, 2011;Sanz-Barrio et al, 2011;Wei et al, 2011). A comparison between the two operon-expression systems in plants indicates that: 1) the IL-60 system is not transgenic while plastid transformation produces transgenic plants, 2) preparation and handling of the IL-60 system is much simpler than plastid transformation protocols, and 3) IL-60 delivery into plants circumvents the need to use selectable markers.…”

Section: Discussionmentioning

confidence: 89%

Expression of an Entire Bacterial Operon in Plants

et al. 2012

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Multigene expression is required for metabolic engineering, i.e. coregulated expression of all genes in a metabolic pathway for the production of a desired secondary metabolite. To that end, several transgenic approaches have been attempted with limited success. Better success has been achieved by transforming plastids with operons. IL-60 is a platform of constructs driven from the geminivirus Tomato yellow leaf curl virus. We demonstrate that IL-60 enables nontransgenic expression of an entire bacterial operon in tomato (Solanum lycopersicum) plants without the need for plastid (or any other) transformation. Delivery to the plant is simple, and the rate of expressing plants is close to 100%, eliminating the need for selectable markers. Using this platform, we show the expression of an entire metabolic pathway in plants and delivery of the end product secondary metabolite (pyrrolnitrin). Expression of this unique secondary metabolite resulted in the appearance of a unique plant phenotype disease resistance. Pyrrolnitrin production was already evident 2 d after application of the operon to plants and persisted throughout the plant's life span. Expression of entire metabolic pathways in plants is potentially beneficial for plant improvement, disease resistance, and biotechnological advances, such as commercial production of desired metabolites.

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Section: Discussionmentioning

confidence: 89%

Expression of an Entire Bacterial Operon in Plants

et al. 2012

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“…In alfalfa, for example, gabaculine is a non-lethal toxin that inhibits both cell proliferation and regeneration: when cultured with gabaculine, cells are not killed, show very little proliferation and never produce green embryos (Rosellini et al 2007;Ferradini et al 2011). There is only one report of alfalfa plastome transformation using the aadA SMG (Wei et al 2011). In the Authors' as well as in other labs, however, several unsuccessful attempts to obtain transplastomic alfalfa have been made using both the aadA and aphA-6 genes (Rosellini et al 2004) SMGs.…”

Section: Discussionmentioning

confidence: 99%

A mutant Synechococcus gene encoding glutamate 1-semialdehyde aminotransferase confers gabaculine resistance when expressed in tobacco plastids

et al. 2015

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A mutant glutamate 1-semialdehyde aminotransferase gene from the Synechococcus , inserted into tobacco plastid DNA by means of particle bombardment and antibiotic selection, conferred gabaculine resistance allowing to attain homoplasmy. Many plant species are recalcitrant to plastid genome transformation. New selections systems may help to overcome this limitation and to extend the application of this technology. A mutant hemL gene from the photosynthetic cyanobacterium Synechococcus, encoding a gabaculine-insensitive glutamate 1-semialdehyde aminotransferase (GSA), is an efficient selectable marker gene for nuclear transformation of tobacco, alfalfa and durum wheat. Since GSA functions in the plastid, we introduced the mutant hemL gene into the tobacco plastid genome along with the conventional antibiotic resistance aadA gene, in the attempt to develop a new selection system for plastome transformation. Although we were unable to directly regenerate gabaculine resistant transplastomic plants, we demonstrated the functionality of hemL in tobacco plastids by using gabaculine selection in the second and third rounds of in vitro selection that permitted to obtain the homoplasmic state in transgenic plants. Thus, the mutant hemL gene functions as a secondary selection marker in tobacco plastids. Our results encourage further attempts to test gabaculine resistant GSA for plastome transformation of crop plants in which gabaculine has stronger regeneration-inhibiting effects with respect to tobacco.

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“…However, obtaining homoplasmic transplastomics is quite challenging, and several additional rounds of selection adversely affect the regeneration efficiency of recalcitrant species. Even after several rounds of antibiotic selection, complete intraorganellar homoplasmy is difficult to achieve in species with poor regeneration capacity (Bock 2001) and upon transfer to the selection-free media, segregation of plastids with wild-type plastid genomes reoccurs, thereby resulting in the heteroplasmy of transplastomics (Wei et al 2011). …”

Section: Limitationsmentioning

confidence: 99%

Plastid genetic engineering in Solanaceae

Venkatesh

Park

2012

Protoplasma

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Plastid genetic engineering has come of age, becoming today an attractive alternative approach for the expression of foreign genes, as it offers several advantages over nuclear transformants. Significant progress has been made in plastid genetic engineering in tobacco and other Solanaceae plants, through the use of improved regeneration procedures and transformation vectors with efficient promoters and untranslated regions. Many genes encoding for industrially important proteins and vaccines, as well as genes conferring important agronomic traits, have been stably integrated and expressed in the plastid genome. Despite these advances, it remains a challenge to achieve marked levels of plastid transgene expression in non-green tissues. In this review, we summarize the basic requirements of plastid genetic engineering and discuss the current status, limitations, and the potential of plastid transformation for expanding future studies relating to Solanaceae plants.

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Transformation of alfalfa chloroplasts and expression of green fluorescent protein in a forage crop

Cited by 35 publications

References 34 publications

Expression of an Entire Bacterial Operon in Plants

Expression of an Entire Bacterial Operon in Plants

A mutant Synechococcus gene encoding glutamate 1-semialdehyde aminotransferase confers gabaculine resistance when expressed in tobacco plastids

Plastid genetic engineering in Solanaceae

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