Elsevier

Biotechnology Advances

Volume 29, Issue 6, November–December 2011, Pages 703-714
Biotechnology Advances

Research review paper
Suitability of non-lethal marker and marker-free systems for development of transgenic crop plants: Present status and future prospects

https://doi.org/10.1016/j.biotechadv.2011.05.019Get rights and content

Abstract

Genetically modified crops are one of the prudent options for enhancing the production and productivity of crop plants by safeguarding from the losses due to biotic and abiotic stresses. Agrobacterium-mediated and biolistic transformation methods are used to develop transgenic crop plants in which selectable marker genes (SMG) are generally deployed to identify ‘true’ transformants. The commonly used SMG obtained from prokaryotic sources when employed in transgenic plants pose risks due to their lethal nature during selection process. In the recent past, some non-lethal SMGs have been identified and used for selection of transformants with increased precision and high selection efficiency. Considering the concerns related to bio-safety of the environment, it is desirable to remove the SMG in order to maximize the commercial success through wide adoption and public acceptance of genetically modified (GM) food crops. In this review, we examine the availability, and the suitability of wide range of non-lethal selection markers and elimination of SMG methods to develop marker-free transgenics for achieving global food security. As the strategies for marker-free plants are still in proof-of-concept stage, adaptation of new genomics tools for identification of novel non-lethal marker systems and its application for developing marker-free transgenics would further strengthen the crop improvement program.

Introduction

Plant breeding is a classical method to improve agronomically important traits in cultivated crops. However, traditional plant breeding is limited by constraints like narrow gene pool in many crop plants, besides being laborious and time consuming process. Plant biotechnology can complement traditional plant breeding through the deployment of molecular markers for indirect selection of traits and mobilization of useful genes into the crop gene pool from alien sources through plant transformation. Genetic engineering is considered as the best tool to introduce useful genes from heterologous/homologous species for improvement of agronomically important traits in many crops due to their advantages like rapid, precise and consistent as compared to conventional breeding which rely on stringent selection process for its accuracy.

In the early 1980s feasibility of introducing foreign genes into the plant genome was attempted, giving birth to the new and powerful technology of transgenesis. Genetic engineering relies on the introduction of foreign gene into plant nuclear or plastid genome along with a selection marker gene (SMG). SMGs are mostly either antibiotic/herbicide resistance genes that produce a protein which permit only the transformed cells/tissues to grow so that transformants get a competitive advantage and thus assist in identification of transformed cells from the non-transformed ones. The methodology has been successfully applied for developing transgenics for various traits such as resistance to biotic and/or abiotic stresses in different crops which is otherwise considered impossible through conventional breeding.

The first transgenic plant was developed in tobacco with amino glycoside resistance gene (targeting amino glycoside antibiotics like neomycin and kanamycin) used as the first antibiotic SMG for identification of transformants (Bevan et al., 1983, Carrer et al., 1993, Fraley et al., 1983). Later transgenics have been successfully developed with other plant species like celery (Catlin et al., 1988), Brassica (Moloney et al., 1989), tomato (Horsch et al., 1985), wheat (Hess et al., 1990), rice (Toriyama et al., 1988), etc. Hygromycin phosphotransferase (hpt) is the second major antibiotic resistant gene used as SMG in various crops such as rice (Datta et al., 1992), grape (Perl et al., 1996) and cassava (P. Zhang et al., 2003). The other conventional herbicide SMGs like phosphinothricin N-acteyl transferase (ppt) or bialophos resistance (bar), 5-Enolpyruvylshikimate-3-phosphate synthase (EPSP) and glyphosate oxidase (GOX), Acetolactate synthase (ALS) were also deployed in various crops. The popularity of this selection system is based on a broad range of applications of these markers for a variety of plant species and the availability of a repeatable tissue culture system (Miki and McHugh, 2004). Ever since 1980's, most of the transgenic events were selected based on either antibiotic (e.g. nptII and hpt) or herbicide (e.g. bar) resistance genes, which are frequently used as selection markers in various crops (Balachandran et al., 2009, Miki and McHugh, 2004). There are some concerns regarding the biosafety of antibiotic and herbicide resistance genes and efforts are being made for development of alternative marker systems and to standardize marker-free selection systems.

Although several reviews on SMG and marker elimination strategies are already available (Darbani et al., 2007, Hare and Chua, 2002, Miki and McHugh, 2004, Sundar and Sakthivel, 2008), we made an attempt to review the topic with the recent updates on use of non lethal marker genes by grouping them into plant and non plant based SMG with either conditional or non conditional positive selection system and to assess their suitability for development of commercial transgenics.

Section snippets

Selectable marker gene (SMG)

In plant transformation technology, SMGs play a vital role for rapid selection of transgenic plants from the non-transformed event. Selectable marker genes encode proteins that confer resistance to antibiotic/herbicide selection agents and identify the transformed cells from non-transformed cells. The non-transformed cells get killed due to the presence of the selection agents after the process of genetic transformation. Based on their application, SMG is classified into positive and negative

Elimination of selection marker gene from transgenic plants

Development of transgenic crop plants is generally carried out by either Agrobacterium-mediated transformation or through biolistic particle bombardment, involving selection markers to identify transgenic plants from non-transgenic ones. Commercialization of such transgenic plants (particularly food crops) may have public concerns regarding the persistence of marker gene in the crop system and its environmental implications (Bajaj and Mohanty, 2005, Kuiper et al., 2001, Miki and McHugh, 2004).

Regeneration of direct marker-free plants

Alternative methods for direct marker-free transformation of crop plants have been reported. Vector devoid of marker gene was used to develop marker free potato for amylase-free transformants with 0.8% to 5.6% transformation frequency. After transformation, very low frequency of chimerism was observed (De Vetten et al., 2003). Agroinfiltration of tobacco leaf disk and production of direct marker-free transgenic plants using marker-less vector was demonstrated successfully with 15%

Future prospects

The application of transgenic technology is necessary for trait-enhancement in many agronomically important crops. Most of the agri-horticultural crops can be protected through this technology, by introducing the genes resistance/tolerance to various biotic and abiotic stresses and also quality improvement. Plant genetic engineering has enabled scientists to produce transgenic plants in various crop species using first generation SMGs like nptII, hpt, ppt etc. Though, SMGs are definitely useful

Acknowledgement

The authors thank Dr. B.C. Viraktamath, Project Director, Directorate of Rice Research for providing all the facilities and Department of Biotechnology, Govt. of India for financial assistance. The authors also thank Dr. S.K. Datta, Deputy Director General (Crop Sciences), ICAR for his constant encouragement and support while preparing the manuscript.

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