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HORTSCIENCE 34(5):922–927. 1999. tatively inherited trait. A study by Vallejo et al. (1994a, 1994b) produced low heritability val- ues for density of both types of trichomes and Colorado Potato Resistance in determined that additive genetic variance for trichome densities was low. Mehlenbacher et Somatic Hybrids of Diploid al. (1983) were able to recover parental tri- chome densities in F2 and backcross genera- Interspecific Solanum Clones tions, indicating that these traits are controlled by relatively few genes. Yencho et al. (1996) Shelley Jansky have identified quantitative trait loci associ- ated with trichome-based resistance in S. Department of Biology, University of Wisconsin–Stevens Point, Stevens Point, berthaultii. WI 54481 A major problem with control of the CPB is its genetic flexibility. Resistance to insecti- Sandra Austin-Phillips cides may develop quickly and has been re- University of Wisconsin Biotechnology Center, Madison, WI 53706 ported widely (French et al., 1992; Heim et al., 1990; Roush and Tingey, 1991; Tisler and Corine McCarthy Zehnder, 1990). The also has the poten- Department of Biology, University of Wisconsin–Stevens Point, Stevens Point, tial to overcome host plant resistance. Man- WI 54481 agement of transgenic plants that produce the Bacillus thuringiensis endotoxin is being care- Additional index words. insect resistance, protoplast fusion, somaclonal variation, Solanum fully monitored to limit the development of chacoense, S. berthaultii, Leptinotarsa decemlineata resistant to the toxin (Everich et al., 1992; Ferro, 1993). The possibility also exists Abstract. The (CPB) is a major insect that is controlled mainly for the CPB to overcome glandular trichome- through the use of . Development of potato clones with multiple forms of host based host plant resistance (Franca et al., 1994; plant resistance may provide a stable alternative or supplemental form of CPB control. Groden and Casagrande, 1986). Tetraploid hybrids were developed by somatic fusion of diploid interspecific Solanum One way to delay the development of resis- clones with different forms of resistance to CPB. Hybrids were created between a clone tant CPB populations is to combine two or containing leptine glycoalkaloids and four clones producing glandular trichomes. One more types of resistance via somatic fusion. fusion produced vigorous hybrids that were analyzed for CPB resistance traits. Somaclonal Numerous reports exist describing somatic variation among hybrids was detected for trichome density and resistance to feeding by hybrids among Solanum species (Austin et al., adult and larval . Somatic hybrids were less resistant than the parents in adult 1985a; Deimling et al., 1988; Helgeson et al., feeding preference trials, but several were more resistant than either parent in larval 1988). Hybrids have generally been created to feeding trials. Future studies are needed to determine whether clones producing both access novel disease resistance genes from glandular trichomes and leptines express resistance that is more stable than that of clones species that are sexually incompatible with S. with only one resistance factor. tuberosum L. (Barsby et al., 1984). For ex- ample, somatic hybrids have been created that The Colorado potato beetle (CPB) There are two major documented sources are resistant to potato leaf roll virus (Austin et (Leptinotarsa decemlineata Say) is the major of host plant resistance to the CPB. The first, al., 1985b; Gibson et al., 1988), potato virus Y defoliator of potato and, in some cases, can be found rarely among selections of S. chacoense (Gibson et al., 1988; Novy and Helgeson, the limiting factor for potato production. This Bitt., is production of high levels of leptine 1994a, 1994b), Phytophthora infestans (Mont.) insect is largely controlled by chemical means, glycoalkaloids (Sinden et al., 1986a), which de Bary (Helgeson et al., 1988; Menke et al., which is expensive and potentially damaging are effective feeding deterrents and are syn- 1996), and Erwinia soft rot (Austin et al., to the environment. Furthermore, some CPB thesized only in foliage (Lawson et al., 1992). 1988) in wild Solanum species. Gibson et al. populations have developed resistance to sev- The inheritance of the ability to produce (1988) observed intermediate levels of resis- eral major classes of (Mowry and glycoalkaloids is complex because plants gen- tance to potato leaf roll virus and potato virus Sandvol, 1995). Utilization of host plant resis- erally contain a mixture of glycoalkaloid types Y in somatic hybrids between a resistant and a tance offers a feasible alternative or supple- (Lawson et al., 1993). Sinden et al. (1986b) susceptible clone. ment to chemical insect control, but is difficult suggested, based on observations of low fre- In addition to combining genomes of in- because modern potato cultivars have a nar- quencies of high leptine clones among sibs in compatible species, somatic fusions can be row genetic base and do not offer adequate S. chacoense populations, that high levels of useful with sexually compatible species, such genetic variability for selection of insect-re- leptines may be produced by recessive alleles. as S. berthaultii and S. chacoense. Somatic sistant types (Sanford et al., 1984). However, The second major resistance mechanism in hybridization between two highly selected sources of resistance have been found in some wild Solanum species is glandular trichomes, diploid clones may result in tetraploids with wild Solanum species. which have been most thoroughly studied in S. the entire genetic complement of both parents. berthaultii Hawkes. Gibson (1976) identified This method, therefore, allows one to bypass two types of glandular trichomes that are ef- normal sexual recombination and segrega- fective against insect pests. Type A trichomes tion. Somatic fusions between two clones with are short, each with a four-lobed gland. Type different forms of insect resistance, for ex- Received for publication 28 Sept. 1998. Accepted B trichomes are longer, with single droplets at ample, could produce hybrids with genes for for publication 15 Jan. 1999. This work was funded the tips. Pelletier and Smilowitz (1991) re- both forms of resistance. In addition, tetrap- by USDA/ARS SCA #58-5114-0-1011 and the Univ. ported that deterrents influencing host accep- loid clones so created can be crossed directly of Wisconsin–Stevens Point Univ. Personnel De- tance and initiation of feeding occur in S. with cultivars. velopment Committee. Wild species germplasm berthaultii. According to Mehlenbacher et al. Reports of somatic hybrids with insect was obtained from the U.S. Potato Genebank, NRSP- (1984), heritability of droplet size of type B resistance are rare. Recently, Cheng et al. 6. We gratefully acknowledge the assistance of J.P. Helgeson’s laboratory for RAPD analysis. The cost trichomes is high, of density of type B tri- (1995) reported the production of somatic of publishing this paper was defrayed in part by the chomes intermediate, and of density of type A hybrids between S. tuberosum and a S. payment of page charges. Under postal regulations, trichomes low to intermediate. They suggested chacoense clone with high levels of leptine. this paper therefore must be hereby marked adver- that the resistance mechanism due to trichomes The somatic hybrids were not preferred as a tisement solely to indicate this fact. is complex and should be handled as a quanti- host for the CPB, but leptine levels were

922 HORTSCIENCE, VOL. 34(5), AUGUST 1999 reduced compared to the high leptine parent. sette B-2A (blue excitation 450–490 nm, bar- lection to ensure maximum trichome droplet This study was initiated to combine two rier 520 nm). Protoplasts stained with size. Healthy, fully expanded terminal leaflets forms of CPB resistance, glandular trichomes rhodamine fluoresce bright using filter were collected from the fifth node below the and leptine glycoalkaloids, in somatic hy- cassette G-2A (green excitation 510–560 nm, apex of the plant. They were maintained on brids. The resulting tetraploids should contain barrier 590 nm). Heterokaryons therefore will moist filter paper in petri dishes for a maxi- genes for both resistance mechanisms. The fluoresce both green and red when viewed mum of 3 h. Trichome counts were made using goal was to produce clones with stable host under the two appropriate conditions. The a dissecting microscope containing a 25-mm2 plant resistance to CPBs, which would be location of the fused cells on the plates was ocular grid. Only the adaxial surface, near the valuable in potato breeding programs. determined using a Nikon object marker on an base of the leaflet, was used for counts. Counts inverted microscope. The marker stamps a were made near the midvein, avoiding lateral Materials and Methods small ink circle (diameter ≈1.5 mm) on the veins. underside of the plate. Calli appearing within Glycoalklaloid analysis. Leaflets of each Plant material. Eleven diploid (2n = 2x = these circles were picked off as they appeared greenhouse-grown clone were freeze-dried and 24) clones with high levels of glandular tri- (2–3 weeks later) and transferred to CUL sent to A.R. Miller (Ohio Agricultural Re- chomes, developed by Mooney (1989), were plates as described by Haberlach et al. (1985), search and Development Center, Wooster) for used in fusion attempts. They were selected with t-zeatin (1.0 mg·L–1) replacing 6- glycoalkaloid analysis. Thirty-six clones, in- based on the ability to suppress development benzylaminopurine (BA). When calli were cluding the parents, were analyzed for solani- of neonate CPB larvae. The fusion partners light green and at least 2 mm in diameter (2–6 dine, leptinidine, and acetylleptinidine as de- were two high leptine diploid S. chacoense weeks) they were transferred to differentiation scribed by Lawson et al. (1992, 1997). clones (8379-1, 8380-6), supplied by L. medium SA4 [MS salts with NH4NO3 at half- Resistance to CPB adults. About 500 first- Sanford (U.S. Dept. of Agriculture, Beltsville, strength, 100 mg myoinositol, 2.5 g sucrose, generation adult CPBs were collected from Md.). Plants were grown in vitro on MS me- 35 g mannitol, N&N vitamins (Sigma Chemi- fields in central Wisconsin in 1995 and again dium (Murashige and Skoog, 1962). cals, product #N8764), 2.0 mg t-zeatin, 0.5 mg in 1997. They were maintained in mesh cages

Protoplast isolation. Attempts were made kinetin, 0.5 mg gibberellin (GA3), 0.1 mg and fed greenhouse- and field-grown potato to isolate protoplasts from all 11 trichome indole-3-acetic acid (IAA), 100 mg casein cultivars. To measure resistance to adult feed- clones and from the three leptine clones. The hydrolysate, and 10 g noble agar per L, filter ing, a preference test was used (Cantelo et al., goal was to identify clones that gave sufficient sterilized]. Any calli with shoot initials were 1987). Three leaflet pieces of a test clone, each viable protoplasts for fusion. For fusions using further proliferated by transfer to a high gib- measuring 13 × 25 mm, were cut and weighed, polyethylene glycol (PEG), protoplast isola- berellin medium (Austin and Cassells, 1983). as were three pieces of S. tuberosum (cv. tion procedures were essentially those de- Shoots were excised when they were ≈1 cm in Atlantic) leaflets. The S. tuberosum control scribed by Haberlach et al. (1985). If proto- height and established on Prop medium leaf pieces and somatic hybrid test leaf pieces plasts were to be used for electrofusion, the (Haberlach et al., 1985). were placed alternately on moist filter paper in modified isolation procedure of Novy and Culture of hybrids. Putative hybrids, along 9-cm petri dishes. Three female adult beetles, Helgeson (1994a) was followed. In both cases, with their parents and the cultivar Atlantic which had been starved overnight, were then the protoplasts were labeled with fluorescent were grown in soilless mix in 15-cm-diameter placed into the dish. The petri dishes were dye after release to facilitate heterokaryon pots, and fertilized with a standard timed- maintained under diffuse light at room tem- selection. After the initial centrifugation in release fertilizer. They were placed outdoors perature. When ≈50% of the cultivar leaf area babcock bottles (enzyme medium), protoplast in 1995 and grown in a greenhouse under a had been consumed, the beetles were removed bands were transferred to a 100-mL Erlenm- natural daylength in Summer 1997. These and the leaf pieces weighed. Six replications eyer flask containing 50 mL of the appropriate plants were used for verification of hybridity, were completed in 1995 and five replications rinse medium. Fluorescein diacetate (FDA) male fertility analysis, glycoalkaloid analysis, in 1997. (500 mg) was added to each flask containing trichome density evaluation, and CPB feeding Resistance to CPB larvae. To measure the one set of protoplasts and rhodomine assays. effect of somatic hybrids on larvae, a larval isothiocyanate (750 mg) was added to the Analysis of hybridity. Randomly amplified development method was used (Cantelo et al., other set of protoplasts. Flasks were placed in polymorphic DNA (RAPD) was used to verify 1987). Egg masses were collected from the the dark for 1 h and then transferred to babcock hybridity; DNA from young leaf tissue was field and maintained in petri dishes with moist bottles and centrifuged. Protoplasts were cen- extracted by the method of Deragon and Landry filter paper under low light until they hatched. trifuged twice more in fresh rinse medium. (1992). Each 25-µL amplification reaction After neonate larvae had consumed the egg Protoplast concentration was determined us- contained ≈25 ng template DNA in 10 mM chorion, camel’s hair brushes were used to × 6 ing a hemocytometer and adjusted to 1 10 Tris-HCl pH 8.3, 50 mM KCl, 3.4 mM MgCl2, transfer them to petri dishes containing test mL–1 for PEG fusions and 5 × 105 for 0.01% gelatin, 100 µM each of dATP, dCTP, leaflets on moist filter paper. Ten larvae were electrofusion. This step gave two populations dGTP, and dTTP, 250 µM decameric oligo- placed in each dish, which was were main- of protoplasts uniformly labeled with different nucleotide primer (Operon Technologies) and tained under diffuse light at room temperature fluorescent stains. 1 unit AmpliTaq DNA polymerase (Perkin- for 4 d. Leaflets were replaced and filter paper Protoplast fusion and plating. The PEG Elmer Cetus, Norwalk, Conn.). The following was moistened as necessary. At the end of the fusion procedure used was essentially that of cycling parameters were used: 94 °C 0.5 min. incubation period, the stage of development Austin et al. (1985a) and subsequent plating (1 cycle); 92 °C 0.5 min.; 35 °C 0.5 min., 72 °C and survival of each larva was noted. Five and procedures were as described by Austin et al. 2 min. (40 cycles); 72 °C 5 min. (1 cycle). six replications were completed in 1995 and (1993). Electrofusion and subsequent plating Amplification products were separated on 0.6% 1997, respectively. of protoplasts were performed as described by agarose and 1% synergel (Diversified Biotech, Statistical analysis. Trichome, glycoalka- Novy and Helgeson (1994a), except that the Newton Centre, Mass.). loid, and feeding data were analyzed using the final plating was at a concentration of 10,000 Pollen stainability. Pollen from 10 air- General Linear Model in SAS (SAS Institute, protoplasts per mL. dried flowers per clone from 18 somatic hy- 1994). Duncan’s multiple range test was used Selection and regeneration of heterokary- brids and both parents was stained in 1% to compare means. Transformations of the ons. Fused cells were identified 1 to 4 d after acetocarmine (1% carmine in 45% v/v acetic data were performed to reduce variance het- plating using dual fluorescence microscopy. acid) and observed at 200× magnification. erogeneity. Data for proportion of first instar Plates were viewed using a Nikon inverted Percentage of plump, uniformly stained pol- larvae in the larval feeding analysis were arc- microscope (Diaphot-TMD; Nikon, Inc., Gar- len was estimated as a measure of male fertil- sine transformed. Adult feeding data in 1997 den City, N.Y.) equipped with epifluorescence. ity. were transformed by calculating the square Protoplasts stained with FDA are bright green Analysis of glandular trichomes. Plants root of the percent S. tuberosum leaf mass when viewed under UV light using filter cas- were watered the evening prior to leaflet col- consumed.

HORTSCIENCE, VOL. 34(5), AUGUST 1999 923 BREEDING, CULTIVARS, ROOTSTOCKS, & GERMPLASM RESOURCES

Results and Discussion Table 1. Pedigrees of high trichome, somatic Solanum hybrid parents. Clone Female parentz Male parentz Protoplast fusion and culture. There was C [(tbr haploid x chc) x ber PI265858-7]-10 tar PI458394-17 considerable variation in the yield and viabil- E [(tbr haploid x tar) x ber PI265857-1]-20 tar PI458394-17 ity of protoplasts from the parent clones. Over- G [tbr haploid x tar) x ber PI265857-1]-20 tar PI458394-17 all yields were 10% to 40% of those expected J [tbr haploid x chc) x ber PI265858-7]-14 ber PI310926-9 from cultivated S. tuberosum cultivars such as zAbbreviations: tbr = S. tuberosum; chc = S. chacoense; tar = S. tarijense; ber = S. Katahdin. Only one of the leptine clones (K) berthaultii. consistently produced viable protoplasts in sufficient quantity to use in fusion studies and Table 2. Parents of somatic hybrids of Solanum and number of hybrids produced. also gave protoplasts that were capable of regeneration into whole plants. Of the 11 tri- “Family” Leptine parent Trichome parent Number of clones chome clones, eight yielded protoplasts, and KC K C 13 of these, six regenerated plants after subse- KE K E 21 quent tissue culture. Fluorescent labeling typi- KG K G 16 cally reduced viability by 10% to 50% in the KJ K J 50 trichome clones, but had no effect on the viability of the leptine clone. Initial attempts to produce somatic hybrids Table 3. Pollen stainability, indicating level of male via PEG fusion were not successful. The clones fertility, of parents and somatic hybrids of did not survive the fusion procedure well and Solanum in 1995 and 1997. viability of protoplasts dropped to essentially Clone 1995 1997 zero over the next 3 to 7 d. Electrofusion was J Highz High more successful. Survival rates after fusion K High High were 45% to 60% for the trichome clones and KJ5 Sterile ---y 60% to 70% for the leptine clone. Putative KJ57 Sterile Sterile somatic hybrids were produced from four high KJ59 --- Sterile trichome clones combined with the same leptine KJ67 Low Low clone (K). Typically one of 150 to 250 viable KJ68 Low High KJ69 --- Sterile cells was dual labeled 1 to 3 d after fusion. KJ81 Sterile --- These cells were circled as described above KJ82 Moderate High and any resulting microcalli were cultured KJ87 --- Sterile further. In most experiments two to 12 KJ123 Sterile Moderate microcalli were recovered from each plate. KJ125 --- Moderate Subsequent regeneration from these calli was KJ130 Low Moderate dependent on the trichome clone used in the KJ138 Low --- study, and ranged from 2% to 18%. Many of KJ139 Low --- the regenerated plants from selected calli were KJ187 —- Sterile KJ192 Low Sterile not vigorous compared to protoclones from KJ193 Sterile --- parental lines. About 50% failed to root suc- KJ194 Sterile --- cessfully either during initial establishment in zSterile = 0%; low = 1% to 20%; moderate = 21% to culture or through subsequent subculturing. 50%; high = 51% to 100% stainable pollen. Somatic fusions were obtained with four of yFailed to flower. the 11 high trichome clones attempted. Their pedigrees are outlined in Table 1. Only one of Table 4. Analyses of variance for A and B trichome density in somatic hybrids of Solanum, including (incl.) the leptine-producing S. chacoense clones and excluding (excl.) parents. (8380-6 from PI 458310) successfully pro- duced somatic hybrids. It will be identified in A trichomes B trichomes this paper as clone “K.” The best pair of clones Incl. parentsz Excl. parentsy Incl. parents Excl. parents for somatic hybrid production, based on num- Source df MS df MS df MS --- ber of fusion clones produced and vigor of Clone 34 894.90** 32 50.55** 34 1247.95** --- those clones, was the high trichome clone J Replication 1 5.18 1 4.54 1 102.67 --- with the leptine clone K (Table 2). In fact, most zParents included in the analysis. clones from other fusions lacked sufficient yParents not included in the analysis. vigor to be used for replicated trials. Most of **Significant at P ≤ 0.01. the J + K fusions were vigorous, so these clones are described in this paper. Rokka et al. effective. Several published reports have also ported in protoplast-derived potato plants (Karp (1994) and Deimling et al. (1988) also re- verified somatic fusions through DNA analy- et al., 1982). Therefore, some sterility may be ported differences in success rates among fu- sis (Baird et al., 1992; Menke et al., 1996; due to structural chromosome changes or aneu- sion partners. Provan et al., 1996; Rokka et al., 1994; ploidy induced by protoplast culture. Eight of Analysis of hybridity. Five of the six prim- Takemori et al., 1994; Xu et al., 1993). This 18 clones exhibited low to moderate, and ers surveyed (Z10, Z12, Z13, Z18, and Z19) method is especially valuable because small occasionally high pollen stainability. How- produced parent-specific RAPD bands. Primer amounts of leaf tissue are required, and hy- ever, Ehlenfeldt and Helgeson (1987) reported Z7 did not produce unique bands. Primers Z13 brids can therefore be identified at an early a poor correlation between pollen stainability and Z18 were chosen to verify hybridity be- stage in development. and crossability of tetraploid Solanum hy- cause they consistently produced bands that Pollen stainability. Parents J and K were brids. were unique to each parent and easily scored. highly fertile, but pollen stainability was mark- Analysis of glandular trichomes. Signifi- Twenty-eight putative hybrids were surveyed edly reduced in the fusion hybrids (Table 3). cant differences were observed among clones, and all produced the bands of both parents. Reduced fertility is common in somatic hy- but not replications, for density of type A Therefore, the process of selecting for hybrids brids (Rokka et al., 1994). Chromosome aber- trichomes, whether or not parents were in- during the production of somatic fusions was rations, especially aneuploidy, have been re- cluded in the analysis (Table 4). The high

924 HORTSCIENCE, VOL. 34(5), AUGUST 1999 trichome parent (J10) had a significantly higher Table 5. Mean type A trichome density in somatic hybrids of Solanum and their parents. type A trichome density than did any hybrid or Clone Trichome density (no./25 mm2) Clone Trichome density (no./25 mm2) the leptine parent (Table 5). One hybrid, KJ62, J10 75.2 az KJ125 5.0 c–e had a higher trichome density than all other KJ62 17.0 b KJ76 5.0 c–e clones except J10. Twenty-nine clones had KJ64 8.3 c KJ126 4.8 c–e low type A densities and did not differ statis- KJ192 7.5 cd KJ81 4.8 c–e tically from the leptine parent. Narrow-sense KJ191 7.5 cd KJ188 4.7 c–e heritability and, therefore, additive genetic KJ129 7.2 c–e KJ193 4.7 c–e variance for type A trichome density, has been KJ59 7.0 c–e KJ186 4.5 c–e reported to be low (Vallejo et al., 1994a). In KJ83 6.3 c–e KJ85 4.2 c–e this study, somatic hybrids exhibited moder- KJ127 6.2 c–e KJ82 4.0 c–e ate to low type A trichome densities compared KJ196 6.2 c–e KJ58 4.0 c–e KJ67 5.8 c–e KJ130 3.7 c–e with the high trichome parent. Therefore, the KJ194 5.7 c–e KJ57 3.3 c–e alleles controlling trichome density appear to KJ187 5.5 c–e KJ136 2.0 c–e be acting in a more dominant than additive KJ78 5.3 c–e KJ87 0.3 de fashion. KJ142 5.3 c–e KJ197 0.0 e All hybrids were derived from the same KJ69 5.2 c–e J11 0.0 e two clones. Therefore, assuming that both KJ139 5.2 c–e KJ123 0.0 e parental genomes were transmitted intact, KJ68 5.2 c–e somaclonal variation would account for dif- zMean separation by Duncan’s multiple range test, P ≤ 0.05. ferences in type A trichome density (Table 5). This type of variation in protoplast-derived Table 6. Analyses of variance for adult and larval feeding trials across 2 years (1995 and 1997), including potato plants has been reported extensively (incl.) and excluding (excl.) parents. (Belknap et al., 1994; Karp et al., 1982; Shepard, 1981; Shepard and Totten, 1977). Adult trial Larval trial Scowcroft and Larkin (1982) suggest that Incl. parentsz Excl. parentsy Incl. Parents Excl. parents somaclonal variation may be caused by epige- Source df3 MS4 df MS df MS df MS netic variations, karyotypic changes, muta- Clone 36 0.0542** 34 0.0481** 46 0.5753** 44 0.5058** tions in nuclear and cytoplasmic DNA, chro- Year 1 0.0322 1 0.0494 1 5.2093** 1 5.4584** × * mosomal changes, and non-conventional mu- Clone Year 27 0.0276 25 0.0282 26 0.2276 24 0.2320 tations, such as gene amplification or trans- zParents included in the analysis. posable elements. Williams et al. (1993) pro- yParents not included in the analysis. *, ** ≤ vided evidence for deletions in S. brevidens Significant at P 0.05 or 0.01, respectively. Phil. + S. tuberosum somatic hybrids. Al- though somaclonal variation has been reported Roddick and Melchers (1985) reported that for vine, flower and tuber traits, along with plastomes do not influence the biosynthesis disease resistance, it has not been documented and distribution of α-solanine and α-chaconine for trichome traits in potato. in potato-tomato somatic hybrids. The somatic Significant differences were also observed hybrids produced the same glycoalkaloids as among clones, but not replications, for density their parents, supporting Melchers’ report. of type B trichomes (Table 4). The high tri- Sanford et al. (1997) found that somatic dou- chome parent (J10) contained a high density of bling of a high leptine clone produced Table 7. Comparison of inhibition of larval develop- type B trichomes (85.33 trichomes/25 mm2). tetraploids with significantly lower leptine ment by somatic hybrid clones of Solanum in However, no type B trichomes were observed levels. Therefore, it is not surprising that leptine 1995 and 1997. J = trichome parent; K = leptine parent; KJ = hybrid. Asterisks indicate hybrids in the other parent or any of the hybrids. levels were not higher in the somatic hybrids that were more inhibiting than both parents in Therefore, ANOVA could not be performed than in the diploid S. chacoense parent. 1995 and 1997 at P ≤ 0.05. when parents were excluded. Previous reports Resistance to the CPB. In the combined indicate that density of type B trichomes is analysis, significant differences were observed Year complexly inherited, with low levels of addi- among somatic hybrids in both adult and lar- 1995 Parent 1997 Parent tive genetic variance (Mehlenbacher et al., val feeding trials, whether or not parents were Clone J K J K 1984; Vallejo et al., 1994a). Our results indi- included in the analysis (Table 6). Therefore, KJ127 * * * cate that the inheritance of type B trichomes somaclonal variation among clones was ob- KJ129 * * may be recessive, since these trichomes were served in both adult and larval trials. The KJ130 * * not observed in any somatic hybrids. Alterna- effects of year were nonsignificant in the adult KJ136 * * tively, cytoplasmic DNA may affect the ex- trial, but were highly significant in the larval KJ139 * KJ183 * * pression of type B trichomes, as suggested by trial. A probable explanation for the year ef- KJ186 * * Vallejo et al. (1994b). Somatic hybrids pre- fect on larval resistance is that somatic hybrids KJ187 * * sumably contain equal quantities of mitochon- were grown in the greenhouse in 1997 and KJ191 * * drial and chloroplast DNA from both parents, outdoors in 1995. Trichomes on the plants KJ192 * * in contrast with sexual hybrids. Therefore, the grown outside may have been damaged more KJ193 * * S. chacoense cytoplasm in the somatic hybrids by wind, blowing dust, and rain than were KJ194 * * may have inhibited the expression of genes for those on plants in the greenhouse (Flanders et KJ196 * * type B trichomes. al., 1992). Compared with 1995, fewer larvae KJ197 * * * Glycoalkaloid analysis. Although the S. in the 1997 study developed past the first KJ199 * * KJ57 * * * * chacoense clone K produced leptines, their instar. These results would be expected if KJ59 * levels were low. Analysis of variance re- trichomes were more intact in 1997 and they KJ68 * * vealed no significant difference among are a major factor in resistance to larval feed- KJ76 * * clones for total glycoalkaloids, solanidine ing. The clone by year interaction was not KJ79 * (solanine and chaconine glycoalkaloids), significant in either the adult or the larval trial, KJ83 * * * leptinidine (leptinine glycoalkaloids), and except when parents were included in the KJ87 * * acetylleptinidine (leptine glycoalkaloids). latter.

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Adult beetles always preferred leaves of S. Literature Cited cal associations. Euphytica 61:83–111. tuberosum to those of the parents or the so- Franca, F.H., R.L. Plaisted, R.T. Roush, S. Via, and Austin, S., M.A. Baer, M. Ehlenfeldt, P.J. matic hybrids. However, no somatic hybrids W.M Tingey. 1994. Selection response of the Kazmierczak, and J.P. Helgeson. 1985a. In- Colorado potato beetle for adaptation to the were more resistant than both parents. Clones traspecific fusions in Solanum tuberosum. Theor. that were among the most resistant included resistant potato, Solanum berthaultii. Entomol. Appl. Genet. 71:172–175. Expt. Appl. 73:101–109. KJ74, KJ69, and KJ81. Note that clones varied Austin, S., M.A. Baer, and J.P. Helgeson. 1985b. French, N.M. II, D.C. Heim, and G.G. Kennedy. in resistance to feeding by adult beetles even Transfer of resistance to potato leaf roll virus 1992. resistance among Colorado though they did not vary in glycoalkaloid from Solanum brevidens into Solanum tuberosum potato beetle, Leptinotarsa decemlineata (Say) levels. In addition, the somatic hybrid with the by somatic fusion. Plant Sci. Lett. 39:75–82. (Coleoptera: Chrysomelidae), populations in highest level of type A trichomes was not Austin, S. and A.C. Cassells. 1983. Variation be- North Carolina. Sci. 36:95–100. among the most resistant clones, and the most tween plants regenerated from individual calli Gibson, R.W. 1976. Trapping of the spider mite produced from separated potato stem callus cells. resistant clones did not differ significantly Tetranychus urticae by glandular hairs on the Plant Sci. Lett. 31:107–114. wild potato Solanum berthaultii. Potato Res. from less resistant ones in type A trichome Austin, S., E. Lojkowska, M.K. Ehlenfeldt, A. density. Some other resistance mechanism must 19:179–182. Kelman, and J.P. Helgeson. 1988. Fertile inter- Gibson, R.W., M.G.K. Jones, and N. Fish. 1988. be influencing the acceptance of the somatic specific somatic hybrids of Solanum: A novel Resistance to potato leaf roll virus and potato hybrid leaves by adult beetles. The contents of source of resistance to Erwinia soft rot. Phyto- virus Y in somatic hybrids between dihaploid the trichomes may vary among clones. An pathology 78:1216–1220. Solanum tuberosum and Solanum brevidens. alternative explanation may be suggested by Austin, S., J. D. Pohlman, C.R. Brown, M. Mojtahed, Theor. Appl. Genet. 76:113–117. the study of Yencho et al. (1996), in which a G.S. Santo, D.D. Douches and J.P. Helgeson. Groden, E. and R.A. Casagrande. 1986. Population strong and consistent quantitative trait locus 1993. Incorporation of nematode resistance in dynamics of the Colorado potato beetle, interspecific somatic hybrids between S. was reported for CPB resistance that was not Leptinotarsa decemlineata (Coleoptera: tuberosum and S. bulbocastanum. Amer. Potato Chrysomelidae), on Solanum berthaultii. J. Econ. associated with any trichome traits in S. J. 70:485–495. berthaultii hybrids. Entomol. 79:91–97. Baird, E., S. Cooper-Bland, R. Waugh, M. DeMaine, Haberlach, G.T., B. Cohen, N. Reichert, M. Baer, L. The larval trial focused on first instar lar- and W. Powell. 1992. Molecular characteriza- Towill, and J.P. Helgeson. 1985. Isolation, cul- vae because they would presumably be most tion of inter-and intra-specific somatic hybrids ture and regeneration of protoplasts from potato affected by chemically-based resistance of potato using randomly amplified polymor- and several related Solanum species. Plant Sci. mechanisms. In fact, few clones except tbr phic DNA (RAPD) markers. Mol. Gen. Genet. 39:67–74. allowed development of larvae past the second 233:469–475. Heim, D.C., G.G. Kennedy, and J.W. VanDuyn. instar. According to Dimock and Tingey Barsby, T.L., J.F. Shepard, R.J. Kemble, and R. 1990. Survey of insecticide resistance among Wong. 1984. Somatic hybridization in the genus (1987), large larvae tend to accumulate exu- North Carolina Colorado potato beetle (Co- Solanum: S. tuberosum and S. brevidens. Plant leoptera: Chrysomelidae) populations. J. Econ. date on more of their tarsi than do young larvae Cell Rpt. 3:165–167. and are, therefore, more likely to be affected Entomol. 83:1229–1235. Belknap, W.R., D. Corsini, J.J. Pavek, G.W. Snyder, Helgeson, J.P., G.T. Haberlach, J. Pohlman, and S. by the mechanical impediments conferred by D.R. Rockhold, and M.E. Vayda. 1994. Field Austin. 1988. Somatic fusion of Solanum spe- glandular trichome-based resistance. These performance of transgenic Russet Burbank and cies. Plant Cell Tiss. Org. Cult. 12:185–187. observations indicate that the major resistance Lemhi Russet potatoes. Amer. Potato J. 71:285– Helgeson, J.P., J.D. Pohlman, S. Austin, G.T. factor affecting larvae in these somatic hy- 296. Haberlach, S.M. Wielgus, D. Ronis, L. brids is chemically-based and present in either Cantelo, W.W., L.W. Douglass, L.L. Sanford, S.L. Zambolim, P. Tooley, J.M. McGrath, R.V. James, glandular trichomes or leaf tissue. Sinden, and K.L. Deahl. 1987. Measuring resis- and W.R. Stevenson. 1998. Somatic hybrids tance to the Colorado potato beetle (Coleoptera: Several somatic hybrids inhibited larval between Solanum bulbocastanum and potato: a Chrysomelidae) in potato. J. Entomol. Sci. new source of resistance to late blight. Theor. development significantly more effectively 22:245–252. than one or both parents (Table 7). The great- Appl. Genet. 96:738–742 Cheng, J., J.A. Saunders, and S.L. Sinden. 1995. Karp, A., R.S. Nelson, E. Thomas, and W.J. Bright. est number of differences between clones and Colorado potato beetle resistant somatic hy- 1982. Chromosome variation in protoplast-de- parents were observed in 1997. As discussed brid potato plants produced via protoplast rived potato plants. Theor. Appl. Genet. 63:265– previously, trichomes may have been dam- electrofusion. In Vitro Cell. Dev. Biol. 31:90– 277. aged more extensively in 1995, when the clones 95. Kumar, A. and E.C. Cocking. 1987. Protoplast fu- were grown outdoors. Chemical resistance fac- Deimling, S., J. Zitzlsperger, and G. Wenzel. 1988. sion: A novel approach to organelle genetics in tors in the trichomes may have been responsible Somatic fusion for breeding of tetraploid pota- higher plants. Amer. J. Bot. 74:1289–1303. toes. Plant Breeding 101:181–189. Lawson, D.R., W.A. Erb, and A.R. Miller. 1992. for the greater resistance of clones vs. their Deragon, J.M. and B.S. Landry. 1992. RAPD and parents in 1997. Clone KJ57 was more resistant Analysis of Solanum alkaloids using internal other PCR-based analyses of plant genomes standardization and capillary gas chromatogra- than both of its parents in both years of trials. using DNA extracted from small leaf disks. phy. J. Agr. Food Chem. 40:2186–2191. Several clones were more resistant than PCR Methods Applic. 1:175–180. Lawson, D.R., T.P. Green, L.W. Haynes, and A.R. either parent in the larval feeding trials. These Dimock, M.B. and W.M. Tingey. 1987. Mechanical Miller. 1997. Nuclear magnetic resonance spec- clones may be expressing resistance mecha- interaction between larvae of the Colorado po- troscopy and mass spectroscopy of solanidine, nisms from both parents, enhancing their abil- tato beetle and glandular trichomes of Solanum leptinidine, and acetylleptinidine. Steroidal al- ity to inhibit larval development. Alternatively, berthaultii Hawkes. Amer. Potato J. 64:507– kaloids from Solanum chacoense Bitter. J. Agr. they may have the potential to express hetero- 515. Food Chem. 45:4122–4126. Ehlenfeldt, M.K. and J.P. Helgeson. 1987. Fertility sis more extensively than do their diploid Lawson, D.R., R.E. Veilleux, and A.R. Miller. 1993. of somatic hybrids from protoplast fusions of Biochemistry and genetics of Solanum chacoense parents. Another factor may be the opportu- Solanum brevidens and S. tuberosum. Theor. steroidal alkaloids: Natural resistance factors to nity for novel recombination products because Appl. Gen. 73:395–402. the Colorado potato beetle. Current Topics Bot. cytoplasmic DNA from both parents is present Everich, R.C., G.P. Dively, and J.J. Linduska. 1992 Res. 1:335–352. in the somatic fusions (Kumar and Cocking, Baseline monitoring of Colorado potato beetle Mehlenbacher, S.A., R.L. Plaisted, and W.M. Tingey. 1987). Finally, the resistance mechanisms that sensitivity to Bacillus thuringiensis and asso- 1983. Inheritance of glandular trichomes in impede larval development may be expressed ciations with pyrethroid resistance. Resistant crosses with Solanum berthaultii. Amer. Potato more effectively at the tetraploid than at the Pest Mgt. 4:14–15. J. 60:699–708. diploid level. Because these hybrids have not Ferro, D.N. 1993. Potential for resistance to Bacil- Mehlenbacher, S.A., R.L Plaisted, and W.M. Tingey. lus thuringiensis: Colorado potato beetle 1984. Heritability of trichome density and drop- been challenged with CPBs for several gen- (Coloeptera: Chrysomelidae)—A model sys- let size in interspecific potato hybrids and rela- erations, it is not known whether their resis- tem. Amer. Entomol. 39:38–44. tionship to aphid resistance. Crop Sci. 24:320– tance is more durable than that of the parents. Flanders, K.L., J.G. Hawkes, E.B. Radcliffe, and 322. Future research could help elucidate the long- F.I. Lauer. 1992. Insect resistance in potatoes: Menke, U., L. Schilde-Rentschler, B. Ruoss, C. term effectiveness of resistance factors in the Sources, evolutionary relationships, morpho- Zanke, V. Hemleben, H. Ninnemann. 1996. somatic hybrids. logical and chemical defenses, and ecogeographi- Somatic hybrids between the cultivated potato

926 HORTSCIENCE, VOL. 34(5), AUGUST 1999 Solanum tuberosum L. and the 1 EBN wild lines and S. brevidens by species-specific RAPD Sinden, S.L., L.L. Sanford, and K.L. Deahl. 1986b. species Solanum pinnatisectum Dun.: Morpho- patterns and assessment of disease resistance of Segregation of leptine glycoalkaloids in Solanum logical and molecular characterization. Theor. the hybrids. Euphytica 80:207–217. chacoense Bitter. J. Agr. Food Chem. 34:372– Appl. Genet. 92:617–626. Roush, R.T. and W.M. Tingey. 1991. Evolution and 377. Mooney, J.J. 1989. Utilization of wild Solanum management of resistance in the Colorado po- Takemori, N., K. Shinoda, and N. Kadotani. 1994. species for resistance to the Colorado potato tato beetle, Leptinotarsa decemlineata, p. 60– RAPD markers for confirmation of somatic hy- beetle and the green peach aphid. MS Thesis, 74. In: I. Denholm, A.L. Devonshire, and D.W. brids in the dihaploid breeding of potato (Solanum North Dakota State Univ. Hollomon (eds.). Resistance ’91, Achievement tuberosum L.). Plant Cell Rpt. 13:367–371. Mowry, T.M. and L.E. Sandvol. 1995. Survey of and developments in combating pesticide resis- Tisler, A.M. and G.W. Zehnder. 1990. Insecticide Colorado potato beetle insecticide resistance in tance. Elsevier, London. resistance in the Colorado potato beetle (Co- Idaho. Amer. Potato J. 72:551–558. Sanford, L.L., R.S. Kobayashi, K.L. Deahl, and S.L. leoptera: Chrysomelidae) on the Eastern shore Murashige, T. and F. Skoog. 1962. A revised me- Sinden. 1997. Diploid and tetraploid Solanum of Virginia. J. Econ. Entomol. 83:666–671. dium for rapid growth and bioassays with to- chacoense genotypes that synthesize leptine Vallejo, R.L., W.C. Collins, and R.H. Moll. 1994a. bacco tissue cultures. Physiol. Plant. 15:473– glycoalkaloids and deter feeding by Colorado Inheritance of A and B glandular trichome 497. potato beetle. Amer. Potato J. 74:15–21. density and polyphenol oxidase activity in dip- Novy, R.G. and J.P. Helgeson. 1994a. Somatic Sanford, L.L., T.L. Ladd, S.L Sinden, and W.W. loid potatoes. J. Amer. Soc. Hort. Sci. 119:829– hybrids between Solanum etuberosum and dip- Cantelo. 1984. Early generation selection of 832. loid, tuber bearing Solanum clones. Theor. Appl. insect resistance in potato (Empoasca fabae, Vallejo, R.L., W.C. Collins, and R.D. Schiavone. Genet. 89:775–782. Leptinotarsa decemlineata, Myzus persicae, 1994b. Genetics and incorporation of glandular Novy, R.G. and J.P. Helgeson. 1994b. Resistance to Macrosiphum euphorbiae). Amer. Potato J. trichomes and polyphenol oxidase activity into potato virus Y in somatic hybrids between 61:405–418. an advanced Solanum phureja–S. stenotomum Solanum etuberosum and S. tuberosum x S. SAS Institute. 1994. Version 6.10 for Windows. diploid potato population. J. Amer. Soc. Hort. berthaultii hybrid. Theor. Appl. Genet. 89:783– SAS Inst., Cary, N.C. Sci. 119:824–828. 786. Scowcroft, W.R. and P.J Larkin. 1982. Somaclonal Willliams, C.E., S.M. Wielgus, G.T. Haberlach, C. Pelletier, Y. and Z. Smilowitz. 1991. Feeding be- variation: A new option for plant improvement, Guenther, H. Kim-Lee, and J.P. Helgeson. 1993. havior of the adult Colorado potato beetle, p. 159–178. In: K. Vasil, W.R. Skowcroft, and RFLP analysis of chromosomal segregation in Leptinotarsa decemlineata (Say), on Solanum K.J. Frey (eds.). Plant improvement and somatic progeny from an interspecific hexaploid so- berthaultii Hawkes. Can. Ent. 123:219–230. cell genetics. Academic Press, New York. matic hybrid between Solanum brevidens and Provan, J., A. Kumar, L. Shepherd, W. Powell, and Shepard, J.F. 1981. Protoplasts as sources of disease Solanum tuberosum. Genetics 135:1167–1173. R. Waugh. 1996. Analysis of intra-specific so- resistance in plants. Annu. Rev. Phytopath. Xu, Y.-S., M. Clark, and E. Pehu. 1993. Use of matic hybrids of potato (Solanum tuberosum) 19:145–166. RAPD markers to screen somatic hybrids be- using simple sequence repeats. Plant Cell Rpt. Shepard, J.F. and R.E. Totten. 1977. Mesophyll cell tween Solanum tuberosum and S. brevidens. 16:196–199. protoplasts of potato. Isolation, proliferation, Plant Cell Rpt. 12:107–109. Roddick, J.G. and G. Melchers. 1985. Steroidal and plant regeneration. Plant. Physiol. 60:313– Yencho, G.C., M.W. Bonierbale, W.M. Tingey, glycoalkaloid content of potato, tomato, and 316. R.L. Plaisted, and S.D. Tanksley. 1996. Mo- their somatic hybrids. Theor. Appl. Genet. Sinden, S.L., L.L. Sanford, W.W. Cantelo, and K.L. lecular markers locate genes for resistance to 70:655–660. Deahl. 1986a. Leptine glycoalkaloids and resis- the Colorado potato beetle, Leptinotarsa Rokka, V.-M., Y.-S. Xu, J. Kankila, A. Kuusela, S. tance to the Colorado potato beetle (Coloeptera: decemlineata, in hybrid Solanum tuberosum x S. Pulli, and E. Pehu. 1994. Identification of so- Chrysomelidae) in Solanum chacoense. Environ. berthaultii potato progenies. Entomol. Expt. matic hybrids of dihaploid Solanum tuberosum Entomol. 15:1057–1062. Appl. 81:141–154.

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