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Propagation in vitro of Chinese gooseberry (Actinidia chinensis) through the development of axillary buds
Scientia Horticulturae, 42 (1990) 45-54 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
45
Propagation in vitro of Chinese Gooseberry (Actinidia chinensis) Through the Development of Axillary Buds XIAO-SHAN SHEN, JUE-ZHEN WAN and WEI-YI LUO
Guizhou Pomological Institute, Guiyang, Guizhou Province 550022 (The People's Republic of China) (Accepted for publication 19 May 1989 )
ABSTRACT Xiao-Shan Shen, Jue-Zhen Wan and Wei-Yi Luo, 1990. Propagation in vitro of Chinese gooseberry (Actinidia chinensis) through the development of axillary buds. Scientic Hortic, 42: 4554. Cultures were initiated from single-node explants on Murashige and Skoog medium containing 2 ttM benzyladenine (BA). Shoot proliferation from axillary buds was achieved on media supplemented with BA or zeatin (5-10 #M). Formation of axillary shoots was influenced by clonal identity, type and concentration of cytokinin and by photoperiod. Sixty to ninety percent of the microcuttings longer than 20 mm rooted on the media containing 3-indole acetic acid or y-indole butyric acid in 3-4 weeks. Approximately 60% of the rooted microcuttings were established successfully as container-grown plants. A bacterium surviving in the explants was not an obstacle to propagation in vitro, if healthy shoots were used as the explants in every subculture. Keywords: Actinidia chinensis; axillary bud; Chinese gooseberry; in vitro; kiwifruit; propagation. Abbreviations: BA--benzyladenine; D--difference; GA3--gibberellic acid; IAA = 3-indole acetic acid; IBA--y-indole butyric acid; LSD--least significant difference; NAA--a-naphthalene acetic acid; MS = Murashige and Skoog (1962).
INTRODUCTION
Hundreds of good clones and tens of superclones of Chinese gooseberry (kiwifruit) have been selected from the wild since the early seventies (Zhu, 1983; Zhou, 1987). In tissue-culture studies to date, plant regeneration has been uniform by adventitious organogenesis from callus, although different explants were used (Gui et al., 1979; Hong, 1981; Huang et al., 1982; Wang and Li, 1982; Yu, 1983; Zhou et al., 1984). The plants which resulted from adventitious organogenesis were genetically aberrant and more variable in characters and re0304-4238/90/$03.50
© 1990 Elsevier Science Publishers B.V.
46
XIAO-SHAN SHEN ET AL.
verted to the juvenile phase (Murashige, 1974; Navarro et al., 1975; Romberger, 1976; Styer, 1983). Adventitious organogenesis from callus cannot be applied in the propagation of Chinese gooseberry if variation is encountered. Plants from axillary bud development were more stable in characteristics and remained in the adult phase (Murashige, 1974; Navarro et al., 1975; Styer, 1983). Propagation in vitro of kiwifruit abroad has been confined to A. deliciosa (Brossard-Chriqui and Tripathi, 1984; Standardi and Catalano, 1984; Wessels et al., 1984; Monette, 1986; Barbieri and Morini, 1987). These reports prompted us to investigate the propagation in vitro ofA. chinensis Planch. var. chinensis C.F. Liang (soft-hair Chinese gooseberry) and A. chinensis Planch. vat. hispida C.F. Liang (bristly-hair Chinese gooseberry) through axillary bud development. MATERIALSAND METHODS Actively growing shoots were excised from plants of soft-hair Chinese gooseberry, Clones 1, 2, 3, 8 and M1 (male) and bristly-hair Chinese gooseberry, Clones 4, 5 and M2 (male), grown in the field and in the greenhouse. Singlenode explants were made from excised shoots. These initial explants were surface sterilized in a solution containing 0.1 To (v/v) Tween 20 as a wetting agent. After several rinses with sterilized distilled water, the explants were placed into test tubes containing 10 ml MS medium solidified with agar (1%, w/v) and supplemented with 2/~M BA. The cultures were incubated at 25 + 2 ° C with 16h and 12-h photoperiods of fluorescent lamps. The irradiance at the level of the cultures was 4-7 W m -2. Bud burst in the initial explants of all clones occurred within 2 weeks and each bud subsequently gave rise to a single extension shoot (Fig. 1 ) which was used as the explant for shoot multiplication. This multiplication was achieved by subcultures at monthly intervals in 100-ml conical flasks or 500-ml jars containing 30 or 80 ml shoot multiplication medium, respectively. Five healthy shoots selected from prior subculture were inoculated in a conical flask and 12 in a jar at each subculture. The medium for subcultures contained MS basal medium and the following growth substances, alone or in combination: BA (230/~M ), kinetin (5-20 #M), zeatin (2-20/~M), NAA (2-4/~M), IBA (2-5 #M), and GA3 (5 mg 1-1 ). Shoots longer than 20 m m were used as microcuttings and were grown in conical flasks or jars, as before on MS basal medium containing either IAA (020/~M ) or IBA (0-20/~M) for rooting. Rooted microcuttings were transferred to a plastic container ( 4 7 0 × 3 3 0 × 1 2 0 m m ) holding a mixture of sterilized perlite and vermiculite ( v / v - - 1 / 1 ). The plantlets were grown initially under glass and were gradually exposed to normal greenhouse conditions. After a 3week period of hardening-off, the plantlets were transplanted into pots. Other details are given with the results.
IN VITROPROPAGATIONOF ACTINIDIA CHINENSIS
47
Fig. 1. Initial explant of Clone 3, cultured on MS medium containing BA 2 #M. Photographed after 3 weeks. Bar--= 2 cm. Fig. 2. Axillary shoots of M2 formed on MS medium supplemented with 10 # M zeatin. Photographed after 1 month subculture. Bar = 2 cm. Fig. 3. Adventitious roots formed by microcuttings of Clone 4 on MS medium containing IAA 10 #M. Photographed 3 weeks after microcutting. Bar-- 2 cm. Fig. 4. Container-grown plant of Clone 3. Photographed 4 months after transplanting. Bar = 4 cm.
Statistical evaluation of results was done by analysis of variance and LSD or by standard error of means. RESULTS
Effect of sterilizing agents and origin of explants on surface sterilization of explants. - Single-node explants of the clones exhibited various responses when different sterilizing agents were applied. Only when explants were sterilized with a solution of 0.1% HgC12 for 3-8 min, could cultures be established (Table 1). Origin of explants also exerted great influence on their surface sterilization. A total of 282 explants, taken in the fall, from field-grown shoots of soft-hair
48
XIAO-SHANSHENET AL.
TABLE 1 The response of single-node explants to different sterilizing agents Agent
Concentration (%)
Time (min)
Result
Sodium hypochlorite
1% available chlorine
5-15
Hydrogen peroxide
30
10
Hydrogen peroxide
10
20
Bromine
1, 2 or 3
5-20
HgC12
0.1
15
HgC12
0,1
3-8
90% of the explants died in 5 days, the remaining died afterwards 70% of the explants showed microbial growth or died after 2 weeks, the remaining failed to grow The same as 30% hydrogen peroxide 60% of the explants showed microbial growth, 40 % blackened and died in 2 weeks 70% of the explants were sterile, but the buds failed to break. Some explants were sterile and capable of forming shoots, culture could be established
clones were treated with 0.1% HgC12, but none were sterile or capable of bursting bud. Of 195 explants of bristly-hair clones treated similarly, only seven were sterile and gave rise to shoots. Of explants which were made from new growth and excised in the spring, 121 of 200 soft-hair clones and 18 of 55 bristlyhair clones appeared sterile and formed shoots when they were treated with 0.1% HgCI2. If explants were taken from greenhouse-grown shoots, surface sterilization of the explants was much easier: of 20 such explants of a bristly-hair clone, 19 were sterile and gave rise to shoots when they were treated in the same way as those made from field-grown shoots. Effect of various factors on axillary shoot formation. - Shoot multiplication was achieved through axillary bud growth in every subculture. Callus was formed on the basal parts of the explants in all clones. These calli, some of which differentiated shoots easily, interfered with axillary shoot development, so that the calli had to be cut off at each subculture. Clones 4 and M2 were chosen for initial experiments to study axillary shoot formation. Addition of either NAA or IBA to the media did not accelerate axillary shoot growth, but made the calli on the basal parts of the explants overgrow, thus the shoot growth and the axillary bud development slowed down. Rapid shoot elongation was obtained with the addition of 5 mg 1-1 GA3 to the
IN VITROPROPAGATIONOFACTINIDIA
49
CHINENSIS
medium, however the axillary buds did not develop well and the multiplication rate of shoots was decreased in the next subculture. Media containing 30 #M BA inhibited the elongation and killed the explants occasionally. Axillary buds developed well and the axillary shoots proliferated on the media supplemented with 10 #M BA. When the concentration of BA decreased to 2 #M, a few axillary shoots formed. Explants were unable to grow and leaves yellowed on the media containing kinetin (5-20 #M). W h e n 10 #M zeatin was added to the media, growth and proliferation of the shoots were unaffected (Fig. 2), but the leaves were smaller compared with those on media supplemented with 10 #M BA. Smaller leaves allowed dense inoculation of explants in culture. The results of shoot proliferation of the explants cultured on media containing 5, 10, 15 or 20 #M zeatin with a 16-h photoperiod are presented in Table 2. Differences in the rate of shoot multiplication in Clone M2 for both total shoots and long shoots between 5 and 10 #M, and between 10 and 15 #M were significant, but in Clone 4 only the difference for total shoot between 5 and 10 #M is significant (Table 2). When explants of Clones 4 and M2 were cultured on MS medium containing 10 #M zeatin with a 16-h photoperiod for half a year, shoot multiplication decreased slightly, but did not continue decreasing during the next 2 years. Extensive experiments were conducted with Clones 1, 2, 3, 5, 8 and M1 on TABLE 2 The effect of zeatin concentration on shoot production of Clones 4 and M2. When no LSDs are given, there are no significant differences Zeatin concentration (ltM) 5 Clone 4 Explant number Mean number of total shoots per explant Significant difference Mean number of long shoots ( > 10 mm) per explant Clone M2 Explant number Mean number of total shoots per explant Significant difference Mean number of long shoots ( > 10 mm) per explant Significant difference
10
61
81
15
20
22
12
2.8 3.6 3.1 D ( 5 - 1 0 / t M ) = L-0.81 > L S D (0.01)=0.56 2.4 55
2.9 145
2.5 21
2.6
2.2 16
3.5 4.6 3.5 D ( 5 - 1 0 # M ) = i - l A D > L S D (0.01)=0.64 D ( 1 0 - 1 5 # M ) = i + l . l l > L S D (0.05)=0.99
4.3
2.8 3.6 2.8 D ( 5 - 1 0 # M ) = I - 0 . 8 i > L S D (0.01)=0.53 D ( 1 0 - 1 5 # M ) = I +0.81 > L S D (0.05)=0.79
3.5
50
XIAO-SHAN SHEN ET AL.
TABLE3 The effect of zeatin concentration and photoperiod on shoot production of Clones 1, 2, 3, 5, 8 and M1. Details as in Table 2 Photoperiod
12 h
Zeatin concentration
5/~M
Clone 1 Explant number Mean number of total shoot per explant Significant difference Mean number of long shoots ( > 10 mm) per explant Significant difference Clone 2 Explant number Mean number of total shoots per explant Mean number of long shoots ( > 10 mm) per explant Clone 3 Explant number Mean number of total shoots per explant Mean number of long shoots ( > 10 mm) per explant Significant difference Clone 5 Explant number Mean number of total shoots per explant Mean number of long shoots ( > 10 mm) per explant Clone 8 Explant number Mean number of total shoots per explant Significant difference Mean number of long shoots ( > 10 mm) per explant Significant difference Clone M1 Explant number Mean number of total shoots per explant Significant difference Mean number of long shoots ( > 10 mm) per explant
81
16 h 10 #M 85
5 ~M 202
10/~M 68
3.8 3.4 4.0 D (12-16 h ) = i - 0 . 4 1 i > L S D (0.05)=0.40
4.3
2.1 1.7 2.8 D (5-10/~M)= ] +0.63] > L S D (0.01)=0.36 D (12-16 h ) = ]-0.77] > L S D (0.01)=0.36
2.3
87
112
95
85
3.2
3.4
3.1
3.1
2.2
2.2
1.9
2.2
47 3.5
74 3.6
25 3.6
2.0 2.1 1.8 D (12-16 h ) = ] + 0 . 4 1 ] > L S D (0.05)=0.34 31
86
22
28 3.0 1.5
129
3.2
3.3
3.4
3.1
2.7
2.8
2.7
2.6
57
21
58
30
2.5 3.8 3.2 D (5-10 t~M) = -0.89[ > LSD (0.01) =0.74 D ( 1 2 - 1 6 h ) = ]-0.521 > L S D (0.05)=0.51
3.7
2.0 2.2 1.9 D (5-10/~M)= ] -0.42] = L S D (0.O5)=0.42
2.5
51
34
50
2.6 2.7 2.4 D (5-10/~M)= ] -0.54] > L S D (0.05)=0.42 1.8
1.7
1.5
68 3.2
1.9
IN VITRO PROPAGATION OF ACTINIDIA CHINENSIS
51
the basis of the above results obtained from Clones 4 and M2. Explants of these six clones were cultured on media supplemented with 2, 5, 10 or 15/tM zeatin. Few axillary shoots were formed on the medium containing 2 ]IM zeatin. With 15 ]IM zeatin in the medium, shoot elongation was restricted, especially in softhair Clones 1, 2, 3, 8 and M1. Further experiments were carried out with 5 or 10 ~M zeatin and photoperiods of 16 or 12 h (Table 3). On the medium containing 10 ~M zeatin the rate of shoot multiplication for total shoots was greater than on 5 ltM zeatin in Clones 8 and M1. The rate for long shoots in Clone 1 was smaller, while in Clone 8 it was bigger. Under 16-h photoperiod the rates of shoot multiplication for both total shoots and long shoots in Clone 1 were bigger than under a 12-h TABLE4
The effect of IAA and IBA on adventitious root formation of eight clones Clone
1
2
3
4
5
8
M1 M2
Medium
M S ÷ IAA ½M S + IAA MS+IBA M S + IAA ½MS + I A A MS+IBA M S + IAA ½MS + IAA M S + IBA ½M S ÷ IBA M S + IAA ½MS + IAA MS+IBA M S + IAA ½MS + IAA M S ÷ IBA M S ÷ IAA ½M S + IAA M S ÷ IBA MS÷IAA M S + IBA M S ÷ IAA ½MS + IAA ½MS + IBA
10/~M 10 # M 2ttM 10 # M 10 # M 10/~M 10 # M 10 # M 5 #M 5 #M 10 # M 10 BM 5BM 10 # M 10 # M 5 #M 10 # M 10 ItM 5 #M 10 # M 5 ~M 10 # M 10 BM 10 # M
Number of microcuttings
Percentage of rooted microcuttings
Mean root number per rooted microcutting ( m e a n +- SE )
275 275 123 193 134 124 122 132 261 80 281 240 135 272 151 75 96 118 70 128 75 340 540 72
41.1 44.4 84.6 10.9 6.0 66.7 8.2 5.3 66.3 72.5 56.2 71.7 77.1 39.0 62.9 80.0 7.3 11.0 58.6 11.7 68.0 80.6 87.6 90.3
2.4 ± 1.67 3.3 __2.16 7.1_+4.10 1.5 + 0.65 1.5 ± 0.33 8.5 +_4.16 3.1 +- 1.97 1.3 +- 0.49 5.9 + 4.59 6.4 + 5.24 5.0 _+3.68 4.6 + 3.19 5.6+-4.19 1.9 ± 1.66 2.5 _+1.24 4.4 + 3.42 1.3 + 0.73 1.8 _+0.82 4.7 + 3.46 1.3 +-0.38 4.6 _+3.80 5.4 _+3.77 5.4 +_3.12 11.9 +- 4.00
52
XIAO-SHAN SHEN ET AL.
photoperiod, while in Clone 8 the rate for total shoots only differed, and in Clone 3 the rate for long shoots was smaller. In Clones 2 and 5 no differences occurred (Table 3). P l a n t establishment. - Microcuttings longer than 20 mm rooted in 2-3 weeks with bristly-hair Clones 4, 5 and M2 (Fig. 3 ) and in 3-4 weeks with soft-hair Clones 1, 2, 3, 8 and M1. A microcutting could give rise to 1-35 roots. The microcuttings of some clones could not root on media without IAA and IBA, while others could, but the percentages of rooted microcuttings remained below 20% and only a few roots were formed on one microcutting. IAA or IBA in media promoted rooting of all 8 clones. In all clones IBA yielded a higher percentage of rooted microcuttings and a higher mean root number per rooted microcutting than IAA (Table 4). Full sun had to be avoided during hardening-off. The rooted microcuttings died if 1-2 h of full sun was given to them. After hardening-off, containergrown plants could be established by transplanting the surviving plantlets into pots (Fig. 4). Survival of rooted microcuttings was closely related to the environmental conditions during hardening-off and after transplanting. Under conditions where humidity and irradiance were not controlled 59.9% of 3211 rooted microcuttings survived after transplanting for a month. Forty in vitro plants of Clones 4 and M2 were planted in a field in June 1987 and have been growing vigorously. Morphological aberration has not been found. E f f e c t of a bacterium s u r v i v i n g in explants on in vitro propagation. - Although the initial explants were surface sterilized, cultures had three symptoms of infection, i.e. shoot tip death, blackened leaves, and rotting of leaves and shoots. Similar symptoms appeared in cultures of other clones and cultivars which were used in our laboratory, including cultivars 'Hayward' and 'Tomuri' from New Zealand. A bacterium was found in the excretion of infected tissues. If healthy shoots were used in every subculture as explants, the bacterium surviving in the explant was not an obstacle to in vitro propagation of Chinese gooseberry. If not, the bacterium not only infected most shoots, but also greatly reduced the rooting of microcuttings. DISCUSSION
In vitro propagation through the development of axillary buds is quicker than conventional methods and needs a small quantity of starting material, the in vitro plants propagated in this way in many species have proved to be true to type (Murashige, 1974; Navarro et al., 1975; Styer, 1983). Therefore, it is the best way for propagation of new cultivars or clones for fruit production. Before this in vitro propagation technique is to be used for propagation of
IN VITROPROPAGATIONOFACTINIDIA CHINENSIS
53
Chinese gooseberry, the plants of this origin must be evaluated, but can be used immediately for breeding purposes, to which our institute pays much attention. Axillary shoot multiplication in vitro required different conditions for each of the clones. In relation to the response of axillary shoot formation of the clones to the zeatin concentration and the photoperiod, Chinese gooseberry might be divided into three types: (1) sensitive to cytokinin, the axillary shoot formation is significantly influenced by cytokinin concentration change; (2) sensitive to photoperiod, the axillary shoot formation is significantly affected by photoperiod; (3) inactive, axillary shoot formation does not differ significantly with the changes of cytokinin concentration and photoperiod. Monette (1986) reported that after standard surface sterilization of the New Zealand cultivar 'Hayward' two surviving bacteria were found in culture and that their presence did not present a major obstacle to in vitro propagation. Similar results were obtained in more clones of Chinese gooseberry in our study.
REFERENCES Barbieri, C. and Morini, S., 1987. Plant regeneration from Actinidia callus culture. J. Hortic. Sci., 62: 107-109. Brossard-Chriqui, D. and Tripathi, B.K., 1984. Comparison of morphogenetic ability in fertile or sterile stamens of A. chinensis cultured in vitro. Can. J. Bot., 62: 1940-1946. Gui, Y.L., An, H.Q., Cai, D.Y. and Wang, J.R., 1979. Tissue culture of Chinese gooseberry. Kexuetongbao, 4:188-190 (in Chinese). Hong, S.Y., 1981. Induction of callus and plantlets from segments of tender shoots and young leaves in Actinidia. Hupei Agric. Sci., 9:28-30 (in Chinese). Huang, Z.G., Huangpu, Y.L. and Xu, Y.Y., 1982. Regeneration of triploid plants from endosperm culture of Chinese gooseberry. Kexuetongbao, 27:247-250 (in Chinese). Monette, P.L., 1986. Micropropagation of kiwifruit using non-axenic shoot tips. Plant Cell, Tissue Organ Cult., 6: 73-82. Murashige, T., 1974. Plant propagation through tissue culture. Annu. Rev. Plant Physiol., 25: 135-166. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant, 15: 473-497. Navarro, L., Roistacher, C.N. and Murashige, T., 1975. Improvement of shoot tip grafting in vitro for virus-free Citrus. J. Am. Soc. Hortic. Sci., 100: 471-479. Romberger, J.A., 1976. An appraisal of prospects for research on juvenility in woody perennials. Acta Hortic., 56: 301-317. Standardi, A. and Catalano, F., 1984. Tissue culture propagation of kiwifruit. Combined Proceedings of International Plant Propagators' Society, 34: 236-243. Styer, D.J., 1983. Meristem and shoot-tip culture for propagation, pathogen elimination, and plasm preservation. Hortic. Rev., 5: 221-277. Wang, J.X. and Li, S.Z., 1982. Propagation ofA. chinensis by tissue culture. Liaoning Agric. Sci., 1:32-34 (in Chinese). Wessels, E., Nel, D.D. and von Staden, D.F.A. 1984. In vitro propagation of A. chinensis P1. cultivar Hayward. Deciduous Fruit Grower, 34: 453-457. Yu, S.Z., 1983. Regeneration of plantlets by cotyledon culture of A. chinensis. Plant Physiol. Commun., 2:37-38 (in Chinese).
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XIAO-SHANSHENET AL.
Zhou, H.Y., 1987. Introduction of super clones of Chinese gooseberry selected in China. Fruit Tree, 3:24-26 (in Chinese). Zhou, D.Z., Gao, H. and Liu, G.P., 1984. Induction of callus and regeneration of plants from segments of tender shoots of Chinese gooseberry. J. Jiangxi Agric. Univ., 1:83-84 (in Chinese). Zhu, H.Y., 1983. Production of Chinese gooseberry. Shanghai Scientific Press, pp. 13-16 (in Chinese ).
45
Propagation in vitro of Chinese Gooseberry (Actinidia chinensis) Through the Development of Axillary Buds XIAO-SHAN SHEN, JUE-ZHEN WAN and WEI-YI LUO
Guizhou Pomological Institute, Guiyang, Guizhou Province 550022 (The People's Republic of China) (Accepted for publication 19 May 1989 )
ABSTRACT Xiao-Shan Shen, Jue-Zhen Wan and Wei-Yi Luo, 1990. Propagation in vitro of Chinese gooseberry (Actinidia chinensis) through the development of axillary buds. Scientic Hortic, 42: 4554. Cultures were initiated from single-node explants on Murashige and Skoog medium containing 2 ttM benzyladenine (BA). Shoot proliferation from axillary buds was achieved on media supplemented with BA or zeatin (5-10 #M). Formation of axillary shoots was influenced by clonal identity, type and concentration of cytokinin and by photoperiod. Sixty to ninety percent of the microcuttings longer than 20 mm rooted on the media containing 3-indole acetic acid or y-indole butyric acid in 3-4 weeks. Approximately 60% of the rooted microcuttings were established successfully as container-grown plants. A bacterium surviving in the explants was not an obstacle to propagation in vitro, if healthy shoots were used as the explants in every subculture. Keywords: Actinidia chinensis; axillary bud; Chinese gooseberry; in vitro; kiwifruit; propagation. Abbreviations: BA--benzyladenine; D--difference; GA3--gibberellic acid; IAA = 3-indole acetic acid; IBA--y-indole butyric acid; LSD--least significant difference; NAA--a-naphthalene acetic acid; MS = Murashige and Skoog (1962).
INTRODUCTION
Hundreds of good clones and tens of superclones of Chinese gooseberry (kiwifruit) have been selected from the wild since the early seventies (Zhu, 1983; Zhou, 1987). In tissue-culture studies to date, plant regeneration has been uniform by adventitious organogenesis from callus, although different explants were used (Gui et al., 1979; Hong, 1981; Huang et al., 1982; Wang and Li, 1982; Yu, 1983; Zhou et al., 1984). The plants which resulted from adventitious organogenesis were genetically aberrant and more variable in characters and re0304-4238/90/$03.50
© 1990 Elsevier Science Publishers B.V.
46
XIAO-SHAN SHEN ET AL.
verted to the juvenile phase (Murashige, 1974; Navarro et al., 1975; Romberger, 1976; Styer, 1983). Adventitious organogenesis from callus cannot be applied in the propagation of Chinese gooseberry if variation is encountered. Plants from axillary bud development were more stable in characteristics and remained in the adult phase (Murashige, 1974; Navarro et al., 1975; Styer, 1983). Propagation in vitro of kiwifruit abroad has been confined to A. deliciosa (Brossard-Chriqui and Tripathi, 1984; Standardi and Catalano, 1984; Wessels et al., 1984; Monette, 1986; Barbieri and Morini, 1987). These reports prompted us to investigate the propagation in vitro ofA. chinensis Planch. var. chinensis C.F. Liang (soft-hair Chinese gooseberry) and A. chinensis Planch. vat. hispida C.F. Liang (bristly-hair Chinese gooseberry) through axillary bud development. MATERIALSAND METHODS Actively growing shoots were excised from plants of soft-hair Chinese gooseberry, Clones 1, 2, 3, 8 and M1 (male) and bristly-hair Chinese gooseberry, Clones 4, 5 and M2 (male), grown in the field and in the greenhouse. Singlenode explants were made from excised shoots. These initial explants were surface sterilized in a solution containing 0.1 To (v/v) Tween 20 as a wetting agent. After several rinses with sterilized distilled water, the explants were placed into test tubes containing 10 ml MS medium solidified with agar (1%, w/v) and supplemented with 2/~M BA. The cultures were incubated at 25 + 2 ° C with 16h and 12-h photoperiods of fluorescent lamps. The irradiance at the level of the cultures was 4-7 W m -2. Bud burst in the initial explants of all clones occurred within 2 weeks and each bud subsequently gave rise to a single extension shoot (Fig. 1 ) which was used as the explant for shoot multiplication. This multiplication was achieved by subcultures at monthly intervals in 100-ml conical flasks or 500-ml jars containing 30 or 80 ml shoot multiplication medium, respectively. Five healthy shoots selected from prior subculture were inoculated in a conical flask and 12 in a jar at each subculture. The medium for subcultures contained MS basal medium and the following growth substances, alone or in combination: BA (230/~M ), kinetin (5-20 #M), zeatin (2-20/~M), NAA (2-4/~M), IBA (2-5 #M), and GA3 (5 mg 1-1 ). Shoots longer than 20 m m were used as microcuttings and were grown in conical flasks or jars, as before on MS basal medium containing either IAA (020/~M ) or IBA (0-20/~M) for rooting. Rooted microcuttings were transferred to a plastic container ( 4 7 0 × 3 3 0 × 1 2 0 m m ) holding a mixture of sterilized perlite and vermiculite ( v / v - - 1 / 1 ). The plantlets were grown initially under glass and were gradually exposed to normal greenhouse conditions. After a 3week period of hardening-off, the plantlets were transplanted into pots. Other details are given with the results.
IN VITROPROPAGATIONOF ACTINIDIA CHINENSIS
47
Fig. 1. Initial explant of Clone 3, cultured on MS medium containing BA 2 #M. Photographed after 3 weeks. Bar--= 2 cm. Fig. 2. Axillary shoots of M2 formed on MS medium supplemented with 10 # M zeatin. Photographed after 1 month subculture. Bar = 2 cm. Fig. 3. Adventitious roots formed by microcuttings of Clone 4 on MS medium containing IAA 10 #M. Photographed 3 weeks after microcutting. Bar-- 2 cm. Fig. 4. Container-grown plant of Clone 3. Photographed 4 months after transplanting. Bar = 4 cm.
Statistical evaluation of results was done by analysis of variance and LSD or by standard error of means. RESULTS
Effect of sterilizing agents and origin of explants on surface sterilization of explants. - Single-node explants of the clones exhibited various responses when different sterilizing agents were applied. Only when explants were sterilized with a solution of 0.1% HgC12 for 3-8 min, could cultures be established (Table 1). Origin of explants also exerted great influence on their surface sterilization. A total of 282 explants, taken in the fall, from field-grown shoots of soft-hair
48
XIAO-SHANSHENET AL.
TABLE 1 The response of single-node explants to different sterilizing agents Agent
Concentration (%)
Time (min)
Result
Sodium hypochlorite
1% available chlorine
5-15
Hydrogen peroxide
30
10
Hydrogen peroxide
10
20
Bromine
1, 2 or 3
5-20
HgC12
0.1
15
HgC12
0,1
3-8
90% of the explants died in 5 days, the remaining died afterwards 70% of the explants showed microbial growth or died after 2 weeks, the remaining failed to grow The same as 30% hydrogen peroxide 60% of the explants showed microbial growth, 40 % blackened and died in 2 weeks 70% of the explants were sterile, but the buds failed to break. Some explants were sterile and capable of forming shoots, culture could be established
clones were treated with 0.1% HgC12, but none were sterile or capable of bursting bud. Of 195 explants of bristly-hair clones treated similarly, only seven were sterile and gave rise to shoots. Of explants which were made from new growth and excised in the spring, 121 of 200 soft-hair clones and 18 of 55 bristlyhair clones appeared sterile and formed shoots when they were treated with 0.1% HgCI2. If explants were taken from greenhouse-grown shoots, surface sterilization of the explants was much easier: of 20 such explants of a bristly-hair clone, 19 were sterile and gave rise to shoots when they were treated in the same way as those made from field-grown shoots. Effect of various factors on axillary shoot formation. - Shoot multiplication was achieved through axillary bud growth in every subculture. Callus was formed on the basal parts of the explants in all clones. These calli, some of which differentiated shoots easily, interfered with axillary shoot development, so that the calli had to be cut off at each subculture. Clones 4 and M2 were chosen for initial experiments to study axillary shoot formation. Addition of either NAA or IBA to the media did not accelerate axillary shoot growth, but made the calli on the basal parts of the explants overgrow, thus the shoot growth and the axillary bud development slowed down. Rapid shoot elongation was obtained with the addition of 5 mg 1-1 GA3 to the
IN VITROPROPAGATIONOFACTINIDIA
49
CHINENSIS
medium, however the axillary buds did not develop well and the multiplication rate of shoots was decreased in the next subculture. Media containing 30 #M BA inhibited the elongation and killed the explants occasionally. Axillary buds developed well and the axillary shoots proliferated on the media supplemented with 10 #M BA. When the concentration of BA decreased to 2 #M, a few axillary shoots formed. Explants were unable to grow and leaves yellowed on the media containing kinetin (5-20 #M). W h e n 10 #M zeatin was added to the media, growth and proliferation of the shoots were unaffected (Fig. 2), but the leaves were smaller compared with those on media supplemented with 10 #M BA. Smaller leaves allowed dense inoculation of explants in culture. The results of shoot proliferation of the explants cultured on media containing 5, 10, 15 or 20 #M zeatin with a 16-h photoperiod are presented in Table 2. Differences in the rate of shoot multiplication in Clone M2 for both total shoots and long shoots between 5 and 10 #M, and between 10 and 15 #M were significant, but in Clone 4 only the difference for total shoot between 5 and 10 #M is significant (Table 2). When explants of Clones 4 and M2 were cultured on MS medium containing 10 #M zeatin with a 16-h photoperiod for half a year, shoot multiplication decreased slightly, but did not continue decreasing during the next 2 years. Extensive experiments were conducted with Clones 1, 2, 3, 5, 8 and M1 on TABLE 2 The effect of zeatin concentration on shoot production of Clones 4 and M2. When no LSDs are given, there are no significant differences Zeatin concentration (ltM) 5 Clone 4 Explant number Mean number of total shoots per explant Significant difference Mean number of long shoots ( > 10 mm) per explant Clone M2 Explant number Mean number of total shoots per explant Significant difference Mean number of long shoots ( > 10 mm) per explant Significant difference
10
61
81
15
20
22
12
2.8 3.6 3.1 D ( 5 - 1 0 / t M ) = L-0.81 > L S D (0.01)=0.56 2.4 55
2.9 145
2.5 21
2.6
2.2 16
3.5 4.6 3.5 D ( 5 - 1 0 # M ) = i - l A D > L S D (0.01)=0.64 D ( 1 0 - 1 5 # M ) = i + l . l l > L S D (0.05)=0.99
4.3
2.8 3.6 2.8 D ( 5 - 1 0 # M ) = I - 0 . 8 i > L S D (0.01)=0.53 D ( 1 0 - 1 5 # M ) = I +0.81 > L S D (0.05)=0.79
3.5
50
XIAO-SHAN SHEN ET AL.
TABLE3 The effect of zeatin concentration and photoperiod on shoot production of Clones 1, 2, 3, 5, 8 and M1. Details as in Table 2 Photoperiod
12 h
Zeatin concentration
5/~M
Clone 1 Explant number Mean number of total shoot per explant Significant difference Mean number of long shoots ( > 10 mm) per explant Significant difference Clone 2 Explant number Mean number of total shoots per explant Mean number of long shoots ( > 10 mm) per explant Clone 3 Explant number Mean number of total shoots per explant Mean number of long shoots ( > 10 mm) per explant Significant difference Clone 5 Explant number Mean number of total shoots per explant Mean number of long shoots ( > 10 mm) per explant Clone 8 Explant number Mean number of total shoots per explant Significant difference Mean number of long shoots ( > 10 mm) per explant Significant difference Clone M1 Explant number Mean number of total shoots per explant Significant difference Mean number of long shoots ( > 10 mm) per explant
81
16 h 10 #M 85
5 ~M 202
10/~M 68
3.8 3.4 4.0 D (12-16 h ) = i - 0 . 4 1 i > L S D (0.05)=0.40
4.3
2.1 1.7 2.8 D (5-10/~M)= ] +0.63] > L S D (0.01)=0.36 D (12-16 h ) = ]-0.77] > L S D (0.01)=0.36
2.3
87
112
95
85
3.2
3.4
3.1
3.1
2.2
2.2
1.9
2.2
47 3.5
74 3.6
25 3.6
2.0 2.1 1.8 D (12-16 h ) = ] + 0 . 4 1 ] > L S D (0.05)=0.34 31
86
22
28 3.0 1.5
129
3.2
3.3
3.4
3.1
2.7
2.8
2.7
2.6
57
21
58
30
2.5 3.8 3.2 D (5-10 t~M) = -0.89[ > LSD (0.01) =0.74 D ( 1 2 - 1 6 h ) = ]-0.521 > L S D (0.05)=0.51
3.7
2.0 2.2 1.9 D (5-10/~M)= ] -0.42] = L S D (0.O5)=0.42
2.5
51
34
50
2.6 2.7 2.4 D (5-10/~M)= ] -0.54] > L S D (0.05)=0.42 1.8
1.7
1.5
68 3.2
1.9
IN VITRO PROPAGATION OF ACTINIDIA CHINENSIS
51
the basis of the above results obtained from Clones 4 and M2. Explants of these six clones were cultured on media supplemented with 2, 5, 10 or 15/tM zeatin. Few axillary shoots were formed on the medium containing 2 ]IM zeatin. With 15 ]IM zeatin in the medium, shoot elongation was restricted, especially in softhair Clones 1, 2, 3, 8 and M1. Further experiments were carried out with 5 or 10 ~M zeatin and photoperiods of 16 or 12 h (Table 3). On the medium containing 10 ~M zeatin the rate of shoot multiplication for total shoots was greater than on 5 ltM zeatin in Clones 8 and M1. The rate for long shoots in Clone 1 was smaller, while in Clone 8 it was bigger. Under 16-h photoperiod the rates of shoot multiplication for both total shoots and long shoots in Clone 1 were bigger than under a 12-h TABLE4
The effect of IAA and IBA on adventitious root formation of eight clones Clone
1
2
3
4
5
8
M1 M2
Medium
M S ÷ IAA ½M S + IAA MS+IBA M S + IAA ½MS + I A A MS+IBA M S + IAA ½MS + IAA M S + IBA ½M S ÷ IBA M S + IAA ½MS + IAA MS+IBA M S + IAA ½MS + IAA M S ÷ IBA M S ÷ IAA ½M S + IAA M S ÷ IBA MS÷IAA M S + IBA M S ÷ IAA ½MS + IAA ½MS + IBA
10/~M 10 # M 2ttM 10 # M 10 # M 10/~M 10 # M 10 # M 5 #M 5 #M 10 # M 10 BM 5BM 10 # M 10 # M 5 #M 10 # M 10 ItM 5 #M 10 # M 5 ~M 10 # M 10 BM 10 # M
Number of microcuttings
Percentage of rooted microcuttings
Mean root number per rooted microcutting ( m e a n +- SE )
275 275 123 193 134 124 122 132 261 80 281 240 135 272 151 75 96 118 70 128 75 340 540 72
41.1 44.4 84.6 10.9 6.0 66.7 8.2 5.3 66.3 72.5 56.2 71.7 77.1 39.0 62.9 80.0 7.3 11.0 58.6 11.7 68.0 80.6 87.6 90.3
2.4 ± 1.67 3.3 __2.16 7.1_+4.10 1.5 + 0.65 1.5 ± 0.33 8.5 +_4.16 3.1 +- 1.97 1.3 +- 0.49 5.9 + 4.59 6.4 + 5.24 5.0 _+3.68 4.6 + 3.19 5.6+-4.19 1.9 ± 1.66 2.5 _+1.24 4.4 + 3.42 1.3 + 0.73 1.8 _+0.82 4.7 + 3.46 1.3 +-0.38 4.6 _+3.80 5.4 _+3.77 5.4 +_3.12 11.9 +- 4.00
52
XIAO-SHAN SHEN ET AL.
photoperiod, while in Clone 8 the rate for total shoots only differed, and in Clone 3 the rate for long shoots was smaller. In Clones 2 and 5 no differences occurred (Table 3). P l a n t establishment. - Microcuttings longer than 20 mm rooted in 2-3 weeks with bristly-hair Clones 4, 5 and M2 (Fig. 3 ) and in 3-4 weeks with soft-hair Clones 1, 2, 3, 8 and M1. A microcutting could give rise to 1-35 roots. The microcuttings of some clones could not root on media without IAA and IBA, while others could, but the percentages of rooted microcuttings remained below 20% and only a few roots were formed on one microcutting. IAA or IBA in media promoted rooting of all 8 clones. In all clones IBA yielded a higher percentage of rooted microcuttings and a higher mean root number per rooted microcutting than IAA (Table 4). Full sun had to be avoided during hardening-off. The rooted microcuttings died if 1-2 h of full sun was given to them. After hardening-off, containergrown plants could be established by transplanting the surviving plantlets into pots (Fig. 4). Survival of rooted microcuttings was closely related to the environmental conditions during hardening-off and after transplanting. Under conditions where humidity and irradiance were not controlled 59.9% of 3211 rooted microcuttings survived after transplanting for a month. Forty in vitro plants of Clones 4 and M2 were planted in a field in June 1987 and have been growing vigorously. Morphological aberration has not been found. E f f e c t of a bacterium s u r v i v i n g in explants on in vitro propagation. - Although the initial explants were surface sterilized, cultures had three symptoms of infection, i.e. shoot tip death, blackened leaves, and rotting of leaves and shoots. Similar symptoms appeared in cultures of other clones and cultivars which were used in our laboratory, including cultivars 'Hayward' and 'Tomuri' from New Zealand. A bacterium was found in the excretion of infected tissues. If healthy shoots were used in every subculture as explants, the bacterium surviving in the explant was not an obstacle to in vitro propagation of Chinese gooseberry. If not, the bacterium not only infected most shoots, but also greatly reduced the rooting of microcuttings. DISCUSSION
In vitro propagation through the development of axillary buds is quicker than conventional methods and needs a small quantity of starting material, the in vitro plants propagated in this way in many species have proved to be true to type (Murashige, 1974; Navarro et al., 1975; Styer, 1983). Therefore, it is the best way for propagation of new cultivars or clones for fruit production. Before this in vitro propagation technique is to be used for propagation of
IN VITROPROPAGATIONOFACTINIDIA CHINENSIS
53
Chinese gooseberry, the plants of this origin must be evaluated, but can be used immediately for breeding purposes, to which our institute pays much attention. Axillary shoot multiplication in vitro required different conditions for each of the clones. In relation to the response of axillary shoot formation of the clones to the zeatin concentration and the photoperiod, Chinese gooseberry might be divided into three types: (1) sensitive to cytokinin, the axillary shoot formation is significantly influenced by cytokinin concentration change; (2) sensitive to photoperiod, the axillary shoot formation is significantly affected by photoperiod; (3) inactive, axillary shoot formation does not differ significantly with the changes of cytokinin concentration and photoperiod. Monette (1986) reported that after standard surface sterilization of the New Zealand cultivar 'Hayward' two surviving bacteria were found in culture and that their presence did not present a major obstacle to in vitro propagation. Similar results were obtained in more clones of Chinese gooseberry in our study.
REFERENCES Barbieri, C. and Morini, S., 1987. Plant regeneration from Actinidia callus culture. J. Hortic. Sci., 62: 107-109. Brossard-Chriqui, D. and Tripathi, B.K., 1984. Comparison of morphogenetic ability in fertile or sterile stamens of A. chinensis cultured in vitro. Can. J. Bot., 62: 1940-1946. Gui, Y.L., An, H.Q., Cai, D.Y. and Wang, J.R., 1979. Tissue culture of Chinese gooseberry. Kexuetongbao, 4:188-190 (in Chinese). Hong, S.Y., 1981. Induction of callus and plantlets from segments of tender shoots and young leaves in Actinidia. Hupei Agric. Sci., 9:28-30 (in Chinese). Huang, Z.G., Huangpu, Y.L. and Xu, Y.Y., 1982. Regeneration of triploid plants from endosperm culture of Chinese gooseberry. Kexuetongbao, 27:247-250 (in Chinese). Monette, P.L., 1986. Micropropagation of kiwifruit using non-axenic shoot tips. Plant Cell, Tissue Organ Cult., 6: 73-82. Murashige, T., 1974. Plant propagation through tissue culture. Annu. Rev. Plant Physiol., 25: 135-166. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant, 15: 473-497. Navarro, L., Roistacher, C.N. and Murashige, T., 1975. Improvement of shoot tip grafting in vitro for virus-free Citrus. J. Am. Soc. Hortic. Sci., 100: 471-479. Romberger, J.A., 1976. An appraisal of prospects for research on juvenility in woody perennials. Acta Hortic., 56: 301-317. Standardi, A. and Catalano, F., 1984. Tissue culture propagation of kiwifruit. Combined Proceedings of International Plant Propagators' Society, 34: 236-243. Styer, D.J., 1983. Meristem and shoot-tip culture for propagation, pathogen elimination, and plasm preservation. Hortic. Rev., 5: 221-277. Wang, J.X. and Li, S.Z., 1982. Propagation ofA. chinensis by tissue culture. Liaoning Agric. Sci., 1:32-34 (in Chinese). Wessels, E., Nel, D.D. and von Staden, D.F.A. 1984. In vitro propagation of A. chinensis P1. cultivar Hayward. Deciduous Fruit Grower, 34: 453-457. Yu, S.Z., 1983. Regeneration of plantlets by cotyledon culture of A. chinensis. Plant Physiol. Commun., 2:37-38 (in Chinese).
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Zhou, H.Y., 1987. Introduction of super clones of Chinese gooseberry selected in China. Fruit Tree, 3:24-26 (in Chinese). Zhou, D.Z., Gao, H. and Liu, G.P., 1984. Induction of callus and regeneration of plants from segments of tender shoots of Chinese gooseberry. J. Jiangxi Agric. Univ., 1:83-84 (in Chinese). Zhu, H.Y., 1983. Production of Chinese gooseberry. Shanghai Scientific Press, pp. 13-16 (in Chinese ).