logo of IAMRevitalisation of old austrian fruit tree cultivars

Hanzer V., Weiss H., Weiss B., da Câmara Machado A., Knapp E., Pühringer H., Katinger H. and Laimer da Câmara Machado M.

Institute of Applied Microbiology, University of Agriculture, Vienna/AUSTRIA
URL: http://www.boku.ac.at/iam iam@mail.boku.ac.at
Nussdorfer Lände 11, A-1190 Vienna, AUSTRIA (+43 1 3692924 - 402 FAX: 400 )

Abstract

In austria some cultivars of apple and pear have been already mentioned in farm descriptions about 400 years ago and for some cultivars individiuals of 250 years are still present today. Out of more than 300 characterized cultivars only about a dozen are sold on market. They already proved to be well adapted to the environmental conditions, but it is unknown, whether - grafted on modern rootstocks - they could give yields comparable to modern cultivars. The old local cultivars partly disappeared because of their bad phytosanitary conditions and the subsequently inefficient yields. Many of this cultivars are not cultivated in commercial orchards, but are of great interest for an extensive plantation system, which in some parts of Austria still plays an important role, in some other parts is regaining importance. The fruits are not only used as fresh fruits for the market, but also for cooking and cider production. Above all, the old genotypes of fruit trees are determinant components of the austrian landscape.
Also a lot of this cultivars have natural resistance genes against different pathogens, so a great breeding potential remains unused. Therefore it is evident to conserve this valueable genetic material of this locally selected cultivars. Tissue culture methods include the most suitable techniques for The processes of plant micropropagation involve a complexe sequence of growth stages (Murashige 1974, Wilkins and Dodds 1982). Protocols are based on the four phases of Murashige (1974): induction, shoot proliferation, rooting and acclimatisation (figure 1). Great differences between the requirements of the cultivars even of the same species make the use of a general protocol impossible, this means the necessity to adapt in each step the media composition and culture conditions for each cultivar. All the optimisation work of culture media and conditions was carried out with about one hundred cultivars of Malus and Prunus sp. (Hanzer et al. 1993).

Figure 1: Different phases of fruit tree micropropagation
Figure 2: Method for initiating cultures of mature apple trees using 8-HQS

Initiation and establishment of tissue cultures

The establishment of woody plants in vitro is a difficult step. The unsatisfactory yield of sterile actively growing shoots of fruit trees in vitro using conventional methods of surface sterilisation has been documented by several workers (Laimer et al 1988a, Webster & Jones 1989). Keeping the donor plants under glasshouse conditions and pretreating them with antimicrobial substances decreases the infection pressure (Enjalric et al 1988), and for Prunus explants this treatment is sufficient. Tissue culture initiation of apple cultivars is presents major difficulties. The most frequent problems are infections because of the hairy surface, endogenous bacteria and browning of explants. During preparation polyphenolic compounds are oxidized by polyphenoloxidases (PPO), inducing a strong browning of the cut surfaces and leading to subsequent death of the explant (Skirvin et al. 1986).

To obtain satisfactory results we developed a new method that allows a faster and safer initiation and establishment of adult material. The application of 8-hydroxy-quinolinol-sulfate (8-HQS), an old antimiotic and bactericidal substance reduced infection rate and browning. 8-HQS seems to inhibit polymerisation of phenols into typical brown polymers, possibly by chelating essential metal ions of enzymes (R.Ebermann, pers. comm..).

Applying the new method (figure 2) actively growing shoot tips of Malus domestica were cleaned and disinfested, dissected to 2-3 mm and placed on a modified MS medium with 4.4 uM BA. Explants were covered for 24 h with 200 ul of a 0.1 % solution of 8-HQS and then moved to fresh medium. After 2 weeks the explants were transferred to a medium containing both auxin and cytokinin (Laimer et al. 1991b). A yield of 50 -90 % sterile explants were obtained in compared to the formerly 100 % losses of untreated shoot tips (figure 3+4).

Figure 3: Comparison between untreated (above) and with 8-HQS treated (below) shoot tips of Malus sp. var. Jonathan
Figure 4: In vitro shoot of Malus sp. 4 weeks after sucessful initiation

Multiplication

When we started our work, only limited information was available on micropropagation of Malus and Prunus species. Especially for those economically less important cultivars all tissue culture protocols were missing. We first set out to elaborate these basic steps (Laimer et al. 1988a, 1988b, 1991a, Weiss et al. 1993). Culture media and conditions for the different genotypes had to be improved in order to get vigorously growing plantlets without any morphological changes like vitrification, callus production and stunting internodes. Multiplication should take place only via axillary budding. The chosen low amount of growth regulators guaranteed an average multiplication rate of 3-5 per subculture and therefore the danger of somaclonal variation was minimized (figure 5+6).

Figure 5: Culture of Malus sp. during multiplication phase
Figure 6: Culture of Prunus sp. during multiplication phase

Virus elimination

The combination of in vitro thermotherapy and meristem culture is the most efficient method for virus elimination. Actively growing plant material is placed into a thermotherapy chamber (figure 7). The exposure should be for 3 weeks or longer with 16 h light at 38 oC and 8 h dark at 36 oC. The temperature and time of exposure are limited by the heat tolerance of the host plant, depending on species and variety. Among fruit trees the pomaceous plants tolerate heat better than stone fruits. After successful heat treatment meristems had to be excised immediately to avoid a new increase of the virus titre. A careful preparation of the meristem with a minimum size under the stereomicroscope is essential. The hight of the explant is usually 0,3 - 0,7 mm. The growing point is excised together with one or two leaf primordia and explanted with the base resting on the surface of the medium, which only contains cytokinin for initiation (figure 8). After 1 month meristem tips have elongated and the resulting shoots are put on the proliferation medium for multiplication.
The success of elimination of viruses depends on the types of plant viruses, particular host and virus combinations and if the plant is single or mixed infected (Knapp et al. 1995a). During the heat treatment there are unsuitable conditions for virus replication and so the virus titre is lowered. There exists no definite explanation for the frequent occurrence of virusfree meristems, but some viruses tend to accumulate preferentially at the base of shoots (Knapp et al. 1995b). Hirth (1958) supposed that there is competition in the metabolically highly active meristem between on one hand production of virus particles and on the other cell production. In meristematic tissue during cell division the capacity for nucleic acid synthesis is being utilizd for cell production to the detriment of virus multiplication. Another explanation why the virus concentration is so low in meristems is that the absence of vascular elements greatly hinders the transport of virus particles into the meristematic dome (Brant et al. 1962). (figure 7+8)

Figure 7: Thermotherapie chamber.
Figure 8: Meristem tip one week after preparation.

Rooting

Essential for any kind of sucessful rooting procedure is the use of well formed shoots of at least 2 cm of size. Also in this phase pome and stone fruit cultivars showed strong genotypic differences in the requirements, of culture conditions, media composition and especially growth regulator concentration.
Based on the perception that auxins on the one hand stimulate root induction, but on the other hand are inhibitory for root development we applied a rooting protocol with two working steps (Zimmerman and Fordham 1985): root induction on liquid culture media with auxin for one week in darkness, and root elongation in hormonfree liquid medium with perlite (figure 9).

Figure 9: In vitro plantlet of Malus sp. after the rooting treatment.

Acclimatisation

Acclimatisation of in vitro plants to normal atmosphere includes usually two major problems: Best results were obtained in perlite during the first two weeks followed by a mixture of earth and perlite. A stepwise reduction of relatively humidity was carried out under plastic covers. After the sucessful acclimatisation plants are to be grown in an insect proof screenhouse. Virus-free plants are mother plants for the distributen of virusfree plant material and for germplasm collection (figure 10+11+12).

Figure 10+11: Acclimatisated shoots under greenhouse conditions.
Figure 12: Virus-free tissue culture plants in an insect proof greenhouse as source of propagation material and for collection germplasm.

References

Brants, D. H., Graafland, W. and Kerling, C. P. (1962) The distribution of tobacco mosaic virus in excised tomato roots cultivated in vitro. T. Pl. Ziekten 68: 198-207
Enjalric, F., Carron M. P. and Lardet L. (1988) Contamination of primary cultures in tropical areas: the case of Hevea brasiliensis. Acta Hort. 225: 57-65
Hanzer, V., Weiss, H., Knapp, E., da Câmara Machado A., Katinger, H. and Laimer da Câmara Machado M. (1993) In vitro Methoden zur Erhaltung und Virusfreimachung von Apfelsorten. Tagungsbericht Nutzbarmachung genetischer Resourcen für Züchtung und Landschaftsgestaltung. Pillnitz, 1993, 322-326.
Hirth, L., (1958) Evolution de la concentration du virus de la mosaique du tabac en fonction des constituants biochimiques-cellularis au cours de la croissance de tissue de tabac cultives in vitro. C. R. Acad. Sci. 247: 1795-1797
Laimer, M., da Câmara Machado, A., Hanzer, V., Weiss, H., Mattanovich, D., Himmler, G. und Katinger, H. (1988a) In vitro Vermehrung der alten Lokalsorte Malus domestica „Graf Uhlhorns Augustkalvill“. Mitt. Klosterneuburg 38: 105-107.
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Laimer da Câmara Machado, M., da Câmara Machado, A., Hanzer, V., Kalthoff, B., Weiss, H., Mattanovich, D., Regner, F. and Katinger, H. (1991a) In vitro Methoden zu Verbesserung von Obstgehölzen. Bioengeneering 4: 37-42
Laimer da Câmara Machado, M., da Câmara Machado, A., Hanzer, V., Kalthoff, B., Weiss, H., Mattanovich, D., Regner, F. and Katinger, H. (1991) A new, efficient method for the initiation and establishment of tissue cultures of apple from adult material. PCTOC 27, 155-160.
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Knapp, E., Hanzer, V., Weiss, H., da Câmara Machado, A., Wang, Q., Weiss, B., Katinger, H. and Laimer da Câmara Machado, M. (1995b) Distribution of apple chlorotic leafspot virus in apple shoots cultivated in-vitro. Acta Hort., in press. Skirvin R. M., Kouider M., Joung A. and Korban S. S. (1986) The tissue culture of apple Malus domestica Borkh. in: Biotechnology in Agriculture and Forestry. Bajaj Y. P. S. (ed.) 1: 183-197
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Weiss, H., Hanzer, V., Knapp, E., da Câmara Machado A., Katinger, H. and Laimer da Câmara Machado M. (1993) In vitro Vermehrung von Prunus armeniaca. Tagungsbericht Nutzbarmachung genetischer Resourcen für Züchtung und Landschaftsgestaltung. Pillnitz, 1993, 322-326. Wilkins C.P. and Dodds J.H. (1982) The application of tissue culture techniques to plant genetic conservation. Sci. Prog. (Oxf.) 68: 281-307
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Acknowledgement

This work was supported by the Austrian Bundesministerium für Land- und Forstwirtschaft, Projekt Nr. 472/87 and we would like to especially thank Fam. Brenninkmeyer (Company C&A) for their support.
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04/11/95 C by IAM