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In vitro rootability of clonal apple microcuttings, derived from rooted and unrooted shoots
SClEN’llA HORllCULluM ELSEVIER
Scientia Horticulhuae 68 (1997) 227-230
Short communication
In vitro rootability of clonal apple microcuttings, derived from rooted and unrooted shoots Javier Puente, Juan A. Marh
*
Depurtment oj’Pomology, E.E. Ada Dei (CSIC), Aptdo. 202, E-50080
Zuragozn. Spain
Accepted 8 October 1996
Abstract In order to see if the differences in rooting ability of individual shoots were stable after successive subcultures, apical portions of rooted and unrooted Jork 9 apple microcuttings were induced to proliferate and the resulting shoots were used for new rooting experiments. No consistent differences were found between either line, showing that some shoots root and some fail to do so, depending on a short-lived physiological state. 0 1997 Published by Elsevier Science B.V. Keywords: h4alus X domestica;
Micropropagation; Rhizogenesis; Rootstock
1. Introduction In vitro rooting is very convenient for propagation of fruit tree rootstocks, since rooting percentages are often higher than those obtained with conventional methods (for a recent review see Bajaj, 1992). In our standard conditions, Jork 9 (J9) apple (M&s X domestica Borkh.) rootstock forms adventitious roots in 64.7% of the microcuttings, although rooting percentages can vary from 20% to 95% in successive batches in a short period of time (unpublished data). The reason why some microcuttings root while others fail to do so is not clear, nor is it known how long this competence may last, i.e. how long it takes for a shoot that can root to produce a shoot that cannot, or vice versa. If the shoots that are competent to root could be propagated to produce easy-to-root cultures, this would be a very convenient way to increase the rooting rate. In this work, we aimed to ascertain whether the difference between rooted and unrooted J9 shoots was consistent over five subcultures or was purely a short-lived physiological difference.
* Corresponding author. Tel: + 34-76-5765 11;Fax: + 34-76-575620, E-mail: [email protected] 03044238/97/$17.00 0 1997 Published by Elsevier Science B.V. All rights reserved. P/I SO304-4238(96)00987-9
228
J. Puente, JA. Marin / Scientia Horticulturue 68 (1997) 227-230
2. Materials and methods
Five-year-old in vitro shoot cultures of the J9 apple rootstock, established from buds of potted trees, were used. The shoot multiplication medium consisted of Murashige and Skoog’s (Murashige and Skoog, 1962) medium with 164.6 mM D-sorbitol instead of sucrose, 4.44 PM 6-benzylaminopurine (BAP) and 0.49 PM indole-3-butyric acid (IBA) as potassium salt. All media were autoclaved at 100 kPa for 20 min and kept in darkness until needed. Cultures were grown at 24 f 1°C with 16 h photoperiod provided by cool-white fluorescent lamps at a photon flux density of 35 pmol me2 s- ’ . Subcultures were carried out at 6-week intervals, since shoots on this medium grew slowly and after 6 weeks they were long and healthy. Shoots longer than 2 cm were used in the rooting experiments. Five microcuttings, obtained by cutting off all leaves except those in the apical part (1 cm), were placed into each baby food jar (Sigma V-8630) with 30 ml of rooting medium and a polypropylene lid (Sigma B-8648). Apparently homogeneous shoots were used to avoid variability. The rooting medium was that of Quoirin et al. (1977), with the macronutrients at half-strength, 0.98 ,uM IBA, 2.66 PM riboflavin and 868.8 PM L - proline. Shoots were incubated for 7 days in the dark and then exposed to the light, so that riboflavin triggers the photo-oxidation of the remaining auxin (Van der Krieken et al., 1992). This was the standard protocol for the COST 87 ‘Root regeneration’ working group. After 1 month of rooting treatment, five rooted shoots and five shoots that had failed to root on the same medium were cut into two pieces. The basal parts, with or without roots, were discarded and the apical portions were subcultured onto fresh shoot multiplication medium, and referred to as ‘rooted’ and ‘unrooted’ lines. Five rooting trials were conducted over the next five subcultures with the newly proliferated shoots. The whole experiment was repeated three times, beginning each time with the establishment of both lines. On the first establishment of the culture lines, more branched shoot cultures were observed in the rooted than in the unrooted line. So, the number of shoots produced in the first subculture was recorded in the following replications. Some of the rooted shoots from both lines were acclimatized on a peat/perlite (1: 1, v/v) mixture, either under a plastic tunnel in the greenhouse, or in plastic propagators within the growth chamber. Fertilizer was supplied with the water, and the plastic or the lids were removed gradually.
3. Results and discussion The ‘rooted’ line produced, in the first subculture, more shoots longer than 1 cm (13.9 f 1.2) than the ‘unrooted’ line (10.6 f 1.0). The difference was significant at P < 0.05(z-test) and was probably due to a carry-over effect of the cytokinins supplied by the roots of rooted shoots in the previous subculture. On most occasions the rooting percentages were similar in both rooted and unrooted lines, with some subcultures yielding much better results than others (Fig. 1) both in
J. Puente, JA. Marin / Scientia Horticulturae 68 (1997) 227-230
229
b
2 -O1
2
3
No. of subculture
4
I
0 I
5
1
I 2
3
4
5
No. of subculture
Fig. I. In vitro rooting percentage (a) and root number (b) from five consecutive subcultures of J9 apple lines established from rooted (W) and unrooted (0) shoots. Bars in (b) indicate standard errors. The number of explants is indicated in each case.
rooting percentage and root number. These differences, though common in this kind of experiments, cannot be easily explained. The three replications were not pooled because the results were sometimes significantly different comparing the three replicates in every subculture and for every culture line. The parallel response in the ‘rooted’ and ‘unrooted’ lines indicated that some external factors were affecting equally the final response in both lines, while the initial difference between them had disappeared. After 1 month, 80.6% of the transferred shoots (n = 284) were successfully acclimatized in the greenhouse. We can, therefore, conclude that the differences in rooting behaviour of individual J9 shoots were only due to a short-term physiological state. Successive in vitro culturing induces an increase in the rooting ability that has sometimes been considered as ‘rejuvenation’ (Webster and Jones, 1989). However, this increase in competence for adventitious root formation is the average of a large number of shoots and it is unpredictable what shoots will be the competent ones in the next subculture. Thus, it does not seem possible to select J9 shoots that are going to give a better result than the average.
Acknowledgements Financial support was received from the AGF 94-1030-CO3-01 project and a fellowship (PN91-25439704) to J. Puente, both from the CICYT.
References Bajaj, Y.P.S. (Editor), 1992. Biotechnology in Agriculture and Forestry, Vol. 18. High-tech and Micropropagation, II. Springer-Verlag, Berlin, 505 pp. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant., 15: 473-497.
230
J. Puente, JA. Marin / Scientia Horticulturae 68 (1997) 227-230
Quoirin, M., Lepoivre, P. and Boxus, P., 1977. Un premier bilan de 10 a&es de recberches sur les cultures de mCristemes et la multiplication “in vitro” de fmitiers ligneux. In: Compte rendu des recherches 1976-1977 et rapports de synthbse. Stat. Cult. Fruit. et Maraich, Gembloux, pp. 93-117. Van der Krieken, W.M., Breteler, H., Visser, M.H.M. and Jordi, W., 1992. Effect of light and riboflavin on indolebutyric acid-induced root formation on apple in vitro. Physiol. Plant., 85: 589-594. Webster, CA. and Jones, O.P., 1989. Micropropagation of the apple rootstock M.9: effect of sustained subculture on apparent rejuvenation in vitro. 3. Hortic. Sci., 64: 421-428.
Scientia Horticulhuae 68 (1997) 227-230
Short communication
In vitro rootability of clonal apple microcuttings, derived from rooted and unrooted shoots Javier Puente, Juan A. Marh
*
Depurtment oj’Pomology, E.E. Ada Dei (CSIC), Aptdo. 202, E-50080
Zuragozn. Spain
Accepted 8 October 1996
Abstract In order to see if the differences in rooting ability of individual shoots were stable after successive subcultures, apical portions of rooted and unrooted Jork 9 apple microcuttings were induced to proliferate and the resulting shoots were used for new rooting experiments. No consistent differences were found between either line, showing that some shoots root and some fail to do so, depending on a short-lived physiological state. 0 1997 Published by Elsevier Science B.V. Keywords: h4alus X domestica;
Micropropagation; Rhizogenesis; Rootstock
1. Introduction In vitro rooting is very convenient for propagation of fruit tree rootstocks, since rooting percentages are often higher than those obtained with conventional methods (for a recent review see Bajaj, 1992). In our standard conditions, Jork 9 (J9) apple (M&s X domestica Borkh.) rootstock forms adventitious roots in 64.7% of the microcuttings, although rooting percentages can vary from 20% to 95% in successive batches in a short period of time (unpublished data). The reason why some microcuttings root while others fail to do so is not clear, nor is it known how long this competence may last, i.e. how long it takes for a shoot that can root to produce a shoot that cannot, or vice versa. If the shoots that are competent to root could be propagated to produce easy-to-root cultures, this would be a very convenient way to increase the rooting rate. In this work, we aimed to ascertain whether the difference between rooted and unrooted J9 shoots was consistent over five subcultures or was purely a short-lived physiological difference.
* Corresponding author. Tel: + 34-76-5765 11;Fax: + 34-76-575620, E-mail: [email protected] 03044238/97/$17.00 0 1997 Published by Elsevier Science B.V. All rights reserved. P/I SO304-4238(96)00987-9
228
J. Puente, JA. Marin / Scientia Horticulturue 68 (1997) 227-230
2. Materials and methods
Five-year-old in vitro shoot cultures of the J9 apple rootstock, established from buds of potted trees, were used. The shoot multiplication medium consisted of Murashige and Skoog’s (Murashige and Skoog, 1962) medium with 164.6 mM D-sorbitol instead of sucrose, 4.44 PM 6-benzylaminopurine (BAP) and 0.49 PM indole-3-butyric acid (IBA) as potassium salt. All media were autoclaved at 100 kPa for 20 min and kept in darkness until needed. Cultures were grown at 24 f 1°C with 16 h photoperiod provided by cool-white fluorescent lamps at a photon flux density of 35 pmol me2 s- ’ . Subcultures were carried out at 6-week intervals, since shoots on this medium grew slowly and after 6 weeks they were long and healthy. Shoots longer than 2 cm were used in the rooting experiments. Five microcuttings, obtained by cutting off all leaves except those in the apical part (1 cm), were placed into each baby food jar (Sigma V-8630) with 30 ml of rooting medium and a polypropylene lid (Sigma B-8648). Apparently homogeneous shoots were used to avoid variability. The rooting medium was that of Quoirin et al. (1977), with the macronutrients at half-strength, 0.98 ,uM IBA, 2.66 PM riboflavin and 868.8 PM L - proline. Shoots were incubated for 7 days in the dark and then exposed to the light, so that riboflavin triggers the photo-oxidation of the remaining auxin (Van der Krieken et al., 1992). This was the standard protocol for the COST 87 ‘Root regeneration’ working group. After 1 month of rooting treatment, five rooted shoots and five shoots that had failed to root on the same medium were cut into two pieces. The basal parts, with or without roots, were discarded and the apical portions were subcultured onto fresh shoot multiplication medium, and referred to as ‘rooted’ and ‘unrooted’ lines. Five rooting trials were conducted over the next five subcultures with the newly proliferated shoots. The whole experiment was repeated three times, beginning each time with the establishment of both lines. On the first establishment of the culture lines, more branched shoot cultures were observed in the rooted than in the unrooted line. So, the number of shoots produced in the first subculture was recorded in the following replications. Some of the rooted shoots from both lines were acclimatized on a peat/perlite (1: 1, v/v) mixture, either under a plastic tunnel in the greenhouse, or in plastic propagators within the growth chamber. Fertilizer was supplied with the water, and the plastic or the lids were removed gradually.
3. Results and discussion The ‘rooted’ line produced, in the first subculture, more shoots longer than 1 cm (13.9 f 1.2) than the ‘unrooted’ line (10.6 f 1.0). The difference was significant at P < 0.05(z-test) and was probably due to a carry-over effect of the cytokinins supplied by the roots of rooted shoots in the previous subculture. On most occasions the rooting percentages were similar in both rooted and unrooted lines, with some subcultures yielding much better results than others (Fig. 1) both in
J. Puente, JA. Marin / Scientia Horticulturae 68 (1997) 227-230
229
b
2 -O1
2
3
No. of subculture
4
I
0 I
5
1
I 2
3
4
5
No. of subculture
Fig. I. In vitro rooting percentage (a) and root number (b) from five consecutive subcultures of J9 apple lines established from rooted (W) and unrooted (0) shoots. Bars in (b) indicate standard errors. The number of explants is indicated in each case.
rooting percentage and root number. These differences, though common in this kind of experiments, cannot be easily explained. The three replications were not pooled because the results were sometimes significantly different comparing the three replicates in every subculture and for every culture line. The parallel response in the ‘rooted’ and ‘unrooted’ lines indicated that some external factors were affecting equally the final response in both lines, while the initial difference between them had disappeared. After 1 month, 80.6% of the transferred shoots (n = 284) were successfully acclimatized in the greenhouse. We can, therefore, conclude that the differences in rooting behaviour of individual J9 shoots were only due to a short-term physiological state. Successive in vitro culturing induces an increase in the rooting ability that has sometimes been considered as ‘rejuvenation’ (Webster and Jones, 1989). However, this increase in competence for adventitious root formation is the average of a large number of shoots and it is unpredictable what shoots will be the competent ones in the next subculture. Thus, it does not seem possible to select J9 shoots that are going to give a better result than the average.
Acknowledgements Financial support was received from the AGF 94-1030-CO3-01 project and a fellowship (PN91-25439704) to J. Puente, both from the CICYT.
References Bajaj, Y.P.S. (Editor), 1992. Biotechnology in Agriculture and Forestry, Vol. 18. High-tech and Micropropagation, II. Springer-Verlag, Berlin, 505 pp. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant., 15: 473-497.
230
J. Puente, JA. Marin / Scientia Horticulturae 68 (1997) 227-230
Quoirin, M., Lepoivre, P. and Boxus, P., 1977. Un premier bilan de 10 a&es de recberches sur les cultures de mCristemes et la multiplication “in vitro” de fmitiers ligneux. In: Compte rendu des recherches 1976-1977 et rapports de synthbse. Stat. Cult. Fruit. et Maraich, Gembloux, pp. 93-117. Van der Krieken, W.M., Breteler, H., Visser, M.H.M. and Jordi, W., 1992. Effect of light and riboflavin on indolebutyric acid-induced root formation on apple in vitro. Physiol. Plant., 85: 589-594. Webster, CA. and Jones, O.P., 1989. Micropropagation of the apple rootstock M.9: effect of sustained subculture on apparent rejuvenation in vitro. 3. Hortic. Sci., 64: 421-428.