PlantCell Reports

Plant Cell Reports (1991) 9:531-534

9 Springer-Verlag1991

Interspecific somatic hybridization between lettuce (Lactuca sativa) and wild species L. virosa Etsuo Matsumoto Nagano Vegetable and Ornamental Crops Experiment Station, 2206 Oomuro, Matsushiro, Nagano 381-12, Japan Received March 21, 1990/Revised version received September 8, 1990 - Communicated by A.R. Gould

ABSTRACT Somatic hybrids between cultivated lettuce (Lactuca sativa) and a wild species L. virosa were produced by p r o t o p l a s t electro fusion. Hybrid s e l e c t i o n was based on inactivation of L. sativa with 20mM iodoacetamide for 15 min, and the inability of L. virosa protoplasts to divide in the culture conditions used. Protoplasts were cultured in agarose beads in a revised MS media. In all 71 calli were formed and 21 of them d i f f e r e n t i a t e d shoots on LS m e d i u m containing 0.1mg/l NAA and 0.2mg/l BA. Most r e g e n e r a t e d plants e x h i b i t e d intermediate morphology. These plants were confirmed as hybrids by isoenzyme analysis. The majority of somatic hybrids had 2n=4x=36 chromosomes, and had more vigorous growth than either parent. Hybrids had normal flower morphology, but all were sterile. INTRODUCTION Lettuce ( Lactuca sativa ), a member of the Compositae, is a widely cultivated leafy v e g e t a b l e crop. Several m o r p h o l o g i c a l types of lettuce such as crisphead, butterhead, leaf, etc., have d e v e l o p e d in a long cultiv a t i o n history. In each of these types many cultivars with desirable agronomic characters have been developed through conventional breeding programs. Resistance to pests and diseases is an important breeding objective and r e s i s t a n c e to several pests and diseases has been introduced into commercial varieties by the c o n v e n t i o n a l sexual cross (Ryder,1979, 1986). But the variation of resistance w i t h i n the same species is so limited that novel sources of resistance are needed (Ryder, 1986). The wild species L. virosa has been recognized as a potential source of desirable traits with a high degree of r e s i s t a n c e to the leaf aphid N a s o n o v i a ribis nigri (Eenink et al., 1982), powdery mildew caused by Erysiphe c i c h o r a e e a r u m (Lebeda, 1985), and also bacterial rot caused by P s e u d o m o n a s cichorii and E r w i n i a carotovora (Tsukada, 1986, unpublished). Attempts to introduce b a c t e r i a l rot r e s i s t a n c e into lettuce cultivars by sexual h y b r i d i z a t i o n w i t h L. virosa have been unsuccessful. Viable hybrid plants were o b t a i n e d only w h e n L. s e r r i o l a was used

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as an intermediate parent (Eenink et al., 1982). Somatic h y b r i d i z a t i o n can be an effective method for overcoming the difficulty in making a sexual cross. Somatic hybrids have been produced in various species, and some of them are being utilized in breeding programs (Austin et al., 1986; Fish et al., 1988; Kubo et al., 1988; Primard et al., 1988). As for Lactuca species , plant regeneration from protoplasts has been reported in L. sativa (Berry et al., 1982; Engler and Grogan, 1983; Brown et al., 1986 ) and L. saligna ( Brown et al., 1987), and somatic hybrids have been obtained between L. sativa and L. serriola (Matsumoto, 1987). This report describes the successful somatic h y b r i d i z a t i o n between L. sativa and L. virosa including m o r p h o l o g i c a l and cytological c h a r a c t e r i z a t i o n of the hybrids. M A T E R I A L S AND METHODS Seeds of L. sativa cv. Shinano green (crisphead type) and cv. Lollo rossa (leaf type), and L.virosa (provided by INRA-France) were surface-sterilized by 9% calcium hypochlorite for 10 min, and then rinsed three times with sterile distilled water. Seeds were aseptically sown on Murashige & Skoog (MS) m e d i u m (1952) in 200 ml conical flasks, germinated and grown for one month at 23~ with 3,000 lux and a 14h photoperiod. Leaves from month old plants were dissected into 1-2 mm strips and floated on enzyme solution (pH 5.6) containing 0.4% Macerozyme RI0 (Yakult Honsha Co.), 0.5% Cellulase RS (Yakult Honsha Co.), CPW salts, and 0.4M mannitol as an osmotic stabilizer. The enzyme treatment was for 18h at 25~ in darkness without shaking. Isolated protoplasts were washed three times with 0.4M mannitol solution by resuspending and centrifuging at 100g for 3 min. Protoplasts of L. sativa were treated with 5, i0, 15, 20 mM iodoacetamide (IOA) at 4, 15, 20, 25~ for 15 min, followed by three washings with 0.4M mannitol. Protoplasts of both species were mixed (i:i ratio) at a density of about 5xl0~/ml, then the fusion was performed with an electric somatic-hybridizer Shimazu SSH-I (Shimazu Co., Japan) by t r a n s f e r r i n g the

532 p r o t o p l a s t m i x t u r e into a f u s i o n chamber FTC03 (volume 0.8mi) composed of r i n g e d p a r a l l e l e l e c t r o d e s (distance 2 mm). For p r o t o p l a s t alignment, AC field at 100V/cm, IMHz for 5-15 s was applied to the p r o t o p l a s t mixture. Subsequently DC ~ q u a r e pulses at 0.5, 1.0, 1.5, 2.0, 2.5 KV for 2 0 - 3 0 # s were a p p l i e d to induce protoplast fusion. Fusion-treated protoplasts were kept at room t e m p e r a t u r e (approx. 25~ for 2h, then collected by c e n t r i f u g a t i o n at 120g for 2 min and suspended in liquid culture medium. The culture m e d i u m was based on revised MS, A and B (Engler and Grogan, 1983) which were 1/10, 1/2 strength MS respectively, lacking NH4NO3, and s u p p l e m e n t e d with 0-0.1 mg/l 2 , 4 - d i c h l o r o p h e n o x y a c e t i c acid (2,4-D), 0 - 0 . 4 m g / l ~ - n a p h t h a l e n e a c e t i c acid (NAA) and 0.3-0.7 mg/l 6-benzylaminopurine (BA). The agarose m e d i u m A c o n t a i n i n g e m b e d d e d protoplasts was floated on the liquid medium B a c c o r d i n g to the agarose (Sigma Type ~ ) bead culture method ( Shillito et al., 1983 ). P l a t i n g density was 0 . 5 - 1 . 0 • Culture dishes ( 6 0 x 1 5 mm) were m a i n t a i n e d at 25~ in darkness. Small p r o t o p l a s t - d e r i v e d calli were transferred to L i n s m a i e r & Skoog (LS) agar m e d i u m (1965) with 0.1mg/l NAA, 0.2mg/l BA, lmM 2 - ( N - m o r p h o l i n o ) e t h a n e s u l f o n i c acid (MES) and 3% sucrose in 9 0 • mm dishes, cultured at 23~ w i t h 1,000 lux and a 14h photoperiod. Shoots d i f f e r e n t i a t e d from calli were transferred to LS agar m e d i u m with 0.1 mg/l BA, then to hormone free LS m e d i u m for rooting. Regenerated plants were transferred to v e r m i c u l i t e in 12 cm pots in the greenhouse, then to soil after one month, to grow to maturity. For d e t e r m i n a t i n of the c h r o m o s o m e numbers, root tips were pretreated w i t h 0.002M 8-hydroxyquinoline for 4h, fixed in 1:3 acetic a c i d / e t h a n o l for 24h at room t e m p e r a t ure and stained a c c o r d i n g to Feulgen method ( F e u l g e n and Rossenbeck, 1924 ). Isoenzyme analysis was carried out on young leaves of regenerated plants. Leaf materials were h o m o g e n i z e d in a 50mM Tris-HCl buffer, pH7.5, c o n t a i n i n g i% 2 - m e r c a p t o e t h a n o l , 10mM dithiothreitol and 20% glycerol. E s t e r a s e and leucine aminopeptidase were separated by e l e c t r o p h o r e s i s on p o l y a c r y l a m i d e gels, and stained (Wetter and Dyck,1983). RESULTS

(Terada et ai.,1987) of n o n - t r e a t e d L. v i r o s a protoplasts. In the h y b r i d i z a t i o n s between cv. Shinano green and L. virosa no escape colonies were observed. There seems to be g e n o t y p i c d i f f e r e n c e s in p r o t o p l a s t interaction b e t w e e n two species. Protoplast fusion rate was greatly affected by the strength of AC field and DC square pulses. The combined t r e a t m e n t of 100 V/cm AC field and 1.5-2 KV DC pulses (2-3 times) induced f u s i o n rates above 10 %. An optimum dielectrophoresis AC field interval of 10 s with 5• protoplasts/ml was determined by the appearance of p r o t o p l a s t alignment. AC field intensities h i g h e r than 100 V/cm shortened the time for adhesion, but caused protoplast destruction w h e n DC pulses were added. Four to five DC pulse shocks at b e l o w 1 KV or 2-3 shocks at 1.5-2 KV e n h a n c e d the f u s i o n rate. The f u s i o n - t r e a t e d p r o t o p l a s t s plated in the agarose m e d i u m had a plating e f f i c i e n c y of about 0.05%, and formed visible colonies after 15-25 days of culture. There were 50 calli formed in the fusion e x p e r i m e n t b e t w e e n cv. Shinano green and L. virosa, 21 calli b e t w e e n cv. Lollo rossa and L. virosa. The largest n u m b e r (40) of calli were o b t a i n e d in the medium containing 0.1mg/l 2,4-D, 0.4mg/l BA (A) and 0.2mg/l NAA, 0.7mg/l BA (B). Small calli (l-3mm in diameter) were transferred to the shoot differentiation medium containing NAA and BA. A f t e r one month, 16 putative Shinano g r e e n + L , v i r o s a hybrids and 3 putative Lollo r o s s a + L , v i r o s a hybrids calli formed shoots. Shoots were transferred to hormone free LS medium and roots d i f f e r e n t i a t e d w i t h i n one month.

and D I S C U S S I O N

L. sativa p r o t o p l a s t s d i v i d e d efficiently to form calli and shoots under the culture conditions used in this study. Plating e f f i c i e n c i e s of cv. Shinano green and Lollo rossa after 15 days of culture were 15.5 and 34.4 %, r e s p e c t i v e l y . L. v i r o s a p r o t o p l a s t s , however, were not able to divide in such conditions. T h e r e f o r e IOA was applied to L. sativa to f a c i l i t a t e hybrid selection. The IOA t r e a t m e n t at c o n c e n t r a t i o n s below 10mM did not completely inhibit division of L. sativa p r o t o p l a s t s . The t r e a t m e n t of 20mM, 1 5 m i n at 4~ inhibited protoplast division completely. At h i g h e r t e m p e r a t u r e s (20, 25~ the 20mM t r e a t m e n t i n f l i c t e d serious damage on p r o t o p l a s t s . In the fusion experiments between cv. Loll rossa and L. virosa, IOA treated protoplasts of LolIo rossa o c c a s i o n a l l y divided. Two out of five r e g e n e r a n t s were Lollo rossa escapes. Such L. sativa escapes may be due to the "nurse effect"

Fig.l. Plants of L. sativa cv. Shinano green (A), L. v i r o s a (B) and somatic hybrid (C) b e t w e e n these species. A l l the somatic hybrids between Shinano green and L. v i r o s a had i n t e r m e d i a t e charact e r i s t i c s of both parents (Fig. i) including intermediate leaf morphology, color and q u a l i t y (Fig. 2-a). These hybrid plants had stiff hairs on the mid rib and stem surface w h i c h is a c h a r a c t e r i s t i c of L,virosa. Most hybrids had vigorous growth and the f l o w e r of them was larger than that of parents (Fig.2-b). These plants had the mitotic chromosome number of 2n=4x=36 (2n=18 from each parent, Fig. 3-a). A few plants had abnormal stunted growth, and asymmetric s l e n d e r leaves. The stunted plants had 40-53

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Fig.4. Isoenzyme patterns of esterase, i. L. sativa cv. Shinano green, 2. L. virosa, 3. somatic hybrid

Fig.2-a,b. Leaves (a), flowers (b) of cv. Shinano green (A), L. virosa (C) and somatic hybrid (B).

S p e c i e s - s p e c i f i c differences in the electrophoretic patterns of isoenzymes, esterase and leucine aminopeptidase, were observed between L. sativa and L. virosa. The regenerated plants with intermediate morphology had the banding patterns found in both parents (Fig. 4), which confirmed the plants as somatic hybrids. Hybrid plants bolted after 3-4 months in the greenhouse and flowered. The flower of these plants exhibited normal morphology, but all were sterile. Most of the somatic hybrids had traits (hard hairs, bitter taste, etc.) which were derived from the wild species. It is n e c e s s a r y to eliminate such undesirable traits by backcrossing the hybrids with L. sativa cultivars in order to develop useful breeding lines. The first backcrosses between somatic hybrids and L. sativa are now being carried out.

ACKNOWLEDGEMENTS Fig.3-a,b. Chromosomes of somatic hybrids. a. p o l y p l o i d with 36 chromosomes between cv. Shinano green and L. virosa b. a n e u p l o i d w i t h 28 chromosomes between cv. Lollo rossa and L. virosa chromosomes. Three somatic hybrids between Lollo rossa and L. virosa had an intermediate morphology. All of these plants had shoots with red pigment w h i c h is c h a r a c t e r i s t i c of Lollo rossa. Two of the hybrids w i t h intermediate m o r p h o l o g y had 36 chromosomes, and one w h i c h grew very slowly was an a n e u p l o i d with 28 chromosomes (Fig. 3-b). The hybrid plants had a range of chromosome numbers (28-53), but 16 out of 19 hybrids had 36 chromosomes. Such stable polyploidy may be due to the genetic a f f i n i t y of both genomes and may be related to the use of m e s o p h y l l p r o t o p l a s t s (Sihachakr et ai.,1989). The plants with more than 40 chromosomes possibly resulted from multiple fusion of protoplasts (Terada et al., 1987). I n c r e a s e d vigor of some hybrids might be due to the combination of the genomes from both species, resulting in heterosis.

I am grateful to M. Tsukada, M. Fujimori and Y. M i y a s a k a for helpful advice and discussion, and to K. Y a m a n i s h i for technical assistance. This work was supported in part by a Grant-in-Aid from the M i n i s t r y of Agriculture, Forestry and Fisheries of Japan. REFERENCES A u s t i n S, Ehlenfeldt MK, Baer MA, H e l g e s o n JP (1986) Theor. Appl. Genet. 71: 682-690 Berry SF, Lu DY, Pental D, Cocking EC (1982) Z. Pflanzenphysiol. Bd 1 0 8 : 3 1 - 3 8 Brown C, Lucas JA, Crute IR, Walkey DGA, Power JB (1986) Ann. Appl. Biol. 109: 391407 Brown C, Lucas JA, Power JB (1987) Plant Cell Reports 6: 180-182 Eenink AH, G r o e n w o l d R, D i e l e m a n FL (1982) E u p h y t i c a 31: 291-300 Engler DE, Grogan RG (1983) Plant Sci. Lett. 28: 223-229 Feulgen R, Rossenbeck H (1924) Zeitshr. Physik. Chem. 135: 203-248 Fish N, Steele SH, Jones MGK (1988) Theor. Appl. Genet. 7 6 : 8 8 0 - 8 8 6

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