Benzyladenine-Stimulated Rooting in Fruit-Tree Rootstocks Cultured in vitro

Institute of Plant Physiology, Biological Research Center Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary

Benzyladenine-Stimulated Rooting in Fruit-Tree Rootstocks Cultured in vitro G. NEMETH'f) With 1 figure Received April 10, 1979 . Accepted June 27, 1979

Summary Root formation was induced by 10-6 M benzylaminopurine in Prunus myrobalan, Prunus avium, Prunus persica X Prunus amygdalus and Cydonia oblonga shoot cultures maintained without exogenous auxin. Other cytokinins, such as kinetin, 2iPA, 2iP and zeatin were not active in inducing root formation. The auxins indolebutyric acid (IBA), l-naphthaleneacetic acid (NAA), ,8naphthoxyacetic acid (NOA) also stimulated rooting; IBA at 5 X 10-6 M was the most active. Light also seems to stimulate root formation in these species. Key words: rootstocks, cytokinins, auxins, roo'ting, light.

Introduction In recent years there has interest been in the in vitro propagation of woody plants. Successful large-scale propagation of trees has so far been limited to a few species (PIERIK, 1975; BUTTON and KOCHBA, 1977). As for the fruit trees of the temperate zone, success has been reported in cherry, plum, quince (Boxus and QUOIRIN, 1977), apple (JONES and HATFIELD, 1976; JONES et ai., 1977; Boxus and QUOIRIN, 1977) and almond (KESTER et ai., 1977). One of the crucial steps in propagation work is root induction on the shoots obtained by multiple shoot induction from shoots in aseptic culture. Auxins have been shown to stimulate root initiation, elongation and lateral root formation (STREET, 1969; SCOTT, 1972). The role of cytokinins in the inception and development of roots in cultured tissues is not clear (MILLER, 1961; Fox, 1969; SKOOG and ARMSTRONG, 1970; TORREY, 1976). Exogenous cytokinins were shown to be deleterious to the initiation and the elongation of the main axis of roots of both monocotyledonous and dicotyledonous plants (SKINNER and SHIVE, 1955; DE Ropp, 1956; McMANUS, 1960; KAMINEK, 1968). ") On leave from SASAD Horticultural co-operative, Budapest. Z. Pf/anzenphysiol. Bd. 95. S. 389-396. 1979.

390

G.

NEMETH

In a few cases, however, cytokinins stimulated growth (ROBBINS and HERVEY, 1971), lateral root formation (FRIES, 1960; TORREY, 1962), or caused an increase in the diameter of the primary root (SKOOG and MILLER, 1957). In this paper the effect of different growth regulators on the rooting of fruit-tree rootstocks is described. Materials and Methods Cultures from virus-free rootstocks of Prunus myrobalan (myrobalan) GF 31, Prunus avium (cherry) F 12/1, Prunus persica X Prunus amygdalus (peach X almond) GF 557 and Cydonia oblonga (quince) BA 29 were initiated from 0.1-0.5 mm shoot tips following the method described by Boxus and QUOIRIN (1974) on WALKEY's (1972) medium. Regenerated plantlets were maintained and propagated on the basal medium of DUDITS et al. (1975) containing 40 mgll adenine, 10-6 or 5 X 10-6 M benzyl adenine, 30 gil sucrose and 0.7 gil agar-agar, pH adjusted to 5.0 before autoclaving. The following growth regulators were used: indole-3-acetic acid (IAA; Merck), 1naphthaleneacetic acid (NAA; Fluka), ,8-naphthoxyacetic acid (NOA; Sigma), indole-3butyric acid (IBA; Merck), N 6-benzyladenine (BA; Fluka or Sigma), gibberellic acid (GA3; Phylaxia), zeatin (Sigma), 2-isopentenyl-adenine (2iP; Sigma), 2-isopentenyl-adenosine (2iPA; Sigma), kinetin (Fluka). Cultures were kept at a temperature of 25 ± 1 °C under continuous illumination (2000 lux) from fluorescent tubes (Floralux, Airam, Finland) and subcultured at 4-week intervals. Each treatment was tested on at least 10-15 cultures and the experiments were performed twice.

Results

Effect of benzyladenine On multiple shoot induction medium containing 10- 6 M BA alone, root formation was observed in myrobalan, cherry and peach X almond cultures. The same phenomenon was found in quince with 5 X 10-6 MBA. These preliminary observations were extended by testing 0-5 X 10- 6 MBA. At the end of ,the second week of treatment, roots emerged from the brown, lignified base of the branching main shoot. The experiments were scored by counting the number of roots induced, and proportion of root-forming shoots. Data are shown in the Table 1. Of the concentrations tried, 10- 6 M proved to be the most effective for myrobalan, cherry and peach X almond and 5 X 10-6 M for quince. Adenine had no significant effect on rooting. Light intensity seems to have a promotive effect on BA-induced rooting, as shown by data from cultures illuminated at 2000, 1000 and 800 lux, respectively (Table 2.). Roots formed on BA-containing medium were elongated and had a well-developed lateral root system, in contrast to those formed on hormone-free medium by myrobalan. Other cytokinins - 2iP, 2iPA and zeatin at a concentra:tion range between 10- 7 to 5 X 10-6 M - had no effect on rooting. Z. Pflanzenphysiol. Bd. 95. S. 389-396. 1979.

'" ;cl

.....

""'"?'

00

""l'

~

'" ~

!"-

ttl

'" ~

~

;:$

'" ":;

N

~ i> ;:$

N

-+-

-+

-+-

-+-

Adenine, 3 X 10-4 M C

0 0 0 0 0

C

4.5 5.0

0 0 0 0

9.09 5.0 0 0

RIS Ofo R

RIS = average number of roots per shoot 0/0 R = percent of shoots producing roots 0/0 S = percent of shoots producing new shoots

P. persica / P. amygdalus GF 557 Cydonia oblonga BA 29

F12/1

P. myrobalan GF 31 Prunus avium

Rootstock

0 0 0 0 0 0

22.7 35.0

OfoS 4.4 4.1 1.0 1.0 1.0 1.0 C 0 25.0 27.2 6.45 6.6 3.3 6.9 0 0

RIS Ofo R 85.0 90.9 67.7 70.0 36.7 44.8 17.5 30.9

OfoS

5 X 10-7

BA,M

4.8 4.9 1.87 2.4 3.1 3.0 1.0 1.0

45.5 47.6 26.6 30.0 41.7 50.0 5.0 5.0

RIS Ofo R

10-6 S

95.4 90.4 66.6 83.3 69.0 83.3 40.0 40.0

0/0

Table 1: Stimulation of rooting and multiple shoot formation by BA without exogenously applied auxin.

OfoS 0 100.0 0 95,2 0 0 6.6 83.3 1.0 93.5 1.5 6.4 64.6 0 0 0 70.0 0 97.6 1.4 34.8 1.53 34.8 100.0

RIS OfoR

5 X 10-6

V>

.......

'-D

""

g 5·

0

...0.....

~ o· ::l

~

s·c::

5·

'"::l ...'"

'<1 ~ p..

t::d

'"::lN

392

G.

NEMETH

Table 2: Effect of light intensity on rooting in the presence of BA alone. Light intensity, lux 1000

2000 average No. of roots

No. of plants rooted, scored 0/0 plants

average No. of roots

46.65

3.3

551

203

36.48

3.1

157

36.09

2.5

318

88

27.6

2.2

165

32.16

1.4

581

54

Rootstock

No. of plants rooted, scored % plants

P. myrobalan GF 31

851

397

P. avium F 12/1

435

Cydonia oblonga BA 29

513

9.29

1.3

800 No. of plants rooted, scored % plants

average No. of roots

P. myrobalan GF 31

601

148

24.62

3.2

P. avium F 12/1

329

73

22.18

2.7

Cydonia oblonga AB 29

587

29

4.8

1.1

P. myrobalan and P. avium propagated at lO-6 MBA Cydonia propagated at 5 X lO-6 M BA plants were scored on the 5th week of subculture

Effect of auxins The effectiveness of several auxins in inducing root formation was compared by inserting elongated shoots induced by 10-6 and 5 X 10- 6 MBA, IAA, NAA, NOA and IBA were tested at a range of concentrations up to 10-5 M. Similar reactions were given by the different species in each case. IAA and NOA had only a slight effect on rooting; NAA at concentrations higher than 10- 6 M induced intensive callusing of the roots that had formed. IBA at 5 X 10-6 M proved to be the most efficient (Table 3.). GA3 in combination with IBA had a promotive effect on the frequency of rooting in myrobalan but it w:as inhibitory in quince cultures. Synergistic effects of BA and IBA were examined in myrobalan cultures. Low concentration (5 X 10- 7 M and 10-6 M) of BA increased the frequency of rooting of the shoots, as compared with their control (10- 7 and 10- 6 M IBA alone). Higher concentrations of BA and IBA added together decreased the number of rooted plantlets and callus was induced at the base of the axis. Z. P/lanzenphysiol. Rd. 95. S. 389-396. 1979.

Benzyladenine stimulation of rooting

393

Table 3: Root induction on IBA-containing medium with and without 3 X 10-6 M GAa. IBA,M Rootstock P. myrobalan GF 31 P. avium F12/1

P. persica X P. amygdalus GF 557 Cydonia oblonga BA 29

10-7

0

10-6

5 X 10-6

10-5

GA3

RIS

°io R

RIS

+

2.50 4.00

9.60 14.50

0 3.50

0 15.00

1.00 3.20

5.00 20.80

4.00 3.50

29.20 61.60

3.50 2.00

14.10 10.00

+

0 0

0 0

1.0 1.5

10.0 10.0

1.9 1.75

50.0 54.5

3.33 3.06

75.0 75.2

1.33 1.66

14.2 15.0

+

0 0

0 0

1.0 1.0

1.49 1.41

30.0 35.0

2.05 2.20

70.8 71.35

1.25 1.49

15.0 28.6

+

0 0

0 0

0 0

1.25 1.0

35.0 18.1

1.28 1.0

30.4 10.0

OfoR

RIS

OfoR

RIS

°io R

RIS

OfoR

8.65 8.65 0 0

1.0 1.0

9.52 5.0

RIS = average number of rootslshoot °/0 R = percent of shoots forming roots

Discussion

In the literature there are only a few cases reported in which cytokinins alone promoted the initiation of roots. Rooting on sporeling leaves of the water fern Marsilea drummondii was induced by 0,01 mg/l kinetin (ALLSOPP and SZWEYKOWSKA, 1960). An enhancement of root initiation in maize callus cultures by 0.1-5,uM zeatin was found by LINSMAIER-BEDNAR and BEDNAR (1972). With Limnophila chinensis SANGWAN et al. (1976) found that roots were formed nearly at all the levels of kinetin used (0-5000,ug/I), but the intensity of root formation varied slightly with the concentration. To my knowledge, consistent root formation in the presence of cytokinins, without exogenously applied auxin, has hitherto not been reported in woody plants. In our case, rooting could be induced only by BA, although other cytokinins were tried. The response to different BA concentrations varied between the different species. In myrobalan .and the peach X almond hybrid, rooting could be regularly induced only at low concentrations (5 X 10-7 and 10-6 M). BA at 5 X 10- 6 M was almost completely inhibitory to cherry, whereas with quince this level of BA proved to be the most effective. Of course, at the time of root emergence the BA content of the medium might have been decreased to some extent. The low frequency of rooting of myrobalan on hormone-free medium and the lack of rooting in the absence of other exogenous growth regulators in the other species demonstrate that root induction in this case is due to cytokinin. Data in the literature suggest that in looking for stimulatory effects of cytokinins on the formation of roots, one should always include low concentrations in the series of treatments because at high concentrations a simple inhibition of root elongation Z. PJlanzenphysiol. Bd. 95. S. 389-396. 1979.

394

G.

NEMETH

Fig. 1: Plandets of rootstocks rooted on BA alone: a, b) Prunus myrobalan (la- 8 M); c) Prunus avium (10- 6 M); d) Cydonia oblonga (5 X 10-8 M). Z. Pjlanzenphysiol. Bd. 95. S. 389-396. 1979.

Benzyladenine stimulation of rooting

395

can occur (WEBSTER and RADIN, 1972; CHANDRA et al., 1977). Endogenous auxin and other growth regulators possibly playa role in BA-induced rooting. A question that arises is whether there is any correlation between BA-promoted rooting and the lignification process. In our case BA-induced rooting was observed earliest when the lignified base of the main shoot was not removed during subculture. Rooting never occurs before the thickening and browning of the base of subcultured cxplants. TORREY reported (1962) that in isolated pea root segments at low concentrations of kinetin and adenine-sulphate together, a stimulation of lateral root initiation could be observed. In our rootstocks ,adenine had no noticeable effect on rooting. In the present experiment, when a great number of shoots from rootstocks were grown in the presence of BA alone under different intensities of continuous illumination, different frequencies of rooting were observed. DUTCHER and POWELL (1972) reported root formation in nearly one third of apple explants grown under continuous low light intensity on IAA-containing medium. Evidence for a cytokinin-light interaction in root development has also been reported in rice callus (NISHI et al., 1968) and maize callus (LINSMAIER-BEDNAR and BEDNAR, 1972). The frequency of rooting was higher on BA than on IAA, NOA and NAA, but was lower than on IBA (5 X 10-6 M). In quince the activities of BA and IBA were similar. Gibberellic acid has been reported to stimulate rooting (BUTCHER and STREET, 1960; KOCHBA et al., 1974). QUOIRIN et al. (1974) obtained viable rooted plantlets of Prunus in the presence of GAs + IBA. With the exception of quince, our data seem to confirm this effect. Acknowledgements I thank Dr. P. MALIGA for suggestion and encouragement and Mrs. E. KISS, Miss I. DEBRECZENI, E. VIGH and KATALIN HuszAR for the technical assistance. This research was supported from a grant by the SASAD Horticultural Co-operative to Dr. P. MALIGA.

References ALLSOPP, A. and A. SZWEYKOWSKA: Nature, 186, 813-814 (1960). BLAKELY, L. M., S. J. RODAWAY, L. B. HOLLEN, and S. G. CROKER: Plant Physiol., 50, 35-42

(1972).

Boxus, PH:. and M. QUOIRIN: Bull. Soc. Roy. Bot. Be1g., 107, 91-101 (1974). -- - Acta Hort., No. 78, 373-379 (1977). BUTCHER, D. N. and H. E. STREET: J. Exptl. Bot., 11,206-216 (1960). BUTTON, J. and J. KOCHBA: Tissue culture in the Citrus industry. In: REINERT, J. and Y. P. S. BAJA] (Eds.): Applied and Fundamental Aspects of Plant Cell, Tissue and Organ Culture, 70-92. Springer Verlag, Berlin, Heidelberg, New York, 1977. CHANDRA, G. R., J. F. WORLEY, L. E. GREGORY, and H. D. CLARK: Plant and Cell Physiol.,

14, 1209-1212 (1973). DE Ropp, R. S.: Plant and Cell Physiol., 31, 253-254 (1956). DUDITS, D., G. NEMETH, and Z. HAYDU: Canad. J. Bot., 53, 957-963 (1975).

z.

P/lanzenphysiol. Bd. 95. S. 389-396. 1979.

396

G. NEMETH

DUTCHER, R. D. and L. E. POWELL: J. Amer. Soc. Hort. Sci., 97,511-514 (1972). Fox, J. E.: The cytokinins. In: WILKINS, M. B. (Ed.): Physiology of Plant Growth and Development, 85-123. McGraw-Hili, New York, 1969. FRIES, N.: Physio!. Plant., 13,468-481 (1960). JONES, O. P. and S. G. S. HATFIELD: J. Hort. Sci., 51, 495-499 (1976). JONES, O. P., M. E. HOPGOOD, and D. O'FARRELL: J. Hort. Sci., 52, 235-238 (1977). KAMINEK, M.: Biologia Plantarum, 10,462-471 (1968). KESTER, D. E., L. TABACHNIK, and J. NEGUEROLS: Acta Hort., No. 78,95-101 (1977). KOCHBA, J., J. BUTTON, J. SPIEGEL-Roy, C. H. BORNMAN, and M. KOCHBA: Ann. Bot., 38, 795-802 (1974). LINSMAIER-BEDNAR, E. AM. and T. W. BEDNAR: Development, Growth and Differentiation, 14,165-174 (1972). McMANUS, M. A.: Nature, 185, 44-45 (1960). MILLER, C. 0.: Ann. Rev. Plant Physio!., 12,395-407 (1961). NANDI, S., G. FRIDBORG, and T. ERIKSSON: Hereditas, 85, 57-62 (1977). NISHI, T., Y. YAMADA, and E. TAKAHASHI: Nature, 219, 508-509 (1968). PIERIK, R. L. M.: Acta Hort., 54, 71-82 (1975). QUOlRIN, M., PH. Boxus, and T. GASPAR: Physio!. Veg., 12, 165-174 (1974). RoOBBINS, W. J. and A. HERVEY: Proc. Nat!. Acad. Sci. USA, 68, 347-348 (1971). SANGWAN, R. S., B. NORREEL, and H. HARADA: Biologia Plantarum, 18, 126-131 (1976). SCOTT, T. K.: Ann. Rev. Plant Physio!., 23, 235-258 (1972). SKINNER, CH. G. and W. SHIVE: J. Amer. Chern. Soc., 77,6692-6693 (1955). SKOOG, F. and C. O. MILLER: Symp. Soc. Expt!' Bio!., 11, 118-131 (1957). SKOOG, F. and D. J. ARMSTRONG: Ann. Rev. Plant Physio!., 21, 359-384 (1970). STREET, H. E.: Factors influencing the initiation and activity of meristems In roots. In: Root Growth, 20-41, Butterworths, London, 1969. TORREY, J. G.: Physio!. Plant., 15, 177-185 (1962). - Ann. Rev. Plant Physio!., 27, 435-459 (1976). WALKEY, D. G. A.: Canad. J. Plant Sci., 52, 1085-1087 (1972). WEBSTER, B. D. and J. W. RADIN: Amer. J. Bot., 59, 744-751 (1972). G. NEMETH, Institute of Plant Physiology, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, P.O.B. 521, Hungary.

Z. P/lanzenphysiol. Bd. 95. S. 389-396. 1979.