Somatic Embryogenesis and Plant Regeneration from Cotyledon Protoplasts of Cucumis sativus Wisconsin 2843

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Cucurbit Genetics Cooperative Report 15:40-44 (article 14) 1992

B. Garcia-Sogo, M. Dabauza, L.A. Roig, and V. Moreno
Plant Cell Tissue Culture Lab, Biotechnology Department, Universidad Politecnica de Valencia, Cno. de Vera, 14. 46020-Valencia, Spain

Wisconsin 2843 is a cucumber breeding population with multiple disease resistance (12). The population is homozygous for resistance to cucumber mosaic virus (CMV), Pseudoperonospora cubensis (Berk & Curt) Rostow (downy mildew), Cladosporium cucumerinum Ellis & Arthur (scab spot rot), Pseudomonas lacrymans (E.F. Smith & Bryan) Carsner (angular leaf spot), and Colletorichum lagenarium (Ross.) Ellis & Halst (anthracnose). It is segregating for two levels of resistance to Sphaerotheca fuliginea (Schl. ex. Fr.) (powdery mildew, PM). One level is the intermediate type as seen in Cy 14; the other is the high resistance of WI 1983. The population is heterozygous for resistance to Erwinia tracheiphila (E.F. Smith) Holland (bacterial wilt) and to Corynespora cassiicola (Berk & Curt) Wei (target leaf spot). In addition, it is gynoecious with non-bitter fruits (lacking cucurbitacins) and has genes for parthenocarpy (12).

The transfer of other disease resistance to Wisconsin 2843 would be of great importance to the crop. However, the existence of sexual incompatibility barriers has hindered the exploration of sources outside Cucumis sativus. Protoplast fusion could help to overcome those incompatibility barriers. However, before that biotechnological alternative can be applied it is necessary to develop good and reproducible methods of plant regeneration from protoplasts of the recipient cucumber line.

In this work, we present a method for obtaining cultures of cucumber protoplasts with high mitotic activity in liquid media, a culture system which has presented problems in other research (1, 11, 13). In addition, we show how to obtain embryogenic calli at an acceptable rate and to regenerate normal plants from somatic embryos.

Plant material. Seeds of Wisconsin 2843 were obtained from Dr. C.E. Peterson. Cotyledons from 5- to s-day-old seedlings, axenically germinated in MG medium (8) were used as starting material. Before protoplast isolation, cotyledon explants were precultured for 3 days in darkness at 28 ˚ C in NB 2/5/1.0 solid medium consisting of 2.5 mg/l NAA, 1.0 mg/l BA, M&S (10) mineral salts, 100 mg.l myo-inositol, 1 mg/l thiamine-ClH, SH-vitamins (16), 30 m/l gentamycin, 30g/l sucrose and 8 g/l agar (Technical No. 3, Oxoid). The pH was adjusted at 5.7 before autoclaving at 115 ˚ C.

Protoplast isolation. The method was previously described for melon (4, 7) with some modification. Briefly, cotyledons were cut into 1 to 2 mm wide strips and placed in enzymatic medium at the ratio of 1 g per 8 ml of medium for 12 to 14 hours at 28 ˚ C in darkness in a reciprocal shaker at 100 strokes/min (amplitude 20 mm). Enzyme solution consisted of 1.5% cellulase Onozuka-R-10 (Yakult-Honsha Co., Ltd.) in the washing medium LG-0.5 (M&S macronutrients, 0.4-mannitol, 0.1 M-glycine, 0.5 mM MES). The pH was adjusted to 5.7 and the solution was sterilized by filtration. Crude protoplast suspension was filtered through 85 μ m nylon mesh and purified by centrifugation at 75g for 10 min, and resuspended in the purification medium F-0.65 (consisting of M&S macronutrients, 0.6M-sucrose, 0.5 M-glycine, 0.5 mM MES). An overlay of protoplast ring which formed at the interface between the sucrose solution and the overlaid mannitol solution was collected with a Pasteur pipette and resuspended in the washing medium. After two rinses in that medium by centrifugation at 75g for 10 min, the protoplasts were resuspended in the culture medium DNB 1.0/0.5/0.5 at the final density of 0.75 to 1 x 105 protoplasts/ml and distributed into 55 mm glass petri dishes at the rate of 2.5 ml/dish.

Protoplast culture. The culture medium DNB 1.0/0.5/0.5 consisted of 1.0 mg/l 2,3,-D, 0.5 mg/l NAA, 0.5 mg/l BA, B5 mineral salts (3), 100 mg/l myo-inositol, SH-vitamins, 100 mg/l MES, 100 mg/l carbenicillin, 20 g/l glucose and 3.8 g/l xylitol. The medium was osmotically stabilized up to 0.5M with mannitol and the pH adjusted at 5.7 before autoclaving at 115 ˚ C. Medi8um osmolarity was gradually reduced throughout the culturing period by further additions of fresh mannitol-free culture medium: 0.5 ml after 5 days in culture and 1 ml each 5 days for two weeks until microcalli formation. Incubation was always at 28 ˚ C in darkness.

Embryogenic response. Microcalli (multicellular colonies) grown 20 to 25 days after protoplast isolation and culture were transferred to the embryogenic media and cultured by the double layer method (7) in 90 mm plastic Petri dishes at 28˚ C in darkness for 25 days. Embryogenic response in diverse culture media was studied following a factorial design with three factors under control: 1) type of auxin (2,4-D and NAA); 2) concentration of auxin (1.0 and 2.5 mg/l); 3) concentration of BA (1.0 and 2.5 mg/l). In this case the basal medium consisted of M&S mineral salts, 250 mg/l glutamine, 100 mg/l myo-inositol, SH-vitamins, 100mg/l carbenicillin, 30 g/l sucrose and either 6 g/l (semicolid layer) or 8 g/l (solid layer) agar. (Technical No. 3, Oxoid). The pH was adjusted to 5.7 before autoclaving.

Minicalli grown in these semisolid media were transferred after 25 days to the same solid culture medium (with 8 g/l agar), distributed in 250 ml glass culture vessels filled with 40 ml solid medium, and incubated under the same conditions to obtain proembryogenic calli. Proembryogenic calli were transferred to new culture media with a reduced concentration of auxin and cytokinin to study their effect on formation and development. A factorial design was carried out with two factors: NAA (0.01; 0.1; 0.5 mg/l; and BA (0.01; 0.1; 0.5 mg/l). The basal medium was M&S mineral salts, 100 mh/l myo-inositol, SH-vitamins, 30 mg/l gentamycin, 30 g/l sucrose and 8 g/l agar (Technical No. 3, Oxoid). Cultures were incubated under 16 h photoperiod (1000 lux = 45 μ E*s-1 *m-2) for 25 days.

Embryonic lines and plant regeneration. The embryogenic calli obtained in any of the preceding media were kept as embryogenic lines by subculturing every 20 days in media NB 0.1/0.1 or NB 0.01/0.01. It was possible to regenerate normal plants by culturing on basal medium without regulators the cotyledonary somatic embryos developed in media with a low concentration of NAA and BA.

Results. The yield from culture was 0.5 to 1.0 x 106 protoplasts/g of precultured cotyledonary tissue. The culture in medium DNB 1.0/0.5/0/5 was as follows. There was a high rate of cell wall regeneration (80 to 90% of cultured protoplasts) on the 2nd or 3rd day followed by intense mitotic activity leading to a plating efficiency (percentage of dividing cells) of nearly 80% after 4 to 5 days in culture. That mitotic capacity was maintained during culture so that after 25 days, the dishes were covered by a mass of microcalli with the liquid medium almost consumed. Periodic addition of fresh medium facilitated the high mitotic rate of the cells and prevented browning problems.

Others have reported methods for cucumber protoplast culture (1, 6, 11, 13, 17). In order to attain a reasonable plating efficiency, most of them have made use of solid media or gelled microdroplets for culturing the protoplasts since they obtained low results when liquid culture media were used (1, 11, 13). Only Trulson and Shahin (17) reported high plating efficiencies (40 to 60%) with Wisconsin 2843. That increase in the mitotic activity did not seem to be a function of genotype used, since we obtained similar results when using other genotypes. That suggests the most important factor was the preculture of the cotyledon explants before isolation of protoplasts, always used as a routine protocol in our experiments.

The adventageous effect of preculture of plant material on the latter cell division rate of the cultured protoplasts had already been observed in previous experiments carried out in our laboratory where the same medium (DNB 1.0/0.5/0.5) was used for culturing leaf and cotyledon protoplasts from melon (7, 8, 15) and from other Cucumis Species (2,14).

Culture of p-microcalli in media with NAA / BA. Growth type and intensity were quite similar in the four assayed media (NB 1.0/1.0; NB 1.0/2.5; NB 2.5/1.0 and NB 2.5/2.5). In all cases, minicalli of 3 to 4 mm in diameter, white-cream in color, and most of them friable were formed. In any case visible proembryogenic structures developed. With those microcalli were subcultured in the corresponding solid media and incubated under darkness, calli of approximately 1 cm diameter, light cream color, friable (and which disintegrated in globular structures) were formed. Creamy calli with proembryogenic structures appeared only sporadically in media NB 1.0/2.5, NB 2.5/1.0 and NB 2.5/2.5 (Table 1). The remaining non-embryogenic calli were transferred to media with reduced levels of NAA and BA and no embryogenic response was obtained in any case.

Table 1. Influence of combination of 2,4 D/BA and NAA/NA on cultural and embryogenic response from cucumber cotyledon p-minicalli.

Culture medium

Growth indexz

% of embryogenic calliy
NB 1.0/1.0 1.69 + 0.05 0.0 + 0.0
NB 1.0/2.5 2.71 + 0.05 3.3 + 1.6
NB 2.5/1.0 1.86 + 0.06 3.3 + 1.6
NB 2.5/2.5 1.81 + 0.06 0.8 + 0.8
DB 1.0/1.0 2.00 + 0.10 8.4 + 3.6
DB 1.0/2.5 2.08 + 0.08 6.7 + 3.2
DB 2.5/1.0 1.67 + 0.09 11.5 + 4.4
DB 2.5/2.5 1.54 + 0.07 15.0 + 5.8

z Growth index was calculated by assigning an arbitrary value, ranging fro 0 to 3, to each callus growth estimated qualitatively: 0 = no growth, 1 = little growth, 2 = moderate growth, 3 = much growth (mean + SE).
y Frequency of embryogenic calli giving whole plants + SE; SE = √ P (1 – P) / N , where p = % of calli with somatic embryos and n = total number of calli.

Culture of p-microcalli in media with 2.4D/BA. In the four semisolid media studied (DB 1.0/1.0; DB 1.0/2.5; DB 2.5/1.0 and DB 2.5/2.5) growth type and intensity were also quite similar. Most of the minicalli grown on those media were friable, light cream colored, without visible morphogenic structures, and of a reduced size (2 to 3 mm in diameter). Both the slow growing the the absence of light seemed to be advantageous in the prevention of browning. Besides this, although only sporadically, some proembryogenic minicalli had already appeared at that premature level. That response was, however, more generalized and at higher frequencies after subculturing in the same solid media (Table 1). In the four solid media, two kinds of calli were formed: non-embryogenic calli presenting friable, cream-white colored zones and glomerular structures (potentially embryoids); and soft, yellow colored embryogenic calli, with proembryos and white embryos similar to, but bigger than those obtained in the semisolid media. The highest frequency of embryogenic calli able to regenerate plants (15%) was achieved in medium DB 2.5/2.5.

Both the morphogenic potential and the plant regeneration capability of those calli are not lost by successive subcultures in media with reduced concentrations of NAA and BA. Moreover, the number of embryo-producing calli can be increased if the non-proembryogenic calli grown on the 2,4-D/BA media are transferred to those media. With that procedure, an additional 18% of calli with somatic embryos could be obtained.

Plant regeneration. Whole plants were obtained without problem, since practically every embryogenic callus developed in any of the above-mentioned media gave rise to normal cotyledonary embryos which germinated and grew easily on the basal medium without plant regulators. The average of normal plants coming from each embryogenic callus ranged between 1 and 3.

Many have reported embryogenesis from cucumber protoplasts (1, 6, 11, 13, 17). Frequencies of enbryogenic calli, when reported, were comparable (1, 13, 17) to those described here. However, when plant regeneration was taken into account the results differed. Trulson and Shahin (17) reported frequencies of embryogenic calli giving whole plants less than 1%; Punja et al. (13) reported 18 plants regenerated and Colijn-Hooymans et al. (1) reported problems with plant regeneration in their work on cotyledon-derived cucumber protoplasts. In our study, the frequency rose to 15% in the first phase and was increased another 18% by the second phase of subculturing. In previous works carried out with protoplasts from leaves and cotyledons of other cucumber genotypes, we observed that the frequency of calli with embryos was much higher than the frequency of embryogenic calli capable of giving whole plants. Fortunately, in this study we did not have those problems, perhaps due tot he fortunate choice of the cucumber genotype, or to the fortuitous choice of cultural parameters.

The effectiveness of the regeneration procedures was, of course, too far from the ones previously achieved by us for melon protoplasts (4, 7). However, we are improving this protocol in successive experiments. The results of this study permit the setting up of a method for culturing cotyledon protoplasts of cucumber in liquid medium with a high mitotic activity. Moreover, the induction of somatic embryogenesis, the production of embryogenic lines and the regeneration of whole plants from somatic embryos in cotyledon protoplasts of Wisconsin 2843 over the relatively short period of 3 months had also been achieved.

Those results are valuable since they permit the use of this cucumber population as the recipient parent in a biotechnology program aimed at the introduction of new resistance to plagues and diseases by fusion of protoplasts. genetic transformation by electroporation of protoplasts should also be a valuable alternative in the achievement of interesting improvements in that population.

Acknowledgements. The authors express their appreciation to CICYT (Commission Interministerial de Ciencia y Tecnologia, Ministry of Education and Science, Spanish Government) for financial support (Project BIO89-0446). M. Dabauza and B. Garcia-Sogo are grateful for their Grants from the Autonomous Government of the Valencian Community (Spain) and from the Spanish Government, respectively.

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