Program 4

Developing Weevil Resistance in Sweetpotato with Genetic Transformation
D.P. Zhang¹, A. Golmirzaie¹, G. Cipriani¹, A. Panta¹, M. Ghislain¹, N. Smit¹, I. Rety², and D. Michaud²

Regeneration and Transformation System
Identification of Appropriate Protease Inhibitors
Production of Transgenic Plants
Conclusions
Selected Reading

Crop loss of sweetpotato (Ipomoea batatas (L.) Lam.) caused by insect damage is the most important problem in the field. Although the importance of pest species varies regionally, sweetpotato weevil (Cylas spp.) is the most important threat worldwide. The weevils C. formicarius (Fabricius) and C. puncticollis (Boheman) are the most destructive. Larvae of this pest tunnel through storage roots, resulting in major damage and economic yield loss. Production losses often surpass 60% and can reach 100%.

Weevil damage imparts a characteristic turpentine odor to the roots, which renders even slightly damaged roots unfit for human consumption. Larvae also feed inside the vines.

The cryptic feeding habit of the sweetpotato weevil larvae and the nocturnal activity of the adults make it difficult to detect sweetpotato weevil infestations. Approximately 80-90% of the weevil population within vines and roots is distributed below the soil surface. These factors limit the effectiveness of chemical insecticides applied for weevil management.

Host resistance is an important component in successful integrated pest management (IPM). CIP’s experience in Caribbean countries demonstrated that use of less susceptible cultivars significantly contributed to the effect of IPM. The search for weevil resistance started 50 years ago, but the level of resistance found in sweetpotato and its wild relatives is rather low. A large genotype by environment interaction and low heritability generally preclude the use of conventional breeding techniques.

Genetic transformation with genes that encode protease inhibitors (PI) is a novel approach to creating weevil resistance in sweetpotato. Foreign genes that encode protease inhibitors have been introduced into tobacco, tomato, strawberry, potato, and many other crops to control a range of insect pests. These proteins bind to proteases in the midgut of the insect, thus affecting the insect’s metabolism.

In this article, we summarize research at CIP and its collaborating institutions to develop a transgenic sweetpotato with protease inhibitor genes. This work is divided into three parts:

1. Developing a regeneration and transformation system protocol for sweetpotato,
2. Identifying appropriate protease inhibitors for sweetpotato weevil, and
3. Producing transgenic plants that carry foreign protease inhibitors.

Regeneration and Transformation System

Sweetpotato plant regeneration
We have developed an efficient regeneration protocol and Agrobacterium-mediated transformation system. Using this system, we have been able to regenerate and transform most of the sweetpotato cultivars tested.

The bottleneck in developing transgenic sweetpotato has been the lack of a reliable regeneration system, because regeneration is strongly genotype dependent. Cultivars vary significantly in their regeneration successes. Therefore, we conducted a series of experiments at CIP to modify the regeneration media and to standardize protocol to improve regeneration efficiency.

Two pathways for sweetpotato regeneration—somatic embryogenesis and organogenesis—have been explored. So far, our regeneration is achieved mainly by shoot organogenesis from leaves, roots, and stem internodes. We have regenerated most cultivars tested. Table 1 lists the ones that have been successfully regenerated at CIP, even though the regeneration rate remains genotype dependent. Meanwhile, we are actively exploring use of somatic embryogenic suspension cultures, which will eventually improve the efficiency of regeneration.

All tested cultivars were obtained from the in vitro gene bank maintained at CIP. Plants were propagated in vitro at 25 ± 2ºC under fluorescent light (45 uEm^-2s^-1) with 16 h photoperiod. Using explants of stem segments and complete leaves, we achieved direct regeneration through shoot organogenesis using a two-stage regeneration protocol. In general, we found that the protocol using complete leaves gave the best regeneration rate.

Transformation through A. tumefaciens

Plasmid description. We tested the transformation system using two plasmids. One was pBI121 plasmid containing GUS (b-glucuronidase), cecropin (encoding cecropin proteins that have activity against bacteria), and NPTII (neomycin phosphotransferase) genes. The other was pCIP5 plasmid containing NPTII and WCI-3 genes. WCI-3 encodes chymotrypsin inhibitor from winged bean (Psophocarpus tetragonolobus). Plasmids were transferred to A. tumefaciens by electroporation.

Culture of bacteria. Agrobacterium tumefaciens was subcultured and incubated at 28ºC for 48 h. Isolated colonies were transferred to a yeast-mannitol liquid medium and incubated at 28ºC for another 48 h. Bacteria concentration in the liquid medium was determined by spectrophotometry. Bacteria were then inoculated in Erlenmeyer flasks containing 25 ml of liquid potato propagation medium.

Transformation. Explants were excised from the top of young in vitro plants and inoculated in the liquid potato propagation medium at 25ºC. Inoculated explants were transferred to batata propagation media and co-cultured at 25ºC for 2-3 d. After co-culture, explants were transferred to different regeneration media depending on the type of explants.

Using this transformation system, we have obtained 70 putative transgenic lines with the cecropin gene from cultivars Huachano, Regal, and Jewel. The GUS assay and Southern blot have confirmed the insertion of the GUS gene into the genome of sweetpotato (Figures 1 and 2). About 300 putative transgenic lines with WCI-3 genes were obtained from cultivars Chugoku, Huachano, Huarmeyano, PI-318846-3, Jewel, Mabrouka, Morada Inta, and Tanzania.

 

These results show that the A. tumefaciens-mediated transformation system works efficiently on sweetpotato. We now have the technical ability to transform and regenerate most sweetpotato cultivars, including the ones most widely grown in Asia and Africa.

Identification of Appropriate Protease Inhibitors
To select efficient protease inhibitors for sweetpotato weevil control, we need to know which proteolytic enzymes are used by these weevil species for the hydrolysis of dietary proteins. As a first step, midgut proteases were analyzed in larvae and adults of three Cylas species, C. formicarius, C. brunneus, and C. puncticollis. C. formicarius was collected in southern China and Indonesia, whereas C. brunneus and C. puncticollis were sampled in Uganda. Characterization of the weevil digestive proteases was conducted at the Horticultural Research Center (CRH), Laval University, Quebec, Canada. The study was done using standard quantitative assays and electrophoretic techniques developed at CRH for the analysis of protease-protease inhibitor interactions.

Digestive protease activity in all three species was partly accounted for by sets of stage-specific protease species. Larvae and adults of all three species were shown to use a mixture of exopeptidases and endopeptidases active primarily in the alkaline pH range.

High caseinase activity was detected in the insect samples at pH 10-11, whereas barely detectable activity was measured in mildly acidic conditions (pH from 5 to 7), even in the presence of reducing agents such as l-cysteine or di-thiothreitol. This observation, which suggests the predominance of serine-type proteases in the midgut of all three species, was confirmed by inhibition assays.

Whereas inhibitors such as cystatins (cysteine-type inhibitors) and pepstatin (aspartyl-like inhibitor) showed no effect, a significant fraction of the caseinase activity measured at pH 10.5 was inhibited in reducing conditions by the serine-type protease inhibitors phenylmethylsulfonyl fluoride (PMSF) and soybean (Phaseolus angularis) trypsin inhibitor (SBTI). Interestingly, the inhibitory spectrum of SBTI against the midgut proteases was apparently larger than that of endogenous inhibitors in sweetpotato (Figure 3), suggesting the potential usefulness of this inhibitor in weevil control.

 

At this point, we are not clear whether all the serine endopeptidase activity in weevil was blocked by SBTI, because the insect may have certain proteases that are insensitive to the plant trypsin inhibitors. A partial inhibition, however, is enough to cause developmental alterations as demonstrated in many other Coleopteran insects. Foreign (nonhost) inhibitors such as recombinant SBTI and cowpea (Vigna unguiculata) trypsin inhibitor (CpTI) are promising candidates for the development of transgenic sweetpotato with resistance to Cylas weevils. Therefore, SBTI and CpTI genes were selected and are being used to develop weevil-resistant transgenic sweetpotatoes.

Production of Transgenic Plants
The first transgenic sweetpotato with the cowpea trypsin inhibitor was developed by Axis Genetics Ltd., U.K. Since the gene construct CpTI encodes a Bowman-Birk trypsin inhibitor, our objective is to determine whether the transgenic lines with CpTI will demonstrate enhanced resistance to C. formicarius.

Ten transgenic lines of cv. Jewel were obtained through a collaborative project between Axis Genetics Ltd. and CIP, and seven of them had the cowpea trypsin inhibitor. The other three lines had both the CpTI gene and PCG gene, which encodes snowdrop lectin.

We have propagated these transgenic plants in an isolated screenhouse following Peruvian biosafety guidelines. These transgenic lines will be evaluated in Cuba and China using a screenhouse and field bioassay.

Meanwhile, we are incorporating a new plasmid, pAD1289, into A. tumefaciens LBA4404. It enhances transformation efficiency as reported in tobacco, cotton, and other crops. We are using three PI genes for sweetpotato transformation: OCI, which encodes a cystatin of rice; CpTI, which encodes a Bowman-Birk trypsin PI of cowpea; and SBTI, which encodes a Kunitz-type trypsin PI from soybean. The spectrum of these inhibitors covers the major digestive proteases of sweetpotato weevils (CpTI and SBTI), and of other pests of sweetpotato (OCI).

Biochemical analyses provided evidence that sweetpotato weevil larvae, which feed mainly on storage roots, and adults, which feed mainly on foliage, both use similar digestive proteolytic systems. The use of root-specific promoters appears unnecessary and a constitutive expression of the recombinant inhibitors is preferred. Therefore, CIP is using the cauliflower mosaic virus 35S promoter (CaMV35S) for all gene constructions. The expression of this promoter should lead to a constitutive expression of the PI transgenes in all tissues of the plant.

Conclusions
Serine protease inhibitors such as SBTI from soybean and CpTI from cowpea were identified as candidate inhibitors for transformation. Transgenic cv. Jewel sweetpotato expressing CpTI is being propagated for field evaluation for weevil resistance. Co-transformation with several serine PI and cystatin PI genes will be applied to construct a combined defense mechanism against sweetpotato pests.

Genetic transformation plays an important role in enhancing sweetpotato germplasm. Recent progress in developing a transformation system and identifying candidate protease inhibitors has established an essential base for creating host resistance for sweetpotato weevil. The combination of several serine PI in sweetpotato through transformation would most likely produce a durable defense mechanism against sweetpotato weevil.

Selected Reading
Chalfant, R.B, R.K. Jansson, D.R. Seal, and J.M. Schalk. 1990. Ecology and management of sweetpotato insects. Annu. Rev. Entomol. 35:157-180.

Michaud, D., N. Bernier-Vadnais, S. Overney, and S. Yelle. 1995. Consti- tutive expression of digestive cysteine proteinase forms during development of the Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Insect Biochem. Mol. Biol. 25:1041-1048.

Newell, C.A., J.M. Lowe, A. Merryweather, L.M. Rooke, and W.D.O. Hamilton. 1995. Transformation of sweetpotato (Ipomoea batatas (L.) Lam.) with Agrobacterium tumefaciens and regeneration of plants expressing cowpea trypsin inhibitor and snowdrop lectin. Plant Sci. 107:215-227.

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1 CIP, Lima, Peru.
2 Horticultural Research Center, Laval University, Quebec, Canada.