*Corresponding author

Keywords: Biotechnology, Propagation, rol gene, Transformation

Financial Support: National Plan for Plant Biotechnology, Ministry of Agriculture

The micropropagation is a non-conventional technique that allows plants to vegetatively multiplicate in shorter time and little spaces. With this method the plants are cultured in an aseptic environment (covered glass jars), furnishing the optimal growth conditions by the use of cultural media containing salts, vitamins, sugars, plant growth regulators and other organic compounds, under defined temperature, humidity, light intensity and light quality. In this way it is possible to rapidly stimulate the proliferation of new plants from a single bud, ensuring the health of the plant material and the preservation of the genetic uniformity. Since each bud can produce several shoots, in few months it occurs an exponential increase in the plant number. Varying the culture conditions, particularly using certain plant hormones called auxins; it is possible to induce the in vitro root formation in micropropagated cuttings.

This is a crucial step in the micropropagation, because only a good rooting permits a successful transfer from the in vitro to the greenhouse and then to the field conditions. However the induction of roots in micropropagated plants is not always so easy.

Several woody plants often show poor rooting efficiency both in conventional (layering, cutting) and in vitro propagation. In the latter case it was possible to improve the in vitro rooting with hormonal application, dark treatment, use of organic compounds, etc. (Damiano et al., 1991; Rugini et al.,1991); however, the difficulty of rooting is still one of the obstacle to successful micropropagation. Nowadays biotechnology is providing new soft methodology to overcome the use of synthetic auxins in inducing rooting. This is the case of infecting the base of the cuttings with Agrobacterium rhizogenes.

This bacterium is widely spread throughout the soils; it was selected for its capability to induce the infected cells to form roots in a large number of plants (Chilton et al., 1982). This is due to the transfer, the integration and the subsequent expression of a portion of bacterial DNA (T-DNA) from a little ring of bacterial DNA named Ri (Root Inducing) plasmid, to that of the plant. In fact this portion of DNA contains the necessary informations to change the normal cell growth programme towards the roots production. The process occurs naturally and is called transformation. Several authors reported a successful rooting by Agrobacterium rhizogenes-mediated transformation both in fruit trees as almond (Damiano et al., 1995), apple (Sutter and Luza, 1993), kiwi (Rugini et al., 1991), walnut (Caboni et al, 1996), and in woody plants as the genus Pinus (McAfee et al., 1993; Magnussen et al, 1994; Mihalievic et al, 1996; Burns and Schwarz, 1996; Tzfira et al., 1996), Larix (McAfee et al., 1993), Eucalyptus (MacRae and Van Staden, 1993). The aim of this work was to determine the ability of A. rhizogenes to induce the root formation on different species and cultivars of micropropagated fruit trees, both recalcitrant and easy-to-root ones, in presence or not of rhizogenic hormones, and to evaluate the nature of the adventitious roots in order to obtain a successful transfer to the field.

The in vitro micropropagated cuttings of the almond cultivars Fascionello, Ferragnes, Tuono; the almond selections M49, M50, M51, M52, M53, M55; the apple cvs. Gala and McIntosh; three seedlings of Pyrus pyraster (clones P8, P38 and P50); the plum cultivar Ontario, and two hybrid rootstocks, Citation (plum X peach) and GF677 (almond X peach), were infected with an Agrobacterium rhizogenes culture, dipping the basal part of the microcutting for 24 hours in darkness into the bacterial suspension. After the infection the explants were transferred to the appropriate co-cultivation root induction medium, with or without hormone, and finally transferred to the same medium containing the antibiotic cefotaxime (250 mg/l) to eliminate the bacteria. Plants not infected were maintained in the same cultural conditions as a control. After 35 days from the beginning of the experiments data regarding the number of rooted plants, the number of roots per plant and the length of roots in mm, were collected. Results were expressed as percentages.

A molecular analysis of DNA was carried by a technique called PCR (Polymerase Chain Reaction) to verify the integration of the bacterial DNA in plant tissues.

In the Table the rooting percentages of the genotypes exposed to different treatments are reported. According to their rooting ability, the plants can be divided into three groups: a) those rooting without auxins: Citation, GF677, M51, Ontario, Gala, McIntosh; b) those rooting only with auxins: M49, M50, M52, M53, M55, P38, P50; c) those rooting only after A. rhizogenes infection: P8, Fascionello, Ferragnes, Tuono. A general positive trend was observed in the group (a) after the addition of the auxins to the media.

After the bacterial infection all the genotypes gave roots. However, some differences could be detected. For group (a) no substantial differences were found between hormone free treatments, while a tendency to decrease the percentage of rooting was observed within the combination auxin plus infection. Concerning the group (b) it should be noticed the positive answer of infection on hormone free media and the synergism between the hormones in the media and the infection in the cases of P38 and P50. No comments are needed for the results of group (c).

Some alterations in the roots number and root length occurred, but the more evident was a reduced gravitropic response, i.e. the roots develop upwards, detected in several infected plants, particularly in almond; this growth behaviour was reported as typical in transformed roots (Legué et al., 1996). However the molecular analysis did not allow to confirm this correlation because not all the roots growing upwards appeared to be transformed.

The results shown in the Fig. 1 indicate that the use of A. rhizogenes is a successful approach to improve rooting of fruit trees, although different responses to the infection can occur (Damiano and Monticelli, 1998). From our experiments, it is possible to individuate two principal behaviours. While for the difficult-to-root genotypes the infection has always a positive effect, sometimes also in association with a hormonal treatment, for the easy-to-root ones the responses can be more differentiated. Most of the species do not appear really affected by the infection and the hormonal supply can be sufficient to have a good rooting, as in the hybrid genotypes and in the apple cultivars. Furthermore, the infection has a detrimental effect on rooting in presence or not of the hormone, as it occurred in the plum cv Ontario. However the infection with the bacterium becomes essential in some cultivars and species.

Figure 1. Responses of different genotypes to the localized infection.

An interesting result emerging from the molecular analysis is that only few roots show to be transformed. Our results seem to suggest also that the rooting process does not involve only the transformed cells: according with several authors (McAfee et al., 1993; Guivarc'h et al., 1996a; Guivarc'h et al., 1996b) possible changes in the root environment (organic acid secretions and pH lowering) and the presence of diffusible factors associated with the expression of the bacterial genes integrated in the plant cells, could play a role on the roots induction.

Two further considerations are that there is no risk of DNA transfer to other plant through pollen dissemination since the transformation of vegetative cells is confined to some roots; finally the bacterium is naturally occurring and is not genetically manipulated. The transfer from the glass containers to the soil does not require any special care (Fig. 2).

Figure 2. Different phases of acclimation in almond. a) 1 month after the transfer to the greenhouse: plant rooted after the infection with A. rhizogenes (left) and not infected control plant (right); b) acclimation; c-d) further development of the plants in the acclimation after 3 and 5 months respectively. (Reproduced from Damiano et al. 1997. In vitro rooting of fruit trees by A. rhizogenes, in: "Biology of root formation and development: the case of almond and apple", edited by Altman and Waisel, Plenum Press, New York, pp. 289-296 with the permission of the authors)

Table. Rooting percentages (± S.E.) of microcuttings infected with A. rhizogenes (Ar+) and not infected (Ar-) in hormone free medium (HF) or in the hormone supplemented medium (WH)

Plants rooting naturally without auxins:
	Auxin free treatment	Treatment with hormone	Auxin free treatment after infection	Treatment with hormone after infection
	% ± S.E.	% ± S.E.	% ± S.E.	% ± S.E.
Citation	69.1± 5.6	82.2 ± 3.8	63.6 ± 5.7	69.2 ± 7.7
GF677	58.7 ± 5.7	84.8 ± 5.2	56.2 ± 7.3	71.8 ± 12.3
M51	42.0 ± 11.2	50.0 ± 4.1	52.6 ± 6.3	51.9 ± 3.3
Ontario	35.2 ± 4.1	49.4 ± 10.3	16.5 ± 5.6	16.5 ± 8.0
Gala	26.2 ± 8.1	55.4 ± 5.1	15.7 ± 3.4	34.4 ± 3.2
McIntosh	22.1 ± 8.1	35.7 ± 3.6	25.3 ± 12.5	31.4 ± 13.2
Plants rooting only with auxins
	Auxin free treatment	Treatment with hormone	Auxin free treatment after infection	Treatment with hormone after infection
	% ± S.E.	% ± S.E.	% ± S.E.	% ± S.E.
M49	0.0 ± 0.0	26.1 ± 3.5	41.1 ± 4.2	28.1 ± 4.9
M50	0.0 ± 0.0	26.0 ± 6.8	27.7 ± 6.5	6.9 ± 4.4
M52	0.0 ± 0.0	33.8 ± 3.8	75.0 ± 15.0	33.8 ± 3.8
M53	0.0 ± 0.0	70.2 ± 3.8	79.4 ± 3.2	80.9 ± 3.3
M55	0.0 ± 0.0	65.0 ± 8.8	57.8 ± 4.3	67.8 ± 8.2
P38	0.0 ± 0.0	32.1 ± 6.0	26.3 ± 8.6	73.4 ± 5.8
P50	0.0 ± 0.0	16.7 ± 5.1	27.7 ± 9.0	63.7 ± 8.7
Plants rooting only after infection with Agrobacterium rhizogenes
	Auxin free treatment	Treatment with hormone	Auxin free treatment after infection	Treatment with hormone after infection
	% ± S.E.	% ± S.E.	% ± S.E.	% ± S.E.
P8	0.0 ± 0.0	0.0 ± 0.0	50.8 ± 2.8	15.0 ± 6.4
Fascionello	0.0 ± 0.0	0.0 ± 0.0	30.0 ± 4.5	25.3 ± 4.6
Ferragnes	0.0 ± 0.0	0.0 ± 0.0	52.3 ± 3.9	55.0 ±3.4
Tuono	0.0 ± 0.0	0.0 ± 0.0	46.4 ± 6.2	33.3 ± 4.9

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