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number of resistant diploid sexual hybrids and tetraploid/hexaploid somatic hybrids were selected. Backcross progenies were obtained through 2x x 4x crosses ...
Potato Research 43 (2000) 135 - 142

Erwinia resistance in backcross progenies of Solanum tuberosum • S. tarijense and S. tuberosum (+) S. commersonii

hybrids D. C A R P U T O I , B. B A S I L E l, T. C A R D I 2 and L. F R U S C I A N T E t 1 Department of Agronomy and Plant Genetics, Via Universith 100, 80055 Portici, Italy 2 CNR - IMOF, Research Institute for Vegetable and Ornamental Plant Breeding, Via Universith 133, 80055 Portici, Italy Accepted for publication: 12 January 2000 Additional keywords: tuber soft rot, 2n gametes, sexual hybridization, somatic hybridization, germplasm exploitation, potato

Summary The resistance to tuber soft rot caused by Erwinia carotovora of Solanum tuberosum x S. tarijense and S. tuberosum (+) S. commersonii hybrids and their backcrosses is reported. A number of resistant diploid sexual hybrids and tetraploid/hexaploid somatic hybrids were selected. Backcross progenies were obtained through 2x x 4x crosses involving a resistant diploid hybrid and tetraploid S. tuberosum, and through 4x x 4x crosses between a resistant somatic hybrid and S. tuberosum. The hybrids showed high variability in terms of resistance to tuber soft rot. The resistance of progeny from 2x x 4x backcrosses was similar to that of the parental sexual hybrid. By contrast, the resistance of genotypes deriving from 4x x 4x backcrosses was reduced compared with the resistant somatic hybrid. In general, tuber characteristics of the backcross hybrids improved considerably as compared with their parents, and tuber yield per plant was good.

Introduction Exploitation of wild potato species as source of genes is fundamental for potato breeding. The easiest way to introgress genes from diploid wild species into Solanum tuberosum form is to produce hybrids between S. tuberosum haploids (2n=2x=24) and the wild species. If the hybrids are fertile and produce 2n gametes, they can be easily crossed with cultivars to produce tetraploid genotypes by sexual polyploidization (Peloquin & Ortiz, 1991). The poor differentiation between the genomes of Solanum species, and the widespread occurrence of 2n gametes in Solanum strongly favour this approach. Through the use of hybrids between wild species and S. tuberosum haploids and 2n gametes, resistance genes have already been transferred from diploid species to tuberosum gene pool (Iwanaga et al., 1989; Watanabe et al., 1992; Ortiz et al., 1997). However, somatic hybridization may be used when differences in the Endosperm Balance N u m b e r (EBN) between diploid species (which have been classified either 1 or 2EBN) and tuberosum haploids (which are 2EBN) hamper sexual hybridization. In the potato each species has an E B N value, and a 2:1 maternal to paternal E B N ratio in the hybrid endosperm is a necessary condition for normal endosperm Potato Research 43 (2000)

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development (Johnston et al., 1980). Interspecific somatic hybridization has been successfully used to transfer resistance to biotic stresses (Austin et al., 1988; Mattheij et al., 1992; Rokka et al., 1994) as well as abiotic stresses (Cardi et al., 1993) from incongruent 1EBN species into the cultivated gene pool. Among the useful traits possessed by wild species, much attention has been given to the resistance to tuber soft rot caused by Erwinia carotovora subsp, atroseptica (Eca) and E. c. subsp, carotovora (Ecc). These bacteria may seriously hamper potato cultivation in several environmental conditions, causing losses in the field and during tuber storage. The control of soft rot is difficult due to the the wide range of hosts and of temperatures at which Erwinia spp. can grow, and to the fact that tubers may be latently infected by bacteria located in the vascular system or in suberized lenticels. Unfortunately, the frequency and degree of resistance is very low in the cultivated potato, and there is no effective method of chemical control. By contrast, sources of resistance have been identified in diploid Solanum species (Hanneman & Bamberg, 1986; Rousselle-Bourgeois & Priou, 1995). The 2EBN species S. phureja (Wolters & Collins, 1994) and S. chacoense (Zimnoch-Guzowska & Lojkowska, 1993) were used in crosses with 2EBN genotypes to generate resistant diploid hybrids. Similarly, 1EBN S. brevidens was used by Austin et al. (1988) to produce hexaploid somatic hybrids with S. tuberosum. Recently, we screened 1EBN and 2EBN diploid genotypes for resistance to Erwinia spp. We found sources of resistance in 2EBN S. tari]ense (Carputo et al., 1996) and 1EBN S. commersonii (Sirianni, 1997), two species carrying multiple resistances. S. tari]ense is also resistant to Verticillium wilt, aphids and the Colorado potato beetle; S. commersonii is resistant to low temperatures, Verticillium wilt and PVX (Hanneman & Bamberg, 1986). In this paper we report on the characterization of interspecific hybrids deriving from a) sexual hybridization between 2EBN S. tuberosum haploids and 2EBN S. tarijense, and b) somatic hybridization between 2EBN S. tuberosum haploids and 1EBN S. commersonii. Materials and methods

Production of hybrid plants. Table 1 reports the characteristics of genotypes used as parental clones. Diploid hybrids were generated from interspecific crosses between S. tuberosum haploid W730 and a clone of PI 414149 of S. tarijense (Carputo et al., 1997). Tetraploid, hexaploid, and aneuploid interspecific hybrids were produced by somatic fusion between S. tuberosum haploid SVP11 and a clone of PI 243503 of S. commersonii (Cardi et al., 1993). The chromosome number of hybrids produced was checked by counting mitotic chromosomes in root tips, which were treated with 8hydroxyquinoline (0.29 g/l) for 5 hours, and then fixed for at least 48 hours in Carnoy solution.

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Table 1. Diploid (2n=2x=24) and tetraploid (2n=4x=48) genotypes used in somatic fusion and sexual crosses. Genotypes

Species

EBN

Tuber soft rot resistance a

Reference

Diploids W 730 SVPll tar 11 cmm 1

S. S. S. S.

2 2 2 1

S S R R

Carputo et al., 1996 Carputo et al., 1996 This study Sirianni, 1997

Tetraploids Carmine Tollocan LT5, LT7

S. tuberosum S. tuberosum S. tuberosum

4 4 4

S S na

Carputo et al., 1998a Carputo et al., 1998a

tuberosum tuberosum tarijense (P1414149) commersonii (P1243503)

aR=resistant; S=susceptible; na=not available.

Following hydrolysis for 55 min in 5N HCI, root tips were gently squashed in 45% acetic acid before observation. Sexual and somatic hybrids were further used to generate the BC I progeny. In particular, and based on the results of screening tests for resistance to Erwinia and for 2n gamete production, a diploid sexual hybrid was used in 4x x 2x and 2x x 4x crosses with cv. Toliocan to generate a tetraploid backcross progeny (coded BCDT) through sexual polyploidization. For somatic hybrids, the fertile resistant tetraploid clone SH9A was used both as male and as female parent in backcrosses with cvs Tollocan and Carmine, and with tetraploid breeding lines LT5 and LT7 to generate the so coded BCSH backcross progeny. Based on stolon length, eye depth and on general tuber appearance, only selected genotypes within the B C D T and BCSH populations (5 and 31 genotypes, respectively) were tested for the resistance to Erwinia spp. Bacterial strains. Erwinia strains used were Eca 121 and Ecc 009 obtained from the International Potato Center. They were stored at 4 ~ in sterile bi-distilled water, and bacterial suspensions of lxl07 colony-forming units/ml were obtained from cultures on nutrient agar at 25 ~ for 48 hours. Evaluation o f resistance to tuber soft rot. Diploid sexual hybrids, somatic hybrids and BC 1 progenies were screened for the resistance to tuber soft rot. All the tests were performed in the autumn, about one month after the harvest, with tubers stored at 8 ~ The screening technique reported by Austin et al. (1988) was used. After sterilization with 0.5% sodium hypochloride for 20 min, from 3 to 5 holes (2 mm wide and 2 cm deep) were drilled into each tuber. One hole was inoculated with sterile water as control, and all the others were inoculated with 20 Ill of bacterial suspension of either Eca or Ecc (lxl07 cfu/ml). Ten tubers of each clone were inoculated with each strain. After 72 h incubation at either 18 ~ (Eca) or 24 ~ (Ecc) in a dew Potato Research 43 (2000)

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chamber, the tubers were cut vertically through the injection points and the diameter of decay was measured. Following the scale reported by Carputo et al. (1997), genotypes with an average diameter of rotted area smaller than 4 mm were classified resistant, those with diameter of decay between 4 and 6 mm intermediate, and those with diameter of rotted area larger than 6 mm susceptible. Tubers of cultivars Carmine, D6sir6e, Draga, Majestic and Tollocan were used as control. Three independent sets of experiments were performed, including 1) the sexual and somatic hybrids, 2) the BCDT backcrosses and 3) the BCSH backcrosses. The first set only was screened with both strains. Evaluation of tuber yield and specific gravity of tubers of BC t. Eighteen tubers of each BCSH hybrid and five tubers of each BCDT hybrid were planted at Camigliatello Silano, southern Italy, in June 1997. For each genotype, tubers were spaced 30 cm in the row and 70 cm between rows. Plots were irrigated and standard culture practices of the area were carried out during the growing season (Carputo et al., 1994). Pollen stainability of each hybrid was estimated by staining pollen grains with acetocarmine. Tubers of each plant were harvested separately after 120 days, and for each plant those with a diameter larger than 35 mm were weighted. The specific gravity of tubers was determined weighting a sample (1 kg) of tubers according to the weight in airweight in water method. Results and discussion

The results of tuber inoculation of the F 1 hybrids are reported in Table 2. They indicate that a number of S. tuberosum x S. tarijense sexual hybrids and S. tuberosum (+) S. commersonii somatic hybrids expressed resistance to Eca and Ecc. The diameter of rotted area after a three-day incubation ranged from 2.5 mm (somatic hybrid SH5A, 2n=70) to 7.3 mm (somatic hybrid SH25A, 2n=72) when tubers were inoculated with Ecc, and from 2.7 mm (hybrid SH5A) to 5.2 mm (hybrid SH25A) following inoculation with Eca. Of 11 genotypes tested against Ecc, 5 (1 S. tuberosum x S. tarijense and 4 S. tuberosum (+) S. commersonii hybrids) were classified resistant, 5 intermediate and 1 susceptible. For Eca, 1 S. tuberosum (+) S. commersonii genotype (SH5A) was resistant and all the others were intermediate. Draga was the best control, with intermediate resistance. To produce backcross generations through conventional breeding, it is necessary that developed genotypes flower and are fertile. The resistant diploid sexual hybrid ESC1 was fertile and, more importantly, produced 2n eggs. Through 2x x 4x crosses it was possible to generate a tetraploid offspring. ESC1 was also used in 4x x 2x crosses as the male parent, but these crosses did not set berries, probably due to the low frequency (1.7%) of 2n pollen produced. With the resistant somatic hybrids, one genotype (SH9A) was male and female fertile and had 48 chromosomes. Thus it was chosen as parent in backrosses with tuberosum cultivars (Carputo et al., 1998b). The high fertility of SH9A is felt to be very important considering that frequently the limiting factor of somatic hybridization is the sterility of hybrids produced. 138

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Table 2. Chromosome number and diameter of rotted area after artificial inoculation with Erwinia carotovora subsp, carotovora (Ecc) and E. c. subsp, atroseptica (Eca) of Solanum tuberosum x S. tarijense sexual hybrids and S. tuberosum (+) S. commersonii somatic hybrids. Hybrid

Somatic chromosomes, no.

Mean rotted area (mm) _+SE Ecc

Eca

3.8_+0.2 4.7• 5.3_+0.4

4.1_+0.1 4.3• 4.4•

66 70 69 48 48 70 72 72

4.6_+0.6 2.5_+0.0 3.7_+0.1 3.2_+0.1 3.2+0.1 4.4_+0.3 4.3_+0.3 7.3•

_a 2.7• 4.7_+0.2 4.6_+0.2 4.6_+0.2

48 48 48

12.9+1.0 4.1_+0.2 14.2_+1.4

5.1• 4.8_+0.2 7.5_+0.3

S. tuberosum x S. tarijense hybrids

ESC1 ESC2 ESC3

24 24 24

S. tuberosum (+) S. commersonii hybrids

SH4A SH5A SH8A SH9A SH9B SH 11C SH14A SH25A

5.2_+0.4

S. tuberosum varieties

D6sir6e Draga Majestic anot tested.

The BC 1 progeny deriving from the somatic hybrid (previously coded as BCSH) had a different behaviour compared with that deriving from the sexual hybrid (coded BCDT). In general, the average resistance of BCSH was very much reduced in comparison with the F 1 somatic hybrid SH9A it came from (Table 3), and segregation for this trait was observed. In particular, the average diameter of decay of the BCSH progeny was 11.4 mm, ranging from 9.5 mm (LT5 • SH9A cross combination) to 13.1 mm (Tollocan • SH9A cross combination), with no maternal effects in the reciprocal crosses available. Two genotypes from SH9A • Carmine crosses with intermediate resistance (diameter of decay of 4.2 mm and 5.8 mm) were selected. All the others BCSH genotypes were classified as susceptible to tuber soft rot. By contrast, the average resistance of the B C D T progeny (6.2 mm) was much closer to that of the sexual hybrid ESC1 it came from. Of five genotypes tested, two were classified as resistant (average diameter of rotted area of 3.6 mm), two intermediate and only one susceptible. The difference in the number of genotypes present in the BCSH and the B C D T populations makes comparisons difficult. However, hypotheses can be drawn to understand why resistance was transmitted to most progenies from the sexual hybrid and not from the somatic hybrid. It is possible that normal meiosis in the somatic hybrid SH9A caused disruption of chromosomes and reassortment of alleles. However, modified meiosis of sexual hybrid ESC1 leading to 2n egg production not Potato Research 43 (2000)

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only allowed the transmission of the entire genome of the hybrid in the BC 1 populations, but could also restrict rearrangement of alleles, which is related to the gene-centromere map distance (Peloquin, 1983).

Table 3. Diameter of rotted area after artificial inoculation with Erwinia carotovora subsp. carotovora, tuber yield and specific gravity of tetraploid hybrids obtained from backcrosses between diploid sexual hybrid ESC1 and Tollocan (BCDT progeny) and between tetraploid somatic hybrid SH9A and Carmine, LT5, LT7 and Tollocan (BCSH progeny). Data are grouped for cross combination. Progeny analyzed

Genotypes

Rotted area a (mm) Tuber yield (g/plant)

Tuber specific gravity

no.

mean (range)

mean (range)

mean (range)

BCDT ESC1 x Tollocan

5

6.2 (3.6-12.8)

630 (420-980)

1.099 (1.090-1.106)

BCSH LT5 • SH9A LT7 x SH9A SH9A x LT7 Tollocan x SH9A SH9A xTollocan Carmine xSH9A SH9A x Carmine

5 2 2 3 3 9 7

11.6 (7.3-15.8) 9.5 (8.3-10.8) 12.3 (12.312.3) 13.1 (11.5-15.4) 12.6 (10.9-14.4) 10.9 (6.9-14.1) 10.1 (4.2-15.0)

710 (360-980) 690 (530-840) 580 (540-620) 900 (280-1340) 550 (350-880) 650 (400-990) 620 (220-1040)

1.058 (1.049-1.066) 1.075 (1.074-1.076) 1.072 (1.071-1.073) 1.074 (1.065-1.080) 1.074 (1.071-1.086) 1.062 (1.056-1.070) 1.063 (1.056-1.070)

a Solanum tuberosum controls Carmine, Draga, D6sir6e, Majestic and Tollocan showed a

diameter of rotted area of 7.6, 6.3, 10.7, 9.1, and 9.7 mm, respectively.

Tuber yield and specific gravity of BC 1 genotypes, grouped by cross combinations, are presented in Table 3. In general, tuber characteristics of BC 1 genotypes improved considerably as compared to their hybrid parents ESC1 and SH9A, which had long stolons and relatively small tubers. Variability was found for tuber shape, skin colour, tuber yield per plant and specific gravity of tubers. Preliminary data on tuber yield showed a high variability among the genotypes. Tuber yield ranged from 200 g/plant (a genotype from SH9A x Carmine) to 1300 g/plant (a genotype from Tollocan x SH9A). This variability may well be the expression of different levels of ailelic diversity present in each hybrid. It is well known tuber yield mainly depends upon intra- and inter-locus interactions ( T a r n & Tai, 1977), and wild species may provide an allelic diversity significantly greater than that of S. tuberosum. It is also remarkable that all the B C D T hybrids showed a specific gravity of tubers higher than 1.080, the minimum value requested for processing. Backcross interspecific hybrids often have undesired traits inherited from the wild parent. Instead, a number of our BC1 genotypes showed good tuber traits and yield. These positive characteristics will probably save time and resources during further backcrosses. Most of BC 1 genotypes flowered profusely and shed high frequencies (>50%) of 140

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stainable pollen (Carotenuto, 1997). In general, BCSH hybrids with S. tuberosum cytoplasm were male sterile. This is probably due to nuclear-cytoplasmic male sterility, a widespread phenomenon in S o l a n u m (Hermundstat & Peloquin, 1985). Fertile BC l hybrids with the highest degree of resistance to Ecc have already been crossed with S. t u b e r o s u m cultivars to produce the BC 2 population. Genotypes obtained are also being evaluated for other noteworthy traits (resistance to Verticillium wilt and low temperatures, and capacity to cold acclimate), with the final goal being the identification of hybrids with multiple resistances.

Acknowledgments Contribution no. 196 from CNR - IMOF, Research Institute for Vegetable and Ornamental Plant Breeding. Thanks are due to Prof. Astolfo Zoina for supplying the Erwinia strains used in this study and for supervising the screening tests.

References Austin, S., E. Lojkowska, M.K. Ehlenfeldt, A. Kelman & L.P. Helgeson, 1988. Fertile interspecific somatic hybrids of Solanum: a novel source of resistance to Erwinia soft rot. Phytopatology 78: 1216--1220. Cardi, T., F. D'Ambrosio, D. Consoli, K.J. Puite & K.S. Ramulu, 1993. Production of somatic hybrids between frost-tolerant Solanum commersonii and S. tuberosum: characterization of hybrid plants. Theoretical and Applied Genetics 87: 193-200. Carotenuto, N., 1997. Valutazione agronomica e caratterizzazione di cloni derivanti da ibridazione somatica tra Solanum commersonii e S. tuberosum. Ph.D. Thesis, University of Naples, pp. 96. Carputo, D., A. Barone, D. Consoli & L. Frusciante, 1994. Use of seedling tubers from TPS in Southern Italy. American Potato Journal 71:29-38. Carputo, D., M. Speggiorin, P. Garreffa, A. Raio & L.M. Monti, 1996. Screening for resistance to tuber soft rot and blackleg in diploid Solanum species and S. tuberosum haploids. Journal o f Genetics & Breeding 50: 221-226. Carputo, D., T. Cardi, M. Speggiorin, A. Zoina & L. Frusciante, 1997. Resistance to blackleg and tuber soft rot in sexual and somatic interspecific hybrids with different genetic background. American Potato Journal 74: 161-172. Carputo, D., T. Cardi, L. Frusciante, P. Sirianni, S. Vega & J.P. Palta, 1998a. Transfer of resistance genes from Solanum Commersonii (2n = 24, 1EBN) to S. Tuberosom (2n = 48, 4EBN) through ploidy and EBN manipulation. Proceedings international symposium "Breeding research on potatoes" Rostock, Germany, June 23-26 1999. Carputo, D., P. Garreffa, M. Mazzei, L. Monti & T. Cardi, 1998b. Fertility of somatic hybrids Solanum commersonii (2n=24, 1EBN) (+) S. tuberosum (2n=24, 2EBN) in intra- and interEBN crosses. Genome 41: 776-781. Hanneman, R.E. Jr & J.B. Bamberg, 1986. Inventory of tuber-bearing Solanum species. Bullettin 533 of Research Division of the College of Agriculture and Life Sciences, University of Wisconsin-Madison, pp. 216. Hermundstat, S.A. & S.J. Peloquin, 1985. Male fertility and 2n pollen production in haploidwild species hybrids. American Potato Journal 62: 479-487. Iwanaga, M., P. Jatala, R. Ortiz & E. Guevara, 1989. Use of FDR 2n pollen to transfer resistance to root-knot nematodes into cultivated 4x potatoes. Journal o f American Society o f Horticultural Science 114: 1008-1013.

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D. CARPUTO,B. BASILE,T. CARDIAND FRUSCIANTE Johnston, S.A., T.M. den Nijs, S.J. Peloquin & R.E. Hanneman Jr, 1980. The significance of genic balance to endosperm development in interspecific crosses. Theoretical and Applied Genetics 57: 5-9. Mattheij, W.M., R. Eijlander, J.R.A. de Koning & K.M. Louwes, 1992. Interspecific hybridization between the cultivated potato Solanum tuberosum subspecies tuberosum L. and the wild species S. circaeifolium subsp, circaeifolium Bitter exhibiting resistance to Phytophthora infestans (Mont.) de Bary and Globodera pallida (Stone) Behrens. 1. Somatic hybrids. Theoretical and Applied Genetics 83: 459-466. Ortiz, R., J. Franco & M. lwanaga, 1997. Transfer of resistance to potato cyst nematode (Globodera pallida) into cultivated potato Solanum tuberosum through first division restitution 2n pollen. Euphytica 96: 339-344. Peloquin, S.J., 1983. Genetic engineering with meiotic mutants. In: D.L. Mulchay & E. Ottaviano (Eds), Pollen: Biology and implications for plant breeding. Elsevier Science Publishing Co., pp. 311-316. Peloquin, S.J. & R. Ortiz, 1991. Techniques for introgressing unadapted germplasm to breeding populations. In: H.T. Stalker & J.P. Murphy (Eds), Plant breeding in the 1990's. CAB International, Wallingford, UK, pp. 485-507. Rokka, V.M., Y.S. Xu, J. Kankila, A. Kuusela, S. Pulli & E. Pehu, 1994. Identification of somatic hybrids of dihaploid Solanum tuberosum lines and S. brevidens by species specific RAPD patterns and assessment of disease resistance of the hybrids. Euphytica 80: 207-217. Rousselle-Bourgeois, F. & S. Priou, 1995. Screening tuber-bearing Solanum spp. for resistance to soft rot caused by Erwinia carotovora subsp, atroseptica (van Hall) Dye. Potato Research 38: 111-118. Sirianni, P., 1997. Superamento di barriere di incompatibilith interspecifica attraverso la manipolazione della ploidia e dell'"Endosperm Balance Number" per I'introgressione di geni utili in Solanum tuberosum L. (2n=4x=48). M.S. Thesis, University of Naples, pp. 77. Tarn, T.R. & G.C.C. Tai, 1977. Heterosis and variation of yield components in F 1 hybrids between Group Tuberosum and Group Andigena potatoes. Crop Science 17: 517-521. Watanabe, K., H.M. EI-Nashaar & M. Iwanaga, 1992. Transmission of bacterial wilt resistance by First Division restitution (FDR) 2n pollen via 4x x 2x crosses in potatoes. Euphytica 60: 21-26. Wolters, P.J. & W.W. Collins, 1994. Evaluation of diploid potato clones for resistance to tuber soft rot induced by strains of Erwinia carotovora subsp, atroseptica, Erwinia carotovora subsp, carotovora and E. chrysanthemi. Potato Research 37: 143-149. Zimnoch-Guzowska, E. & E. Lojkowska, 1993. Resistance to Erwinia spp. in diploid potato with a high starch content. Potato Research 36: 177-182.

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