A PROTOCOL FOR RAPID CLONAL MICROPROPAGATION IN VITRO OF PRIMOCANE-FRUITING RED RASPBERRY CULTIVARS

MISCELLANEOUS REPORTS 95 ISSN 0253-6749 A PROTOCOL FOR RAPID CLONAL MICROPROPAGATION IN VITRO OF PRIMOCANE-FRUITING RED RASPBERRY CULTIVARS G.J. Minas and D. Neocleous AGRICULTURAL RESEARCH INSTITUTE MINISTRY OF AGRICULTURE, NATURAL RESOURCES AND THE ENVIRONMENT LEFKOSIA CYPRUS MAY 2007

Editor - in Chief Dr C. Papachristoforou, Agricultural Research Institute, Lefkosia, Cyprus. All responsibility for the information in this publication remains with the author(s). The use of trade names does not imply endorsement of or discrimination against any product by the Agricultural Research Institute. 2

A PROTOCOL FOR RAPID CLONAL MICROPROPAGATION IN VITRO OF PRIMOCANE-FRUITING RED RASPBERRY CULTIVARS G.J. Minas and D. Neocleous SUMMARY A method has been developed for the rapid in vitro micropropagation of the primocane-fruiting red raspberry cultivars Autumn Bliss and Polana (Rubus idaeus L.). Red raspberries can profitably be used for off season production with better market opportunities, as several crops per year may be harvested in warmer climates as in Cyprus. Since red raspberry is a totally new crop for Cyprus, the developed micropropagation method could significantly contribute to the rapid expansion of this crop. Starting from in vitro culturing of apical and nodal segment explants, 0.5 to 1 cm long microplants were produced of the primocane-fruiting red raspberry cultivars Autumn Bliss and Polana (Rubus idaeus L.). For the explant establishment, proliferation and shoot growth, a modified MS medium was used. The cultures were incubated for the first week in the dark at 22+2 C to avoid any blackening of the tissues and were subsequently transferred to a growth room. With this method the propagation material increased three fold every 6-8 weeks. This means that from each individual explant, it is possible to produce up to 18 000 microplants per year. Rooting of microshoots was achieved by deliberate delay of the transfers for more than 4 weeks or by transfer of 3 cm long microshoots to a specific rooting medium. Plantlets produced were subjected in two forms of hardening, in vitro by increasing the light intensity and ex vitro in a microfog system where relative humidity was progressively reduced. The utility of this method for the rapid in vitro micropropagation of cvs. Autumn Bliss and Polana (Rubus idaeus L.) either for establishing mother stock plantations or for direct release to the growers is discussed. ΠΕΡΙΛΗΨΗ Αναπτύχθηκε µέθοδος για τον ταχύ µικροπολλαπλασιασµό in vitro των δίφορων κόκκινων σµέουρων των ποικιλιών Autumn Bliss και Polana (Rubus idaeus L.). Η εκτός εποχής παραγωγή κόκκινων σµέουρων απολαµβάνει συνήθως αµειπτικοτέρων τιµών στην αγορά και µπορεί να δώει περισσότερες σοδειές το χρόνο σε θερµότερες κλιµατικά ζώνες όπως η Κύπρος. Καθώς η καλλιέργεια των κόκκινων σµέουρων είναι εντελώς νέα για την Κύπρο, η µέθοδος µικροπολλαπλασιασµού που αναπτύχθηκε µπορεί να συµβάλει στην ταχεία διάδοση αυτής της καλλιέργειας. Ξεκινώντας από καλλιέργεια in vitro εκφύτων ενός οφθαλµού µήκους 0.5 µέχρι 1 εκ. που πάρθηκαν από φυτά δίφορων κόκκινων σµέουρων (Rubus idaeus L.) των ποικιλιών Autumn Bliss και Polana, παρήχθηκε µεγάλος αριθµός µικροφύτων. Χρησιµοποιήθηκε το θρεπτικό υπόστρωµα MS µε ορισµένες τροποποιήσεις. Οι καλλιέργειες κατά την διάρκεια της πρώτης εβδοµάδας τοποθετήθηκαν σε σκοτάδι στους 22+2 C για να αποφευχθεί η δηµιουργία µαυρίσµατος στους ιστούς και στην συνέχεια σε δωµάτιο ανάπτυξης καλλιεργειών. Το φυτικό υλικό τριπλασιαζόταν in vitro κάθε 6-8 εβδοµάδες. Αυτό σηµαίνει ότι από ένα και µόνο έκφυτο µπορούν να παραχθούν σε ένα χρόνο µέχρι και 18 000 µικρόφυτα. Η ριζοβολία των µικροβλαστών επιτυγχανόταν µε την καθυστέρηση της µεταφύτευσης για 4 εβδοµάδες ή µε τη µεταφορά σε υπόστρωµα για ριζοβολία όπου ρίζωναν µέσα σε 8 µε 10 εβδοµάδες. Οι παραγόµενοι έρριζοι µικροβλαστοί σκληραγωγούνταν µε δύο τρόπους: in vitro, µε αύξηση του φωτισµού και ακολούθως ex vitro σε συνθήκες υδρονέφωσης µε σταδιακή µείωση της σχετικής υγρασίας. Στην εργασία συζητείται η χρήση της µεθόδου για µαζικό µικροπολλαπλασιασµό των δίφορων κόκκινων σµέουρων (Rubus idaeus L.) των ποικιλιών Autumn Bliss και Polana για άµεση διάθεση στους παραγωγούς αλλά και για δυνατότητα δηµιουργίας καθαρών µητρικών φυτειών. 3

INTRODUCTION In Cyprus, red raspberry is a totally new crop. It could be economically feasible to use primocane-fruiting red raspberries (Rubus idaeus L.) for off season production that provides better market opportunities as several crops per year might be harvested in tropical and subtropical climates (Lopez- Medina and Moore, 1999). Throughout the world, production of red raspberries outside the main summer cropping season in greenhouses, is increasingly dependent on primocane-fruiting cultivars (Brennan et al., 1999; Pritts et al., 1999; Rosati et al., 1999). Oliveira et al. (1996, 1999), used summer cuttings of primocane-fruiting red raspberries to induce off season fruit production under protected cultivation in mild winter climates. Micropropagation techniques are capable of producing many plants in a short period of time (Snir, 1981), whereas a sufficient number of plants of new mechanical harvesting varieties could not be produced by conventional methods in the same time period (James et al., 1980). Multiplication can be achieved if shoots are cultured on Linsmaier and Scoog (LS) medium (Linsmaier and Scoog, 1965; James et al., 1980) or on Murashige and Scoog (MS) medium (Murashige and Scoog, 1962) with different vitamins (Sobczykiewicz, 1987; Snir, 1981). Some investigators have suggested that better results can be obtained with preparations of lower ionic strength composition. It is normal to start cultures from shoot tips and nodal segments of shoots obtained by meristem culture (Popov and Shchelkunova, 1973; Mikhovska, et al., 1995), but suitable explants can be excised from field grown plants. Anderson (1979; 1980) grew roots in the dark. Shoot tips were excised from the etiolated shoots which grew from nodules on the roots. Snir (1981) multiplied Rubus idaeus L. cultivars by node culture and shoots placed on MS medium containing 5 g/l activated charcoal and twice the normal concentration of Fe-EDTA, but no growth regulators The single shoots produced, when cut into 2-node sections and subcultured on the same medium, gave new shoots from every distal node. Auxiliary shoot proliferation has been induced by adding 0.1-4 mg/l BAP (according to cultivar) to the medium, often in combination with 0.1 mg/l IBA. Desjardins and Gosselin (1987) obtained higher proliferation rates of the red raspberry cultivar Madawaska on MS medium with 1 mg/l BAP and 50 mg/l ascorbic acid. Attempts to elongate in vitro Rubus idaeus L. with GA3 failed because inhibited subsequent rooting (Welander, 1985). The in vitro rooting of red raspberry was reported as being genotype depended (James et al., 1980; Sobczykiewicz, 1987). Rooting can be achieved spontaneously in cultures or on rooting medium with reduced to half MS medium macronutrients and the addition of 0.1-1.6 mg/l IBA (Welander, 1985; Sobczykiewicz, 1987). The further addition of 0.1 mm phloroglucinol (James et al., 1980; Sobczykiewicz, 1987) or 5 g/l activated charcoal (Anderson, 1979; 1982b) may improve root formation in red raspberry. The objective of this study was to investigate the possibility of in vitro clonal propagation of primocane-fruiting red raspberry cultivars Autumn Bliss and Polana for the production of large numbers of healthy and true-to-type plants. MATERIALS AND METHODS Explant preparation Ten cm long actively growing apical shoots were collected from 75% shaded greenhouse plants of the red raspberry cultivars Autumn Bliss and Polana (Rubus idaeus L.). They were placed in new polyethylene bags and transferred immediately to the laboratory. Explants were rinsed for 15 min under running tap water, followed by continuous agitation for 20 min in 15% commercial bleach (0.78% sodium hypochlorite) plus 0.1% v/v Tween20 and three 20 min rinses in sterile distilled water. Media preparation Separate stock solutions of macronutrients, micronutrients, chelate iron, vitamins and amino acids, ascorbic acid and growth regulators were used. The pre-autoclaving ph of the medium was adjusted at 5.8. Media were autoclaved in a commercial pressure cooker at 1.5 atm and 120 C for 20 min. Sixty ml aliquots were transferred in 330 ml light transparent polycarbonate jars or 15 ml aliquots in culture tubes (22x150 mm) with transparent polypropylene caps. Jars 4

and tubes had been sterilised in a commercial pressure cooker at 1.5 atm and 120 C for 20 min. Single node culture Single nodes 0.5 to 1 cm long were excised aseptically in a laminar flow cabinet using alcohol flame sterilized blades no. 11 mounted on scalpel no. 5. Explants were placed on the scratched upper surface in order to facilitate better contact with the medium in the culture tubes (22x150 mm). The modified medium was composed of the MS medium inorganic salts supplemented with 0.5 mg/l nicotinic acid, 0.4 mg/l thiamine-hcl, 0.5 mg/l pyridoxine-hcl, 2.0 mg/l glycine, 55.7 mg/l ascorbic acid, 4.45 mg/l BAP, 0.009 mg/l IBA, 2.5 g/l phytagel and 30 g/l sucrose. Cultures were incubated for one week in the dark at 22+2 C and subsequently transferred to a growth room at 25 C and 1 000 lux fluorescent illumination for establishment, proliferation, and in vitro shoot growth. Shoot development Raspberry shoot clusters 3-5 cm regenerated after 6-8 weeks. Plantlets were separated and transferred to fresh medium where they developed for another 6-8 week cycle. Freshly transferred plantlets developed new branches during the next growing cycle. In subsequent subcultures during the next six months, the number of plantlets increased substantially. A 3-fold increase in multiplication rate was achieved every 6-8 weeks with each transfer. Rooting Shoot clusters were transferred to a fresh medium with no growth regulators added. Cultures rooted after 8-10 weeks and obtained a well formed rooting system. Temperature in the culture rooms was maintained at 25 C with fluorescent light of 2 000 lux for 16 h daily. Shoots were transferred as clusters in an effort to avoid the one by one separation procedure which is time consuming and also to minimize losses at the acclimatization stage. Acclimatization Rooted shoot clusters 3-5 cm long, taken from the rooting medium, were washed under running tap water and immersed in fungicide (benomyl) before planted in pots containing sterile peat moss. The potted plants were placed in a shaded greenhouse where the temperature did not exceed 29 C. To avoid dehydration, plants were covered with inverted plastic beakers, which were progressively removed. After 3 weeks acclimatization, the plants were treated in the usual manner. During the acclimatization stage, the plants were watered when needed, with a solution containing N=6%, P=5%, K=6%, Mg=0.6%, B=0.016%, Cu=0.003%, Fe-DTPA=0.03%, Mn=0.025%, Mo= 0.001%, Zn= 0.0125%. RESULTS AND DISCUSSION Establishment of in vitro culture and shoot multiplication The supplemented modified MS nutrient medium was found suitable for red raspberry single node culture. To eliminate blackening of the tissue due to the diffusion of phenolic compounds from the cut surfaces to the culture medium which eventually are oxidized to tannins (Deberg and Read, 1991), measures were taken based on previous experience (Cassells and Minas, 1983b). These consisted of a) use of the antioxidant ascorbic acid in high concentrations (55.7 mg/l) in the culture media, b) use of phytagel rather than agar into the solidify media, and c) maintenance of the mother plants at low light intensity. When the explants were placed on the medium, the micropropagation process started and the buds developed shoots. With a 3-fold increase in multiplication rate and a 6-8 week cycle, a single bud produced a considerable number of plantlets in a period of 6 months. Klokonos and Solov (1986), found that the best medium for tissue culture of various raspberry cultivars was the MS medium. Welander (1985) reported that very poor proliferation rate was obtained from shoot tips but better results were obtained with nodal segments. The present results corroborate both statements. Shoot development, rooting and acclimatization After a 6-8 week cycle, raspberry shoot clusters were created, ready to be divided or transferred as 3-5 cm long clusters to the rooting medium (Fig. 1a). Divided shoots or 5

a b Figure 1. a) red raspberry shoot clusters growing in vitro, b) rooted red raspberry shoot clusters growing ex vitro. shoot clusters rooted within 8-10 weeks. Shoot clusters were shown to be better for transplanting. The survival rate of potted plantlets reached 95% and they were successfully established ex vitro. After 45 days in the greenhouse, plants were 10 cm tall and perfectly capable to be transplanted in the field or in other containers (Fig. 1b). No irregularities in plant development were observed. The main objective of this study was to establish a method for carrying out rapid clonal production of red raspberry plants with tissue culture methods as a contribution to the local red raspberry production system. Our results show that the method is suitable for either rapid in vitro micropropagation of red raspberry plants for immediate release to the growers or for the production of healthy stock plantations. Before release, the plantlets may undergo several pathogen free and true-to-type tests. Future research should concentrate on additional improvements in the productivity rate of this method. REFERENCES Anderson, W.C. 1979. Tissue culture propagation of red raspberry. InVitro 15:177. Anderson, W.C. 1980. Tissue culture propagation of red and black raspberries, Rubus idaeus and occidentalis. Acta Horticulturae 112:69-75. 6 Anderson, W.C. 1982b. Etiolation as an aid to rooting. Combined Proceedings of International Plant Propagation Society 31:138-141. Brennan, R.M., R. McNicol, T. Gillepsie, and S. Raffle. 1999. Factors affecting out-of season Rubus production. Acta Horticulturae 505:115-120. Cassells, A.C., and G.J. Minas. 1983. Plant in vitro factors influencing the micropropagation of Pelargonium cvs. Scientia Horticulturae 21:53-65. Debergh, P.C., and R.A. Read. 1991. Micropropagation. In: Micropropagation (Debergh, P.C. and Zimmerman, R.A., eds) pp. 113. Kluwer Academic Publishers. The Netherlands. Desjardins, Y., and A. Gosselin. 1987. The effect of hormonal concentrations, culture medium and an antioxidant on the shootdoubling time for the raspberry cultivar Madawaska grown in-vitro. Canadian Journal of Plant Science 67:863-869. Ferradini, N., M. Effati, and A. Standardi. 1997. In vitro propagation of some Rubus genotypes. Italus-Hortus 4:3-8. James, D.J., V.H. Knight, and I.J. Thurbon. 1980. Micropropagation of red raspberry and the influence of phloroglucinol. Scientia Horticulturae 12:313-319.

Klokonos, N.P., and I.I. Solov-eva. 1986. Propagation of raspberry by tissue culture method. Vestnik Sel`skokhozyaistvennoi Nauki Kazakhstana 9:44-47 (abstr.). Linsmaier, E.M., and F. Skoog. 1965. Organic growth factors requirements of tobacco tissue cultures. Physiologia Plantarum 18:100-126. Lopez-Medina, J., and N.J. Moore. 1999. Chilling enhances cane elongation and flowering in primocane-fruiting blackberries. HortScience 34:638-640. Mikhovska, B., T. Petkov, K. Dragoinski, and T. Stoevska. 1995. Obtaining and propagation of virus-free planting material of raspberry. Proceedings of the International Conference on Plant Virology, Apriltsi, Troyan, Bulgaria, Rasteniev dni Nauki, 32:142-143. Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15:473-497. Oliveira, B.P., C.M. Oliveira, L. Lopes-da-Fonseca, and A.A. Monteiro. 1996. Off season production of primocane-fruiting red raspberry using summer pruning and polyethylene tunnels. HortScience 31:805-807. Oliveira, P.B., C.M. Oliveira, L. Lopes da-fonseca, and A.A. Monteiro. 1999. Summer pruning intensity affects on off-season production of primocane- fruiting red raspberries. Acta Horticulturae 505:101-106. Popov, Y. G., and. S.E. Shchelkunova. 1973.The regeneration of Rubus idaeus shoot apices in relation to the presence of growth substances in the nutrient medium IBA and IAA activated. Botanicheskii Zhurnal 58:1515-1520. Pritts, M.P., R.W. Langhans, T.H. Whitlow, M.J. Kelly, and A. Roberts. 1999. Winter Raspberry Production in Greenhouses. HortTechnology 9:13-15. Rosati, P., B. Mezzetti, V. Knight, K.Y. Lim, V. Trajkovsky, B. Sjostedt, I. Greco, G. Martelli, S. Lionaki, G. Stavroylakis, M. Ciorda, and A. Polledo. 1999. Expanding the adaptation and production area of Rubus in Europe. Acta Horticulturae 505:39-45. Shchelkunova, S.E. 1974. Seasonal regeneration of plants from meristem tips of raspberry. Fiziologia Rastenii 21:69-74. Snir, I. 1981. Micropropagation of red raspberry. Scientia Horticulturae 14:139-143. Sobczykiewicz, D. 1987. Mass production of raspberry plants by meristem culture. ActaHorticulturae 212:607-609. Welander, M. 1985. In vitro culture of raspberry (Rubus idaeus) for mass propagation. Journal of Horticultural Science 60:493-499. 7

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