Potato Research 42 (1999) 489- 498

In vitro microtuberization: an evaluation of culture conditions for the production of virus-free seed potatoes C.L. LI~ Station F6d6rale de Recherches en Production V6g6tale de Changins, CH-1260 Nyon, Switzerland Additional keywords: Solanum tuberosum L., microtubers, nodal cuttings, in vitro tuberization

Summary A tissue culture technique is described in which nodal cuttings of Solanum tuberosum L. are induced to form usable microtubers in order to produce high health status of the seed potato genotypes cultivated in Switzerland.

Introduction In Switzerland micropropagation through in vitro tissue culture is common practice to provide a high health status of the potato seed (Reust & L6, 1985). However, this practice is severely criticized due to the fact that it presents some disadv/mtages including the under-occupancy of facilities and of personel, work being often concentrated in a few periods of the year. A series of experiments were conducted to evaluate culture conditions leading to the production of usable microtubers in vitro, in order to avoid slack periods in the occupancy of facilities, and to allow a more flexible production planning implemented in the scheme of the pre-base potato seed production.

Material and methods In vitro plantlets of 11 potato cultivars of the Swiss assortment of potato varieties were used to perform the experiments. The process of in vitro microtuberization consisted of two stages: Production of in vitro plantlets. Shoot growth was examined by culturing single node cuttings in three tissue culture containers (glass tube, polypropylene jar (Plastem| and Polycarbonate box (Lifeguard| with liquid and solidified medium containing salt mineral CMS (Collet, 1985). Effect of nitrogen content referring to the Murashige & Skoog (1962) medium, and influence of the position of the explant on the quality of shoot growth were investigated as well. Production of in vitro tubers. Nodal explants were induced to tuberize firstly in basal mineral salt (Charles et al., 1993) containing 3% saccharose under long day conditions (16 h/day) for a month at 18• ~ and/or 8% saccharose-medium (L~, 1990) under short day conditions (8 h/day) for 2 weeks at 18 ~ day/14 night _+2 ~ Afterwards, they were transferred to complete darkness at 19_+1~ until tubers were collected. Potato Research 42 (1999)

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Influence of carbon source (L~, 1990), growth regulators (L8, 1993), growth retardant (Paclobutrazol) added alone or in combination with nitrogen (L~ & Page, 1996) on the quality of microtubers was evaluated with regard to the times of application. Effect of medium renewal was also examined during the darkness period. The length of dormancy period of in vitro microtubers was also determined regarding to the intensity of sprouting after different times of storage at +4 ~ Results and discussion

In vitro growth of plantlets. The type of container and support of culture had an important influence on the growth of the microplants in vitro (Fig. 1). A significant difference can be noted between the microplants cultured in glass-tubes, polypropylene jars or polycarbonate boxes.

Fig. 1. Effect of container on in vitro growth of potato miniplants (cv. Charlotte). Means followed by different letters are significantlydifferent at P = 0.05.

Growth is greatly promoted by the polycarbonate containers compared to the two other types of containers, whereas liquid medium was better than agar solidified medium (Table 1). The plant growth was stimulated when lower contents of nitrogen were added to the basal medium. Increasing the nitrogen content in the medium did not promote the growth of miniplants regarding the height of miniplants obtained after 1 month of culture (Fig. 2) and the number of usable nodal explants (Fig. 3). High contents of nitrogen reduced, on the contrary, the fresh weight of miniplants; particularly, they caused a significant decrease in dry weight as well (Fig. 4). In the present study, the higher growth rate was obtained on the medium supplemented with less nitrogen content (15 meq). The results confirmed the importance of low nitrogen level for the growth of miniplants in vitro as reported by Charles & Rossignol (1992). 490

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EFFECTS OF CULTURE CONDITIONS ON IN VITRO TUBERIZATION Table 1. Effect of support of culture on in vitro growth of potato miniplants (cv. Charlotte). Means within the same row with different letters are significantly different at P = 0.05. Medium

Height (cm) Fresh weight (mg) Usable plants

Agar

Liquid

8.11 • 0.23 a 193 • 11.17 a 4.74 • 0.10 a

10.65 • 0.15 b 250 • 7.91 b 4.80 + 0.07 b

Fig. 2. E f f e c t of nitrogen content on in vitro growth of potato miniplants (cv. Urgenta). Different letters indicate significant difference at P = 0.05.

Fig. 3. Effect of Nitrogen content on the yield of usable node explants (cv. Urgenta). Different letters indicate significant differentce at P = 0.05.

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Fig. 4. Effect of nitrogen content on the yield of fresh and dry weight (cv. Urgenta). Different letters indicate significant difference at P = 0.05.

By using explants originated from different parts on the main shoot, best growth capacity was found in microcuttings excised from the middle part compared to those provided from the top (0) and bottom (6) (Fig. 5). Similar results were also obtained previously with cultivar D6sir6e (L6, 1991) and other species including apple cultivars (Collet & L6, 1987).

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Fig. 5. Effect of position of explant on the growth of potato miniplants in vitro (cv. Bintje). 492

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h7 vitro tuberization. In vitro formation of tubers was effectively obtained on medium containing high content of saccharose alone (Fig. 6) or in combination with 2.5 mg/l benzyladenine in short day conditions (Fig. 7) (L6, 1990). But the tuber size was still small for practical use, despite the use of the tuberization process proposed by Charles & Rossignol (1993) for the synchronous formation of in vitro tubers (Fig. 8). There were significant differences between the percentage of tuberization and the weight of the microtubers obtained in different cultivars.

Fig. 6. Effect of saccarose content on in vitro formation of tubers (cv. Agria).

Fig. 7. Effect of growth regulators on in vitro formation of tubers (cv Agria). BA: Benzyladenine; Kin: Kinetin; CCC: Chlorocholinechloride. Potato Research 42 (1999)

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Fig. 8. Influence of genotype on in vitro formation of tubers. Such variability within clones induced to tuberize in vitro has also been reported with other potato varieties (Ranalli et al., 1994). In order to obtain larger microtubers for a direct planting under field conditions, explants were cultured in liquid medium supplemented with 30 or 60 mM nitrogen. High concentration of nitrogen (60 mM) seemed to be favourable to yield larger tubers (Fig. 9). There was a significant increase in the fresh weight o microtubers if nitrogen was supplied at the beginning or at the latest seven da',s after being transferred to the short day period (Fig. 10).

Fig. 9. Effect of Nitrogen supply on in vitro formation of tubers in 5 cultivars of Solanum tuberosum L.

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EFFECTSOF CULTURECONDITIONSON IN VITROTUBERIZATION

Fig. 10. Effect of Nitrogen supply (60 mM) and periods of application on in vitro formation of tubers in 8 cultivars of Solanum tuberosum L.

Furthermore, larger tubers were also produced by adding 0.3 mg/1 paclobutrazol to the medium, which enhanced significantly both weight and size of microtubers (Table 2). Improvement to the quality of microtubers produced in vitro was clearly obtained when both paclobutrazol (PCL) and nitrogen (N) were applied to the induction medium as mentioned above. The results obtained under our conditions showed that paclobutrazol/nitrogentreated cuttings produced larger tubers with a significantly higher mean fresh weight as compared to the control and to the N or PCL-treated cuttings (Fig. 11). The positive effects of PCL on in vitro tuberization are in agreement with previous results mentioned by Harvey et al. (1991), whereas the beneficial interaction between PCL and N on the quality of microtubers obtained in this study has not been reported previously. In addition, a high proportion of larger tubers can be produced when the culture medium was renewed during the darkness period (Fig. 12). Table 2. Effect of concentrations of Paciobutrazol on the development of potato microtubers (cv. Panda) in vitro. Means within the same column with different letters are significantly different at P = 0.05 by Duncan's multiple range test. Concentration (mg/1)

Weight (mg)

Diameter (mm)

0 0.05 0.10 0.20 0.40

106.31 • 6.61 a 169.10 + 19.90 b 172.73 • 20.56 b 254.81 • 23.65 c 238.61 • 17.15 c

4.35 + 0.17 a 5.27 • 0.31 b 5.36 • 0.35 b 6.17 • 0.34 c 6.63 • 0.27 c

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Fig. 11. Effect of Paclobutrazol and Nitrogen (alone or in combination) on the development of microtubers (cv Sirtema) in vitro.

Fig. 12. Effect of renewal of medium for 1 (lx), 2 (2x) and 3 times (3x) on the yield of larger microtubers (cv. Nicola) in vitro.

Dormancy release. The results obtained on the intensity of sprouting of in vitro microtubers show that the dormancy release increased with the time of storage at low temperature (+4 ~ (Fig. 13). Yet 50% of microtubers developed sprouts on most of cultivars after 3 months at low temperature. The average sprout development varied with cultivar. A maximum of sprouting (ca. 100%) was observed for a period of 16 weeks. These observations correspond to previous findings reported by Ranalli et al. (1994). More than 80% of the in vitro microtubers sprouted after three months under cold storage. 496

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Fig. 13. Intensity of sprouting (%) of microtubers stored for different times at low temperature (+4 ~

Acknowledgements We wish to thank the Direction of R A C - C h a n g i n s for facilities and Delley Seeds and Plants Ltd (Switzerland) for financial support. We are also grateful to the Stauffer Chemicals B V / C a r o u g e (Switzerland) for its generous gift of paclobutrazol.

References Collet, G.F., 1985. Enracinement amflior6 lors de la production in vitro de rosiers. Revue Suisse de Viticulture, Arboriculture et Horticulture 17 (4): 259-263. Collet G.F.& C.L. L~, 1987. Micropropagation de porte-greffe de pommier et de poirier. 1. Etablissement et multiplication in vitro de Pyrus malus L. (M25, M26, M27, MM106, M9 type Jork) et de Cydonia oblonga Mill (A). Revue Suisse de Viticulture, Arboriculture et Horticulture 19 (4): 253-259. Charles, G. & L. Rossignol, 1992. Environmental effects on potato plants in vitro. Journal o f Plant Physiology 139: 708-713. Charles, G., L. Rossignol & M. Rossignol, 1993. A synchronous model perfecting for fundamental studies on the tuberization process. Journal o f Plant Physiology 142: 474-479. L& C.L., 1990. Facteurs influenqant la tub6risation in vitro des microboutures de pomme de terre ( Solanum tuberosum L.). Revue Suisse d'Agriculture 22 (2): 115-116. L& C.L., 1991. Aspects pratiques de la micropropagation de la pomme de terre (Solanum tuberosum L.). Revue Suisse d'Agriculture 23 (6): 357-358. L~, C.L., 1993. Tub6risation h~ vitro de la pomme de terre cultiv6e (Solanum tuberosum L. var. Bintje). Revue Suisse d'Agriculture 25 (6): 365-367. L~, C.L. & D. Page, 1996. In vitro mass tuberization as a means to produce virus-free seed potatoes in Switzerland. In: Fionnbharr & Riordain (Eds.), COST 822 - Development of integrated systems for large-scale propagation of elite plants using in vitro techniques. European Commission, Report of activities, 1996--1997 (in press). Potato Research 42 (1999)

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C.L. L~ Murashige, T. & F. Skoog, 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473--479. Ranalli, P., M. Bizarri, L. Borghi & M. Mari, 1994. Genotypic influence on in vitro induction, dormancy length, advancing age and agronomical performance of potato microtubers (Solanum tuberosum L.). Annals of Applied Biology 125: 161-172. Reust, W. & C.L. L~, 1985. La multiplication rapide des pommes de terre par le microbouturage. Revue Suisse d'Agriculture 17 (1): 11-18.

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