M.E. Arena, G.J. Martinez Pastur/Scientia Horticulturae 72 (19971 73-79 ... to the methodology previously described (Arena and Martinez Pastur, 1995) collected.
SCIENTIA HORTlCuLTuRR ELSEVIER
Scientia Horticulturae 72 (1997) 73-79
Short communication
Adventitious shoot induction from leaf explants of Ribes magellanicum cultured in vitro Miriam E. Arena Centro Austral de Inuestigaciones
*,
Cientifcas
Guillermo J. Martinez Pastur (CADIC), cc 92 (9410), Vshuaia, Tierra de1 Fuego, Argentina
Accepted 29 May 1996
Abstract Several factors were studied on adventitious shoot induction from leaf explants of Ribes cultured in vitro: type of explant, position of the leaves along the stem and growth regulators. Explants with petiole and complete leaf lamina presented the highest percentage of organogenesis. The position of the leaf along the shoot strongly affected the induction capacity. The percentage of explants with adventitious shoots was maximum (53%) when using apical leaves, with an average of three shoots per explant. 6-benzylaminopurine (BA) and N-benzyl-9(2-tetrahydropyranyl)-adenine (BPA) had a significantly higher promotive effect than 6-(T-T-dimethylallylamino) purine (2iP) or kinetin on induction of adventitious shoots. Cytokinin concentration significantly affected shoot production, best responses being obtained with 4.44 to 8.88 PM. No significant differences were observed between cr-naphthaleneacetic acid (NAA) concentrations but 0.5 PM NAA usually promoted the highest number of non-vitrified shoots per explant. The results of this work show that R. magellanicum can be induced to form adventitious shoots from leaf explants cultured in vitro. 0 1997 Elsevier Science B.V.
mugellanicum
Keywords:
Ribes; Adventitious shoot: In vitro culture; Leaf explant; Leaf position; Growth regulators
1. Introduction Ribes magellanicum Poiret is a deciduous shrub often growing in Nothofagus pumilio (Poepp. et. Endl.) Krasser forest clearings and wood margins. It grows spontaneously
Abbreviations: BA = 6-benzylaminopurine; NAA = cu-naphthaleneacetic acid; BPA = N-benzyl-9-(2tetrahydropyranylradenine; 2iP = 6-(T-7-dimethylallylamino) purine; IBA = indole 3.butyric acid; AIA = indole-3-acetic acid * Corresponding author. 0304-4238/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO304-4238(96)00940-5
14
M.E. Arena, G.J. Martinez Pastur/Scientia
Horticulturae 72 (19971 73-79
throughout Tierra de1 Fuego, except the north and west extreme areas, between 0 and 300 m above sea level (Moore, 1983). Its berries are used for jams and syrups (Arena and Martinez Pastur, 1995). The induction of adventitious shoots from leaf explants has been possible in several fruit species, such as M&s (James et al., 1988; Faso10 et al., 1989; Pawlicki and Welander, 1994); Pyrus (Chevreau et al., 1989; Leblay et al., 1991) Rubus (Fiola et al., 1990; Ambrozic Turk et al., 1994) and Vuccinium (Billings et al., 1988). To our knowledge, there have been no reports on adventitious shoot induction from leaf explants of Ribes. The aim of this work was to develop a protocol for shoot induction from leaf explants of R. magellanicum cultured in vitro, for both propagating and for exploiting somaclonal variation. Among the many factors known to affect the induction process, several were tested here: type of explant, position of the leaves along the stem and growth regulators.
2. Materials
and methods
Leaves of R. magellanicum were obtained from in vitro propagated shoots according to the methodology previously described (Arena and Martinez Pastur, 1995) collected 8-18 months after the culture initiation and with subcultures at 4 week intervals. The explants were placed on Murashige and Skoog (1962) medium with half strength macronutrient salts (MS2), 3.0% (w/v) sucrose and 0.7% (w/v) of a regionally produced agar. The pH was adjusted to 5.75 &-0.05 with KOH before autoclaving for 20 min at 0.1 MPa. The media were dispensed into 350 ml glass jars containing 50 ml each. Cultures were grown at 22 + 2°C with a 16 h photoperiod using cool-white fluorescent lamps (57 pmol me2 s-l photosynthetic active radiation). Explants were cultured for 8 weeks, being transferred to the same fresh medium at 4th week. Preliminary experiments were carried out in order to evaluate the effects of some factors on adventitious shoot induction. Different periods of continuous initial dark (7, 14, 21 and 28 days) did not enhance the adventitious shoot induction compared with the normal photoperiod. These results showed that an initial dark period did not favour either adventitious shoot formation or growth of organogenetic callus in the explants. Accordingly, all following experiments were carried out using a 16 h photoperiod at all times. Three auxins were tested in another experiment: a-naphthaleneacetic acid (NAA), indole 3-butyric acid (IBA) and indole-3-acetic acid (AIA) at 0.50 PM in combination with 4.44 PM 6-benzylaminopurine CBA). NAA increased significantly the formation of adventitious shoots compared with the other auxins. Therefore, NAA was used in all following experiments. In these experiments, the number of explants producing shoots and callus at 8th week was higher than the number obtained at 4th week. So, a period of culture of 8 weeks was considered suitable. Three experiments were conducted to study organogenesis: In the first experiment the effect of the presence or absence of the petiole and the leaf lamina (complete or basal half) was evaluated using a factorial design. So, four types of explant were tested: complete leaf lamina with the petiole, basal half of the leaf lamina with the petiole, complete leaf lamina without the petiole and basal half of the leaf lamina without the
M.E. Arena, G.J. Martinez Pastur/Scientia
Horticulturae 72 (1997) 73-79
15
petiole. When using explants with the petiole, it was detached from the shoot taking care to exclude the axillary bud. Explants without the petiole were cut at the union with the leaf lamina. The explants were placed with the abaxial side down on MS2 medium with 4.44 FM BA and 0.50 ,uM NAA. In a second experiment the effect of position of the leaf along the shoot was determined. Explants were obtained from shoots 3 cm long and three positions were tested: basal (three basal leaves), middle (three leaves from the central portion of the shoot) and apical (three expanded leaves just below the apex). A treatment with a pool of leaves of each position was also included as a control. Explants with the complete leaf lamina and petiole were used, while MS2 with 4.44 PM BA and 0.50 PM ANA was employed as culture medium. In the last experiment the effect of the cytokinins BA, N-benzyl-9-(2_tetrahydropyranyl)-adenine (BPA), kinetin and 6-(r-r-dimethylallylamino) purine (2iP) (0.00, 4.44, 8.88 and 22.22 ,uM) at three levels of NAA (0.00, 0.50 and 2.50 PM) on shoot formation was determined using a factorial design with apical explants. After 8 weeks of culture the percentage of explants with shoots, callus, total browning, expanded leaf lamina and roots were recorded. In all experiments, five explants were placed per jar and each treatment had eight replicate jars. Each jar was considered as an experimental unit. Each experiment was repeated twice. The data were subjected to analyses of variance using Fisher’s and Tukey’s tests.
3. Results and discussion The percentage of explants forming shoots was significantly higher on explants with the petiole than on explants without the petiole (Table 1). The percentage of explants forming shoots was highest on the explants with complete leaf lamina, but this difference was not statistically significant. None of the two studied factors affected the percentage of explants with callus, total browning and expanded leaf lamina. In explants with petiole, the callus grew at the base and along the petiole; in explants without petiole, the callus grew on the lamina. Shoots differentiated from the callus. In explants with petiole, shoots were observed only on the petiole, not on the lamina, as has been found in Aruchis (Kanyand et al., 1994). The use of explants with the petiole and complete leaf lamina presented the highest percentage of organogenesis (14.28%), yielding the best response in terms of shoot induction. These results allow to conclude that the presence of the petiole is important in organogenesis, as reported by Jerzy and Lubomski (1991) in Gerber-u. The position of the leaf along the shoot strongly affected the organogenesis capacity. The percentage of explants with adventitious shoots was maximum (53%) when using apical leaves with the petiole and complete leaf lamina (Table 21, with an average of three shoots per explant. The organogenesis decreased from the shoot tip to the base. These results coincide with those obtained in Diunthus (van Altvorst et al., 1992) and in Rubus (Ambrozic Turk et al., 1994). Shoots differentiated at the base and along the petiole from the callus (Fig. 1). The percentage of leaves with callus and expanded leaf lamina was also maximum in the apical leaves, while browning was null. The greater organogenetic ability of the apical leaves might be because they have less differentiated
20
1 1 1
df
14.3 4.0 lO.Oa
15.4ns 15.4ns 275.8ns 327.9 225.9ns 20.7ns O.lns 141.5
B
88.6 80.0 85.0a
test: in each column
C
497.7 * 156.6ns 7.8ns 103.4
11.7a 1.5b
S
Mean squares
8.0 0.0 3.la
Complete lamina
Mean
Complete lamina
Half
C
S
85.0a 83.la
Mean
by different
5.7 12.0 8.3a
Complete lamina
B
5.0a 10.8a
Mean
54.3 68.0 60.0a
51.7a 50.8a
Mean
at P < 0.05;
48.0 40.0 43.la
Half
different
Complete lamina
letters are significantly
4.0 10.0 7.7a
Half
E
l
(B) and expanded leaf lamina (E) on MS2 with 4.44 PM BA and 0.50 PM
and row means followed
39Sns 1821.5ns 706.Ons 1025.3
E
80.0 85.0 83.la
Half
of explants with shoots (S), callus (C), total browning
Mean = main effects. Mean separation by Fisher’s significant at P < 0.05; ns, not significant.
Error
Petiole Leaf lamina Petiole X leaf lamina
Source of variation
Analysis of variance
Mean
+ _
Petiole
Table 1 Effect of explant type on percentage NAA at 8th week
,
ME. Arena, G.J. Martinez Pastur/Scientia
Horticulturae 72 (1997) 73-79
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Table 2 Effect of the position of the leaf along the shoot on percentage of explants with petiole and complete leaf lamina forming shoots (S), callus in the petiole (0, total browning (B) and expanded leaf lamina (I?) on MS2 with 4.44 pM BA and 0.5 PM NAA at 8th week Position Basal Middle Apical Control
S
C
B
E
70.OOb 90.OOa 1OO.OOa 96.00a
33.33a 3.33b O.OOb 3.33b
3.33c 2O.OObc 83.33a 33.33b
O.OOb 3.33b 53.33a 6.66b
Analysis of variance Source of variation
df
Mean squares S
C
B
E
Position Error
3 20
3794.4 * * 110.0
1083.3 * * 116.7
1466.7 * * loo.0
7133.3 * * 220.0
In each column means followed * * Significant at P < 0.01.
by different
letters are significantly
different
at P < 0.05 by Tukey’s
test.
and more metabolically active cells, with a more suitable hormonal and nutritional situation that could improve organogenesis (Famiani et al., 1994). On apical explants with the petiole and complete leaf lamina BA and BPA had a significantly higher promotive effect than 2iP and kinetin on percentage of explants producing adventitious shoots (Table 3). Cytokinin concentration significantly affected shoot production. The percentage of explants producing shoots was significantly higher with 4.44 and 8.88 PM cytokinin than with 0.00 and 22.22 PM (Table 3). No significant differences were observed between NAA concentrations but 0.5 PM NAA
Fig. 1. Adventitious shoot induction from leaf explant of R. magellanicum cultured in vitro using apical leaf explant with the petiole and complete leaf lamina on MS2 with 4.44 /.LM BA and 0.5 pM NAA after 8 weeks culture.
M.E. Arena, G.J. Martinez Pastur/Scientia
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Horticulturae 72 (1997) 73-79
Table 3 Effect of cytokinin type and concentration on percentage of apical explants with petiole and complete leaf lamina forming shoots at 8th week. Values are means from three different NAA concentrations (0, 0.5 and 2.5 FM). NAA effect is significant at P = 0.13 Cytokinin type
Cytokinin 0
BA BPA 2iP Kinetin Mean
0.0 0.0 0.0 0.0 O.Ob
concentration
( yM)
Mean
4.44
8.88
10.43 9.16 0.00 0.00 4.90a
10.00 10.00 0.83 0.00 5.20a
22.22 2.60 4.16 0.00 0.00 1.69b
5.76a 5.83a 0.20b O.OOb
Analysis of variance Source of variation
df
Mean squares
Type
3 3 9 366
1032.4* * 612.0 * * 190.7 * * 58.1
Concentration Type X concentration Error
Mean = main effects. Mean separation by Tukey’s test: in each column and row means followed by different letters are significantly different at P < 0.05; * * significant at P < 0.01.
usually promoted the highest number of non-vitrified shoots per explant. The percentage of explants producing adventitious shoots was maximum (22.5%) with 8.88 PM BA combined with 0.5 NAA. The combination of cytokinins (BA and BPA) with NAA allowed the induction of adventitious shoots in R. magellanicum as was found for Malus (Pawlicki and Welander, 1994). When NAA was added to a medium without cytokinins, 50% of the explants formed adventitious roots. Roots emerged from the callus of the petiole. Also, when 4.44 PM 2iP was combined with 0.50 PM NAA, 30% of the explants formed roots. The variability between the results of the same treatment from experiments carried out at different dates was great (compare data of percentage of explants forming shoots in Tables 2 and 3). This could be explained by a progressive decline in the physiological state of mother plants with repeated subcultures (Leblay et al., 1991). The results of this work show that R. magellanicum can be induced to form adventitious shoots through the in vitro culture of leaf explants with petiole and complete leaf lamina, coming from the apical portion of the shoot. The MS2 culture medium with 4.44 to 8.88 PM BA or BPA and 0.50 PM NAA gave the maximum organogenetic capacity.
Acknowledgements
The authors thank to Dr Osvaldo Caso for his critical reading of the manuscript.
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