Szczerbakowa 2010 somatic hybrydyzation

Acta Physiol Plant (2010) 32:867–873 DOI 10.1007/s11738-010-0472-3

ORIGINAL PAPER

Somatic hybridization between the diploids of S. 3 michoacanum and S. tuberosum Anna Szczerbakowa • Justyna Tarwacka Michał Oskiera • Henryka Jakuczun • Bernard Wielgat

•

Received: 15 July 2009 / Revised: 27 January 2010 / Accepted: 11 February 2010 / Published online: 3 March 2010 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2010

Abstract Interspecific somatic hybrids between a diploid potato clone DG 81-68 susceptible to Phytophthora infestans (Mont.) de Bary and a resistant diploid tuber-bearing species Solanum 9 michoacanum were generated and analyzed. About 30 regenerants displaying an intermediate morphology were obtained as a result of three separate PEG-mediated fusion experiments. The RAPD analysis confirmed the hybridity of all the regenerants. About 50% of the hybrid plants exhibited vigorous growth and were stable in culture, while the rest of them rooted poorly and grew slowly in vitro. Most of the hybrid clones were at the tetraploid level (70%), while 30% of the clones examined were at the hexaploid level. The S. 9 michoacanum (?)

DG 81-68 hybrids with growth anomalies were aneuploid.

The variation in late blight resistance of the hybrid clones was found in detached leaflet tests, with enhanced resistance characteristic for three tetraploid hybrids.

Keywords Somatic hybrids Solanum 9 michoacanum S. tuberosum Chromosome counts Ploidy RAPD

Communicated by J. Sadowski.

A. Szczerbakowa (&) J. Tarwacka B. Wielgat Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawin´skiego 5A, 02-106 Warsaw, Poland e-mail: annasz@ibb.waw.pl M. Oskiera The Emil Chroboczek Research Institute of Vegetable Crops, Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland H. Jakuczun Młocho´w Research Center, Plant Breeding and Acclimatization Institute (IHAR), Platanowa 19, 05-831 Młocho´w, Poland

Introduction Wild Solanum species have proven to be valuable in breeding potatoes for disease resistance, environmental tolerance, and other agronomic traits of interest (Spooner and Hijmans 2001). However, introgression of resistance traits from wild species into cultivated potato is difficult due to sexual incompatibility as well as differences in ploidy and endosperm balance number (EBN). Somatic hybridization is an alternative method that is successfully used for creating potato genotypes with improved tolerance to various biotic and abiotic cues. Interspecific somatic hybridization allows to increase genetic variability as well as to transfer resistance traits from wild species to cultivated potato, e.g. resistance to viruses (Valkonen and Rokka 1998), frost (Preiszner et al.

1991), tuber soft rot and early blight (Tek et al. 2004) from S. brevidens, resistance to Phytophthora infestans and Globodera pallida (Serraf et al. 1991) and to salinity (Bidani et al.

2007) from S. berthaultii, resistance to PLRV and PVY from S. tuberosum (Novy et al. 2007), resistance to bacterial wilt from S. commersonii (Laferriere et al. 1999), as well as resistance to PVY and P. infestans from S. tarnii (Thieme et al. 2008). In case of late blight (LB) disease caused by P. infestans—the most devastating potato disease in the world—wild diploid Mexican species, S. bulbocastanum and S. pinnatisectum, as well as their hybrids obtained in generative cross, were previously used as donors of LB resistance for cultivated potato in somatic hybridization experiments (Thieme et al. 1997; Szczerbakowa et al. 2003, 2005). However, the number of produced somatic hybrids was often low, and the resistance level was far below the immunity of the wild species, being insufficient for their agronomic application. Only in case of the gene blb1 from S. bulbocastanum, its successful introduction into potato breeding lines was achieved by means of somatic

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hybridization, and their field resistance enhanced (Helgeson et al. 1998).

Despite the rapid progress in understanding the evolution and mechanism of pathogenicity of P. infestans, this pathogen still causes significant losses in potato yield worldwide. The extreme variability and adaptability of P. infestans makes the control of LB disease difficult and very expensive. Potatoes with durable resistance to LB are not available to growers (Kamoun and Smart 2005). Hence, novel sources of sustainable genetic resistance to various strains of P. infestans are searched among P. infestansresistant, wild Solanum species, in order to engineer resistance to LB in potato (Hein et al. 2009).

S. 9 michoacanum is a nothospecies originating from a spontaneous cross of S. bulbocastanum and S. pinnatisectum, occurring within the distributional range of both of these species, and is morphologically intermediate between them (Hawkes 1990; Spooner and Hijmans 2001). The flowers of S. 9 michoacanum are stellate and the fruits are usually devoid of seeds (http://www.nhm.ac.uk/ solanaceaesource). S. 9 michoacanum is regarded as an attractive source of valuable agronomic and quality traits, such as LB resistance of both leaflets and tubers (Jakuczun and Wasilewicz-Flis 2004a) and suitability for production of potato chips (Jakuczun and Wasilewicz-Flis 2004b).

Combining LB resistance with potato quality is a very difficult breeding task. As a 1EBN species, S. 9 michoacanum cannot be directly crossed with cultivated potato.

Complicated bridgecross and backcross breeding schemes involving EBN and ploidy manipulation need to be developed to overcome the sexual isolation of S. 9 michoacanum. The biotechnological approaches, such as somatic hybridization and embryo rescue, seem adequate for transferring the useful genes from S. 9 michoacanum into the cultivated gene pool.

The present paper describes the production and characteristics (morphology, ploidy, and resistance to P. infestans) of the somatic hybrids between S. 9 michoacanum and a potato diploid clone DG 81-68.

Materials and methods Plant material The clone 99-12/8 of S. 9 michoacanum (Bitter) Rydb. (blb 9 pnt, 2n = 2x = 24) and the S. tuberosum diploid clone DG 81-68 were from the in vitro collection of the Plant Breeding and Acclimatization Institute (IHAR), Młocho´w Research Center. Clone 99-12/8 was selected from the seedlings of VIR 5763 accession. Clone DG 81-68 is an interspecific hybrid having in its pedigree S. tuberosum (75%), S. chacoense (19%) and S. yungasense (6%).

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Since the input of S. tuberosum is dominating, we refer to this clone as S. tuberosum. All the plants were propagated in vitro on hormone-free MS medium (Murashige and Skoog 1962), with 2% sucrose and 0.6% agar.

Protoplast isolation, fusion and regeneration The applied technique of protoplast isolation and regeneration was as described previously (Szczerbakowa et al.

2000). Parental protoplasts in a 1:1 mixture at final density of 106 ml-1 were fused in a medium consisting of 25% PEG 6000, 0.1 M Ca(NO3)2, 0.3 M mannitol and 10% DMSO, adjusted to pH 9.0.

Morphology of regenerants The morphology of regenerated plants was assessed after embedding the in vitro grown plants in soil. The plants grew in 14-cm pots in growth chamber under 16 h day length at light of 150 lmol m-2 s-1 and 22°C/18°C day/ night temperature. Plants were watered twice a week, and fertilized once a week with a 0.25% solution of Florovit fertilizer (Inco-Veritas S.A., Poland). The morphological characteristics recorded were: vigor, growth habit, size and shape of leaves and flowers.

Ploidy of regenerants In leaves of soil-grown plants, chloroplasts were counted in guard cells on lower surface of the leaf. The mean of at least 20 measurements was indicative of plant ploidy level as shown for potato by Jakuczun et al. (1997).

Cytology For chromosome count, the root tips (5–10 mm) of in vitrocultured hybrid plants were detached 6–9 days after the last passage. The root tips were pretreated with 2 mM 8-hydroxychinoline for 6 h and fixed in Carnoy solution (ethanol:acetic acid, 3:1) for 48 h at room temperature. The fixed tissues were digested for 30 min at 37°C with the following solution: 1% cellulase Onozuka R-10, 1% cellulase from Aspergillus niger, 0.32 units/mg (Serva), 20% pectolyase, 0.70 units/mg (Serva), 1 mM EDTA, and 10 mM citric buffer, pH 4.8. The root tip squashes were stained with DAPI and examined under UV with an inverted microscope Olympus IX-70.

DNA isolation and RAPD analysis For RAPD analysis, DNA was isolated from leaves of in vitro grown plants as described by Szczerbakowa et al.

(2003).

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Polymerase chain reaction (PCR) was performed in Gene Amp PCR System 2400 (Perkin Elmer) in 25 ll of reaction mixture containing: 0.5 U Tag recombinant DNA polymerase, 19 buffer and 3.0 mM MgCl2 (all supplied by Perkin Elmer), 0.4 lM primer, 0.25 mM of each dNTP (Gibco-BRL) and the DNA template. The PCR procedure was that of Naess et al. (2001).

The amplification products (12.5 ll) were separated by electrophoresis in 1.5% agarose in 0.59 Tris–borate– EDTA (TBE) buffer at room temperature and visualized by staining with ethidium bromide. A 100-bp DNA ladder (Fermentas) was used as a molecular marker. Decamer oligonucleotide primers were synthesized on a DNA synthesizer Oligo 1000M DNA (Beckman) and preliminarily tested to detect species-specific amplification products. The RAPD patterns produced by primer OPG-02 (50 ACGGATCCTG30 ) for each parental species were used to verify the hybridity of the after-fusion regenerants.

Assessment of LB resistance The evaluation of resistance to P. infestans in detached leaflet assay was as described by Zarzycka (2001). A very aggressive and virulent P. infestans isolate MP 324 was used as a source of inoculum. Sporangia were collected in deionized water and adjusted to a standard concentration of 50 sporangia mm-3. Five to ten leaflets from each genotype were tested on two different dates. The leaflets were placed upside down on a plastic tray on the wet wood wool, and a drop of inoculum was placed near the midrib on each leaflet. Inoculated leaves were incubated at temperature 16°C under constant illumination of about 30 lmol m-2 s-1. After 6 days of incubation, the resistance was evaluated using a 9-grade scale, where 9 indicated the highest resistance. Cvs Tarpan (susceptible) and Bzura (mid-resistant), as well as a diploid clone DG 94-15 (resistant), were used as standards. The inoculum virulence (on leaflets of Black’s differentials) and aggressiveness (on cvs Bintje and Tarpan carrying no R genes) were examined in each test.

Results Three separate fusion experiments yielded 388 calli, including 31 shoot-producing ones. Due to the effect of heterosis (more vigorous growth of interspecific hybrids), the most rapidly growing and regenerating calli were presumably hybrid. The first shoots were detached for rooting in 3 months after protoplast fusion. The protoplasts of S. 9 michoacanum did not regenerate in separate controls under the conditions applied, as is characteristic for wild Solanum diploids, while the DG 81-68 protoplasts

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regenerated more slowly as tetraploids, as a result of spontaneous fusion or polyploidization occurring during protoplast regeneration. This phenomenon has often been reported in literature and is explained by the process of potato genome stabilization and its return to the most stable tetraploid level (see e.g. Debnath and Wenzel 1987; Greplova´ et al. 2008).

One shoot from each of the 30 regenerating calli was detached for rooting. After a year of in vitro propagation, 26 clones survived whereas 4 clones were lost due to growth abnormalities. Of the 26 clones cultured in vitro, only 7 had normal morphology and growth, while the rest (19) exhibited minor leaf deformations, growth abnormalities, as well as temporal disturbances in rooting capacity and apical domination (Table 1). The after-fusion regenerants differed morphologically from the parents, as judged by the shape of the leaves and the plant habitus. For example, the first detached regenerant MT-1, shown in Fig. 1, was characterized by abnormal leaf shape, stunted apical growth and adventitious shoot formation.

The hybridity of in vitro regenerants was verified by RAPD analysis. Of several decamer primers tested, the primer OPG02 generated polymorphic markers specific for each of the parents, which were combined in the tested regenerants (Fig. 2), proving their hybridity. The RAPD profiles of the hybrids were mostly uniform (Fig. 2, lines 3–7), suggesting similar genotypes of these independent clones. However, the profile of MT-29 (Fig. 2, line 8) contained an additional amplification product (indicated by an arrowhead), implying a distinct genotype of this clone.

Plants of the most vigorous 14 clones were transferred to soil for evaluation of ploidy level and LB resistance. The Table 1 Growth characteristics of the after-fusion regenerants in vitro Clone

Rooting capacity

Phenotype

Clone

Rooting capacity

Phenotype

MT-1

?

AN

MT-14

?

N

MT-2

±

D

MT-15

---

AN

MT-3

?

AN

MT-16

±

D

MT-4

?

N

MT-17

--

AN

MT-5

±

D

MT-18

??

N

MT-6

?

D

MT-19

??

N

MT-7

?

AN

MT-20

--

D

MT-8

---

AN

MT-23

--

AN

MT-9

---

AN

MT-25

??

N

MT-10

?

D

MT-26

--

D

MT-11 MT-12

--

AN AN

MT-27 MT-28

-

D AN

MT-13

?

N

MT-29

??

N

Rooting: from very good ? ? to extremely poor - - -; growth: AN abnormal, D disturbed, N normal

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Fig. 1 The in vitro grown parental clones, a S. tuberosum DG 81-68 and c S. 9 michoacanum, and b their hybrid MT-1

hybrid clones grown in soil differed in leaf morphology (Fig. 3) and growth habit (Fig. 4). The selected four MT hybrid clones are presented in Fig. 2 (leaves) and Fig. 3 (whole plants). The leaf shape of the hybrids was intermediate between those of the parents, and sometimes abnormal. The plant habitus varied from normal type to ‘‘bushy’’, without the main stem and with numerous short lateral shoots.

The results of ploidy measurements by chloroplast counts in leaf guard cells are presented in Table 2. Most of the hybrid clones were tetraploid and hypertetraploid (seven and three clones, respectively), while the remaining 3 clones of the 13 examined were at the hexaploid level.

The leaves of tetraploid hybrids resembled those of S. 9 michoacanum (see MT-18 in Fig. 3a). The hybrid clones with broad, slightly abnormal leaves, such as MT-25 (Fig. 3c), were found to be aneuhexaploid (Table 2).

Chromosome counts in metaphase plates of root meristems confirmed tetraploidy (Fig. 5a) as well as hexaploidy (Fig. 5b) of the hybrids analyzed and proved high aneuploidy of the hybrid MT-25 (Table 3).

The testing for LB resistance of ten hybrids of different ploidy showed that at least three tetraploids (MT-6, MT-18 and MT-20) expressed resistance level higher than that of the parental potato diploid DG 81-68 (Table 2).

Discussion

Fig. 2 RAPD profiles of S. 9 michoacanum (mch) and S. tuberosum diploid DG 81-68 (tbr), and their somatic hybrids (lines 3–8, from left to right: MT-4, MT-6, MT-18, MT-20, MT-25, MT-29). Amplification was performed with primer OPG-02. Arrows indicate the bands specific for S. 9 michoacanum (%) and DG 81-68 (.). An arrowhead indicates a new band in line 8 for MT-29. 100 bp a 100-bp DNA ladder

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The produced mch (?) tbr hybrids varied in morphology and resistance to P. infestans. The phenotypic variation can be partly explained by various ploidy levels of the afterfusion regenerants. All the tested hexaploids were susceptible to P. infestans presumably due to the larger input of the S. tuberosum genome. However, of six tetraploid hybrids tested, only three expressed resistance higher than the potato parent (Table 2). An equal input of both parental genomes does not guarantee the increased resistance of the somatic hybrids. As shown previously, the tetraploid somatic hybrids between the resistant accessions of S. bulbocastanum and S. pinnatisectum and the susceptible potato dihaploid H-8105 were all susceptible to P. infestans (Szczerbakowa et al. 2003, 2005). In blb (?) tbr H-8105 hybrids, the interrelation between parental genomes affected negatively the beneficial trait of LB immunity of S. bulbocastanum (Szczerbakowa et al. 2003). As a result, the complex race of the pathogen P. infestans overcame the

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Fig. 3 Leaves of a S. tuberosum DG 81-68, b somatic hybrid MT-18, and c S. 9 michoacanum

Fig. 4 Somatic hybrids grown in soil. a MT-4, b MT-25, and c MT-29

Table 2 Ploidy and late blight resistance of the parental species and selected hybrids

Clone

* Means marked by the same letter are not significantly different at P = 0.05 (Tukey test) a

Number of guard cells checked in parentheses

7.3 ± 0.6 (22)a

Statistical validness*

Ploidy

Chromosome counts

Resistance to P. infestans, mean ± SD

a

2x

24

7.6 ± 1.2

tbr DG 81-68 MT-1

6.9 ± 1.0 (57) 11.4 ± 0.9 (25)

a b

2x 4x

24 nd

3.6 ± 0.8 3.7 ± 1.6

MT-5

13.1 ± 2.0 (26)

c

4x

nd

3.2 ± 1.2

mch 99-12/8

SD standard deviation, nd not determined

Mean number of chloroplasts in a guard cell ± SD

MT-4

13.8 ± 1.5 (32)

c

4x

48

4.3 ± 1.8

MT-18

14.1 ± 1.3 (26)

cd

4x

48

6.3 ± 2.4

MT-6

14.8 ± 1.5 (37)

de

4x?

nd

5.5 ± 0.6

MT-20

16.2 ± 1.5 (48)

f

4x?

nd

5.3 ± 0.5

MT-10

16.6 ± 1.4 (36)

f

6x

72

3.2 ± 1.6

MT-25

20.1 ± 2.7 (23)

g

6x?

mix 72/78-80

2.0 ± 1.7

MT-29

21.4 ± 3.0 (27)

g

6x?

nd

3.3 ± 1.7

MT-2

21.8 ± 1.7 (16)

g

6x?

nd

3.7 ± 1.6

RB gene in blb (?) tbr H-8105 hybrids in detached leaflet tests. The molecular analysis of the S. bulbocastanum (?)

S. tuberosum H-8105 somatic hybrids with the RAPD markers specific for individual chromosomes of S. bulbocastanum confirmed the presence of all but one (chromosome 2) chromosomes of the wild parent in the combined hybrid genome. It is unclear whether the absence of blb

chromosome 2 contributed to the decreased LB resistance of the blb (?) tbr H-8105 hybrids (Bołtowicz et al. 2005).

The lack of RAPD markers assigned to individual chromosomes of S. bulbocastanum might indicate recombination or deletion of chromosomal regions during callus culture and shoot regeneration in vitro; this could lead to a loss of LB resistance characteristic of the wild species. On

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Fig. 5 The metaphase chromosomes in root meristem cells of the mch (?) tbr hybrids. a MT-4 (2n = 4x = 48) and b MT-25 (2n = 6x = 72) Table 3 Chromosome counts in root meristem cells of the selected mch (?) tbr hybrids Clone

Total number Chromosome Number of Ploidy of mitotic counts mitotic plates plates checked (=100%)

MT-4

21

24

3

4x (48)a

46–48

18 (87%)

48±

4

72

19 (83%)

MT-18 14

24± 48

2 12 (86%)

MT-19 22

42–48

22

4x (48)

MT-25 16

36

1

mixoploid

72

7 (44%)

6x/7x- (72/\84)

78–80

8 (50%)

MT-10 23

a

6x (72) 4x (48)

Number of chromosomes in parentheses

the other hand, the expression of RB gene could be modified in blb (?) tbr H-8105 hybrids in the presence of alien tbr genome.

The S. 9 michoacanum parent was resistant but not immune to the isolates of P. infestans used in detached leaf tests (Table 2). Only three mch (?) tbr hybrids expressed moderate resistance under laboratory conditions, while seven hybrids were susceptible. It is known that the RB gene, which is presumably functional in S. 9 michoacanum as well, codes for horizontal, non-specific resistance in the field (Helgeson et al. 1998). The hybrids should therefore be tested under pathogen pressure in the field, where the resistance level depends on differences in the rate of colonization and on the level of success in defense

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strategy of the plant. Although single R genes are thought to be ineffective in the field over long periods of time, some of them apparently mediate durable resistance, as the RB (=Rpi-blb1) gene of S. bulbocastanum (Song et al.

2003; van der Vossen et al. 2003).

Although the breeding for LB resistance in potato started around 1850, caused by LB epidemics and Irish potato famine, high durable resistance of potato cultivars has not been achieved. Disease resistance processes in plants are diverse, and various types of resistance have different durability in the field. Race or cultivar-specific resistance mediated by single resistance (R) genes turned out to be of limited value in the field because of the rapid evolution of new virulent races of pathogens. The non-host and partial resistances (a quantitative phenotype with a partial reduction in disease severity) appear more durable. However, the extent to which durable non-host or partial resistance involves genetic components that are distinct from the R genes remains unclear (Kamoun et al. 1999; Tan et al. 2008).

Various wild Solanum species are currently being screened for novel resistances to P. infestans that could be exploited in potato breeding programs (Hein et al. 2009).

The mch-derived resistance of the generated hybrids could also be valuable and should be examined under field conditions. The best genotypes (vigorous eutetraploids) of the mch (?) tbr hybrids will be evaluated for their fertility. The modification of ploidy of the selected genotypes by colchicin treatment, as well as by haploidization techniques, will also be attempted.

Acknowledgments The authors are grateful to Ms. I. Dzikowska for propagation of in vitro plants.

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