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The development of virus resistant carrot genotypes using RNAi technology

Nina L. McCormick1 , Rebecca Ford1, Paul W.J. Taylor1 and Brendan Rodoni2.

1 BioMarka, Joint Centre for Crop Innovation, Institute of Land and Food Resources, The University of Melbourne, Vic 3010
www.landfood.unimelb.edu.au/research/biomarka; Email n.mccormick@pgrad.unimelb.edu.au
2 Department of Primary Industry – Knoxfield, Private Bag 15, Ferntree Gully Delivery Centre, Vic 3156
www.nre.vic.gov.au/agvic/ihd/index/htm ; Email Brendan.Rodoni@dpi.vic.gov.au

Abstract

Carrot virus Y (CarVY) is a single-stranded, positive sense RNA potyvirus that infects members of the Apiaceae family. The development of carrot cultivars that are highly resistant or immune to infection will greatly benefit the horticultural industry through reduced inputs into crop protection, increased crop and food quality and yield. There are no known sources of natural resistance to CarVY and therefore a transgenic approach is being employed to speed up conventional resistance breeding programs. An efficient regeneration system was developed based on somatic embryogenesis. Carrot cultivars Stefano and Crusader required 0.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for optimal callus induction from petiole explants. For the induction of viable somatic embryos, callus cultures were grown for 4-8 weeks with a 16 hr photoperiod on a medium devoid of plant growth regulator. Petiole explants were successfully transformed via Agrobacterium-mediated transfer with binary vectors containing the gfp reporter gene. Approximately sixty percent of transformed petioles produced regenerative transgenic callus. A short sequence of the nuclear inclusion protein a (NIa) region of the CarVY genome will be targeted for future RNAi silencing of the virus. A partial sequence of this region has been isolated and characterised.

Media summary

Carrot cultivars with unique virus resistance being developed using RNAi technology will greatly benefit the horticultural industry through reduced inputs into crop protection and will lead to increased quality and yield.

Key Words

RNAi, virus resistance, carrot, somatic embryogenesis, regeneration

Introduction

Carrot virus Y is a potyvirus that infects carrot (Daucus carota). First seen in Western Australia in 1997 (Latham and Jones, 2000), CarVY is fast becoming a growing concern for Australian carrot producers. High infection incidences of up to 95% and yield loss above 35%, cost the Australian carrot industry between $5 and $10 million a year (Latham and Jones 2000). Conventional breeding for virus resistance is not possible due to unavailable resistance sources. RNAi technology offers an alternative practical approach to developing virus resistant carrot genotypes.

Plants defend themselves against viruses by exploiting the requirement of most plant RNA viruses to replicate using a double-stranded, replicative intermediate (dsRNA). This evolutionary conserved plant defence mechanism is referred to as post-transcriptional RNAi. Introduced transgenes that encode viral dsRNA enable the plant to subsequently recognise invading virus. Double-stranded RNA produced from either a transgene or a replicating virus is cleaved into approximately 21 nucleotide fragments by the Dicer enzyme (Wesley et al., 2001). These small fragments, known as short-interfering RNA (siRNA), are used as guides for cleavage of viral homologues on infection by CarVY. In order to harness this technology, to protect valuable carrot cultivars, robust and efficient transformation and regeneration systems are required. Targeted regions of the viral genome must be characterised for targeted gene silencing to allow for engineering of transgenes that can be used for RNA silencing of CarVY on infection.

Methods

Tissue culture systems

Seed was surface sterilised then germinated on Murashige and Skoog (MS) medium (1962). Four weeks following germination, cotyledon, hypocotyl and root explants were harvested and used in subsequent callus initiation experiments. Meristematic shoot tips were harvested, proliferated and used as a stock plant source for further callus induction experiments. Proliferation of meristematic shoot tips was achieved using 100% MS medium. Stock cultures were maintained on 100% MS medium and sub-cultured every eight weeks.

Explants derived from seedlings and stock cultures were tested for their ability to generate viable callus for future somatic embryo production. A range of plant growth regulator (PGR) concentrations and combinations were applied to different explant types. Callus cultures were maintained and proliferated on MS medium containing 0.5 μM 2,4-D. Callus was sub-cultured every four weeks. Callus was moved to suspension cultures and agar plates, containing MS media devoid of PGRs, to produce a large number of viable somatic embryos under a 16 hr photoperiod.

Transformation systems

In order to determine transformation efficiency, petiole explants were transformed via Agrobacterium-mediated transfer with strain LBA4404 carrying Rova77 and Rova78 binary vectors, constructed from the pPZP112 harbouring either the gfp5intron or gfp7intron reporter gene respectively. Tissue was incubated with Agrobacterium for 10 min at RT. Following incubation, explants were co-cultivated in the dark for 3 d before being moved to selection medium (MS + 0.5 μM 2,4-D + 50 μg/mL kanamycin + 150 μg/mL timentin) also in the dark for a period of 8 weeks. The effect of kanamycin and hygromycin was assessed on non-transformed petioles in order to determine the optimal concentration for selective callus proliferation from transformed petiole explants. Following transformation with the Rova vectors, gfp fluorescence was visualised using a Leica MZ6 stereomicroscope.

Viral sequence isolation

To isolate a suitable short viral sequence (~300bp) for RNAi, degenerate PCR primers were designed from conserved regions of the NIa region of viruses closely related to CarVY (Moran et al., 2002). This was done in order to avoid any future potential intellectual property issues that may arise from using previously patented sequenced viral regions (Figure 1).

Figure 1. Potyvirus polyprotein structure including NIa targeted region. Arrows indicate PCR primer binding sites.

Results

Tissue culture systems

An efficient micropropagation and regeneration system was established for carrot cultivars Stefano and Crusader. Callus induction and proliferation was best achieved from both cultivars with a 2,4-D concentration of 0.5 μM (Figure 2).

Figure 2. Effect of 2,4-D concentration on callus proliferation from carrot petiole explants. Mean score: 0 = explant dead; 1 = explant alive, no callus; 2 = callus at ends of explant, yellow and not friable; 3 = some callus along length of explant, appears watery and not friable; 4 = callus along length of explant with more proliferation at ends, becoming friable; 5 = callus in large knobs at explant ends, callus white and friable.

Transformation systems

Carrot petiole explants were successfully transformed via Agrobacterium-mediated transfer with LBA4404 and readily expressed gfp (60% for Rova77 and 50% for Rova78; Figure 3). The optimal concentrations of antibiotic chosen for future transgenic callus selection, that also maintain good callus induction and proliferation, were 25 and 50 μg/mL of kanamycin (Figure 4). Although 5 μg/mL of hygromycin produced the highest score for callus production from petiole explants, this treatment was not chosen due to concerns regarding the downstream inhibition of shoot regeneration from somatic embryos.

Figure 3. A. gfp positive callus emerging from carrot petiole explant; B. Proliferating gfp positive carrot callus; C. gfp positive torpedo-shaped somatic embryo generated from carrot callus.

Figure 4. Effect of antibiotic treatment on callus proliferation from carrot petiole explants after 8 weeks. Mean score: 0 = explant dead; 1 = explant alive, no callus; 2 = callus at ends of explant, yellow and not friable; 3 = some callus along length of explant, appears watery and not friable; 4 = callus along length of explant with more proliferation at ends, becoming friable; 5 = callus in large knobs at explant ends, callus white and friable.

Viral sequence isolation

Approximately 300bp of the 3’ end of the NIa region has been cloned and sequenced. This sequence was found to be highly similar to the same region from Bean yellow mosaic virus, Leek yellow strip virus and Turnip mosaic virus and other members of the ssRNA potyvirus family. This sequence will be incorporated into self-complementary, intron-containing ‘hairpin’ RNA (ihpRNA) silencing constructs (Wesley et al., 2001). These constructs will be integrated into the carrot genome and transgenic carrot plants will be screened for CarVY resistance.

Conclusion

Efficient transformation and regeneration systems have been developed and will facilitate the future integration of CarVY silencing constructs into the carrot genome. This will enable the plant to recognise invading virus and marshal internal defences.

References

Latham LJ and Jones RAC (2000). Yield and quality losses in carrots infected with Carrot virus Y. Proceeding of Carrot Conference Australia, Perth, Western Australia, October 2000.

Moran J, van Rijswijk B, Traicevski V, Kitajima EW, Mackenzie AM and Gibbs AJ (2002). Potyviruses, novel and known, in cultivated and wild species of the family Apiaceae in Australia. Archives of Virology 147, 1855-1867.

Murashige T and Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco cultures. Physiol Plant 15, 473-497.

Wesley SV, Helliwell CA, Smith NA, Wand MB, Rouse DT, Liu Q, Gooding PS, Sing SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG and Waterhouse PM (2001) Construct design for efficient, effective and high-throughput gene silencing in plants, The Plant Journal 27, 581-590.

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