Beta vulgaris - Wiley Online Library

Annals of Applied Biology ISSN 0003-4746

R E V I E W A RT I C L E

Genetic approaches to sustainable pest management in sugar beet (Beta vulgaris) C.-L. Zhang1,2, D.-C. Xu3, X.-C. Jiang4, Y. Zhou1, J. Cui3, C.-X. Zhang4, D.-F. Chen1, M.R. Fowler1, M.C. Elliott1, N.W. Scott1, A.M. Dewar5 & A. Slater1 1 The Norman Borlaug Institute for Plant Science Research/Systems Biology Research Laboratory, Faculty of Health and Life Sciences, De Montfort University, The Gateway, Leicester, UK 2 National Centre for Sugar Crops Improvement, Institute for Sugar Beet Research, Chinese Academy of Agricultural Sciences/Faculty of Agricultural Sciences, Heilongjiang University, Harbin, China 3 Institute of Beet Sugar Industry and Genetic Engineering, Harbin Institute of Technology, Harbin, China 4 Department of Plant Science, Hunan Normal University, Changsha, China 5 Dewar Crop Protection Ltd, Drumlanrig, Great Saxham, Bury St Edmunds, Suffolk, UK

Keywords Insect resistance; molecular breeding; nematode resistance; sugar beet (Beta vulgaris L.); transgenesis; wild Beta germplasms. Correspondence C.-L. Zhang, The Norman Borlaug Institute for Plant Science Research/Systems Biology Research Laboratory, Faculty of Health and Life Sciences, De Montfort University, The Gateway, Leicester, LE1 9BH, UK. Email: [email protected] Received: 5 November 2007; revised version accepted: 13 January 2008. doi:10.1111/j.1744-7348.2008.00228.x

Abstract Sugar beet (Beta vulgaris) is an important arable crop, traditionally used for sugar extraction, but more recently, for biofuel production. A wide range of pests, including beet cyst nematode (Heterodera schachtii), root-knot nematodes (Meloidogyne spp.), green peach aphids (Myzus persicae) and beet root maggot (Tetanops myopaeformis), infest the roots or leaves of sugar beet, which leads to yield loss directly or through transmission of beet pathogens such as viruses. Conventional pest control approaches based on chemical application have led to high economic costs. Development of pest-resistant sugar beet varieties could play an important role towards sustainable crop production while minimising environmental impact. Intensive Beta germplasm screening has been fruitful, and genetic lines resistant to nematodes, aphids and root maggot have been identified and integrated into sugar beet breeding programmes. A small number of genes responding to pest attack have been cloned from sugar beet and wild Beta species. This trend will continue towards a detailed understanding of the molecular mechanism of insect–host plant interactions and host resistance. Molecular biotechnological techniques have shown promise in developing transgenic pest resistance varieties at an accelerated speed with high accuracy. The use of transgenic technology is discussed with regard to biodiversity and food safety.

Introduction Sugar beet (Beta vulgaris L.) is an important arable crop that plays a vital role in the crop rotation systems of northern hemisphere agriculture. It produces about 27% of the total world sucrose production annually (http://www.faostat.fao.org). Recently, this crop has also been developed as an efficient biofuel alternative to fossil fuel energy. The latter development is a response to increasing concerns about global warming, recent energy shortages and consequently, potential fertiliser shortage

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(Barbanti et al., 2007). Traditional breeding has played a vital role for productivity improvement in sugar beet. However, more recently, molecular biotechnological approaches have been developed and are being integrated with the conventional approaches. This should lead to enhanced efficiency of conventional sugar beet breeding through integration of molecular marker-assisted selection and development of novel sugar beet strains through genetic engineering. Sugar beet is attacked by a wide range of pests that cause damage to the leaves and taproot (directly and/or through 143

Breeding and biotechnological development of pest resistance in sugar beet

transmission of pathogens), leading to substantial yield loss (reviewed by Dewar & Cooke, 2006). Although conventional pesticide application has been effective for the majority of the pests, many chemicals are expensive and may be toxic to the environment and deleterious to human health. The continuing removal of highly toxic and environmentally damaging chemicals from the market (e.g. fumigant methyl bromide) and the continuous development of pesticide tolerance in several pests have prompted plant breeders to develop pest-resistant cultivars as an important part of integrated pest management systems. Since the 1980s, programmes for largescale Beta germplasm collections and multiple parameter evaluations including pest tolerance, disease resistance and productivity have been carried out in several laboratories to enable sustainable sugar beet breeding and to broaden its genetic base (Doney & Whitney, 1990; Zhang & Liu, 1998; Asher et al., 2001; Frese et al., 2001; Luterbacher et al., 2005; Panella & Lewellen, 2007). Sources of resistance to pests, for example nematodes, are frequently found in wild Beta species and have been introgressed into sugar beet through interspecific hybridisation (reviewed by Savitsky, 1975; Brandes et al., 1987; Van Geyt et al., 1990; Yu, 2005; Panella & Lewellen, 2007). Valuable information has been accumulated from several worldwide initiatives and made available through printed publications, lectures and/or electronically through the World Wide Web. Although sugar beet is a relatively young crop with only about 300 years history, there have been considerable developments in genetic and biotechnological knowledge and of genetic resources for pest resistance. This review summarised recent progress made on the development of genetic resistance through conventional and molecular approaches to nematodes, root maggot, aphids and leaf-feeding insects and discussed opportunities and concerns regarding the use of transgenic technologies.

Breeding and molecular biology for resistance to nematodes Beet cyst nematode (Heterodera schachtii Schmidt) is one of the most damaging pests of sugar beet cultivation in the Europe and the USA. Conventional chemical control is less effective but sometimes at a high environmental cost. Complete resistance to the cyst nematodes was found in the section Procumbentes, the Beta species most distantly related to sugar beet, in 1950s. The resistance has been transferred into sugar beet although chromosome additions and translocations by several research groups’ efforts (Savitsky, 1975; Lange et al., 1993; Cai et al., 1997; Mu¨ller, 1998; Heijbroek et al., 2002; Yu, 2005). Several strains of nematodes failed to reproduce 144

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in derived resistant accessions (Lange et al., 1993). The resistant genes were located on chromosomes 1 and 7 of Beta procumbens (designated as Hs1pro1 and Hs2pro7, respectively), chromosome 7 of Beta webbiana (designated as Hs1web7) and chromosome 1 of Beta patellaris (designated as Hs1pat1) (Lange et al., 1993; Mesbah et al., 1997). However, the transmission rate for nematode resistance genes was regarded as low because of meiotic disturbances (Brandes et al., 1987). Several undesirable traits including tumour formation in leaves and roots and multiple shoots, also appeared in the derived nematoderesistant stains (Sandal et al., 1997). Further backcrossing with elite sugar beet lines was used to develop resistant materials. DNA markers based on B. procumbens-specific repetitive DNA elements or random amplified polymorphic DNA (RAPD) markers linked to the resistance were developed for marker-assisted selection (Jung et al., 1992; Hallden et al., 1997). Studies of pathotypes of H. schachtii indicates that the Schach 1-derived population was virulent towards translocation lines, Hs1pro1, Hs2pro7 and Hs1web7, while the Schach 0-derived population was unable to break the resistance conferred by translocations (Mu¨ller, 1998). It was reported that the nematode population decreased by 73% with experimental resistant varieties whereas that increased by 35% with a susceptible variety (Werner et al., 1995). A resistant variety called Nematop was developed which showed acceptable performance in nematode-infested soil (Dewar, 2005). However, it yields less than the highest yielding susceptible varieties in the absence of the pest and the resistance could be eroded under high population pressure because of resistance-breaking strains of the pest. Using a positional cloning method, the major gene controlling nematode resistance, Hs1pro-1, has been cloned from the translocation line A906001 and analysed in transformed hairy root cultures (Cai et al., 1997). Recently, a full-length Hs1pro-1 (DQ148271) which includes an additional 176 amino acid N-terminal extension has been reported, which confers about 70% more resistance to soybean cyst nematode (H. glycines Ichinohe) in T1 generation transgenic soybean plants than susceptible control cultivars (McLean et al., 2007). It has been suggested that germin-like proteins are possible key components of the Hs1pro1-mediated resistance response (Cai D., unpublished results). It has been shown that the othologous Arabidopsis proteins, AtHSPROl/2, are implicated in plant pathogen resistance by interacting with the KIS/GBD domains of AKINbc, subunits of SnRKl heterotrimeric complex (Gissot et al., 2006). The search for additional genes continues because it appears that Hs1pro-1 is not the only nematode resistance gene in these sugar beet lines (Jung C., unpublished results). A gene encoding a 317 amino acid polypeptide similar to


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phosphatidylinositol-specific phospholipase C, responded to nematode infection and was isolated by cDNA-AFLP differential screening of nematode-resistant lines (Samuelian et al., 2004). A putative cation transporter gene has been found linked to the Hs1pro-1 and may play a role in the defence response and/or signal transduction (Oberschmidt et al., 2003). Sequencing of a full translocation of B. procumbens in sugar beet translocation line TR363 (which does not carry the Hs1pro-1 gene) has identified about 12 genes involved in biotic stress resistance responses or transcription factors (Schulte et al., 2006). Two of these genes, a MYB DNA binding/transcription factor (ORF125, ABD83304) and a glutathione S-transferase gene (ORF133, ABD83308) have been identified as candidates for the proposed resistance gene, Hs1-1, after comparative expression analysis (www.plantbreeding. uni-kiel.de/project_hs1pro.shtml). Using another approach, further disease resistance gene analogues [nucleotidebinding site (NBS) and leucine-rich repeat (LRR)] including genes similar to nematode- or aphid-resistance genes Hs1pro-1, Mi (Rossi et al., 1998) and Gpa2 (Van der Vossen et al., 2000) have been identified in sugar beet using DNA amplification with polymerase chain reaction (PCR) (Hunger et al., 2003; Tian et al., 2004). One sequence resembles the nematode-resistance genes, Mi and Gpa2, and has been designated as Hs1-1pro-1. These clones are being tested for nematode resistance in transformed sugar beets (Cai et al., 2005). Polygenic and recessive resistance against the cyst nematode was found in B. vulgaris ssp. maritima and advanced breeding lines were developed by crossing sugar beet with further selection (Mesken & Lekkerkerker, 1988). Molecular markers such as RAPD and sequence tagged sites (STS) linked to the nematode resistance have been developed for facilitating genome diagnosis (Weiland et al., 2007). Commercial hybrids based on this partial resistance have been developed and marketed as Beta 8520N in California and KWS Pauletta in Europe (Panella & Lewellen, 2007). For another beet cyst nematode species, Heterodera trifolii (Goff.) f. sp. beta, the Beta species of section Procumbentes were found to be resistant. Some sugar beet accessions initially developed for resistance to H. schachtii by interspecific hybridisation with species of Procumbentes showed partial resistance (reviewed by Van Geyt et al., 1990). Partial resistance to H. trifolii was also found in advanced breeding line of B. vulgaris spp. maritima crossed with sugar beet (Mesken & Lekkerkerker, 1988). Beet root-knot nematodes, for example Meloidogyne spp. cause root gall symptoms and reduce beet production. Wild Beta species have been screened for rootknot nematode resistance and B. procumbens was found to have partial resistance towards six species of these


nematodes (reviewed by Van Geyt et al., 1990). The resistance appears not to have been introduced into sugar beet through interspecies hybridisation. In an intense screening of sea beet (B. vulgaris ssp. maritima) resources, accession WB66 was found to confer high heritable resistance and had already transmitted this into sugar beet (Yu, 1995). Further research showed that the sea beet line was resistant to six species of root nematodes (Meloidogyne incognita, Meloidogyne javanica, Meloidogyne arenaria, Meloidogyne hapla, Meloidogyne chitwoodi and Meloidogyne fallax), and the gene was located on chromosome 4 and designated as R6m-1 (Yu et al., 1999). A molecular marker, NEM06 has been obtained, which encodes a plant bZIP transcription factor (Weiland & Yu, 2003). Further sequence analysis revealed that there are numerous nucleotide substitutions between these genes cloned from resistant and susceptible plants.

Breeding and molecular biology for resistance to root maggot Root maggot (Tetanops myopaeformis) is an important pest in North America. Resistant sugar beet lines showed a low rate of maggot survival and less yield loss than susceptible varieties. Genetic resistance has been found in a few sugar beet lines, red table beet and B. vulgaris ssp. maritima accessions (Doney & Whitney, 1990; Campbell et al., 2000; Panella & Lewellen, 2007). After four rounds of mass selection, the resistance level in several advanced sugar beet lines reached the equivalent of control by insecticides. As a consequence, breeding lines F1015 and F1016 have been released by ARS-USDA (Campbell et al., 2000). A molecular biological approach has been undertaken to identify genes induced in sugar beet after maggot infestation. About 150 sugar beet genes were identified including those involved in the defence/stress response, secondary metabolism including a resistant cultivar specific serine protease inhibitor (BvSTI) and polyphenol oxidase, and signal transduction (Puthoff & Smigocki, 2007). Many of these genes are regulated by signalling molecules such as methyl jasmonate, salicylic acid and ethylene. These genes may have potential to derive molecular markers for beet breeding or genetic engineering. Studies of the digestion system of the root maggot revealed that two predominant classes of proteases, aspartyl and serine proteases, were the key elements (Smigocki, 2004). The same author found that more than 90% of aspartyl protease activity was reduced by pepstatin A and squash aspartyl protease inhibitor, while 90% and 50% of serine protease activities were reduced by soybean trypsin–chymotrypsin inhibitor and PMSF, respectively. It has also been shown that secondary metabolites 145


extracted from cytokinin-overproducing tobacco kill 90% of the maggot. The BvSTI gene has been fused to the CaMV 35S promoter for expression in sugar beet hairy roots to elucidate its functional roles in resistance and root biology (Smigocki et al., 2007). About a twofold to fourfold increase in trypsin inhibitory activity was observed in the BvSTI-transformed hairy roots. Insect feeding assays are in progress to examine the effects of the inhibitor on insect mortality and growth rates.

Breeding and molecular biology for resistance to aphids Breeding for resistance to aphids Aphids cause serious problems to sugar beet production worldwide. Green peach aphid (GPA, Myzus persicae) and black bean aphid (Aphis fabae Scopoli) are phloem-feeding insects, infesting leaves and reducing photosynthesis and sucrose accumulation (Dewar & Cooke, 2006). They also act as vectors for the beet yellowing viruses, beet yellow virus, beet chlorosis virus and beet mild yellowing virus. Recent research has indicated that at least three types of pesticide-resistant M. persicae variants have developed, which render many of the insecticides ineffective (Foster et al., 2002). Identification of genetic resistance to aphids started in the 1960s, and resistance to several aphid species was identified which might be species specific (Russell, 1972; Lowe & Singh, 1985; reviewed by Van Geyt et al., 1990). By analysing GPA response to host quality, aphids feeding on resistant plants showed a stomach precipitate phenotype (Williams et al., 1997; Kift et al., 1998). This may be a useful parameter to further screen Beta germplasm for resistance to the aphids. A range of biochemical parameters including the contents of leaf malonic dialdehyde, chlorophyll, carotenoid and total phenolic compounds and the peroxidase activity in seedlings were suggested as parameters for early identification of sugar beet lines for resistance to the aphids (Pokhiton & Nechiporuk, 1987; Nechiporuk, 1989). High resistance to GPA (M. persicae) colonisation was found in B. vulgaris ssp. maritima (Dale et al., 1985). The multiplication of M. persicae was considered to be slow on Beta corolliflora and low susceptibility to aphids was recorded in species of section Procumbentes (reviewed by Van Geyt et al., 1990). Beet root aphids (Pemphigus spp.) can occur in large numbers on the fibrous roots of sugar beet causing plants to remain stunted, to wilt and show yield losses (reviewed by Dewar & Cooke, 2006). Breeding for sugar beet resistant to the root aphid (Pemphigus fuscicornis Koch) has been one of the targets for breeders in the USA. Two resistance mechanisms, antibiosis and antixenosis, were reported to be important for the breeding 146

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of root aphid-resistant varieties (Campbell & Hutchison, 1995). Resistant sugar beet populations such as FC201 and varieties have been developed based on initial greenhouse screenings and a final field testing (Hein et al., 2001; Panella & Lewellen, 2005). Molecular biology for resistance to aphids GPA has an exceptionally wide host range, which includes model species Arabidopsis thaliana. Several pathways of pest–host plant interactions including jasmonates, ethylene and salicylic acid have been well characterised in several plant species (Ellis et al., 2002; Cipollini et al., 2004; Dong et al., 2004; Rayapuram & Baldwin, 2007). It is known that these pathways interact, sometimes resulting in antagonism between the pathways. Several genes involved in the jasmonates and/or ethylene pathways, such as CEV1 and EIN2, have been shown to confer resistance to GPA through genetic analysis of the cev1 mutant (Ellis et al., 2002) or in combination with in planta treatment with a pathogenic bacterial harpin protein (Dong et al., 2004). Phytoalexin deficient4 (PAD4) and its signalling and stabilising partner enhanced disease susceptibility1 (EDS1) are key players of salicylic acidmediated pathogen resistance. Pegadaraju et al. (2007) showed that PAD4, which is expressed at elevated levels in response to GPA infestation, is required for resistance to GPA in A. thaliana. Electrical monitoring of aphid feeding behaviour revealed that PAD4 modulates a phloembased defence mechanism against GPA. The activity of PAD4 in limiting phloem sap uptake serves as a deterrent in host-plant choice and restricts aphid population size. PAD4-modulated defence against GPA also involves premature leaf senescence but does not involve EDS1. Couldridge et al. (2007) identified 27 genes that are significantly altered during GPA–A. thaliana interactions (3 downregulated and 24 upregulated) using the Arabidopsis whole-gene microarray hybridisation procedure. Those genes identified are involved in cell wall modification, carbon metabolism and signalling, oxidative stress (e.g. GST1 and GST2) and defence against other pathogens (e.g. PAD4) and a gene encoding short-chain dehydrogenase/reductase (SDR). There have been limited research reports on the molecular aspect of aphid–sugar beet interactions. Dimmer et al. (2004) showed that the expression of a xyloglucan endo-transglucosylase/hydrolase was induced during insertion of GPA stylet in leaves. We undertook a search of DNA databases using genes involved in aphid resistance and defence signal transduction as queries and identified sugar beet orthologues of a number of Arabidopsis genes including CEV1, FAD3/7, EIN2, SDR, SGT1, HSP90 and JAR1 (Table 1). Detailed characterisation of those genes in



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Table 1 Sugar beet expressed sequence tags (EST) sequences with similarity to Arabidopsis genes involved in insect resistance and defence signal transductiona Arabidopsis Accession Number

Beet EST Number

Identity (%)

Similarity (%)

E-value

Constitutive expression of JA and ethylene responses 1 (CEV1)

At5g05170

x-3 fatty acid desaturase (FAD3/7)

At2G29980 At3G11170 At2G39940

BQ583301 BI543316 DV501733 BQ586240 BQ587148 BQ588767 BQ589271 BQ584610 BQ583639 TC3578 BQ595680 CV301677 BQ592267 BQ588562 CF542941 BQ490498 BQ593715 BQ487727

79 71 91 70 66 33 48 39 51 28 63 92 84 72 31 38 86 57

85 83 96 85 79 60 67 61 63 47 77 98 92 87 57 58 94 70

5e-89 6e-88 4e-38 1e-85 2e-57 1e-26 4e-22 2e-44 5e-33 1e-17 5e-48 1e-117 1e-107 4e-93 4e-22 6e-22 4e-100 2e-51

Genes Function

Insensitive to JA 1 (COI1) JA resistant 1 (JAR1) Ethylene insensitive 2 (EIN2) Short-chain dehydrogenase (SDR) Suppressor of G2 1A (SGT1) Heat shock protein 90 (HSP90) Mitogen-activated protein kinase 4 (AtMPK4) Vegetative storage protein 1 (VSP1)

At2G46370 At5G03280 At4G13180 At4G23570 At5G56010 At4G01370 At5G24780

Arginase Threonine deaminase

At4G08870 At3G10050

a

Data on identity (%) and similarity (%) indicate the percentages of identical and similar amino acids of the aligned regions, respectively.

genetic mutants or transgenic plants is needed to ascertain their value in sugar beet improvement.

Breeding and molecular biology of resistance to leaf-feeding insects Breeding for resistance to leaf-feeding insects Leaf-feeding insects such as beet armyworm (Spodoptera exigua) and cabbage armyworm (Mamestra brassicae) cause serious damage, particularly in the Asian region including India and China. They tend to infest vigorously growing sugar beet plant stands. Genetic variation in insect tolerance was found between 11 tetraploid breeding lines, 3 of which, AB-19-120, Duojing and 83426, were damaged less than others (Zhang, 1994). These characters are inherited at relatively high rates and have the potential for improvement through selection. Stanescu (1994) noted that B. corolliflora might be resistant to attack by cabbage armyworm and beet webworm (Loxostege stricticalis L.) larvae. Resistance to leaf miner (Pegomyia spp.) was reported in sugar beet cultivars (Luczak, 1996). The complex character of resistance to Pegomyia betae was conditioned by the poor attractiveness of some cultivars for ovipositing females (in cultivars AJ3 and AJ4 that grew smooth leaves in less numbers) and by the inhibition of larval feeding and development in several monogerm cultivars. The effect of the particular mechanisms was not regarded to be uniform, and the reduction of P. betae populations


varied between cultivars. In oviposition, the effect of antixenosis reduced the population of P. betae by 24–30% and conditioned a low level of resistance. A partial (moderate) resistance and a pronounced (at least 50%) reduction in the P. betae population occurred as a result of the antixenotic mechanism in the feeding of larvae (AJ Polycama) and by antibiosis (PN Mono 4). In feeding experiments, AJpoly2 and PN Mono1 had less sugar reduction and were regarded as miner-tolerant varieties. Tolerance against leaf miner was also reported in Beta trigyna and B. patellaris inhibited miner spread because the larvae could not reach second pupae custodiata (reviewed by Van Geyt et al., 1990; Frese et al., 2001). Golev and coworkers claimed the recovery of flea beetle (Chaetocnema concinna) resistant sugar beet accessions after chemical mutagenesis (Golev et al., 1984). Subsequently, Lunin (1986) indicated that the beetle resistance in those accessions was controlled by multiple recessive genes. The same report also indicated that inbreds had been developed after several rounds of individual selection and inbreeding. Molecular biology of resistance to leaf-feeding insects Beet armyworm is regarded as a generalist herbivore. Chemical application studies in Arabidopsis indicated that jasmonic acid (JA) treatment generally increased activity of four defence proteins and reduced S. exigua growth by about 25% (Cipollini et al., 2004). Analysis of Arabidopsis 147


genetic mutants showed that growth of S. exigua was highest on the cep1 mutant, a constitutive expressor of high salicylic acid (SA) levels. However, growth was low on the JA-deficient fad mutant, and the nim1-1 and jar1mutants, which are defective in the SA and JA pathways, respectively. In Nicotiana attenuata plants, S. exigua attack elicited increases in JA and JA-mediated defences but also increased SA levels and expression of non-expressor of PR-1 (NPR1) transcripts (Rayapuram & Baldwin, 2007). Tobacco plants silenced in NPR1 accumulation by RNAi were shown to be highly susceptible to herbivore attack. It was suggested that during herbivore attack, NPR1 negatively regulates SA production, allowing the unfettered elicitation of JA-mediated defences. However, when NPR1 is silenced, the elicited increase in SA production antagonises JA and JA-related defences, making the plants susceptible to herbivores (Rayapuram & Baldwin, 2007). Transgenic rice expressing a mutated OsNPR1(2CA) gene, with alterations to the conserved cysteine residues leading to constitutive localisation in the nucleus, enhanced disease resistance and also abolished herbivore hypersensitivity (Yuan et al., 2007). A sugar beet NPR1 gene has recently been cloned (AY640381) and its full genomic sequence (EF101866) has been analysed (Kuykendall et al., 2007). Bargabus-Larson & Jacobsen (2007) showed that the systemic resistance elicited by Bacillus spp. biocontrol agents against C. beticola in sugar beet is salicylic acid independent and NPR1 dependent. Therefore, it can be concluded that BvNPR1 is a very important defence gene in sugar beet. JA signalling is important in the regulation of woundresponse genes, such as proteinase inhibitors, polyphenol oxidases, threonine deaminase and arginase, which have roles in antagonising caterpillar growth and development (Chen et al., 2005). The JA-regulated VSP1 has been shown to have anti-insect phosphatase activity against corn rootworm (Diabrotica undecimpunctata howardi) and cowpea weevil (Callosobruchus maculatus) (Liu et al., 2005). Harpin-treated vegetables repelled European corn borer (Ostrinia nubilalis) and cucumber stripe beetles (Acalymma vittatum) (Dong et al., 2004). Harpin treatment of sugar beet induced resistance against Aphanomyces root rot and increased beet yield (J. Weiland, unpublished results). It will be interesting to see whether it has impacts on sugar beet insects. It has been found recently that systemins in the plant cell wall mediate defence responses against herbivorous insects, and mitogen-activated protein kinases MPK1, 2 and 3 play vital roles in the process in Solanaceous plants (Kandoth et al., 2007). Potential insect-resistance genes have been identified in sugar beet including a prosystemin homologue (AY839628), and sugar beet expressed sequence tags (ESTs) potentially encoding 148

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MPKs, VSP1, (Table 1).

arginase

and

threonine

deaminase

Genetic transformation for pest resistance There are currently few commercial sugar beet varieties showing resistance to insects. It seems that there has been limited progress made in breeding for insect resistance not only because many of the mechanisms governing the resistance are controlled by multiple genes but also because the resistance is not significantly high and may be overcome in the presence of heavy infestation. There have been few molecular biological studies towards understanding the leaf-feeding insects–sugar beet plant interactions and plant resistance. Hence, molecular biotechnology has been envisaged in response to these deficiencies. Development of transgenic technology The application of transgenic technology to sugar beet improvement requires efficient regeneration and transformation systems, a number of desired genes and knowledge on the recipient genome. Effective regeneration pathways have been exploited for the large-scale production of transformed sugar beet plants (D’Halluin et al., 1992; Hall et al., 1996; Zhang et al., 2004). The guard cell protoplast system for high efficiency generation of transgenic plants has now been utilised in several academic and commercial laboratories (Hall et al., 1996). IvicHaymes & Smigocki (2005) have developed a direct transformation protocol by microprojectile bombardment. Several Agrobacterium tumefaciens-mediated transformation procedures have been widely applied because of the fact that more transformants with simple integration patterns and desired transgene expression levels were produced (Fry et al., 1991; D’Halluin et al., 1992; Zhang et al., 1998, 2001). Modern genomics, proteomics and transcriptomics approaches are being used in sugar beet research. ESTs (21 321) representing 13 618 genes have been cloned from sugar beet (3 February 2005 release, http://compbio. dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=beet; Herwig et al., 2002; Bellin et al., 2007). There are 26 870 ESTs deposited in the GenBank (accessed on 13 November 2007), of which about 20% are estimated to represent defence, signal transduction and secondary product synthetic genes. As discussed above, a search of those databases identified a number of genes involved in insect resistance and defence signal transduction in sugar beet (Table 1). Those genes provide a foundation for further investigation into the development of sustainable insect resistance.


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At De Montfort University, we have constructed sugar beet cDNA and genomic DNA libraries and cloned and characterised a number of genes (Slater et al., 1994; Elliott et al., 1996; Fowler et al., 2000a,b). Several promoters with a pattern of storage root-preferential expression have been identified and characterised in transgenic plants. Those promoters are functional in a wide range of species and, interestingly, one such construct carrying a specific element showed higher pest sensitivity than other constructs carrying various lengths of the promoter region (Scott et al., 2002 and unpublished results). Transgenic pest resistance The Hs1pro21 gene cloned from B. procumbens has been transferred back into sugar beet through transgenic technology, and a transgenic variety is in the latest developmental stage (Cai et al., 2002; W. Lange, personal communication). Further disease resistance analogues including genes similar to the nematode- or aphid-resistance genes Hs1pro-1, Mi-1 and Gpa2 have been identified using PCR (Hunger et al., 2003; Tian et al., 2004). The Mi-1.2 gene of tomato mediates resistance to several root-knot nematodes, including M. incognita, the sweet potato whitefly (Bemisia tabaci) and the potato aphid Macrosiphum euphorbiae through interactions involving SGT1 and HSP90 (Rossi et al., 1998; Bhattarai et al., 2007). In potato, Gpa2 confers resistance to the potato cyst nematode, Globodera pallida, and the Rx1 gene confers resistance to potato virus X. They are highly homologous resistance genes containing leucine zipper, NBS and LRR (LZ–NBS–LRR) motifs at a single potato resistance-gene cluster. It will be very interesting to examine the ability of these beet proteins to confer resistance against several pests to confirm whether they have multiple activities in vivo. These genes are currently used by several groups to genetically engineer resistance to nematodes in sugar beet and other crops. Urwin et al. (2003) showed that expression of an engineered protein variant of a rice cystatin (OcID86) conferred partial resistance to Globodera and full resistance was achieved by pyramiding a cystatin with natural host-based resistance. Transformed potato lines were recovered that expressed the cystatin OcID86 under the control of promoters preferentially expressed in root tissue and strongly upregulated in the galls induced by feeding M. incognita. In a field trial, they showed 70% resistance against Globodera using the ARSK1 promoter and, in containment, 67% resistance against M. incognita using the TUB-1 promoter (Lilley et al., 2004). Sporamins, a group of foreign proteins, which inhibit trypsin activity, have been expressed in beet hairy roots and conditioned resistance to cyst nematodes (Cai et al.,


2003). This approach could deliver non-host-specific resistance against nematodes. Gatehouse et al. (1996) developed transgenic potato lines expressing snowdrop lectin (GNA) and a bean chitinase or wheat a-amylase inhibitor, which had a marked and significant effect on the fecundity of potato-peach aphid. Wang et al. (2001) showed that suppression of a P450 hydroxylase gene in plant trichome glands led to increased cembratriene-ol production and enhanced aphid (Myzus nicotianae Blackman) resistance in tobacco. These strategies also may be feasible for engineering sugar beet for aphid resistance. Several strategies including use of the toxic crystal proteins from Bacillus thuringiensis (Bt) and cowpea trypsin inhibitor (CPTI) have been incorporated in the development of insect-resistant plants (reviewed by Zhang, 1994; Slater et al., 2003). Twelve groups of toxic Bt crystal proteins have been isolated, of which classes I, II and IX Bt crystal proteins are resistant to Lepidoptera insects; cry1Ib-e and cry2Aa(1-10) are resistant to Coleoptera and Diptera, respectively; classes III and VIII are resistant to Coleoptera; classes V and VI are resistant to nematodes and classes IV, X and XI are resistant to Diptera (Slater et al., 2003; Li et al., 2007). The genes for cryIA(b) and cryIC have been transferred into sugar beet through A. tumefaciens-mediated transformation for the development of beet armyworm resistance (Hisano et al., 2004). Further experiments showed that feeding with leaves taken from those transformed sugar beets delayed growth of the larvae with reductions in body weight, and cryIC was more toxic than cryIA(b) (Kimoto & Shimamoto, 2001). McBride et al. (1995) showed highly efficient amplification of a chimeric Bacillus gene crylA(c) in chloroplasts leading to an extraordinary level of an insecticidal protein in tobacco. At Harbin Institute of Technology, we have recently sequenced the whole beet plastidic genome for the development of plastid transformation vectors (GenBank accession number EF534108). Constructs carrying the Bt gene in combination with two sequences encoding the beet rbcL and atpB genes have been developed for sugar beet plastid transformation (Cui et al., 2006). Bacterially expressed protein showed high insecticidal activity towards beet armyworms. Dovzhenko (2001) sequenced the chloroplast DNA trnV-rps7 region of sugar beet and constructed vectors that had the potential for beet plastid transformation. Transplastomics may provide a platform to deliver high toxicity to sugar beet pests. Delaying insect development or altering reproductive behaviour may provide alternative strategies for genetic engineering towards insect resistance. Thomas (1991) suggested the introduction of phytoecdysteroid (plant chemicals similar to insect hormones) to prevent insect 149


development. Several genes have been isolated from armyworms including chitinase (Bolognesi et al., 2005), pheromone (Xu et al., 2007a) and prothoracicotropic hormone (Xu et al., 2007b). Transgenic corn plants engineered to express the western corn rootworm (Diabrotica virgifera virgifera) double-stranded RNAs show a significant reduction in pest feeding damage in a growth chamber assay (Baum et al., 2007). These genes or constructs could be tested in transgenic sugar beet plants for insect resistance. Transgenic technology, biodiversity and food safety It has been suggested that insect-resistant transgenic crops are environmentally beneficial, although there have been arguments that they may have a negative impact on biodiversity, for example to monarch butterfly (Danaus plexippus) (reviewed by Slater et al., 2003; Romeis et al., 2006). The Bt protein has no proven toxicity in mammals (Slater et al., 2003). The phytocystatin for transgenic plant resistance to nematodes was reported to have no toxic risk in the human diet (Atkinson et al., 2004). Most of feeding trials with genetically modified (GM) insect-resistant crops have confirmed that there are no disadvantages from GM food than companion non-GM products. Although there are arguments regarding the scientific data and approaches, Pusztai et al. (1992) showed that there was a negative impact of consuming high dose of CPTI in experimental rats and the expression of the lectin transgene GNA in a GM potato-induced proliferation of the gastric mucosa (Ewen & Pusztai, 1999). Sugar extracted from a GM crop is not regarded as a direct GM product, and there was evidence that both DNA and protein were degraded during the sugar purification process (Klein et al., 1998). By-products from sugar beet processing are used as animal feed in several countries. In this respect, the insect toxic protein may be better deployed through transgene targeting or inducible expression approaches by use of a leaf-specific promoter (Stahl et al., 2004), storage root-specific promoter (Oltmanns et al., 2006), wound-inducible promoter (Dimmer et al., 2004) or nematode-inducible promoter (Thurau et al., 2003). Pest-resistant GM varieties carrying antibiotic- or herbicide-resistant marker genes are also causing concerns in terms of food safety and their wide release to the environment. On the positive side, it has been noted that cultivation of GM herbicide-tolerant sugar beet has resulted in reductions in the populations of potato cyst nematodes (Globodera rostochiensis and G. pallida) after glyphosate application and decreases in peach–potato aphids infestation to the beet crop by delaying weed control (Dewar et al., 2000a,b). However, transgenes 150

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may be spread from GM sugar beets to cultivated forms of beets, for example fodder beet, table beet and leaf beet and wild species B. vulgaris ssp. maritima through pollen contamination or seed dispersal leading to a ‘super weed’ that would be extremely difficult to control (Arnaud et al., 2003). This has been strengthened by the fact that overwintering of sugar beet is possible in the Germany and Holland border (Pohl-orf et al., 1999). It has been advised that commercial GM crops are free of selectable marker genes to avoid transgene escapes (Zhang et al., 2001; Slater et al., 2003).

Prospects on the development of pest-resistant sugar beet The challenge for pest resistance research is to continue enhancing sugar or bio-ethanol yield while minimising pesticide input, making sugar beet a more profitable/ sustainable/greener crop. Recent achievements include the development of nematode-resistant variety, transgenic insect-resistant lines and a number of functional markers for molecular breeding. Various strategies of pest resistance have been defined and several of those were verified in sugar beet. Molecular mechanisms of pest–sugar beet interactions are starting to be elucidated, including characterising the function of specific genes, their expression control elements and regulatory networks. The beet genome projects initiated by scientists in Germany and the USA will provide further biotechnological tools for crop improvement. These are the focuses of current researches and future directions.

Commercialisation of transgenic varieties Molecular biotechnological approaches are being integrated into conventional approaches by the development of novel transgenic sugar beet lines. Many genes conferring pest resistance are being identified from a diverse range of species and deployed for the generation of transgenic sugar beet lines. This will lead to the development of transgenic pest resistance varieties at an enhanced efficiency with accelerated speed and high accuracy. There are currently low levels of acceptance of the GM crops in Europe (Slater et al., 2003). Herbicide-tolerant transgenic sugar beet varieties have been commercially cultivated in the USA since 2006. The approval and deployment of a GM variety depend on the characteristics of specific genes used and their consequences and rigorous environmental risk assessment required. However, we anticipate that pest-resistant sugar beet varieties may be grown commercially within the next decade in China or the USA.


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One of the pitfalls of pest-resistant varieties is that high levels of pest resistance may compromise disease resistance and yield potential. In this case, the integration of several approaches and use of dual resistant breeding lines are encouraged. Monogenic resistance may lead to quick development of insect tolerance for a large number of insects. The development of dual or multiple genes-based resistance mechanisms is vital for success. Innovative germplasm research Worldwide Beta genetic banks have expanded rapidly as results of recent collections and explorations. There is a need to screen for pest tolerance in these enlarged Beta germplasm collections assisted by modern equipment and techniques including molecular biological techniques. Sugar beet lines carrying additions or translocations of extra chromosomes from B. procumbens (Schmidt et al., 1990; Mesbah et al., 1997) or Beta lomatagona and B. corolliflora have been developed and characterised by several groups (Paul et al., 1994; Gao et al., 2001). These lines will be very useful for cytological location and molecular identification of pest-resistance genes. Transformation of those genes to elite sugar beet lines will speed up the development of sustainable pest-resistant varieties.

Sugar beet seed is an important resource for farming and vital part of commercial enterprises. It has been the case that most research activities are carried out by seed companies. To some degree, intellectual property issues had repressed wide collaboration and rapid advances in developing pest-resistant varieties. The intake of emerging technologies, for example transcriptomic, proteomic and metabolomic analyses, has been slow. Various funding policies have led to different prioritised researches and sometimes fragmented research activities in some regions. However, a wide enthusiasm for scientists from public institutes, universities and seed companies to work together to make sugar beet a sustainable crop has developed recently and will hopefully bear fruit in the near future.

Acknowledgements We are grateful to Dr Y.-H. Zhang (Rothamsted Research), J. Drury (Nottingham University Library) and P. Cavanagh (De Montfort University Library) for their help during the preparation of this manuscript. We thank Drs M.J.C. Asher, D.G. Cai, P. Devaux, L. Frese, M. Grimmer, C. Jung, T. Kraft, W. Lange, J.M. McGrath, L. Panella, T. Schmidt and M.H. Yu for fruitful discussions or their assistances. Parts of the work discussed here have been funded by MAFF, EU, KWS, DMU, Chinese Department of Science and Technology and Department of Science and Technology, Heilongjiang Province, China.

Mutagenesis as a supplemental tool While the conventional approach of interspecific hybridisation to transfer pest resistance is possible, it is generally time consuming and very expensive because of the genetic barrier between sugar beet and its related wild species. The lack of sufficient numbers of pest-resistant sugar beet mutants also hindered breeding progress. Mutagenesis in combination with high-throughput DNA sequence inspection (termed targeting induced local lesions in genomes, TILLING) has become an important tool in functional genomics and alternative to transgene technology. This approach is supported by the finding that many disease-resistance genes are derived from DNA base substitutions or deletions (Tian et al., 2004). The bolting gene B was mutated at a frequency of 0.3% by seed treatment with 1% ethylmethanesulphonate mutagen and further characterised by TILLING (Hohmann et al., 2005). Mutagenesis using transposable elements is an effective method to generate a large number of mutants for further phenotyping and functional gene analysis. Such elements have become available for sugar beet (Menzel et al., 2006). These together with earlier investigations indicate that mutagenesis can be a very promising supplemental tool for gene discovery and the production of pest-resistant breeding lines.


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