Plant Cell Reports (t995) 14:666-669 PlantCell Reports �9 Springer-Verlag 1995

Screening for in vitro shoot-forming capacity of seedling explants in bell pepper (Capsicum annuum L.) genotypes and efficient plant regeneration using thidiazuron

Attila Szfisz 1, Giuseppe Nervo 2, and Mikl6s Fhri 1

1 Agricultural Biotechnology Center, H-210t G6d6116, P.O. Box 411, Hungary 2 Research Institute for Vegetable Crops, Via Paulese 28, 20075 Montanaso Lombardo, Italy

Received 11 November 1994/Revised version received 19 December 1994 - Communicated by H. L6rz

Summary. In vitro shoot regeneration ability o f t7 (7 Italian and I0 Hungarian) bell pepper genotypes was investigated using excised cotyledons and rooted hypocotyls as explants. Most o f the Italian genotypes and two o f the Hungarian genotypes responded well, producing shoots from rooted hypocotyls. Only two genotypes (one Italian and one Hungarian) gave a weak response using cotyledons. For direct shoot induction in these explants, in addition to the methods cited in the relevant papers, a new method was applied using thidiazuron as a cytokinin. Shoots were successfully regenerated from cotyledons o f two Italian and two Hungarian genotypes using thidiazuron which were considered to be non responsive to the usual methods,

Abbreviations: BAP: 6-benzylaminopurine; GA3: gibberellic acid; IAA: indole-3-acetic acid; MS medium (Murashige and Skoog 1962); TDZ: N-phenyl-N- thiadiazol-l,2,3-5-ylurea (thidiazuron); PVC: polyvinyl chloride

Introduction

Pepper is an important vegetable crop grown worldwide. There have been several reports on regeneration of plants from adventitious shoot buds induced by hormone treatment in excised cotyledons and hypoeotyls o f pepper (F~i and Czak6 1981, Phillips and Hubstenberger 1985, Sripichitt et al. 1987, Agrawal et al. 1989, Ochoa-Alejo and Ireta-Moreno 1990, Arroyo and Revilla 1991, Ebida and Hu 1993). Cultivars differ markedly in their regeneration requirements (F~i et al. 1990, Ochoa-Alejo and Ireta-Moreno 1990). Regeneration is affected by the developmental stage o f the donor plant (Morrison et al. 1986), location o f the explants (F~iri and Czak6 1981) and environmental factors such as temperature, light intensity and light regime (Phillips and Hubstenberger 1985). Recently, shoot bud induction and successful plant regeneration was obtained using decapitated, rooted hypoc0tyls o f different Mexican chili pepper varieties

Correspondence to: A. Szfisz

cultured for an interval in the upside-down position (Valera-Montero and Ochoa-Alejo 1992). However, application of cell and molecular biology techniques to pepper genetic improvement is limited because of the difficulties in efficient development o f induced buds into complete plantlets (Liu et al. 1990, Wang et al. 1991). We describe investigations carried out on important bell pepper varieties and hybrids grown in the Carpatian basin and in Italy. The most beneficial methods and media described earlier were employed to estimate the shoot-forming capacity o f 17 Italian and Hungarian genotypes. The aim was to select the most responsive genotypes for later genetic manipulation. For this reason a simple and rapid screening method was applied. The shoot forming capacity o f the different genotypes was compared with that o f a highly responsive, standard inbred line (No. 40017 R- 13).

Materials and methods.

Establishment of aseptic plants. Seeds of the cultivars and hybrids were provided as follows, Quadrato d'Asti, Cuneo, Cuneo LS ISO, Nocera, Nocera LS ISO, Mottese (Research Institute for Vegetable Crops, Montanaso, Italy), No. 40017 R-13, Feh6rOz6n, Ttlltos, Suptol, Greygo, Syn. Cecei, TH 160, Novares F1, Novator Fp Rapires FI, Rapidus F](Vegetable Research Institute, Budapest, Hungary), Piknik (Pannon Agricultural University, Keszthely, Hungary). Seeds were surface sterilized by presoaking for a day in distilled water, then treated with 70% (v/v) ethanol for 1 rain. Subsequently, they were immersed for 20 min in a solution of 7.5% (w/v) CaCI202 supplemented with a few drops of Tween 20. Thereafter, they were rinsed three times for 5 rain with sterile distilled water, then were sown in sterile plastic tissue culture containers (Plant Box, Kontaplant Co. Ltd., Szentes, Hungary) containing hormone free MS-medium, pH 5.8, solidified with 0.8% agar-agar (Sigma) and incubated in a culture room at 25~ with a photo period of 16 h/day (cool white fluorescent light, 35 pxnol m2s-1).

Culture Media. MS-medium was used as a basal medium supplemented with B 5 vitamins (Gamborg et al. 1968) and with 10 g1-1 each of sucrose, glucose and maltose as carbohydrate sources (MSBs) except for rooting. Nine different adventitious shoot bud inducing media were utilized: Medium 1: containing 2.0 mg1-1 BAP + 1.0 mg1-1 LAA (Gunay and Rao 1978); Medium 2:5.0 mgl -l BAP + 0.3 mg1-1 IAA (Valera-Montero and Ochoa-Alejo 1992); Medium 3:11.3 mgl "~ BAP + 0.54 mg1-1 IAA (Fhri et al. 1992); Medium 4:0.88 mg1-1 TDZ; Medium 5:1.1 mg1-1 TDZ; Medium 6:1.76 mgl 1 TDZ; Medium 7:2.2 mg1-1 TDZ; Medium 8:2.75 mgl q TDZ; Medium 9:3.3 mg1-1 TDZ

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Two different media were used for elongation of the shoot buds induced on cotyledonary explants: Medium 10:1.65 mg1-1 BAP + 1.0 mgl 1 GA 3 (F~i et al. 1992); Medium 11:1.0 mgl 1 GA 3 Rooting medium: Medium 12: MS-medium supplemented with 0.5 mgl q IAA.

Type of the explants. As explants, cotyledons and rooted hypocotyls of 12-14 day old seedlings were used. Intact cotyledons with petioles were cut from the plants. Rooted hypocotyls were obtained by the methods of Valero-Montero and Ochoa-Alejo (1992). The seedlings were decapitated with a pair of scissors some millimeters below the cotyledonary node. The roots were freed from the gel and trimmed to 8-10 ram.

Inoculation. Cotyledons were put individually in 15x2.5 cm glass tubes containing 10 ml of one of the shoot bud inducing media (Medium 1 to 9). Both the tip of blades and the petiole were put into the medium. Rooted hypocotyls were placed upside-down into the medium in similar glass tubes. Explants remained there and were kept for 21 days under the same conditions as for germination. All the treatments had 5 replicates.

Elongation of vegetative buds. Cotyledons with adventitious shoot buds treated by conventional methods (Medium 1 to 3) were transferred into 100 ml Erlenmeyer flasks containing 25 ml of Medium 10. Cotyledons incubated on TDZ containing media (Medium 4 to 9) were put on the culture medium without BAP (Medium 11). Rooted hypocotyls were cleaned to remove the adherent shoot bud inducing medium and were placed on Medium 12 in plastic containers. Roots were placed in contact with the medium but the rest of the explant was kept away from it.

Rooting of elongated shoots. The regenerated shoots were cut from the explants and transferred into plastic containers containing Medium 12. After rooting they were put into 500 ml jars on sterile perlite:soil (1:1) mixture wetted by sterile distilled water. The glasses were covered by PVC foil, which gradually was removed helping the acclimatization of the plants (F~i et al. 1987).

R e s u l t s

Shoot bud induction and shoot regeneration in cotyledon expIants

Table 1. shows the shoot forming capacity (given in average number o f regenerated shoots/explant) o f cotyledon explants derived from different genotypes treated with various concentrat ions of BAP + IAA or TDZ, respectively. On the media conta in ing BAP + IAA (Medium 1 to 3) in addit ion to the standard inbred line (No. 40017 R-13) only cv. Cuneo LS ISO and cv. Piknik regenerated shoots. On M e d i u m 1, in consequence of the higher IAA concentra t ion (1 mg1-1) strong root and callus formation was found to occur at the cut surface o f the petiole, which inhibi ted the development o f the adventi t ious shoot buds. On Media 2 and 3, this phenomenon was not so apparent. After transferring onto shoot e longat ion med ium (Medium 10), the induced buds developed but they usual ly formed pale, leaflike structures only.

On the media conta in ing T D Z in various concentrat ions (Medium 4 to 9), callus format ion was found to be less frequent and the buds induced on the cotyledons were green and vigorous. After the transfer onto the shoot e longat ion med ium (Medium 11) one Hungar ian variety (cv. Piknik), one hybr id (Rapires F~) and three Italian varieties (cv. Quadrato d'Asti, Cuneo and Mottese) regenerated 0.8-2.6 shoots/cotyledon.

TABLE 1. The effect of different culture media on shoot-forming capacity of cotyledon explants of bell pepper cultivars and hybrids responded to any treatment.

Cultivars and Mean number of shoots per explant hybrids

Culture media* 1 2 3 4 5 6 7 8 9

Quadrato 0 0 0 o.8 0 0 o.8 0 o.8 d'Asti Cuneo 0 0.4 0 0 0 0 0 1.0 0 Cuneo 0.4 0 0 0 0 0 0 0 0 LS ISO Mottese 0 0 0 0 0.8 1.2 0 0 0.5 Piknik 0 0 0 1.0 0.6 1.4 1.4 0 0 Rapires F1 0 0 0 2.6 0.6 0 0 0 0.6 No. 40017 5.6 1.0 0.2 2.0 Z0 R-13

*MSB 5 supplemented with different growth regulators: (1) 2.0 mg1-1 BAP + 1.0 mgl-qAA (6) 1.76 mg1-1 TDZ (2) 5.0 mglqBAP + 0.3 mg1-1 IAA (7) 2.2 mgl l TDZ (3) 11.3 mg1-1BAP + 0.54 mgl "l/AA (8) 2.75 mgl-lTDZ (4) 0.88 mgl 1 TDZ (9) 3.3 mg1-1 TDZ (5) 1.1 mg1-1TDZ

Shoot bud induction and shoot regeneration in rooted hypocotyls

Table 2. shows the shoot forming capacity of rooted hypocotyls o f the investigated pepper genotypes treated with various concentrat ions o f BAP + IAA or TDZ. No shoots were obtained ha the rooted hypocotyls incubated on any media conta in ing TDZ. The cut surface of the hypocotyls were swollen greatly and adventit ious shoot buds were rarely found.

TABLE 2. The effect of different culture media on shoot forming capacity of rooted hypocotyls of bell pepper cultivars and hybrids responded to any treatment

Cultivars and hybrids Mean number of shoots per explant Culture media*

1 2 3 Quadrato d'Asti 0 0.8 0 Cuneo 0 0.8 0 Cuneo LS ISO 0.4 0.2 0 Nocera 0 0.8 0 Mottese 0 0.6 0 Suptol 2.2 0 0 Rapires F 1 0.2 0.6 02 No. 40017 R-13 0 2.4 0 *MSB 5 supplemented with different growth regulators:

(1) 2.0 mgl -l BAP + 1.0 mgl 1 IAA (2) 5.0 mg1-1 BtuP + 0.3 mg1-1 IAA (3) 11.3 mg1-1BAP + 0,54 mg1-11AA

On media conta in ing BAP + IAA (Medium 1 to 3), adventi t ious shoot buds were induced in rooted hypocotyls o f all the investigated genotypes, and eight genotypes were able to regenerate shoots from these buds to different degrees. The best and the most uni form regenerat ion was obtained on M e d i u m 2 (5.0 mgN BAP + 0.3 mgl -~ IAA). On Med ium 1, similar to the cotyledon explants, strong root and callus forrrtation was found to occur at the cut surface o f the rooted hypocotyl explants, which inhibited the development o f the induced buds. The only exception

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Fig.1. In vitro regeneration and e x vi tro R o development of bell pepper genotypes. A. Multiple adventitious shoot buds proliferated at the cut surface of cotyledons after three weeks of incubation in the regeneration medium. (cv. Quadrato d'Asti treated with 0.88 mgl "1 TDZ). B.-C. Elongated shoots derived from the cut surface of cotyledons. B. No.40017 R-13 inbred line used as a standard treated with 11.3 mg1-1 BAP + 0.5 mg1-1 IAA C. cv. Piknik treated with 0,88, 1.1 and 1.75 mg1-1 TDZ, respectively. D.-E. Rooted hypocotyl explants showing regeneration around the periphery of the cut surfaces. The hypocotyls were induced by 5.0 mgl -l BAP + 0.3 mgl "1 IAA D. No 40017 E. cv, Suptol F. Regenerated plant in the greenhouse (Rapires F I induced by 0.88 mgl -I TDZ).

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was cv. Suptol, which was able to regenerate shoots from the induced buds in a number comparable to that of the standard variety (2.2 shoots/explant). From the adventitious shoot buds induced on Medium 3, generally only leafy structures were formed, except for Rapires F~ which gave a weak response.

Rooting and ex vitro plantlet development

Rooting of 90-95% frequency resulted when the shoots formed on the explants were transferred onto Medium 12. The hormone treatments have not shown any negative effect on the rooting of these shoots. By ore" acclimatization method, plants were grown successfully and potted out in the greenhouse. The survival rate approached 100% and they grew into normal fruit bearing plants.

Discussion

For genetic improvement of pepper by methods of plant biotechnology a reproducible in vitro regeneration system is required. The aim of our investigations was to determine, by a rapid screening method, the shoot forming capacity of some pepper genotypes grown in the Central East European region. Methods described earlier for in vitro regeneration in pepper were optimized at different steps in our laboratory (F~ri et al. 1992). Effects of the carbohydrate source, dark treatment during the shoot regeneration and promotional effect of GA 3 on shoot elongation were assessed in successive assays. In addition, an inbred pepper line with excellent regeneration ability was selected (No, 40017 R-13), In our regeneration system we utilized the above mentioned line as a standard for shoot forming capacity, thus a particular pepper genotype can be correctly estimated, even in 5 replicates/treatment. In the evaluation of these experiments the number of regenerated complete shoots/explants only has been considered. Valero-Montero and Ochoa-Alejo (1992) spent about two years with experiments of plant regeneration via direct organogenesis in some Mexican chili pepper cultivars following methods described by other authors. They obtained mainly vegetative buds, which developed into abnormal leafy structures. Normal shoot development was rarely observed.

The responsiveness of the 17 pepper varieties and hybrids investigated turned out to be very different. This was not surprising because the strong genotype dependency on the regeneration ability in excised seedling parts had been known earlier (F~i et al. 1990, Ochoa-Alejo and Ireta-Moreno 1990). Ten genotypes did not respond to any kind of treatment, no shoots were obtained either from cotyledons or rooted hypocotyls (Feh6r6zOn, T~iltos, Greygo, Syn. Cecei, TH 160, Novares F~, Novator F~, Rapidus F v Nocera LS ISO). Seven other genotypes regenerated shoots on different media, but only one hybrid (Rapires F~) using cotyledons, and one variety (Suptol) using rooted hypocotyls, gave a response comparable to the result obtained by the standard line.

Thidiazuron (TDZ) is a substituted phenyl urea developed primarily as a cotton defoliant (Arndt et al. 1976). It has exhibited strong cytokinin like activity in various cytokinin bioassays (Mok et al. 1982). TDZ has a high efficiency in stimulating cytokinin-dependent shoot regeneration from a wide variety of plants (van Nieuwkerk et al. 1986, Malik and Saxena 1992, Huetteman and Preece 1993).To our knowledge, this is the first report on the effects of thidiazuron in direct regeneration of pepper. It has been shown in our results that a fraction of the pepper genotypes not inducible by conventional methods employing BAP and IAA as plant hormones could regenerate shoots in cotyledon explants after TDZ treatment (0.8-2.6 shoots/explant). Gill and Saxena (1993) reported the high efficiency of TDZ inducing direct embryo genesis in leaf disc cultures of tobacco (Nicotiana tabacum L). TDZ altered the shoot regeneration process from organogenesis into embryogenesis. Relying on our preliminary results, it is difficult to decide whether this phenomenon was similar to ours or not.

In conclusion, we have found a promising new way for direct regeneration in cotyledon explants of recalcitrant bell pepper genotypes, that may make it possible to use genetic manipulations in these varieties adapted to specific regions.

Acknowledgements. The authors would like to thank Dr. Gregory C. Phillips fbr critical reading of the manuscript.

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