Aspects of Applied Biology 96, 2010Agriculture: Africa’s “engine for growth” - Plant science and biotechnology hold the key

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Establishment of alternative selection systems for transgenic sugarcane callus

By C VAN DER VYVER, C STANDER, J KOSSMANN and H GROENEWALD

Institute for Plant Biotechnology, Department of Genetics, University of Stellenbosch, Stellenbosch, 7602, South Africa

Summary

Selection-marker genes are routinely used in plant genetic transformation protocols to ensure the survival of transformed cells by limiting the regeneration of non-transgenic cells after transformation. In order to find alternatives to the use of antibiotics as selection agents we focused on so-called positive selection systems. Four alternative systems were investigated for their potential as selection agents in sugarcane genetic transformation. The systems included i) galactose, ii) 2-deoxyglucose iii) NaCl and iv) gluconic acid as selective agents in combination with the galactose-�-phoshate uridyl-transferase (galT), 2-deoxyglucose-6-phosphate-phosphatase (2Dog6PP), “cytosolic” betaine aldehyde dehydrogenase (BADH), and gluconokinase selection genes, respectively. Control sugarcane callus sensitivity towards these selection agents were determined and resulted in gluconic acid being eliminated as a possible selection agent. Transgenic calli were produced for the remaining three selection systems and results monitored. Galactose and 2-deoxyglucose showed the most promise as alternative selection agents in the production of transgenic sugarcane callus.

Key words: Alternative selection, sugarcane, genetic transformation

Introduction

Existing selection systems for use in genetic transformation of plant cells are based mainly on antibiotic or herbicide resistance (Joersbo & Okkels, �996). However, heightened consumer concerns over the presences of antibiotic and herbicide resistance genes in genetically modified plants have prompted development of alterative selection systems. Also, alternative selection systems will allow repeated transformations where more than one selection system is needed for multiple gene transfer into a particular plant. In this study we investigate the suitability of four positive selection systems, namely galactose, 2-deoxyglucose, NaCl and gluconic acid in genetic transformation of sugarcane. Positive selection systems provide the putative transgenic tissue with a metabolic advantage over non-transgenic tissue by converting the selective agent into a fully metabolisable compound by the selective gene product (Joersbo & Okkels, �996; Joersbo, 200�).

Materials and Methods

Plant material, establishment of in vitro cultures and genetic transformation Leaf rolls of sugarcane variety Nco3�0 were isolated from tightly furled inner leaves. The basal part of the leaf rolls were cut into 2 mm thick transverse sections and placed on MS medium

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(Murashige & Skoog, �962) containing 2% sucrose, 3 mg L-� 2,4-D, 0.5 g casein and 0.22% gelrite, pH 6. Embryogenic callus developed within 6 weeks of incubation in the dark at 24°C and was bombarded as described by Franks & Birch (1991). To guarantee the identification of transgenic callus clones expressing an alternative selection gene, callus was co-bombarded with an alternative selection and the pEmuKN plasmid. Putative transgenic clones were first selected for geneticin resistance followed by alternative selection with the various selection agents. Studies showed that at least 85% of transgenic clones will contain both co-bombarded genes (Snyman et al., 2006).

Plasmid DNA Alternative selection genes were cloned into the plant expression vector UBi-5�0 under control of an ubiquitin promoter and CaMV terminator. For co-bombardment plasmid pEmuKN, containing the nptII gene driven by the strong monocot promoter Emu, allowed for clear-cut selection on geneticin (Last, �990). The four selection genes were i) pUBI-galT: galactose-�-phosphate uridyltransferase gene from E. coli (AP009048); ii) pUBI-dogPP: 2-deoxyglucose-6-phosphate phosphatase gene from Saccharomyces cerevisiae (AY5585�6); iii) pUBI-BADH: betaine aldehyde dehydrogenase gene from Amaranthus hypochondriacus, lacking transit peptides for cytosolic expression (Legaria et al., �998); iv) pUBI-ScGNK5�0: gluconokinase gene from S. cerevisiae (NP0�0534).

Results

Sugarcane callus sensitivity towards selection agents

Table �. Sugarcane callus growth on basic MS3 medium (control) compared to medium supplemented with varying selection agents and concentrations

0 = No, + = Limited, ++ = Medium, +++ = Severe inhibiting growth effect

Concentration Sucrose Growth InhibitionSelection Agent (mM) (%) Low sucrose High sucrose

Galactose 5.5�42855

2Or�

++++++++++

0+

++++++

2-DOG 3 6�8

2 ++++++++

NaCl 200230250280300

2 + +

++++ ++

Gluconic acid 80�00�50300

0.5Or0

++++

++++

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Fig. �. Sugarcane callus placed on MS3 medium (control) containing 2% (middle) or �% (bottom) sucrose, supplemented with 5.5 mM (A), �4 mM (B), 28mM (C) or 55 mM (D) galactose.

Fig. 2. Sugarcane callus placed on MS3 medium (control) supplemented with (A) 3 mM, (B) 6 mM or (C) �8 mM 2-DOG, respectively.

Fig. 3. Sugarcane callus placed on MS3 medium (control) supplemented with (Left): (A) 200 mM, (B) 230 mM, (C) 250 mM, (D) 280 mM or (E) 300 mM NaCl, respectively; (Right): (F) �00 mM or (G) 200 mM gluconic acid, respectively.

Discussion

Here we have investigated the potential of four selection systems for identification and isolation of transgenic sugarcane callus. In the past, three of the four systems were successfully used to regenerate transgenic tobacco, potato or carrot plants, respectively (Joersbo et al., 2003; Kunze et al., 200�; Kumar et al., 2004). It is also long since established, in work done as early as the �950’s, that a large pool of plant species are sensitive to galactose, 2-DOG and NaCl (Farkas, �954; Stenlib, �959; Zemek et al., �975). The approach to use gluconokinase as selection gene in combination

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with gluconic acid selection is novel and newly investigated. Gluconokinase catalyze a reaction between gluconate and ATP to form 6-phosphogluconate (Sable & Guarino, �952). Gluconic acid can act as an alternative carbohydrate source. For galactose LD50 levels as low as 0.�3 g L-� were determined for maize (Roberts et al., �97�). However, when galactose is combined with sucrose in the culture medium, higher concentrations of galactose are needed for toxicity (Joersbo et al., 2003). This was also true in the current study where galactose concentrations of �4 and 28 mM combined with � or 2% sucrose, displayed adverse effects on sugarcane callus growth (Fig. �). Therefore, sugarcane callus seems to be sensitive to galactose concentrations, which equals 25% or more of the sucrose concentration in the medium. A number of transformation events, containing the transgene galactose-�-phosphate uridyltransferase, were identified. Previous studies showed that there is a linear correlation between successful transformation events and the galactose concentration used for selection. Higher galactose concentrations lead to lower transformation efficiency (Joersbo et al., 2003). Therefore, the galactose concentration should be high enough for selecting transformed cells effectively but without compromising callus viability. Transgenic clones were able to survive on 50 mM galactose in combination with �% sucrose. Only one transformation event regenerated into a plantlet, which might be due to transient reduction of vigor caused by the selection. Loss of ability to regenerate was not seen in other plant species over expressing galactose-�-phosphate uridyltransferase. Transformation efficiency of sugarcane expressing the 2Dog6PP gene and secondly the BADH gene was low. In this study, for 2-DOG and NaCl selection, three and one transformation events were obtained, bombarding eight plates each of sugarcane starting material, respectively. This is in contrast with the average of one transformation event per plate of starting material as seen with other transgenes (unpublished data). Sugarcane callus started to show visible effects of stress at concentrations as low as 3 mM 2-DOG, which was similar to tobacco and potato data described by Kunze et al. (200�). However, transgenic lines harboring the 2Dog6PP gene tolerated up to 6 mM 2-DOG (Fig. 2). Transgenic sugarcane callus did not show any off-type and easily regenerated into plants, which is in contrast with some studies done on tobacco, potato and cucumber where shoot and root formation was problematic (Kunze et al., 200�). Sugarcane callus growth was affected by concentrations of 200 mM NaCl with more severe growth changes visible at 250 mM NaCl or higher (Fig. 3). This fell within the same concentration range as seen for the sensitivity of carrot cells towards NaCl (Kumar et al., 2004). However, transgenic carrot lines were able to withstand NaCl concentrations of 400 mM, while the transgenic sugarcane could not survive selection on 280 mM NaCl. The expression of the BADH gene in sugarcane seemed to severely impair callus growth and resulted in total loss of capacity for embryogenesis. Very limited increase in biomass was observed over a lengthy time period. Also, sugarcane callus exposed to NaCl does not show color changes or cell death, which makes identification of putative transgenic clones extremely difficult. Following these observations we believe that both the galactose and 2-DOG systems have the potential for use as alternative selections systems to identify transgenic sugarcane. However, we eliminate NaCl and gluconic acid as potential selection agents for transgenic sugarcane callus identification. Expression of the BADH gene seems to impair callus viability and gluconic acid decreases sugarcane callus biomass with no additional traits to distinguish between transgenic and non-transgenic callus clones.

References

Farkas G L. 1954. Die toxische wirkung einiger zucher auf die wurzeln der pflanzen. Zuckerantagonismen Biologisches Zentralblatt 73:506–52�.Franks T, Birch R G. 1991. Gene transfer into intact sugarcane callus using microprojectile bombardment. Australian Journal of Plant Physiology 18:47�–480.

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Joersbo M. 2001. Advances in the selection of transgenic plants using non-antibiotic marker genes. Plant Physiology 111:269–272.Joersbo M, Jorgensen K, Brunstedt J. 2003. A selection system for transgenic plants based on galactose as selective agent and a UDP-glucose: galactose-�-phosphate uridyltransferase gene as selective gene. Molecular Breeding 11:3�5–323.Joersbo M, Okkels F T. 1996. A novel principle for selection of transgenic plant cells: positive selection. Plant Cell Reports 16:2�9–22�.Kumar S, Dhingra A, Daniell H. 2004. Plastid-Expressed Betaine aldehyde dehydrogenase gene in carrot cultured cells, roots and leaves confers enhanced salt tolerance. Plant Physiology 136: �–�2.Kunze I, Ebneth M, Heim U, Geiger M, Sonnewald U, Herbers K. 2001. 2-Deoxyglucose resistance: a novel selection marker for plant transformation. Molecular Breeding 7:22�–227.Last D I, Bretell R I S, Chamberlain D A, Chaudhury A M, Larkin P J, Marsh E L, Peacock W J, Dennis E S. 1990. pEmu: an improved vector for gene expression in cereal cells. Theoretical and Applied Genetics 81:58�–588.Legaria J, Rajsbaum R, Muñoz-Clares RA, Villegas-Sepúlveda N, Simpson J, Iturriaga G. 1998. Molecular characterization of two genes encoding betaine aldehyde dehydrogenase from amaranth. Expression in leaves under short-term exposure to osmotic stress or abscisic acid. Gene 218:69–76.Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15:473–497.Roberts R M, Heishman A, Wicklin C. 1971. Growth inhibition and metabolite pool levels in plants fed D-glu-cosamine and D-galactose. Plant Physiology 48:36–42.Sable H Z, Guarino A J. 1952. Phosphorylation of gluconate in yeast extracts. Journal of Biological Chemistry 196:395–398.Snyman S J, Meyer G M, Richards J M, Haricharan N, Ramgareeb, Huckett B I. 2006. Refining the application of direct embryogenesis in sugarcane: effect of the developmental phase of leaf disc explants and the timing of DNA transfer on transformation efficiency. Plant Cell Reports 25:�0�6–�023.Stenlib G. 1959. Species differences between plant roots in the reaction of inhibitor sugars. Physiologia Plantarum 12:2�8–235.Zemek J, Hricova D, Stremen J, Bauer S. 1975. Effect of 2-deoxy-D-glucose on tissue culture of Nicotiana tabacum L. Zeitschrift fur Pflanzenphysiolie 76:��4–��9.

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