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Micropropagation of Actinidia deliciosa cvs. ‘Hayward’ and ‘Tomuri’
Scientia Horticulturae, 45 (1990) 65-74 Elsevier Science Publishers B.V., A m s t e r d a m
65
Micropropagation ofActinidia deliciosa cvs. 'Hayward' and 'Tomuri' Grazia Marino and Gianpaolo Bertazza lstituto di Cohivazioni Arboree dell'Universitgt, 6 Via F. Re - 40126 Bologna (ltaly) (Accepted for publication 26 March 1990)
ABSTRACT Marino, G. and Bertazza, G., 1990. Micropropagation of Actinidia deliciosa cvs. ,Hayward' and 'Tomuri'. Scientia Hortic., 45: 65-74. Benzyladenine (BA) induced the best proliferation responses, as shoot and node production, in Actinidia deliciosa cultivars 'Hayward' and 'Tomuri', while callus growth was enhanced on zeatin (Z)-enriched media. 'Tomuri' showed a higher proliferation and shoot weight increase than 'Hayward' on most of the culture media tested, but shoots of'Hayward' produced bigger calli. Numerous roots were observed after 25 days in all shoots of both cultivars transferred from proliferation to indole-3-butyric acid (IBA)-enriched media, especially in 'Tomuri'. Rooting percentages on media without auxin were just slightly lower than on IBA-enriched media. Acclimatization was strongly affected by temperature; in summer, plants needed 56 days in the hardening chamber if survival was to remain high (75%) on transfer to the greenhouse. By contrast, ~ 90% survival was easily achieved in spring. Leaf chlorophyll a and b content was influenced by the culture media; however, it was higher in 'Tomuri', particularly in the rooting and hardening phases. Different proliferation, growth, rooting responses and chlorophyll content of in vitro cultures of 'Hayward' and 'Tomuri' suggest that an interaction between culture media and different endogenous hormone concentrations can be expected, i.e. a cytokinin/auxin balance which is higher for 'Tomuri' than for 'Hayward'. Keywords: Actinidia deliciosa; chlorophyll; shoot culture. Abbreviations: BA = N6-benzyladenine; cg= callus fresh weight at shoot base; GA3 = gibberellic acid; IBA=indole-3-butyric acid; K=kinetin; MS=according to Murashige and Skoog (1962); " M S + " = m o d i f i e d MS; pcA=proliferation coefficient A: new shoots/initial shoot; pcB = proliferation coefficient B: new shoots with 1>3 nodes/initial shoot; pcC = proliferation coefficient C: total number of new nodes/initial shoot; P F R = p h o t o n flux rate; sg=final shoot fresh weight less initial fresh weight and cg; Z = zeatin.
INTRODUCTION
In vitro propagation of Actinidia species has been widely tested, starting from many organs and tissues (Bini, 1979; Standardi, 1982, 198,3; Jona and 0304-4238/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
66
G. MARINO AND G. BERTAZZA
Gribaudo, 1983; Wessels et al., 1984; Velayandom et al., 1985; Monette, 1986; Piagnani et al., 1986). However, data from different authors on cultivar 'Hayward' often differ greatly. This suggested further trials; ( 1 ) to verify the proliferation and rooting responses of A c t i n i d i a d e l i c i o s a (A. Chev.) var. deliciosa, formerly A c t i n i d i a c h i n e n s i s Planch. (Liang and Ferguson, 1984), cultivar 'Hayward' and compare it with the behaviour of cultivar 'Tomuri'. Only the proliferation results induced by different N6-benzyladenine (BA) concentrations are known about the latter (Piagnani et al., 1986 ), which revealed higher proliferation rates than three female cultivars; (2) to determine whether in in vitro cultures there are further characters which distinguish cultivars (e.g. shoot weight increase, callus production and chlorophyll content). MATERIALS AND METHODS
Cultures. - Cultures of 'Hayward' and 'Tomuri' were initiated in late spring from shoot tips of 6-year-old vines. After sterilization according to Jones' method (Jones et al., 1977), the apical parts of shoots, ~ 5 m m long, were planted on a medium containing major and minor salts as in Murashige and Skoog (1962) (MS) and (/tM): 555 myo-inositol, 4.06 nicotinic acid, 2.43 pyridoxine-HC1, 26.6 glycine, 2.96 thiamine.HC1, 2.22 BA, 0.29 gibberellic acid (GA3), 58428 sucrose and 7 g 1-1 agar (Bacto-Difco), pH 5.7 (adjusted with 0.1-1 N KOH before autoclaving at 120 ° C for 20 min). The shoots were later transferred to a similar medium, but enriched with (#M) 6.67 BA and 0.25 indole-3-butyric acid ( IBA ) (medium "MS + " ). Shoots used in the trials were taken from cultures maintained on "MS + " medium for a year through 3-weekly subculturing. P r o l i f e r a t i o n . - Six culture media with the same composition as "MS + ", but
with the addition of 4.44 or 8.88/tM BA, or 4.56 or 9.12/zM zeatin (Z), or 4.65 or 9.30 #M kinetin (K), were compared for: proliferation coefficient A (pcA) =newly produced shoots from one initial shoot; proliferation coefficient B (pcB) =newly produced shoots with >I 3 nodes from one initial shoot; proliferation coefficient C ( p c C ) = t o t a l number of newly produced nodes from one initial shoot. At the end of the subculture intervals, tissue growth was also determined and expressed as: shoot growth (sg) = final shoot fresh weight less initial shoot fresh weight and callus fresh weight at shoot base; callus growth (cg) = callus fresh weight. Proliferation and tissue growth were measured for the 3rd, 4th and 5th subcultures, each subculture taking 21 days on the same medium, initially using a randomized block design with 15, 15 and 10 replications ( 500-ml glass jars, each containing 65 ml of medium and five shoots, sealed with a screw metal
MICROPROPAGATIONOF ACTINIDIA DELICIOSA
67
cap, shoots were 10-15 m m long, with four buds, the apex and three nodes) per subculture, respectively. Since no differences existed between subcultures (ANOVA), data were put together having a total average of 200 shoot per medium. and acclimatization. - Single shoots 20-25 m m long were transplanted from "MS + " to similar media, except for the following changes: all MS salts but FeSO4 and Na2EDTA were reduced to half-strength; BA and gibberellic acid (GA3) were lacking and IBA was added at 0 (control), 1.0 and 2.46/tM. Rooting percentage, root n u m b e r and length were determined 14 and 25 days after shoot transfer to the rooting media, using a c o m p l e t e randomized design with 54 replications of one shoot per m e d i u m (shoots ~ 20 m m long were singly cultured in tubes of 26 m m diameter). Cultures were maintained at 2 2 + 2 ° C with a 16-h photoperiod under a p h o t o n flux rate ( P F R ) of ~ 4 5 #E s -1 m -2 (Philips TLM 20/33 cool-white lamps) during all in vitro phases. Acclimatization was carried out in a hardening chamber with a humidity ranging from 70 up to 100% (fog) at 22 + 2 ° C with a 16-h photoperiod under a P F R of ~ 135 #E s -1 m -2 (Philips TLM 20/33 cool-white lamps). Plants were placed in plastic containers with glass covers in a p e a t / s a n d (50%/50%) mixture. Humidity was gradually reduced from 100% by removing covers. After 28 and 56 days in summer, and 15 days in spring, the surviving plants were transferred to the greenhouse.
Rooting
C h l o r o p h y l l c o n t e n t . - Entire leaves without petioles were collected from nonvitreous shoots at the end of the 5th subculture on different proliferation media and at the end of the rooting phase of shoots coming from "MS + " medium. Leaf discs (14 m m diameter) were cut from fully expanded leaves of plants grown in the hardening chamber for 56 days. Both leaves and discs were extracted in 80% ethanol, and chlorophyll a and b were determined by Arnon's ( 1949 ) formula on an average of 50 replicates per medium, using a complete randomized design. RESULTS AND DISCUSSION
As a high n u m b e r of subcultures after explant establishment is required in order to obtain steady proliferation rates of 'Hayward', the initial rate of multiplication on "MS + " m e d i u m was not recorded. Our shoot proliferation rates are comparable with those reported by Piagnani et al. (1986) for 'Hayward' and 'Tomuri', but in reference to "Hayward' they were higher than those reported by Jona and Gribaudo (1983), and slightly lower than those found by Monette (1986) and Standardi ( 1982; 1983). This was probably due to shorter subculture intervals (21 days) in our trials and to the high n u m b e r of shoots tested, which included all shoots Proliferation.
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68
G. MARINO AND G. BERTAZZA
growing on the maintenance medium "MS + " and not purposely those from the most vigorous cultures. No matter what coefficient (pcA, pcB or pcC) was used to express the proliferation rates, the interactions hormone × dose and hormone × cultivar were shown to be highly significant; moreover BA gave the best results for both cultivars (Table 1 ). On the same media, shoots could develop from single nodes and showed proliferation rates comparable with those reported in Table 1 in 3-4 weeks, agreeing with results reported for single-bud cuttings by Piagnani et al. (1986) and Wessels et al. (1984). K often stimulated the growth of vigorous but uniaxial shoots with only 1-2 new nodes developed. However, both BA concentrations sometimes caused vitrification which did not affect proliferation. In fact, only the older leaves became vitrified and not the newly produced shoots. Z, which has been shown to be synthesized by in vitro cultures of Actinidia and other woody plants (Einset, 1986), seldom induced this undesirable effect. K did not have a clear effect: normal and completely vitrified shoots were found in the same jar. Proliferation was higher for 'Tomuri' cultures on most of the six media tested (Table 1 ). This agrees with data reported by other authors for shoots of the same cultivars grown on media with BA (Piagnani et al., 1986 ). The hormone Xcultivar interaction was significant for shoot and callus growth. Z induced the highest shoot growth in 'Hayward' and callus growth in both cultivars, and K always induced the lowest shoot and callus growth TABLE 1 Total number of shoots (pcA), shoots with >/3 nodes (pcB) and total number of nodes (pcC) newly produced from one initial shoot after 21 days culture on different proliferation media Medium composition (#M)
'Hayward'
'Tomuri'
Mean
pcA
pcB
pcC
pcA
pcB
pcC
pcA
pcB
pcC
BA BA
4.44 8.88
3.12aA 3.07aA
0.92aA 0.88aA
6.45 aA 6.47aA
3.63bA 3.78bA
1.42bA 1.30bA
8.18bA 8.35bA
3.40
1.13
7.36
Z Z
4.56 9.12
1.79aA 2.53aB
0.37aA 0.63aB
3.29aA 4.96aB
2.25bA 3.06bB
0.47aA 0.82bB
4.09bA 6.07bB
2.40
0.57
4.60
K K
4.65 9.30
0.94aA 1.32aB
0.00aA 0.01 aA
1.05aA 1.55 aA
1.79bA 1.95bA
0.00aA 0.03aA
2.08bA 2.45bA
1.50
0.01
1.78
2.24
0.47
4.26
2.83
0.67
5.49
Mean
Values with different lower case letters for rows and upper case letters for columns differ statistically at P = 0.01 ( paired comparisons, Student's t, on transformed (x) 1/z values ). Main effects for pcA, pcB and pcC: hormone**, dose**, hormone×dose**, cultivar**, hormone × cultivar**, dose × cultivar n.s.
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MICROPROPAGATION OF ACTINIDIA DELICIOSA
TABLE2
Shoot (sg) and callus (cg) growth after 21 days culture on different proliferation media Medium composition (/tM)
'Hayward'
'Tomuri'
Mean
sg (gfw)
cg (gfw)
sg (gfw)
cg (gfw)
sg (gfw)
cg (gfw)
BA BA
4.44 9.30
0.42 aA 0.44 aA
0. l 1 bA 0.10 aA
0.52 bA 0.51 aA
0.08 aA 0.08 aA
0.47
0.09
Z Z
4.56 9.12
0.51 aA 0.59 aB
0.21bA 0.22 bA
0.50 aA 0.55 aA
0.13 aA 0.11 aA
0.54
0.17
K K
4.65 9.30
0.25 aA 0.28 aA
0.03 aA 0.04 aA
0.37 bA 0.40 bA
0.04 aA 0.04 aA
0.32
0.04
0.40
0.12
0.47
0.08
Mean
fw = fresh weight. Values with different lower case letters for rows and upper case letters for columns differ statistically at P=0.01 (paired comparisons, Student's t). Main effects for sg: hormone**, dose**, hormone × dose n.s., cultivar**, hormone × cultivar**, doseXcultivar n.s.; for cg: hormone**, dose n.s., hormone X dose n.s., cultivar**, hormone × cultivar**, dose × cultivar n.s.
(Table 2). However, 'Tomuri' showed higher values for shoot growth than 'Hayward' on media enriched with 4.44/tM BA or K at both levels. By contrast, callus growth at the base of shoots was greater for 'Hayward' in the presence of BA and especially of Z. R o o t i n g a n d a c c l i m a t i z a t i o n . - The rhizogenic activity of IBA compared with
the control was confirmed. However, no differences in rooting percentages were shown between doses (Table 3 ). Rooting percentages were already high 14 days after shoot transfer to the rooting media, but roots were very short ( 2 - 4 mm; data not reported in the tables). Thereafter they increased up to 100% in both cultivars after 25 days, except in the control. Callus, already present after 14 days on media containing IBA, also increased, especially at the higher auxin level. A small amount of callus also appeared in controls of both cultivars. Root number and length depended on IBA concentrations: the higher auxin level induced more numerous, but shorter roots. Moreover, roots were more numerous and longer in 'Tomuri' than in 'Hayward' on IBA-enriched media. Acclimatization was carried out first in summer and later in spring with plants rooted on the same IBA-enriched media, in order to improve survival results. In both trials, almost all plants of the two cultivars (even those with-
95.06 a
Mean
8.99 a
2.67 aA 10.02 aB 13.35 aC 10.10 a
10.17 aAB 11.14 aB 9.31 aA
Length (mm)
97.53 a
92.59 aA 100 aA 100 aA 10.09 b
2.70 aA 11.78 bB 15.24 bC
Number
Root
12.22 b
ll.18aA 12.82 bB 11.93 bAB
Length (mm) 88.89A 100 B 100 B
Rooting percentage
Mean
2.69A 10.90 B 14.30 C
Number
Root
10.68 A 12.05 B 10.69 A
Length (mm)
Values with different lower case letters for rows and upper case letters for columns differ statistically at P = 0.01 (percentage and root number and length analysed by Z2 and Student-Newman-Keuls test, respectively).
85.18 aA 100 aA 100 aA
Number
Rooting percentage
Rooting percentage
Root
'Tomuri'
'Hayward'
IBA 0.00 IBA 1.00 IBA 2.46
Medium composition (/~M)
Rooting percentage, root number and length after 25 days culture on different rooting media
TABLE 3
N
O
71
MICROPROPAGATION OF ACTINIDIA DELICIOSA
out roots) survived transplanting to the hardening chamber and grew quickly. However, ~ 60% of those which were transferred to the greenhouse in summer after 28 days died (survival rates were not related to different rooting media). The high temperature of the greenhouse (up to 38°C) might have been responsible for these results, as suggested elsewhere (Bini, 1979 ). These plants required a long stay ( 56 days) in the hardening chamber if the survival rate was to remain high (75%) on transfer to the greenhouse. By contrast, plants of 'Hayward' and 'Tomuri' which were transferred to the greenhouse after 15 days in spring showed ~ 90% survival. C h l o r o p h y l l c o n t e n t . - Chlorophyll a and b contents in proliferating shoots of 'Hayward' and 'Tomuri' were influenced by the culture media (Table 4), i.e. highest with Z and lowest with K. It has been reported that Z induced higher chlorophyll contents than BA in callus cultures ofA. c h i n e n s i s P1. (Mannini and Ryugo, 1983 ). However, both chlorophyll a and b levels tended to be higher for 'Tomuri', but seldom with statistically different values. During the rooting phase, average chlorophyll levels were comparable with those of proliferating shoots, notwithstanding the absence of cytokinin. On the contrary, IBA seemed to exert an inhibitory influence on chlorophyll content compared with the control (Table 5 ), agreeing with data reported elsewhere for callus cultures of other species (George and Sherrington, 1984). TABLE 4 Leaf chlorophyll content in proliferating shoots after 21 days culture (5th subculture) on different culture media Medium composition (/tM)
'Hayward' Chlorophyll ( m g g -1 fw)
'Tomuri' Chlorophyll ( m g g -1 fw)
Mean Chlorophyll ( m g g - I fw)
a
b
a
b
a
b
BA BA
4.44 8.88
0.657 aA 0.666 aA
0.286 aA 0.282 aA
0.832 bA 0.783 aA
0.377 aA 0.359 aA
0.734
0.326
Z Z
4.56 9.12
0.751 aA 0.763 aA
0.313 aA 0.308 aA
1.058 bA 1.053 bA
0.467 bA 0.508 bA
0.906
0.399
K K
4.65 9.30
0.383 aA 0.433 aA
0.145 aA 0.178 aA
0.553 aA 0.501 aA
0.241 aA 0.211 aA
0.467
0.194
0.610
0.252
0.796
0.361
Mean
fw = fresh weight. Values with different lower case letters for rows and upper case letters for columns differ statistically at P=0.01 (paired comparisons, Student's t). Main effects for chlorophyll a and chlorophyll b: hormone**, dose n.s., h o r m o n e × d o s e n.s., cultivar**, hormone × cultivar*, dose × cultivar n.s.
72
G. MARINOAND G. BERTAZZA
TABLE 5 Leaf chlorophyll content in rooted shoots after 25 days culture on different rooting media Medium composition (#M)
IBA IBA IBA Mean
0.00 1.00 2.46
'Hayward' Chlorophyll (mg g-1 fw)
'Tomuri' Chlorophyll
Mean Chlorophyll
(mg g-1 fw)
(nag g-1 fw)
a
b
a
b
a
b
0.824 aC 0.541 aB 0.383 aA
0.299 aB 0.269 aB 0.195 aA
1.167 bC 0.759 bB 0.494 bA
0.511 bC 0.364 bB 0.244 aA
0.996 C 0.650 B 0.438 A
0.405 C 0.317 B 0.220 A
0.583 a
0.254 a
0.807 b
0.373 b
fw = fresh weight. Values with different lower case letters for rows and upper case letters for columns differ statistically at P=0.01 (Student-Newman-Keuls test).
Moreover, 'Tomuri' again showed higher chlorophyll (especially a) levels than 'Hayward'. Similar results were found in hardened plants (56 days after transfer) with (mg g-1 fresh weight) average chlorophyll a 1.655 and chlorophyll b 0.842 for 'Tomuri', and chlorophyll a 1.456 and chlorophyll b 0.694 for 'Hayward' (statistically different at P = 0.01 ). CONCLUDING REMARKS
Our results confirm the suitability ofA. deliciosa for shoot micropropagation. The in vitro behaviour of 'Hayward' and 'Tomuri' was influenced by cytokinins and auxins. However, 'Tomuri' showed higher proliferation and shoot growth than 'Hayward' on most of the tested media, which agrees with the higher growth of the former observed in the field. By contrast, callus growth in proliferating cultures was greater for 'Hayward'. 'Tomuri' generally had similar rooting percentages, but more numerous and longer roots than 'Hayward'. Also, the chlorophyll content was consistently higher for 'Tomuri' than for 'Hayward', particularly in the rooting and hardening phases, when exogenous BA was not added. Hormones are known to have regulatory powers over the development of senescence in a number of species, i.e. cytokinins deferred the loss of chlorophyll in Xantium leaves (Leopold and Kriedemann, 1975 ). In conclusion, all these obviously genetic differences in proliferation rates, callus growth and chlorophyll contents suggest that an interaction of culture media with different endogenous hormonal concentrations can be expected in the two cultivars. Tentatively, we can suppose a cytokinin/auxin endogenous balance which is higher for 'Tomuri' than for 'Hayward'. Moreover, the fact that the cultivars compared are of different sexes seems interesting. As
MICROPROPAGATION OF ACTINIDIA DELICIOSA
73
other authors reported higher shoot proliferation rates for 'Tomuri' than for three female cultivars (Piagnani et al., 1986), and sex expression of some other dioecious angiosperms was supposed to be controlled by an endogenous hormonal balance (Heslop-Harrison, 1972 ), further studies are needed to investigate if this is also the case for A. deliciosa. ACKNOWLEDGEMENTS
The research was supported by funds from the Italian Ministry of Education, Roma (M.P.I. 40%).
REFERENCES Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxydase in Beta vulgaris. Plant Physiol., 24:1-15. Bini, G., 1979. Moltiplicazione "in vitro" diActinidia chinensisPl. Atti Incontro SOI: Tecniche di colture "in vitro" per la propagazione su vasta scala delle specie ortoflorofrutticole. Pistoia, 6 October: 211-216. Einset, J.W., 1986. Zeatin biosynthesis from N 6- (2-isopentenyl)adenine in Actinidia and other woody plants. Proc. Natl. Acad. Sci. USA, 83: 972-975. George, E.F. and Sherrington, P.D., 1984. Plant Propagation by Tissue Culture. Exegetics Ltd., Everserley, Basingstoke, pp. 289. Heslop-Harrison, J., 1972. Sexuality of angiosperms. In: Plant Physiology, A Treatise. Vol. 6c. Academic Press, New York. pp. 133-289. Jona, R. and Gribaudo, I., 1983. Comparazione sperimentale di diverse metodologie di micropropagazione dell'actinidia. Atti del II incontro frutticolo sull'actinidia. Udine, 12-13 October: 545-556. Jones, O.P., Hopgood, M.E. and O'Farrell, D., 1977. Propagation in vitro of M 26 apple rootstocks. J. Hortic. Sci., 52: 235-238. Leopold, A.C. and Kriedemann, P.E., 1975. Plant Growth and Development. McGraw-Hill, New York, 157 pp. Liang, C.F. and Ferguson, A.R., 1984. Emendation of the Latin name ofActinidia chinensis var. hispida C.F. Liang. Guihaia, 4: 181-182. Mannini, F. and Ryugo, K., 1983. Relazioni tra il livello endogeno di gibberelline e di clorofilla ed organogenesi in calli di Actinidia chinensis su mezzo di coltura aggiunto di zeatina. Atti del II incontro frutticolo sull'Actinidia. Udine, 12-13 October: 567-578. Monette, P.L., 1986. Micropropagation of kiwifruit using non-axenic shoot tips. Plant Cell Tissue Organ Cult., 6: 73-82. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plant., 15: 473-474. Piagnani, C., Eccher, T. and Castelli, S., 1986. Micropropagation ofActinidia chinensis: effect of growth regulators on proliferation rate. Acta Hortic., 179:887-890. Standardi, A., 1982. Effetti di subcolture ripetute in germogli di Actinidia chinensis (P1.) coltivati "in vitro". Riv. Ortoflorofrutt. It., 66: 419-429. Standardi, A., 1983. La "micropropagazione" nella moltiplicazione dell'actinidia. Frutticoltura, 2: 17-22. Velayandom, L., Hirsch, A.M. and Fortune, D., 1985. Propagation du Kiwi, Actinidia chinensis
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(L.) Planchon, par microbouturage "in vitro" de noeuds. C.R. Acad. Sci. Paris, t. 301, serie III, 12: 597-600. Wessels, E., Nel, D.D. and Van Staden, D.F.A., 1984. "In vitro" propagation ofActinidia chinensis P1. cultivar Hayward. Deciduous Fruit Grow., 34: 453-457.
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Micropropagation ofActinidia deliciosa cvs. 'Hayward' and 'Tomuri' Grazia Marino and Gianpaolo Bertazza lstituto di Cohivazioni Arboree dell'Universitgt, 6 Via F. Re - 40126 Bologna (ltaly) (Accepted for publication 26 March 1990)
ABSTRACT Marino, G. and Bertazza, G., 1990. Micropropagation of Actinidia deliciosa cvs. ,Hayward' and 'Tomuri'. Scientia Hortic., 45: 65-74. Benzyladenine (BA) induced the best proliferation responses, as shoot and node production, in Actinidia deliciosa cultivars 'Hayward' and 'Tomuri', while callus growth was enhanced on zeatin (Z)-enriched media. 'Tomuri' showed a higher proliferation and shoot weight increase than 'Hayward' on most of the culture media tested, but shoots of'Hayward' produced bigger calli. Numerous roots were observed after 25 days in all shoots of both cultivars transferred from proliferation to indole-3-butyric acid (IBA)-enriched media, especially in 'Tomuri'. Rooting percentages on media without auxin were just slightly lower than on IBA-enriched media. Acclimatization was strongly affected by temperature; in summer, plants needed 56 days in the hardening chamber if survival was to remain high (75%) on transfer to the greenhouse. By contrast, ~ 90% survival was easily achieved in spring. Leaf chlorophyll a and b content was influenced by the culture media; however, it was higher in 'Tomuri', particularly in the rooting and hardening phases. Different proliferation, growth, rooting responses and chlorophyll content of in vitro cultures of 'Hayward' and 'Tomuri' suggest that an interaction between culture media and different endogenous hormone concentrations can be expected, i.e. a cytokinin/auxin balance which is higher for 'Tomuri' than for 'Hayward'. Keywords: Actinidia deliciosa; chlorophyll; shoot culture. Abbreviations: BA = N6-benzyladenine; cg= callus fresh weight at shoot base; GA3 = gibberellic acid; IBA=indole-3-butyric acid; K=kinetin; MS=according to Murashige and Skoog (1962); " M S + " = m o d i f i e d MS; pcA=proliferation coefficient A: new shoots/initial shoot; pcB = proliferation coefficient B: new shoots with 1>3 nodes/initial shoot; pcC = proliferation coefficient C: total number of new nodes/initial shoot; P F R = p h o t o n flux rate; sg=final shoot fresh weight less initial fresh weight and cg; Z = zeatin.
INTRODUCTION
In vitro propagation of Actinidia species has been widely tested, starting from many organs and tissues (Bini, 1979; Standardi, 1982, 198,3; Jona and 0304-4238/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
66
G. MARINO AND G. BERTAZZA
Gribaudo, 1983; Wessels et al., 1984; Velayandom et al., 1985; Monette, 1986; Piagnani et al., 1986). However, data from different authors on cultivar 'Hayward' often differ greatly. This suggested further trials; ( 1 ) to verify the proliferation and rooting responses of A c t i n i d i a d e l i c i o s a (A. Chev.) var. deliciosa, formerly A c t i n i d i a c h i n e n s i s Planch. (Liang and Ferguson, 1984), cultivar 'Hayward' and compare it with the behaviour of cultivar 'Tomuri'. Only the proliferation results induced by different N6-benzyladenine (BA) concentrations are known about the latter (Piagnani et al., 1986 ), which revealed higher proliferation rates than three female cultivars; (2) to determine whether in in vitro cultures there are further characters which distinguish cultivars (e.g. shoot weight increase, callus production and chlorophyll content). MATERIALS AND METHODS
Cultures. - Cultures of 'Hayward' and 'Tomuri' were initiated in late spring from shoot tips of 6-year-old vines. After sterilization according to Jones' method (Jones et al., 1977), the apical parts of shoots, ~ 5 m m long, were planted on a medium containing major and minor salts as in Murashige and Skoog (1962) (MS) and (/tM): 555 myo-inositol, 4.06 nicotinic acid, 2.43 pyridoxine-HC1, 26.6 glycine, 2.96 thiamine.HC1, 2.22 BA, 0.29 gibberellic acid (GA3), 58428 sucrose and 7 g 1-1 agar (Bacto-Difco), pH 5.7 (adjusted with 0.1-1 N KOH before autoclaving at 120 ° C for 20 min). The shoots were later transferred to a similar medium, but enriched with (#M) 6.67 BA and 0.25 indole-3-butyric acid ( IBA ) (medium "MS + " ). Shoots used in the trials were taken from cultures maintained on "MS + " medium for a year through 3-weekly subculturing. P r o l i f e r a t i o n . - Six culture media with the same composition as "MS + ", but
with the addition of 4.44 or 8.88/tM BA, or 4.56 or 9.12/zM zeatin (Z), or 4.65 or 9.30 #M kinetin (K), were compared for: proliferation coefficient A (pcA) =newly produced shoots from one initial shoot; proliferation coefficient B (pcB) =newly produced shoots with >I 3 nodes from one initial shoot; proliferation coefficient C ( p c C ) = t o t a l number of newly produced nodes from one initial shoot. At the end of the subculture intervals, tissue growth was also determined and expressed as: shoot growth (sg) = final shoot fresh weight less initial shoot fresh weight and callus fresh weight at shoot base; callus growth (cg) = callus fresh weight. Proliferation and tissue growth were measured for the 3rd, 4th and 5th subcultures, each subculture taking 21 days on the same medium, initially using a randomized block design with 15, 15 and 10 replications ( 500-ml glass jars, each containing 65 ml of medium and five shoots, sealed with a screw metal
MICROPROPAGATIONOF ACTINIDIA DELICIOSA
67
cap, shoots were 10-15 m m long, with four buds, the apex and three nodes) per subculture, respectively. Since no differences existed between subcultures (ANOVA), data were put together having a total average of 200 shoot per medium. and acclimatization. - Single shoots 20-25 m m long were transplanted from "MS + " to similar media, except for the following changes: all MS salts but FeSO4 and Na2EDTA were reduced to half-strength; BA and gibberellic acid (GA3) were lacking and IBA was added at 0 (control), 1.0 and 2.46/tM. Rooting percentage, root n u m b e r and length were determined 14 and 25 days after shoot transfer to the rooting media, using a c o m p l e t e randomized design with 54 replications of one shoot per m e d i u m (shoots ~ 20 m m long were singly cultured in tubes of 26 m m diameter). Cultures were maintained at 2 2 + 2 ° C with a 16-h photoperiod under a p h o t o n flux rate ( P F R ) of ~ 4 5 #E s -1 m -2 (Philips TLM 20/33 cool-white lamps) during all in vitro phases. Acclimatization was carried out in a hardening chamber with a humidity ranging from 70 up to 100% (fog) at 22 + 2 ° C with a 16-h photoperiod under a P F R of ~ 135 #E s -1 m -2 (Philips TLM 20/33 cool-white lamps). Plants were placed in plastic containers with glass covers in a p e a t / s a n d (50%/50%) mixture. Humidity was gradually reduced from 100% by removing covers. After 28 and 56 days in summer, and 15 days in spring, the surviving plants were transferred to the greenhouse.
Rooting
C h l o r o p h y l l c o n t e n t . - Entire leaves without petioles were collected from nonvitreous shoots at the end of the 5th subculture on different proliferation media and at the end of the rooting phase of shoots coming from "MS + " medium. Leaf discs (14 m m diameter) were cut from fully expanded leaves of plants grown in the hardening chamber for 56 days. Both leaves and discs were extracted in 80% ethanol, and chlorophyll a and b were determined by Arnon's ( 1949 ) formula on an average of 50 replicates per medium, using a complete randomized design. RESULTS AND DISCUSSION
As a high n u m b e r of subcultures after explant establishment is required in order to obtain steady proliferation rates of 'Hayward', the initial rate of multiplication on "MS + " m e d i u m was not recorded. Our shoot proliferation rates are comparable with those reported by Piagnani et al. (1986) for 'Hayward' and 'Tomuri', but in reference to "Hayward' they were higher than those reported by Jona and Gribaudo (1983), and slightly lower than those found by Monette (1986) and Standardi ( 1982; 1983). This was probably due to shorter subculture intervals (21 days) in our trials and to the high n u m b e r of shoots tested, which included all shoots Proliferation.
-
68
G. MARINO AND G. BERTAZZA
growing on the maintenance medium "MS + " and not purposely those from the most vigorous cultures. No matter what coefficient (pcA, pcB or pcC) was used to express the proliferation rates, the interactions hormone × dose and hormone × cultivar were shown to be highly significant; moreover BA gave the best results for both cultivars (Table 1 ). On the same media, shoots could develop from single nodes and showed proliferation rates comparable with those reported in Table 1 in 3-4 weeks, agreeing with results reported for single-bud cuttings by Piagnani et al. (1986) and Wessels et al. (1984). K often stimulated the growth of vigorous but uniaxial shoots with only 1-2 new nodes developed. However, both BA concentrations sometimes caused vitrification which did not affect proliferation. In fact, only the older leaves became vitrified and not the newly produced shoots. Z, which has been shown to be synthesized by in vitro cultures of Actinidia and other woody plants (Einset, 1986), seldom induced this undesirable effect. K did not have a clear effect: normal and completely vitrified shoots were found in the same jar. Proliferation was higher for 'Tomuri' cultures on most of the six media tested (Table 1 ). This agrees with data reported by other authors for shoots of the same cultivars grown on media with BA (Piagnani et al., 1986 ). The hormone Xcultivar interaction was significant for shoot and callus growth. Z induced the highest shoot growth in 'Hayward' and callus growth in both cultivars, and K always induced the lowest shoot and callus growth TABLE 1 Total number of shoots (pcA), shoots with >/3 nodes (pcB) and total number of nodes (pcC) newly produced from one initial shoot after 21 days culture on different proliferation media Medium composition (#M)
'Hayward'
'Tomuri'
Mean
pcA
pcB
pcC
pcA
pcB
pcC
pcA
pcB
pcC
BA BA
4.44 8.88
3.12aA 3.07aA
0.92aA 0.88aA
6.45 aA 6.47aA
3.63bA 3.78bA
1.42bA 1.30bA
8.18bA 8.35bA
3.40
1.13
7.36
Z Z
4.56 9.12
1.79aA 2.53aB
0.37aA 0.63aB
3.29aA 4.96aB
2.25bA 3.06bB
0.47aA 0.82bB
4.09bA 6.07bB
2.40
0.57
4.60
K K
4.65 9.30
0.94aA 1.32aB
0.00aA 0.01 aA
1.05aA 1.55 aA
1.79bA 1.95bA
0.00aA 0.03aA
2.08bA 2.45bA
1.50
0.01
1.78
2.24
0.47
4.26
2.83
0.67
5.49
Mean
Values with different lower case letters for rows and upper case letters for columns differ statistically at P = 0.01 ( paired comparisons, Student's t, on transformed (x) 1/z values ). Main effects for pcA, pcB and pcC: hormone**, dose**, hormone×dose**, cultivar**, hormone × cultivar**, dose × cultivar n.s.
69
MICROPROPAGATION OF ACTINIDIA DELICIOSA
TABLE2
Shoot (sg) and callus (cg) growth after 21 days culture on different proliferation media Medium composition (/tM)
'Hayward'
'Tomuri'
Mean
sg (gfw)
cg (gfw)
sg (gfw)
cg (gfw)
sg (gfw)
cg (gfw)
BA BA
4.44 9.30
0.42 aA 0.44 aA
0. l 1 bA 0.10 aA
0.52 bA 0.51 aA
0.08 aA 0.08 aA
0.47
0.09
Z Z
4.56 9.12
0.51 aA 0.59 aB
0.21bA 0.22 bA
0.50 aA 0.55 aA
0.13 aA 0.11 aA
0.54
0.17
K K
4.65 9.30
0.25 aA 0.28 aA
0.03 aA 0.04 aA
0.37 bA 0.40 bA
0.04 aA 0.04 aA
0.32
0.04
0.40
0.12
0.47
0.08
Mean
fw = fresh weight. Values with different lower case letters for rows and upper case letters for columns differ statistically at P=0.01 (paired comparisons, Student's t). Main effects for sg: hormone**, dose**, hormone × dose n.s., cultivar**, hormone × cultivar**, doseXcultivar n.s.; for cg: hormone**, dose n.s., hormone X dose n.s., cultivar**, hormone × cultivar**, dose × cultivar n.s.
(Table 2). However, 'Tomuri' showed higher values for shoot growth than 'Hayward' on media enriched with 4.44/tM BA or K at both levels. By contrast, callus growth at the base of shoots was greater for 'Hayward' in the presence of BA and especially of Z. R o o t i n g a n d a c c l i m a t i z a t i o n . - The rhizogenic activity of IBA compared with
the control was confirmed. However, no differences in rooting percentages were shown between doses (Table 3 ). Rooting percentages were already high 14 days after shoot transfer to the rooting media, but roots were very short ( 2 - 4 mm; data not reported in the tables). Thereafter they increased up to 100% in both cultivars after 25 days, except in the control. Callus, already present after 14 days on media containing IBA, also increased, especially at the higher auxin level. A small amount of callus also appeared in controls of both cultivars. Root number and length depended on IBA concentrations: the higher auxin level induced more numerous, but shorter roots. Moreover, roots were more numerous and longer in 'Tomuri' than in 'Hayward' on IBA-enriched media. Acclimatization was carried out first in summer and later in spring with plants rooted on the same IBA-enriched media, in order to improve survival results. In both trials, almost all plants of the two cultivars (even those with-
95.06 a
Mean
8.99 a
2.67 aA 10.02 aB 13.35 aC 10.10 a
10.17 aAB 11.14 aB 9.31 aA
Length (mm)
97.53 a
92.59 aA 100 aA 100 aA 10.09 b
2.70 aA 11.78 bB 15.24 bC
Number
Root
12.22 b
ll.18aA 12.82 bB 11.93 bAB
Length (mm) 88.89A 100 B 100 B
Rooting percentage
Mean
2.69A 10.90 B 14.30 C
Number
Root
10.68 A 12.05 B 10.69 A
Length (mm)
Values with different lower case letters for rows and upper case letters for columns differ statistically at P = 0.01 (percentage and root number and length analysed by Z2 and Student-Newman-Keuls test, respectively).
85.18 aA 100 aA 100 aA
Number
Rooting percentage
Rooting percentage
Root
'Tomuri'
'Hayward'
IBA 0.00 IBA 1.00 IBA 2.46
Medium composition (/~M)
Rooting percentage, root number and length after 25 days culture on different rooting media
TABLE 3
N
O
71
MICROPROPAGATION OF ACTINIDIA DELICIOSA
out roots) survived transplanting to the hardening chamber and grew quickly. However, ~ 60% of those which were transferred to the greenhouse in summer after 28 days died (survival rates were not related to different rooting media). The high temperature of the greenhouse (up to 38°C) might have been responsible for these results, as suggested elsewhere (Bini, 1979 ). These plants required a long stay ( 56 days) in the hardening chamber if the survival rate was to remain high (75%) on transfer to the greenhouse. By contrast, plants of 'Hayward' and 'Tomuri' which were transferred to the greenhouse after 15 days in spring showed ~ 90% survival. C h l o r o p h y l l c o n t e n t . - Chlorophyll a and b contents in proliferating shoots of 'Hayward' and 'Tomuri' were influenced by the culture media (Table 4), i.e. highest with Z and lowest with K. It has been reported that Z induced higher chlorophyll contents than BA in callus cultures ofA. c h i n e n s i s P1. (Mannini and Ryugo, 1983 ). However, both chlorophyll a and b levels tended to be higher for 'Tomuri', but seldom with statistically different values. During the rooting phase, average chlorophyll levels were comparable with those of proliferating shoots, notwithstanding the absence of cytokinin. On the contrary, IBA seemed to exert an inhibitory influence on chlorophyll content compared with the control (Table 5 ), agreeing with data reported elsewhere for callus cultures of other species (George and Sherrington, 1984). TABLE 4 Leaf chlorophyll content in proliferating shoots after 21 days culture (5th subculture) on different culture media Medium composition (/tM)
'Hayward' Chlorophyll ( m g g -1 fw)
'Tomuri' Chlorophyll ( m g g -1 fw)
Mean Chlorophyll ( m g g - I fw)
a
b
a
b
a
b
BA BA
4.44 8.88
0.657 aA 0.666 aA
0.286 aA 0.282 aA
0.832 bA 0.783 aA
0.377 aA 0.359 aA
0.734
0.326
Z Z
4.56 9.12
0.751 aA 0.763 aA
0.313 aA 0.308 aA
1.058 bA 1.053 bA
0.467 bA 0.508 bA
0.906
0.399
K K
4.65 9.30
0.383 aA 0.433 aA
0.145 aA 0.178 aA
0.553 aA 0.501 aA
0.241 aA 0.211 aA
0.467
0.194
0.610
0.252
0.796
0.361
Mean
fw = fresh weight. Values with different lower case letters for rows and upper case letters for columns differ statistically at P=0.01 (paired comparisons, Student's t). Main effects for chlorophyll a and chlorophyll b: hormone**, dose n.s., h o r m o n e × d o s e n.s., cultivar**, hormone × cultivar*, dose × cultivar n.s.
72
G. MARINOAND G. BERTAZZA
TABLE 5 Leaf chlorophyll content in rooted shoots after 25 days culture on different rooting media Medium composition (#M)
IBA IBA IBA Mean
0.00 1.00 2.46
'Hayward' Chlorophyll (mg g-1 fw)
'Tomuri' Chlorophyll
Mean Chlorophyll
(mg g-1 fw)
(nag g-1 fw)
a
b
a
b
a
b
0.824 aC 0.541 aB 0.383 aA
0.299 aB 0.269 aB 0.195 aA
1.167 bC 0.759 bB 0.494 bA
0.511 bC 0.364 bB 0.244 aA
0.996 C 0.650 B 0.438 A
0.405 C 0.317 B 0.220 A
0.583 a
0.254 a
0.807 b
0.373 b
fw = fresh weight. Values with different lower case letters for rows and upper case letters for columns differ statistically at P=0.01 (Student-Newman-Keuls test).
Moreover, 'Tomuri' again showed higher chlorophyll (especially a) levels than 'Hayward'. Similar results were found in hardened plants (56 days after transfer) with (mg g-1 fresh weight) average chlorophyll a 1.655 and chlorophyll b 0.842 for 'Tomuri', and chlorophyll a 1.456 and chlorophyll b 0.694 for 'Hayward' (statistically different at P = 0.01 ). CONCLUDING REMARKS
Our results confirm the suitability ofA. deliciosa for shoot micropropagation. The in vitro behaviour of 'Hayward' and 'Tomuri' was influenced by cytokinins and auxins. However, 'Tomuri' showed higher proliferation and shoot growth than 'Hayward' on most of the tested media, which agrees with the higher growth of the former observed in the field. By contrast, callus growth in proliferating cultures was greater for 'Hayward'. 'Tomuri' generally had similar rooting percentages, but more numerous and longer roots than 'Hayward'. Also, the chlorophyll content was consistently higher for 'Tomuri' than for 'Hayward', particularly in the rooting and hardening phases, when exogenous BA was not added. Hormones are known to have regulatory powers over the development of senescence in a number of species, i.e. cytokinins deferred the loss of chlorophyll in Xantium leaves (Leopold and Kriedemann, 1975 ). In conclusion, all these obviously genetic differences in proliferation rates, callus growth and chlorophyll contents suggest that an interaction of culture media with different endogenous hormonal concentrations can be expected in the two cultivars. Tentatively, we can suppose a cytokinin/auxin endogenous balance which is higher for 'Tomuri' than for 'Hayward'. Moreover, the fact that the cultivars compared are of different sexes seems interesting. As
MICROPROPAGATION OF ACTINIDIA DELICIOSA
73
other authors reported higher shoot proliferation rates for 'Tomuri' than for three female cultivars (Piagnani et al., 1986), and sex expression of some other dioecious angiosperms was supposed to be controlled by an endogenous hormonal balance (Heslop-Harrison, 1972 ), further studies are needed to investigate if this is also the case for A. deliciosa. ACKNOWLEDGEMENTS
The research was supported by funds from the Italian Ministry of Education, Roma (M.P.I. 40%).
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(L.) Planchon, par microbouturage "in vitro" de noeuds. C.R. Acad. Sci. Paris, t. 301, serie III, 12: 597-600. Wessels, E., Nel, D.D. and Van Staden, D.F.A., 1984. "In vitro" propagation ofActinidia chinensis P1. cultivar Hayward. Deciduous Fruit Grow., 34: 453-457.