PlantCell Reports

Plant Cell R e p o r t s (1991) 1 0 : 7 6 - 8 0

9 Springer-Verlag 1991

Somatic hybridization between selected Lycopersicon and Solanum species A. Guri

1 L.J. Dunbar 2, and K. C. Sink 2

1 D N A Plant Technology Co., 2611 Branch Pike C i n n a m i n s o n N.J. 08077-3723, U S A 2 D e p a r t m e n t o f Horticulture, Michigan State University, East Lansing, M I 48824, U S A Received August 13, t990/Revised version received F e b r u a r y 6, 1991 - C o m m u n i c a t e d by J. M. W i d h o l m

Summary.

Mesophyll protoplasts of an interspecific

Lycopersicon esculentum Mill. (tomato) x Lycopersicon pennellii hybrid plant (EP) were fused with callus-derived protoplasts of Solanum lycopersicoides Dun. using a modified PEG/DMSO procedure. The EP plant was previously transformed by Agrobacterium tumefaciens which carried the NPTII and nopaline synthase genes. Protoplasts were plated at 105/ml in modified KM medium and 16 days post-fusion 25 ug/ml kanamycin was added to the culture medium. During shoot regeneration, 212 morphologically similar putative somatic hybrids were delineated visually from kanamycin resistant EP's. Forty-eight shoots, randomly selected among the 212, were further verified as somatic hybrids by their leaf phosphoglucoisomerase heterodimer isozyme pattern. However, the resulting plants were virtually pollen sterile. In a second fusion, mesophyll protoplasts of Solanum melongena (eggplant) were fused with EP callus-derived protoplasts. Using the same fusion and culture procedure, only two dark green calli were visually selected among the pale green parental EP and verified as somatic cell hybrids by several isozyme patterns. These two calli have produced only leaf primordia in one and half years on regeneration medium. Abbreviations ABA - abscisic acid; BAP 6-benzylaminopurine; 2,4-D 2,4-dichlorophenoxy acetic acid; DMSO - dimethyl sulfoxide; GA 3 gibberellic acid; GOT - glutamate oxaloacetate; IAA - indoleacetic acid; IBA - indolebutyric acid; IDH - isocitrate dehydrogenase; M D H malate dehydrogenase; MES - morpholinoethane-sulfonic acid; PEG polyethylene glycol; 6-PGDH - 6-phosphogluconate dehydrogenase; PGI - phosphoglucoisomerase

Introduction

The genera Solanum and Lycopersicon include the important crops potato (Solanum tuberosum), eggplant Offprint requests to: K . C . Sink

(Solanum melongena) and tomato (Lycopersicon esculentum). In the past decade somatic hybridization has been used in an attempt to incorporate a wide array of agronomic traits, found mainly in wild species, into the above crops (Austin et al. 1986; Guri and Sink 1988; and Sihachakr et at. 1988). The two species Lycopersicon pennellii and Solanum lycopersicoides in particular are sources of valuable agronomic traits (Rick 1982) and both can be sexually crossed to tomato only when serving as the pollen parents (Rick 1979). Previously, protoplasts of S. lycopersicoides and L. pennellii were fused with tomato protoplasts by Handley et al. (1986) and O'Connell and Hanson (1987), respectively, and somatic hybrid plants were verified in both studies. Herein we report the production of somatic hybrid plants that included the genomes of all three of the above species, by fusing protoplasts of the sexual hybrid tomato x L.pennellii (EP) with protoplasts of S. lycopersicoides. In a parallel study, we also fused EP protoplasts with eggplant protoplasts. Eggplant has already been found to be sexually incompatible with tomato (Miwa et at. 1958) and is also probably sexually incompatible with L.

pennellii. Materials and methods Plant material.

Seeds of eggplant (S. rnelongena cv. Black Beauty; 2n=2x=24) were obtained from A.H. Hummert Seed Co., St. Louis, MO, while seeds of S. lycopersicoides (LA 1990); 2n=2x=24 were obtained from C.M. Rick, University of California, Davis, CA. Mieropropagated plants of two sexual hybrids (A54 and A181) between tomato (L. esculentum cv. V F 36) x L. pennellii LA716 (EP) which had been transformed by Agrobacterium tumefaciens by stem inoculation (Chyi et al. 1986) were kindly provided by Richard Jorgensen, DNAP, Oakland, CA. Transformation was by A. tumefaciens strain C58C1/ pGV3850 to introduce kanamycin resistance which was used herein as a seleetable trait for somatic hybrid cells.

Protoplast Isolation. Eggplant leaf-derived protoplasts were isolated from in vitro grown seedlings as in Guri and Izhar (1984) while leaf

77 protoplasts from 7-month-old EP plants, grown in a controlled environmental chamber, were obtained according to Guri et al. (1988). For callus protoplasts of EP, stem sections, 1.0 cm and sliced once longitudinally, were taken from micropropagated plants of A181 and placed on Uchimiya and Murashige (1974) basal medium supplemented with 3% sucrose, 1 mg/l 2,4-D, 0.2 mg/l kinetin, and 0.8% bactoagar (Difco, Detroit, MI) at 30~ in the dark. Primary callus was removed and transferred to the same medium and subcultured at 2-week intervals. Three grams of 3-month-old callus were mixed with 15 ml of enzyme solution containing 0.75% Cellulysin (Calbiochem, San Diego, CA), 0.75% Driselase (Kyowa Ilakko Kogyo Co., Tokyo, Japan), 0.2% Macerase (Calbiochem) dissolved in CPW salts (Frearson et al. 1973) + 0.6 M sorbitol at pH 5.6 and incubated for 12-16 hr in the dark at 26~ on a gyratory shaker (40 rpm). S. lycopersicoides hypocotylderived callus was obtained according to ltandley and Sink (1985). Three grams of 3-month-old callus (approx. 6 subcultures) were mixed with 15 ml of enzyme solution containing 1% Cellulysin, 1% Drisclase and 0.4% Macerase dissolved in CPW salts + 0.6M sorbitol at pI-I 5.6 under the conditions previously stated.

Protoplast Fusion. Protoplasts were prepared for both fusion pairs as in Guri et al. (1988) and mixed at a final density of 107/ml. Callus-derived S. lycopersicoides:leaf-derived EP (plant A54) protoplasts at 3:1, respectively, were used. For the second fusion, eggplant ( + ) EP, the mix was 3:1, leaf-derived ego-plant:callus-derived EP (using kanamycin resistant plant AI81). The fusion procedure in both cases was that described in Guri et al. (1988) except for these modifications: I) the fusions were conducted in test tubes (100 x 16 mm); 2) the fusion solution contained 20% PEG (MW = 8000 Sigma, St. Louis, MO); and 3) dilution with 4 ml of W5 solution + 0.05 M MES was done gently. Each fusion pair was carried out in triplicate experiments. Protoplast Culture. Following fusion of both combinations EP (+) eggplant and EP ( + ) S. tycopersicoides, protoplasts were washed three times with 1/3 strength protoplast culture medium + 0AM sorbitol. They were subsequently cultured at 5 x 104 protoplast/ml in modified liquid Kao and Michayluk (1981) medium in 100 mm x 15 mm Petri dishes as described in Guri et al. (1987). Protoplasts were initially cultured in the dark at 28~ Plating efficiency was calculated as the percent of total protoplasts undergoing divisions 14 days after fusion. Sixteen days post-fusion, 25 /tg/ml of kanamycin was added to the protoplast cultures of both fusion pairs. Four weeks after fusion the plates were exposed to dim light (3-4 #EmZs "1 cool white fluorescent tubes), and three weeks later p-calli (approx. 0.5 cm diameter) were

retrieved individually and placed onto Whatman #1 filter paper laye red on Murashige and Skoog (1962) (MS) basal salts and vitamins medium + 7% sucrose, 2 mg/l zeatin, 1 mg/l IAA and 0.5% Noble agar (Difco). Nine to ten weeks after fusion green p-calli were placed on MS + 3% sucrose (or 1.6% fructose), 2 rag/1 zeatin, 0.8% Noble agar and 50 ttg/ml kanamycin (MS2Z) under 35-60 #Em'2s 1 light intensity on a 16 hr photoperiod for shoot regeneration. Occasionally 0.1 mg/l GA 3 and ABA (both filter sterilized) were added to MS2Z regeneration medium. Regenerated shoots were excised 12 to 15 weeks after fusion, and were inserted in MS + 3% sucrose + 0.9% agar (Sigma) or 0.2% Gelrite (Carolina Biological Co., Burlington, NC). Rooted plants were planted in a 1:1 (v/v) mixture of peat and sand, and covered with clear plastic for two weeks in a controlled envirnoment chamber and eventually transferred to the greenhouse.

Isozyme analysis.

Callus material (approx. 0.2 g) and young leaves (approx 0.5 g) were homogenized as in Guri et al. (1988). The isozymes PGI and GOT were separated on 11% starch gel using the lithium borate-tris citrate system plt 8.3 (Scandalios 1969). The analyses for 6-PGDH, IDH and MD}I were conducted on 11% starch gel using the morpholine citrate buffer system pH 6.1 (Clayton and Tretiak 1972). Staining was done according to Conkle et al. (1982) for 6-PGDH, Soltis et al. (1983) for IDIt, or Vallejos (1983) for PGI, MDH and GOT.

Cytology. Chromosome counts and pollen staining were handled as in Guri et al. (1987).

Results

EP (+) S. lycopersicoides Division of protoplasts within the fusion plates began 6-8 days post-fusion and plating efficiency of visually detected heterokaryons was much higher in comparison to control plates with S. lycopersicoides or EP parental protoplasts (Table 1). The addition of 25 #g/ml of kanamycin, which is still in the selective range (Andre et al. 1986), 16 days post-fusion eliminated the formation of S. lycopersicoides calli and shoots. However, there was no selection against

Table 1. Plating efficiency and percent regeneration of protoplasts of L. esculentum x L. pennellii (EP), eggplant, S. tycopersicoides and fusion combinations. Protoplasts were cultured in modified Kao and Michayluk (1981) medium at 5 x 104 protoplasts/ml. Parent or fusion combination

.... Platinga efficiency (%)

Shoot regeneration (%)

No. somatic hybrids/ total selected calli

Degree of development and morphology

EP (mesophyll)

6 + 2b

12.5

--

Plants

EP (callus)

8 • 2u

15.6

--

Plants

egglant (mesophyll)

22 _+ 4b

87.5

-

Plants

Solanum lycopersicoides

5 -+ 1b

65.6

--

Plants

EP ( + ) eggplant

16 -+ 3

0

2/2000,

No roots Eggplant-like leaf

EP (+) S. lycopersicoides

31 _+ 4

96.0

(callus)

ameans based on 12 replications + S.E. bwithout kanamycin

212/258

Plant with intermediate leaf

78

Fig. 1. LeavesofL. esculentum xL.pennelli (EP) (left),somatichybrid EP (+) SL (center),and S. tycopersicoides.

the formation of EP calli. Subsequently, this fusion experiment yielded 212 kanamycin resistance calli that had a distinctively darker green color than the light green color of the rest of the calIi. Shoot regeneration frequency of these dark green calli was much higher in comparison to the parental caUi (Table 1). All excised shoots from these dark green calli readily rooted and subsequently were transferred to planting medium. The plants all had intermediate leaf morphology (Fig. 1); whereas, flower morphology (Fig. 2) resembled that of S. lycopersicoides. Among the visually determined putative somatic hybrids 48 randomly chosen plants were verified as somatic hybrids using PGI isozyme analysis (Fig. 3). The light green calli regenerated shoots similar to the EP parents and were identified as EP also using PGI analysis (data not shown). The chromosome number of 10 randomly verified hybrid plants showed that 9 had the expected mitotic chromosome number of 2n = 4x= 48 while one plant had only 45 chromosomes. None of these plants has set seed in self pollinations. Pollen viability on those 10 plants averaged 0.6% -+ 0.1. E P ( + ) S. melongena.

Fig. 2. FlowersofL. esculentum xL. pennellii (left),somatichybridEP (+) SL (center)and S. lycopersicoides.

Division of protoplasts began 4-6 days post-fusion and plating efficiency of the heterokaryons was intermediate between that of the parental control dishes (Table 1). The addition of 25 #g/ml of kanamycin 16 days postfusion arrested the formation of S. melongena calli. Only kanamycin resistant calli were placed on shoot regeneration medium. Two weeks later, two dark green spotted calli were phenotypically different from the otherwise light green calli. Isozyme analysis for IDH, MDH, 6-PGDH and GOT (data not shown) verified that

Fig. 3. Phosphoglucoisomerase(PGI) pattern for leaf tissue of: L. esculentum x L. pennellii (EP), S. lycopersicoides (SL), six selected

somatic hybrids (1-6), and EP + SL (mix). Arrow indicates heterodimerbands.

Fig.4. Leafprimordiaproducedbyan EP (+) eggplantsomatichybrid callus.

79 these two calli were somatic cell hybrids while ten randomly sampled light green calli were diagnosed as EP. The two somatic hybrid calli failed to regenerate on

MS2Z with 3% sucrose, but did form leaf-like primordia when 1.6% fructose was used (Fig. 4). Further attempts with GA 3 and ABA added to the regeneration medium to stimulate shoot production have been unsuccessful over a period of one and a half years. Isozyme assays for IDH, MDH and 6-PGDH were repeated 2 months later on leaf primordia (Figs. 5a,b,c) and reconfirmed the somatic hybrid status. IDH and 6-PGDH showed the formation of heterodimer bands while MDH revealed the presence of both pairs of parental bands in the somatic hybrids.

Discussion

Fig. 5a,b,c. Isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), and phosphogluconate dehydrogenase (6-PGDH) patterns respectively for leaf tissue of: somatic hybrids 1 and 2, S. rnelongena (SM), L. esculentum x L. pennellii (EP) and EP + SM (mix). Arrow indicates heterodimer bands.

In the current study the fusion EP (+) S. lycopersicoides yielded more than 200 independently derived somatic hybrid plants. Plating efficiency for EP (+) S . lycopersicoides was considerably higher than that observed for the parents EP or S. lycopersicoides. Likewise, the frequency of shoot regeneration for the somatic hybrid calli exceeded that of the parentals. This hybrid vigor in both traits expressed in vitro undoubtedly led to ease in their selection as was the case with L. esculentum (+) S. ~ycopersicoides hybrids among their parental calli (Handley et al. 1986). The somatic hybrid plants which were obtained apparently retained most of both parental chromosomes since they had the expected chromosome number of 2n=4x=48. Previously, DeVerna et al. (1987) crossed a synthesized [tomato (2x) x S. lycopersicoides (lx) as female parent] with an L. pennellii diploid and obtained hybrids that had the haploid genomes of both tomato and L. pennellii (2x=24) plus one to four extra chromosomes of S. lycopersicoides. The remainder (34%) were strictly tomato x L. pennellii diploids. The difference in cytotypes obtained between the sexual and somatic hybridizations could arise mainly from the fact that the latter obtained in this study received two homologous sets of S. lycopersicoides (2x=24). The hybrids produced by DeVerna et al. (1987) could obtain several or no chromosomes of S. lycopersicoides depending on: 1) random segregation during meiosis and 2) chromosome(s) lagging-loss during subsequent mitosis. With regard to meiosis, DeVerna et al. (1987) reported that in their aneuploids, there was a strong tendency for pairing between tomato and L. pennellii homologous chromosomes while occasionally the remaining chromosomes of S. lycopersicoides paired to both tomato and L. pennellii chromosomes to form multivalents. Furthermore, the number of extra S. lycopersicoides chromosomes was negatively correlated with plant vigor and pollen stainability. In this study, the somatic hybrid plants were vegetatively vigorous and the low pollen fertility may be indicative of meiotic irregularities. Whether pollen viability will increase in time and enable the incorporation of the somatic hybrid germplasm into tomato breeding, remains to be determined.

80

In the second fusion, EP (+) eggplant, where the taxonomic distance is greater than that between EP and S. lycopersicoides, only two somatic hybrid caUi were recovered and their form of regeneration to date has been limited to leaf primordia. Such a recalcitrance could probably be due to strong somatic incompatibility between the eggplant and tomato x L. pennellii genomes. Although only two somatic hybrids were recovered among large numbers of parental p-calli, they were definitely unique by having in one nucleus the chromosomes of both tomato and eggplant coding for the isozymes analyzed. In other studies in which the taxonomic distance is even greater, like fusions between Petunia hybrida (+) L. peruvianum (Tabaeizadeh et al. 1985) or P. hybrida (+) N. tabacum (Pental et al. 1986), entire unilateral chromosome elimination was reported.

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