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A genetic analysis of cell culture traits in tomato

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A genetic analysis of cell culture traits in tomato
  Theor Appl Genet (1987) 74:633-641 9 Springer-Verlag 1987 genetic analysis of cell culture traits in tomato M. Koornneef C.J. Hanhart and L. Martinelli Department of Genetics, Agricultural University Wageningen, 53 Generaal Foulkesweg, NL-6703 BM Wageningen, The Netherlands Received March 7, 1987; Accepted March 20, 1987 Communicated by I. Potrykus Summary. Tomato genotypes superior in regenerating plants from protoplast and callus cultures were ob- tained by transferring regeneration capacity from Lyco- persicon peruvianum into L. esculentum by classical breeding. The genetics of regeneration and callus growth have been studied in selfed and backcross progenies of a selected plant (MsK93) which has 25% L. peruvianum in its ancestry. Segregation data showed that the favourable cell culture traits of L. peruvianum are dominant. Regeneration capacity from established callus cultures was controlled by two dominant genes. Callus growth on primary explants, callus growth of established cultures and shoot regeneration from ex- plants had high heritabilities (0.47, 0.78, 0.87, respec- tively). Callus growth and regeneration capacity were not correlated within the populations studied. Key words: Lycopersicon - Protoplasts - Regeneration - Callus growth Introduction The behaviour of plants in cell and tissue culture is determined to a large extent by medium composition and culture conditions. However, the plant species used and even the genotype within the species are also important additional factors. Intraspecific differences have often been described and analysed for particular tissue culture systems only. Examples of such genetic studies are the induction of callus on maize explants (Nesticky etal. 1983; Tomes and Smith 1985), the morphogenic response of explants in cauliflower (Bui- atti et al. 1974), the response to anther culture in potato (Jacobsen and Sopory 1978) and the regeneration of shootbuds from dedifferentiated callus tissue in Medi- cago (Reisch and Bingham 1980). The interaction of genetic differences with plant hormone composition in the media has been studied in Petunia (Izhar and Power 1977; Skvirsky et al. 1984) and Phaseolus (Mok et al. 1980). For tomato Lycopersicon esculentum Mill.), differences between cultivars and mutants in shoot and callus induction on primary explants have been de- scribed by many authors (Padmanabhan et al. 1974; Behki and Lesley 1976; Tal etal. 1977; Ohki etal. 1978; Frankenberger et al. 1981 a; Morgan and Cocking 1982; Kurtz and Lineberger 1983; Zelcer et al. 1984). Only in two cases (Ohki et al. 1978; Frankenberger et al. 1981b) were the cultivar differences analysed by comparing hybrids with their parents. Differences be- tween cultivars for the regeneration of shoots from subcultured callus, which in general is difficult in tomato (Locky 1983), were reported by Meredith (1979) and Tatchell and Binns (1986). Compared to L. esculentum, the related species L. peruvianum is much easier to regenerate from long- term callus cultures without preorganized areas (Mor- gan and Cocking 1982; Locky 1983) and from proto- plasts (Zapata et al. 1977; Miahlbach 1980; Thomas and Pratt 198la). Also, callus growth is more abundant in L. peruvianum. Thomas and Pratt (1981a) suggested transferring these favourable cell culture traits from this species into L. esculentum. Easy regenerating tomato genotypes allow the efficient application of cell biologi- cal techniques for both basic research in a plant species that genetically is well characterized and for the genetic improvement of this important crop species. In a programme designed to obtain these genotypes (Koorn- neef et al. 1986), callus growth characteristics and the ability of both primary explants and subcultured callus were analysed in segregating populations derived from hybrids ofL. esculentum and L. peruvianum.  634 Materials and methods Plant materials An F~ hybrid population of L. peruvianum• esculentum (IVT741505) and an L. peruvianum strain (PI 128650) were donated by Dr. Hogenboom of the Institute of Horticultural Plant Breeding (IVT), Wageningen, The Netherlands. The pedigree of this material is described in Koornneef et al. (1986). The F3 population was screened for plants with the ability to regenerate shoots on callus that had been in culture for at least 1 year. Upon crossing such plants with the male sterile mutant ms lO 3s of L. esculentum cultivar VFI1, one plant (K93) gave a single offspring. This backcross hybrid plant (FI MsK93) was selfed to give F2 MsK93, and was also backcrossed to VFlh The F2 population was screened for plants that were easy to regenerate from subcultured callus and that were crossable with VF1 h One F2 plant (F2 MsK93- 19) fulfilled both criteria and from its backcross to VF11, two plants were selected for further analysis (MsK8 and MsK9). The pedigree of the material analysed genetically for cell culture traits is shown in Fig. h The male sterile mutant, ms lO 35 in VFI 1 background was a gift from Prof. Rick, Davis, USA and was maintained by seed propagation; the cultivar Bellina was a gift from the Rijk Zwaan Seed Company and the cultivar Moneymaker from Nunhems Seed Company, both in The Netherlands. Plants of K93, MsK93, MsK8 and MsK9 were propagated by cuttings both in vitro and in vivo. Vegetative propagation was facilitated by the apparent resistance to tobacco mosaic virus of this material. Tissue culture and protoplast techniques Leaf discs (5 mm) were punched from surface-sterilized leaves of plants grown in a greenhouse. For shoot induction, these explants were placed on 2Z medium (Thomas and Pratt t981b). Callus was induced on similar explants on R3B medium (Meredith 1979) with vitamins (T) as in Tewes et al. (1984). This medium was used for all callus cultures. Plant regeneration on callus was achieved by repeated transfers to 2Z medium. All cultures were grown at 25 ~ and at 16 h light (approx. 2,000 lx) and transferred to new plates every 4 weeks. In all experiments 9 cm plastic Greiner Petri dishes were used. Several cell-culture traits were assayed per individual donor plant according to the scheme of Fig. 2. Shoot-like structures were counted on the 3 best looking (out of 6) explants placed on 2Z medium for 4 weeks. At the same time, callus weight (plus srcinal explant tissue) was deter- mined for the three (out of six) best looking R3B explants. Six approx. 15 mg calli from the remaining pieces were transferred to new R3B plates. From these cultures, 6 calli were trans- ferred again to R3B medium after 4 weeks; thereafter this established callus was used to assay the relative growth rate (RGR) by determining for the 6 calli the initial weight and that after 4 weeks. From the same source of callus material used for this RGR assay, 10 pieces were placed on 2Z medium. After 4 weeks, the best looking (green or shoot primordia containing) parts were transferred from each of these 10 calli. If after another 4 weeks on 2Z medium at least 1 of these calli showed a clearly visible shoot bud, the srcinal plant was said to be regenerable from an established callus culture. For protoplast isolation, plants were grown sterile in glass containers with Murashige Skoog (MS) salts, vitamins as in Tewes et al. (1984) and 1% sucrose. The plants were kept in the dark I day prior to harvesting of leaflets. These leaflets were floated in the dark at 4~ on a preincubation medium: ~/2 strength MS salts, T vitamins, 0.5 mg 1-1 benzylaminopurine (BA) and 1 mg1-1 2,4-dichlorophenoxyacetic acid (2,4-D). The leaflets were subsequently incubated for 16 h in an enzyme solution: 0.6% Cellulase RS and 0.2% Macerozym dissolved in CPW salts (Zapata 1981), and 73 g1-1 mannitol. L. esculentum cultivar VFII, ms-lO mutant) x K93 plant from F3 L. esculentum VFI___~I, ms-lO/ms-lO x F1 MsK93 one single plant) I I i I I I I I I F2 ltsK93 F3 MmK93-19 x L. peruvianum I F2 MsK93-19 x VFII Ms-lO/ms-lO | j J f i I Fig. 1. Pedigree of MsK93 and its progeny. @ : selfing I I I MsK I--l---q I I I MsK [ l I  635 9 R3B Weight of primary callus per explant R3B R3B Relative f callus growth r te 9 9 Number of shoots per explant Shoot formation on at least one out of i pieces Fig. 2. Assay for cell-culture traits in tomato. Ar rows with R3B or 2Z refer to transfer to R3B and 2Z mediums, respectively at 4 weeks intervals The protoplasts were purified by flotation on a sucrose solution (180 gl-1), using low speed centrifugation. The float- ing protoplasts were then pelleted in W5 wash solution (Menczel etal. 1982) and cultured in 5 ml TM-2 medium (Shahin 1985) with a higher sucrose concentration (102.7 g1-1 sucrose). During the liquid culture, 0.5 ml fresh modified TM- 2 medium was added every 3-5 days. Sucrose (68.5 gl-1), NAA (0.5 mgl -a) and zeatin (0.25 mg1-1) concentrations are different from Shahin s TM-2. After 3 weeks, microcalli were transferred to a solid medium similar to Shahin s TM-3 medium, but with 36.4 gl -~ mannitol, 2.5 gl -~ sucrose, 0.5 mgl -~ BA and 0.1 mg1-1 NAA. Callus pieces from this medium were transferred to 2Z medium from which the most healthy and greenish pieces were transferred to new 2Z plates every 4 weeks until well developed shoots were present. Shoots were rooted on R3B medium with 10 gl -~ sucrose without hormones. esults Repeatability of the tissue culture systems In the successive stages of breeding tomato genotypes with favourable cell culture traits, a number of plants of VFll, MsK93, K93, MsK8 and MsK9 were tested in replicates in different seasons (see Fig. 2 for testing scheme). The averages of the data obtained are presented in Fig. 3. Analyses of variances for VFll and MsK93, which were tested in all four experiments, indicated that the differences between these two genotypes and between the seasons are significant (P<0.01) for the three parameters. The genotype/season interaction was not significant. The relatively low growth rate of callus in the 1984 season can be explained by the larger initial weight of the callus pieces (6 pieces of approx. 100 mg each instead of approx. 15 mg in the other seasons). For each genotype tested in replicates and in different seasons, shoot regeneration from established callus cultures (defined as described earlier) is highly re- producible (Table 1). Genetics of cell culture traits in the progeny of MsK93 From the F2 MsK93, obtained by selfing plant F1 MsK93, 135 randomly chosen plants were tested for cell  636 1.8 1.7 1.6 1. 5 c 1.4 a. 1.3 w 1.2 ~ 1.1 ~ 1.0 ~ 0.9 ca 0.8 L,~ 0.7 l 0.6 ~ 0.5 ~ 0.4 ~ 0.3 0.2 0.1 0 0 3O 28 26 -.J 24 .,-i .~. ~ o ~ 16 ~ a ~ 6 ~ 4 2 0 18 17 16 15 q ~. 13- a,~ 12 ~ ~o 9 Z 7 N 6 ~ 5 4 3 2 1 0 i \ \ \ \ \ \ \ \ \ i [7--7] 84. a4/'a5 85 '86 Fig. 3. Averages of plant values for weight of primary callus, relative growth rate of callus and shoot number per explant. VFll used as seed- lings, the other 4 genotypes as cuttings VF MsK93 K93 MsK6 culture traits in 1984 together with several VF11 and FI MsK93 plants. The frequency distributions are shown in Fig. 4. Shoot regeneration on primary explants is presented as In 2+shoot number/explant) as this transformation resulted in approximately normal distri- butions for genetically identical plants. This was also the case for other seasons data not shown). MsK9 Broad sense heritabilities and genetic correlation coefficients for three cell culture parameters are given in Table 2. The genetic variances and covariances re- quired were obtained indirectly, i.e. by subtracting environmental co) variances from total co) variances of F2 MsK93. The former are estimated by the total co) variances of the isogenics VF11 and MsK93.  637 0 u. 40 0 ~ 3O z 20 10 VF11 MsK93 I 0 CALLUS MsK93 d 0 6 1,2 1,8 2.4 WEIGHT g)/ EXPLANT I i 3.0 VF11 MsK93 MsK93 selfed i i i J i 0 5 10 15 20 25 RELATIVE GROWTH RATE OF CALLUS VF11 MsK93 1 MsK93 selfed //// /11/ //i 1/// /// i i i 1 2 4 Ln OF SHOOT NUMBERIEXPLANT Fig. 4. Frequency distribution of plants for weight of primary callus, relative growth rate and In (2+number of shoots/explant). Hatched areas indicate the number of plants that regenerate from established callus cultures. White areas: plants that do not re- generate from these cultures. Dotted areas. plants not tested for regeneration Table 1. The number of regenerable (R) and non-regenerable (N-R) plants of genotypes that were tested in replicates and in different seasons Season Genotype VF11 MsK93 K93 MsK8 MsK9 R N R R N R R N R R N R R N R 1984 0 20 6 0 1984/85 0 8 5 0 1985 0 9 10 0 9 0 0 1 0 1986 0 7 7 0 7 0 7 0 6 1 Totals 0 44 28 0 16 0 8 0 7 1 The heritabilities were relatively high for all traits, whereas a significant genetic correlation was only observed for the two callus growth parameters and not for shoot regeneration and callus growth. By comparing the numbers of regenerators versus non-regenerators from established callus cultures for the three parameter classes (Fig. 4) a significant positive association (X 2- test; P < 0.01) was observed only with shoot regenera- tion from primary explants. However, this association is far from absolute. Table 2. Heritabilities (italics) and genetic correlation coef- ficients of three cell culture traits in the F2 MsK93 Callus Relative Shoot no. weight growth per explant per explant rate Callus weight/ 0.47 0.53 -0.04 explant Relative growth O. 78 -0.03 rate of callus Shoot no./explant 0.87 From 10 F2 plants with the capacity to regenerate shoots from established callus cultures, F~ progenies (in total 56 plants) were tested in a similar way to the F2. In view of the absence of a correlation between callus growth and shoot regeneration, these 10 F2 plants can be considered a random sample for callus growth. The correlation coefficient between these individual F2 plants and their F3 progeny (weighed by progeny size) was 0.27 for weight of primary callus and 0.77 for the relative growth of established callus cultures. This confirms the higher heritability for the latter trait, which was also found for the F2 population (Table 2). Also, within the F3 there was no significant correlation between callus growth and shoot regeneration.
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