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Induction of adventitious rooting in vitro in difficult-to-propagate cultivars of apple
Plant Science Letters, 24 (1982) 1--9 Elsevier/North-Holland Scientific Publishers Ltd.
1
INDUCTION OF ADVENTITIOUS ROOTING IN VITRO IN DIFFICULT-TO-PROPAGATE CULTIVARS OF APPLE
S. SRISKANDARAJAH*, M.G. MULLINS** and Y. NAIR Department o f Agronomy and Horticultural Science, University of Sydney, N.S.W. 2006 (Australia) (Received February 9th, 1981) (Revision received July 16th, 1981) (Accepted July 16th, 1981)
SUMMARY
Isolated buds of the normally difficult-to-root cultivars Jonathan and Delicious were cultured with 6-benzyladenine (BA) (10 pM) to produce a proliferating shoot culture. The shoots were either (i) made into microcuttings (50 mm), treated either with a-naphthylene acetic acid (NAA) (10 uM) applied to the m e d i u m or with 7-(indole-3)-butyric acid (IBA) (750 pM) applied to the base of the stem and were then tested for formation of adventitious roots in vitro or (ii) were subcultured with BA as before to produce successive generations of shoots. The duration o f each subculture was 5 weeks. When shoot cultures were grown at 26 + 2°C with continuous illumination (90--100 ~E m -2 s -1) there was a progressive improvement in t h e rooting o f microcuttings with increasing numbers of subcultures. Freshly cultured materials did not form roots. After 9 subcultures 95% of Jonathan microcuttings formed roots. With Delicious the percentage rooting was 21 after 4 subcultures and 79 after 31 subcultures.
INTRODUCTION
Pioneer work on the culture in vitro of isolated buds of economicallyimportant fruit species by Nekrasova [ 1 ], Jones [2] and Dutcher and Powell [3] has led to the development of 'micropropagation' procedures for plant multiplication [ 4,5]. Micropropagation involves the culture of isolated buds on cytokinin-rich media and the production of large numbers of shoots *On leave from University of Peradeniya, Sri Lanka. **To whom correspondence may be addressed. Abbreviations: BA, 6-b enzyladenine; NAA, a-naphthalene acetic acid; IBA, 7 -(indole -3)butyric acid. 0304--4211/82/0000--0000/$02.75 © Elsevier/North-Holland Scientific Publishers Ltd.
through the outgrowth of lateral meristems. These shoots are then made to form adventitious roots by subculture on media containing either auxin or an auxin and additives such as phloroglucinol [7--9]. Rooted shoots are transferred to the glasshouse and thence to the field. In the apple, micropropagation has been most successful with juvenile seedlings [ 10] and with apple rootstocks [ 7,8 ], materials which are generally easy-to-root by conventional methods. Most scion cultivars of apple are very difficult to propagate by cuttings and, with a few exceptions [11--14], they have proved to be difficult-to-regenerate by micropropagation [ 3,15-17]. The present paper describes the establishment in vitro of bud cultures of the major apple cultivars Delicious and Jonathan and the induction of a high frequency of adventitious root formation in isolated shoots. MATERIALS AND METHODS
Establishment o f cultures Dormant 1-year-old scions from virus-indexed mother trees of Jonathan were supplied by the N.S.W. Department of Agriculture. Scions of the cultivar Delicious were obtained from commercial sources. Plants of the two cultivars were propagated by grafting onto apple seedlings and were grown (24°C) either in controlled environment chambers (16-h photoperiod, radiant flux density 350/zE m -2 s-1 ) or in the glasshouse with natural illumination. Regrowth from these scions was used as a source of explants for studies on micropropagation. Scion shoots were harvested when approx. 400 mm in length and 5 mm in diameter and were made into segments (80--100 ram). These segments, from which leaves were removed, were surface sterilized by shaking for 15 min with sodium hypochlorite solution (1% available chlorine) containing Tween 20 (0.05%, v/v) as a wetting agent. After repeated washing with sterile distilled water the nodal segments were planted into test tubes (25 ram) containing 15 ml of the basal medium of Murashige and Skoog (1.962, MS). The medium was solidfied with agar {0.8% w/v) and contained BA (10 pM). The sucrose concentration was30 g 1-1 and pH was adjusted to 5.7 before autoclaving. In most experiments the segments were incubated (26 -+ 2°C)in a growth room with continuous illumination provided by Osram MCFE 40 W cool white fluorescent tubes. The radiant flux density at the level of the cultures was 90--100 ~E m -2 s-1. Production o f shoots Bud burst occurred after 7 days and by 14 days each explant had produced a single extension shoot. When these shoots were 50 mm in length they were either made into 'microcuttings' for induction of adventitious roots (see below) or cut into leafy segments (3--4 nodes) and subcultured in larger culture vessels (350 ml) containing 100 ml of the same cytokininmedium as before. There were 3--4 segments per flask and within 5 weeks each segment had produced up to a dozen shoots by outgrowth of axillary
meristems. These shoots were then harvested for use as microcuttings and the remains of the culture was cut into fragments and subcultured with cytokinin medium to produce a new generation of shoots. Experiments to be reported here are concerned with 5--6 such subcultures, each of 5 weeks duration, with the cultivars Jonathan and Delicious. In addition data on root formation in vitro was obtained from some long-established shoot cultures of Jonathan (25 months) and Delicious (60 months). The delicious cultures were grown at 23 + 2°C and with a photoperiod of 16 h (20--30 ~E m -2 s -i) (Table II).
Induction o f adventitious roots Excised shoots from the initial culture and from each subculture, were made into leafy 'microcuttings' (3--4 nodes, length 50 mm). The microcuttings were grown on filter paper bridges in test tubes (25 mm) containing half-strength MS medium (15 ml) with NAA, (10 ~M). There was one microcutting per tube. In some experiments with Delicious the bases of freshly made microcuttings were dipped in IBA (750 #M) and the microcuttings were then grown with basal medium. Microcuttings were cultivated at constant temperature (26 -+ 2°C) and with continuous illumination (Osram MCFE cool white fluorescent tubes, 90 # E m -2 s-1 at the level of the explant). The numbers of rooted cuttings were recorded after 5 weeks. RESULTS
The nodal explants gave rise to a single extension shoot (Fig. la) which, when subcultured with BA (10 #M), produced multiple shoots through the outgrowth of axillary meristems (Fig. lb). In both Jonathan and Delicious growth of this initial shoot, and of the first and second subcultures was more vigorous than that of later subcultures. By the end of the third subculture shoots had become spindly, i.e. diameter approx. 2 m m as compared with 4 mm at the end of the first subculture. The conformation and growth of shoots did not change appreciably in following subcultures, but weakly growing subcultures which gave low yields of shoots did occur from time to time in the long-term culture of Delicious. In all cultivars the leaves produced in vitro were smaller and more highly serrated than the leaves of normallygrown apples. In several preliminary experiments it was found that shoots from newly established cultures of Jonathan had a low regenerative capacity in vitro and that few, if any, microcuttings produced adventitious roots (Table I). Subsequently, it was observed that microcuttings derived from long,established cultures produced adventitious roots with relative ease. At the 4th transfer, for example, up to 60% of microcuttings formed roots when treated either with NAA (10 #M) or with IAA (100 #M). Later subcultures gave progrea~ ively better results and a consistently high frequency (95%) of adventitious rooting was obtained after the 9th transfer (Fig. ld). Microcuttings from the long-term shoot culture of Delicious (established
a
Fig. 1. (a) Single shoot produced by nodal explant, 30 days after culture (× 1.6). (b) Proliferation of Jonathan shoots on MS medium supplemented with BA (10 ~M), 35 days after subculture (×0.9). (c) Callus formation at the bases of microcuttings (x0.8). (d) Rooted microcuttings of Jonathan from culture No. 27 (xl.0).
O1
105 112 92
100 62 105 117 106 166 149
No. microcuttings tested
100 108 87
0 5 34 51 55 102 105
T o t a l No. rooted microcuttings
± 0.16 ± 0.13 ± 0.19 -+ 0.24 -+ 0.27 + 0.24
9.4 ± 0.56 12.1 -+ 0.59 12.0 -+ 0.25
0 1.1 2.2 2.3 5.2 6.7 6.5
M e a n No. roots -+ S.E.
aNodal c u t t i n g s f r o m w h o l e p l a n t t e s t e d in vitro. bInitial c u l t u r e . CFirst s u b c u l t u r e . dCulture lost.
9 25 28
0a Ib 1c 2 3 4 5
Culture No.
Jonathan
95 96 95
0 8 32 44 52 62 70 31 32
0 I 1 2 3 4
% rooted Culture m i c r o c u t t i n g s No.
Delicious
24 20
34 21 85 86 90 86
No. microcuttings tested
79 70
21
18
19 14
0 5 15 20 _.d
% rooted microcuttings
0 1 13 17
T o t a l No. rooted microcuttings
A D V E N T I T I O U S R O O T F O R M A T I O N IN V I T R O IN A P P L E C U L T I V A R S : E F F E C T S O F S U B C U L T U R E
TABLE I
O~
TABLE II SUMMARY OF THE CONDITIONS UNDER WHICH CULTURES OF APPLE CULTIVARS WERE GROWN AND TESTED FOR ADVENTITIOUS ROOT FORMATION Culture
Temperature (°C)
Photoperiod (h)
Radiant flux density (uE m -2 s -~)
Long-established Delicious (60 months in culture)
23 ~ 2
16
20--30
Newly-established Delicious (7 months in culture)
26 ± 2
24
90--100
Jonathan (25 months in culture)
26 ± 2
24
90--100
in 1975) could not be induced to form roots until the growing conditions were changed to those of the Jonathan culture (Table II). At the 30th transfer the illumination was extended from 16 to 24 h, the radiant flux density at the level of the cultures was increased from 25 to 100 ~E m -2 s -~ and the temperature was raised from 22°C to 26°C. Shoots produced under these conditions gave microcuttings which were easy to propagate. Up to 79% of microcuttings produced roots when treated with IBA (750 ~M) by the dipping procedure. Shoots from subsequent transfers have retained the ability to form adventitious roots. The promotion of adventitious rooting by serial subculture has been confirmed in a systematic study with newly~stablished shoot cultures of both Jonathan and Delicious. The cultures were grown with continuous illumination and at high temperature (26 -+ 2°C) and the microcuttings were cultured with NAA as before. The shoots produced by the initial explants, and the shoots of the first subculture, gave microcuttings in which adventitious rooting occurred with very low frequency (Table I). With later subcultures there was a marked increase in root formation. In Jonathan, for example, the rooting percentage at the 4th subculture (62%) was almost 8 times greater than the rooting percentage at the initial culture (8%). Microcuttings from the initial culture (I) of Delicious seldom formed adventitious roots but they formed callus at their bases (Fig. lc). In some experiments initial cultures were transferred to basal medium after 3--5 days of exposure to auxin. This treatment led to a reduction in callus formation but the cuttings still failed to form roots. Up to 20% of microcuttings produced roots at the 3rd subculture. DISCUSSION
Difficult-to-propagate plants such as Jonathan and Delicious apples were
transformed into easily-propagated materials by a simple procedure. First, isolated buds were cultured on a cytokinin (BA, 10 pM) enriched medium to produce a proliferating shoot culture. The cultures were grown with continuous illumination and at a relatively high temperature (26 + 2°C). Next, the shoot cultures were used as a source of propagation material (microcuttings) and were subcultured at regular intervals. With these treatments there was a progressive improvement in the ability of microcuttings from successive generations of shoots to produce adventitious roots (Table I). To achieve these results it was necessary to apply a combination of treatments. Repeated subculture alone did not give improved adventitious rooting. The treatment became effective only when combined with continuous illumination and with the cultivation of explants at 26°C. The mechanisms involved in these responses are not yet clear. In view of the gradual change in behaviour of microcuttings it is unlikely that genetic modifications are involved. An analysis of effects of irradiance, photoperiod, temperature and repeated subculture is in progress, as are studies of auxin metabolism and phenolic compounds in apple microcuttings from successive transfers. Studies so far indicate that continuous illumination (90--100 ~E m -2 s-1) from cool white fluorescent tubes, together with a constant temperature of 26 + 2°C, are the optimum conditions for shoot growth and root formation in cultures of Jonathan. The subculture procedure provides easy-to-root and difficult-to-root forms of the same genotype. Hitherto, such material has been available only in species like Hedera helix where individual plants exhibit well-defined juvenile (rooting) and adult (non-rooting) phases. Again, it is not yet clear if the subculture treatment brought about a 'rejuvenation' of Delicious and Jonathan, that is, a reversion from the normally persistent adult condition of the cultivar to the juvenile, non-flowering, condition of the original seedling [18]. The juvenile phase of woody perennials is associated with a high degree of regenerative competence but many species, including the apple, seem to lose the capacity to form adventitious roots once they achieve the adult condition [ 19]. Prolonged cultivation in vitro led to a reduction in shoot diameter and to a change in leaf shape in cultures of Jonathan and Delicious, but wellrecognized juvenile characteristics such as presence of thorns have not been observed. Plants of Jonathan and Delicious which were established from microcuttings are still small and it remains to be seen whether they have retained the ability to form flowers, or indeed, the fruit characters of the cultivars concerned. The technique described here for the induction of a high frequency of adventitious root formation in isolated shoots of apple cultivars has practical applications in apple propagation, production of virus-symptomless plants and in the production and recovery of somatic mutants. It is also possible that the subculture procedure could be applied to other species which are normally regarded as difficult-to-propagate.
REFERENCES
1 T.V. Nekrasova, Fiziol. Rast., 1 (1964) 127. 2 0 . P . Jones, Nature, 215 (1967) 1514. 3 R.D. Dutcher and L.E. Powell, J. Am. Soc. Hort. Sci., 97 (1972) 511. 4 O.P. Jones, Sci. Hort., 30 (1979) 44. 5 R.L.M. Pierik, Acta Hort., 54 (1975) 71. 6 A.J. Abbott, Sci. Hort., 28 (1977) 155, 7 O.P. Jones and S.G.S. Hatfield, J. Hort. Sci., 51 (1976) 495. 8 D.J. James and I.J. Thurbon, J. Hort. Sci., 54 (1979) 309. 9 D.J..James, J. Hort. Sci., 54 (1979) 273. 10 D.W. Lane, Plant Sci. Left., 13 (1978) 281. 11 O.P. Jones, C.A. Pontikis and M.E. Hopgood, J. Hort. Sci., 54 (1979) 155. 12 A.J. Abbott and E. Whiteley, Sci. Hort., 4 (1976) 183. 13 S. Sriskandarajah and M.G. Mullins, J. Hort. Sci., 56 (1981) 71. 14 R.H. Zimmermann and O.C. Broome, in: Proceedings of the Conference on Nursery Production of Fruit Plants through Tissue Culture-Applications and Feasibility. USDA. Science and Education Administration Agricultural Research Results, ARRNE-11, Beltsville, Maryland, 1980, pp. 54--58. 15 D.G, Walkey, Can. J. Plant Sci., 52 (1972) 1085. 16 R.F. Elliot, N.Z.J. Bot., 10 (1972) 254. 17 M. Quoirin, Bull. Rech. Agron. Gembloux, 9 (1974) 189. 18 W.W. Schwabe, Acta Hort., 56 (1976) 45. 19 F.E. Gardner, Proc. Am. Soc. Hort. Sci., 26 (1929) 101.
1
INDUCTION OF ADVENTITIOUS ROOTING IN VITRO IN DIFFICULT-TO-PROPAGATE CULTIVARS OF APPLE
S. SRISKANDARAJAH*, M.G. MULLINS** and Y. NAIR Department o f Agronomy and Horticultural Science, University of Sydney, N.S.W. 2006 (Australia) (Received February 9th, 1981) (Revision received July 16th, 1981) (Accepted July 16th, 1981)
SUMMARY
Isolated buds of the normally difficult-to-root cultivars Jonathan and Delicious were cultured with 6-benzyladenine (BA) (10 pM) to produce a proliferating shoot culture. The shoots were either (i) made into microcuttings (50 mm), treated either with a-naphthylene acetic acid (NAA) (10 uM) applied to the m e d i u m or with 7-(indole-3)-butyric acid (IBA) (750 pM) applied to the base of the stem and were then tested for formation of adventitious roots in vitro or (ii) were subcultured with BA as before to produce successive generations of shoots. The duration o f each subculture was 5 weeks. When shoot cultures were grown at 26 + 2°C with continuous illumination (90--100 ~E m -2 s -1) there was a progressive improvement in t h e rooting o f microcuttings with increasing numbers of subcultures. Freshly cultured materials did not form roots. After 9 subcultures 95% of Jonathan microcuttings formed roots. With Delicious the percentage rooting was 21 after 4 subcultures and 79 after 31 subcultures.
INTRODUCTION
Pioneer work on the culture in vitro of isolated buds of economicallyimportant fruit species by Nekrasova [ 1 ], Jones [2] and Dutcher and Powell [3] has led to the development of 'micropropagation' procedures for plant multiplication [ 4,5]. Micropropagation involves the culture of isolated buds on cytokinin-rich media and the production of large numbers of shoots *On leave from University of Peradeniya, Sri Lanka. **To whom correspondence may be addressed. Abbreviations: BA, 6-b enzyladenine; NAA, a-naphthalene acetic acid; IBA, 7 -(indole -3)butyric acid. 0304--4211/82/0000--0000/$02.75 © Elsevier/North-Holland Scientific Publishers Ltd.
through the outgrowth of lateral meristems. These shoots are then made to form adventitious roots by subculture on media containing either auxin or an auxin and additives such as phloroglucinol [7--9]. Rooted shoots are transferred to the glasshouse and thence to the field. In the apple, micropropagation has been most successful with juvenile seedlings [ 10] and with apple rootstocks [ 7,8 ], materials which are generally easy-to-root by conventional methods. Most scion cultivars of apple are very difficult to propagate by cuttings and, with a few exceptions [11--14], they have proved to be difficult-to-regenerate by micropropagation [ 3,15-17]. The present paper describes the establishment in vitro of bud cultures of the major apple cultivars Delicious and Jonathan and the induction of a high frequency of adventitious root formation in isolated shoots. MATERIALS AND METHODS
Establishment o f cultures Dormant 1-year-old scions from virus-indexed mother trees of Jonathan were supplied by the N.S.W. Department of Agriculture. Scions of the cultivar Delicious were obtained from commercial sources. Plants of the two cultivars were propagated by grafting onto apple seedlings and were grown (24°C) either in controlled environment chambers (16-h photoperiod, radiant flux density 350/zE m -2 s-1 ) or in the glasshouse with natural illumination. Regrowth from these scions was used as a source of explants for studies on micropropagation. Scion shoots were harvested when approx. 400 mm in length and 5 mm in diameter and were made into segments (80--100 ram). These segments, from which leaves were removed, were surface sterilized by shaking for 15 min with sodium hypochlorite solution (1% available chlorine) containing Tween 20 (0.05%, v/v) as a wetting agent. After repeated washing with sterile distilled water the nodal segments were planted into test tubes (25 ram) containing 15 ml of the basal medium of Murashige and Skoog (1.962, MS). The medium was solidfied with agar {0.8% w/v) and contained BA (10 pM). The sucrose concentration was30 g 1-1 and pH was adjusted to 5.7 before autoclaving. In most experiments the segments were incubated (26 -+ 2°C)in a growth room with continuous illumination provided by Osram MCFE 40 W cool white fluorescent tubes. The radiant flux density at the level of the cultures was 90--100 ~E m -2 s-1. Production o f shoots Bud burst occurred after 7 days and by 14 days each explant had produced a single extension shoot. When these shoots were 50 mm in length they were either made into 'microcuttings' for induction of adventitious roots (see below) or cut into leafy segments (3--4 nodes) and subcultured in larger culture vessels (350 ml) containing 100 ml of the same cytokininmedium as before. There were 3--4 segments per flask and within 5 weeks each segment had produced up to a dozen shoots by outgrowth of axillary
meristems. These shoots were then harvested for use as microcuttings and the remains of the culture was cut into fragments and subcultured with cytokinin medium to produce a new generation of shoots. Experiments to be reported here are concerned with 5--6 such subcultures, each of 5 weeks duration, with the cultivars Jonathan and Delicious. In addition data on root formation in vitro was obtained from some long-established shoot cultures of Jonathan (25 months) and Delicious (60 months). The delicious cultures were grown at 23 + 2°C and with a photoperiod of 16 h (20--30 ~E m -2 s -i) (Table II).
Induction o f adventitious roots Excised shoots from the initial culture and from each subculture, were made into leafy 'microcuttings' (3--4 nodes, length 50 mm). The microcuttings were grown on filter paper bridges in test tubes (25 mm) containing half-strength MS medium (15 ml) with NAA, (10 ~M). There was one microcutting per tube. In some experiments with Delicious the bases of freshly made microcuttings were dipped in IBA (750 #M) and the microcuttings were then grown with basal medium. Microcuttings were cultivated at constant temperature (26 -+ 2°C) and with continuous illumination (Osram MCFE cool white fluorescent tubes, 90 # E m -2 s-1 at the level of the explant). The numbers of rooted cuttings were recorded after 5 weeks. RESULTS
The nodal explants gave rise to a single extension shoot (Fig. la) which, when subcultured with BA (10 #M), produced multiple shoots through the outgrowth of axillary meristems (Fig. lb). In both Jonathan and Delicious growth of this initial shoot, and of the first and second subcultures was more vigorous than that of later subcultures. By the end of the third subculture shoots had become spindly, i.e. diameter approx. 2 m m as compared with 4 mm at the end of the first subculture. The conformation and growth of shoots did not change appreciably in following subcultures, but weakly growing subcultures which gave low yields of shoots did occur from time to time in the long-term culture of Delicious. In all cultivars the leaves produced in vitro were smaller and more highly serrated than the leaves of normallygrown apples. In several preliminary experiments it was found that shoots from newly established cultures of Jonathan had a low regenerative capacity in vitro and that few, if any, microcuttings produced adventitious roots (Table I). Subsequently, it was observed that microcuttings derived from long,established cultures produced adventitious roots with relative ease. At the 4th transfer, for example, up to 60% of microcuttings formed roots when treated either with NAA (10 #M) or with IAA (100 #M). Later subcultures gave progrea~ ively better results and a consistently high frequency (95%) of adventitious rooting was obtained after the 9th transfer (Fig. ld). Microcuttings from the long-term shoot culture of Delicious (established
a
Fig. 1. (a) Single shoot produced by nodal explant, 30 days after culture (× 1.6). (b) Proliferation of Jonathan shoots on MS medium supplemented with BA (10 ~M), 35 days after subculture (×0.9). (c) Callus formation at the bases of microcuttings (x0.8). (d) Rooted microcuttings of Jonathan from culture No. 27 (xl.0).
O1
105 112 92
100 62 105 117 106 166 149
No. microcuttings tested
100 108 87
0 5 34 51 55 102 105
T o t a l No. rooted microcuttings
± 0.16 ± 0.13 ± 0.19 -+ 0.24 -+ 0.27 + 0.24
9.4 ± 0.56 12.1 -+ 0.59 12.0 -+ 0.25
0 1.1 2.2 2.3 5.2 6.7 6.5
M e a n No. roots -+ S.E.
aNodal c u t t i n g s f r o m w h o l e p l a n t t e s t e d in vitro. bInitial c u l t u r e . CFirst s u b c u l t u r e . dCulture lost.
9 25 28
0a Ib 1c 2 3 4 5
Culture No.
Jonathan
95 96 95
0 8 32 44 52 62 70 31 32
0 I 1 2 3 4
% rooted Culture m i c r o c u t t i n g s No.
Delicious
24 20
34 21 85 86 90 86
No. microcuttings tested
79 70
21
18
19 14
0 5 15 20 _.d
% rooted microcuttings
0 1 13 17
T o t a l No. rooted microcuttings
A D V E N T I T I O U S R O O T F O R M A T I O N IN V I T R O IN A P P L E C U L T I V A R S : E F F E C T S O F S U B C U L T U R E
TABLE I
O~
TABLE II SUMMARY OF THE CONDITIONS UNDER WHICH CULTURES OF APPLE CULTIVARS WERE GROWN AND TESTED FOR ADVENTITIOUS ROOT FORMATION Culture
Temperature (°C)
Photoperiod (h)
Radiant flux density (uE m -2 s -~)
Long-established Delicious (60 months in culture)
23 ~ 2
16
20--30
Newly-established Delicious (7 months in culture)
26 ± 2
24
90--100
Jonathan (25 months in culture)
26 ± 2
24
90--100
in 1975) could not be induced to form roots until the growing conditions were changed to those of the Jonathan culture (Table II). At the 30th transfer the illumination was extended from 16 to 24 h, the radiant flux density at the level of the cultures was increased from 25 to 100 ~E m -2 s -~ and the temperature was raised from 22°C to 26°C. Shoots produced under these conditions gave microcuttings which were easy to propagate. Up to 79% of microcuttings produced roots when treated with IBA (750 ~M) by the dipping procedure. Shoots from subsequent transfers have retained the ability to form adventitious roots. The promotion of adventitious rooting by serial subculture has been confirmed in a systematic study with newly~stablished shoot cultures of both Jonathan and Delicious. The cultures were grown with continuous illumination and at high temperature (26 -+ 2°C) and the microcuttings were cultured with NAA as before. The shoots produced by the initial explants, and the shoots of the first subculture, gave microcuttings in which adventitious rooting occurred with very low frequency (Table I). With later subcultures there was a marked increase in root formation. In Jonathan, for example, the rooting percentage at the 4th subculture (62%) was almost 8 times greater than the rooting percentage at the initial culture (8%). Microcuttings from the initial culture (I) of Delicious seldom formed adventitious roots but they formed callus at their bases (Fig. lc). In some experiments initial cultures were transferred to basal medium after 3--5 days of exposure to auxin. This treatment led to a reduction in callus formation but the cuttings still failed to form roots. Up to 20% of microcuttings produced roots at the 3rd subculture. DISCUSSION
Difficult-to-propagate plants such as Jonathan and Delicious apples were
transformed into easily-propagated materials by a simple procedure. First, isolated buds were cultured on a cytokinin (BA, 10 pM) enriched medium to produce a proliferating shoot culture. The cultures were grown with continuous illumination and at a relatively high temperature (26 + 2°C). Next, the shoot cultures were used as a source of propagation material (microcuttings) and were subcultured at regular intervals. With these treatments there was a progressive improvement in the ability of microcuttings from successive generations of shoots to produce adventitious roots (Table I). To achieve these results it was necessary to apply a combination of treatments. Repeated subculture alone did not give improved adventitious rooting. The treatment became effective only when combined with continuous illumination and with the cultivation of explants at 26°C. The mechanisms involved in these responses are not yet clear. In view of the gradual change in behaviour of microcuttings it is unlikely that genetic modifications are involved. An analysis of effects of irradiance, photoperiod, temperature and repeated subculture is in progress, as are studies of auxin metabolism and phenolic compounds in apple microcuttings from successive transfers. Studies so far indicate that continuous illumination (90--100 ~E m -2 s-1) from cool white fluorescent tubes, together with a constant temperature of 26 + 2°C, are the optimum conditions for shoot growth and root formation in cultures of Jonathan. The subculture procedure provides easy-to-root and difficult-to-root forms of the same genotype. Hitherto, such material has been available only in species like Hedera helix where individual plants exhibit well-defined juvenile (rooting) and adult (non-rooting) phases. Again, it is not yet clear if the subculture treatment brought about a 'rejuvenation' of Delicious and Jonathan, that is, a reversion from the normally persistent adult condition of the cultivar to the juvenile, non-flowering, condition of the original seedling [18]. The juvenile phase of woody perennials is associated with a high degree of regenerative competence but many species, including the apple, seem to lose the capacity to form adventitious roots once they achieve the adult condition [ 19]. Prolonged cultivation in vitro led to a reduction in shoot diameter and to a change in leaf shape in cultures of Jonathan and Delicious, but wellrecognized juvenile characteristics such as presence of thorns have not been observed. Plants of Jonathan and Delicious which were established from microcuttings are still small and it remains to be seen whether they have retained the ability to form flowers, or indeed, the fruit characters of the cultivars concerned. The technique described here for the induction of a high frequency of adventitious root formation in isolated shoots of apple cultivars has practical applications in apple propagation, production of virus-symptomless plants and in the production and recovery of somatic mutants. It is also possible that the subculture procedure could be applied to other species which are normally regarded as difficult-to-propagate.
REFERENCES
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