Adult Plants and Juvenile Seedlings of Persimmon (Diospyros kaki L.)

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1 J. Japan. Soc. Hort. Sci. 63(3) : Comparison of Growth Rooting Characteristics of Micropropagated Adult Plants Juvenile Seedlings of Persimmon (Diospyros kaki L.) Ryutaro Tao, Jun Ito Akira Sugiura Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto Summary This study describes the influence of the number of subcultures on the rooting ability of persimmon (Diospyros kaki L. cv. Jiro) shoots cultured in vitro, also investigates the growth characteristics of the acclimatized plants which might be affected by rejuvenation. 1. The percentages of rooted shoots from adult plants increased with repeated subcultures. The rooting percentage was only 3.3% after three subcultures increasing to 90% after 38 subcultures. In contrast, shoots from juvenile seedlings had as high as 70% rooting after three subcultures. 2. In terms of shoot root dry weights, the micropropagated plants grew more vigorously than seedling plants in the first growing season. Some of the micropropagated trees bore their first flower in the second growing season, suggesting that partial rejuvenation or reinvigoration, but not true rejuvenation, was responsible for the early flowering the juvenile characteristics observed in the micropropagated plants. Introduction Japanese persimmon (Diospyros kaki L.) is considered to be one of the most difficult-to-root fruit tree species propagation by cuttings has proven to be very difficult. Thus commercial cultivars have been propagated by grafting or budding onto seedling rootstocks (Kitagawa Glucina, 1984). Recently clonal propagation through tissue culture has become a practical means of rapid large scale multiplication for a wide variety of fruit nut species (Debergh Zimmerman, 1991). We have already described successful methods of shoot tip culture for several persimmon cultivars (Murayama et al., 1989; Sugiura et al., 1986; Tao Sugiura, 1992). Even in vitro, however, rooting is difficult for persimmon especially when using zeatin as the cytokinin to be added in the media for shoot multiplication (Tao Sugiura, 1992; Tetsumura et al., 1991). Since zeatin is particularly effective for persimmon tissue culture some cultivars can be established in culture multiplied only with zeatin, low rooting ability Received for publication 17 February in the shoots cultured with zeatin is a big problem (Tao Sugiura, 1992; Tetsumura et al., 1991). For many woody plant species, repeated subcultures of adult shoots have been reported to result in rejuvenation, by which shoot multiplication rooting ability are improved (Pierik, 1990). The number of subcultures needed to approach maximum rooting depends on the species even cultivar. Azalea required only four subcultures to reach over 90% of rooting (Economou Read, 1986), while an apple cultivar required 31 subcultures before sufficient rooting was obtained (Striskarajah et al., 1982). So far, there has been no data available for the relation between number of subcultures rooting ability of persimmon shoots cultured in vitro. This study describes the influence of the number of subcultures on the rooting ability of persimmon shoots cultured in vitro, also investigates the growth characteristics of the acclimatized plants which might be affected by rejuvenation. Materials Methods Dormant one-year-old shoots were taken from more than 30 year old trees of 'Jiro' in December 537

538 R. Tao, J. Ito A. Sugiura stored in plastic bags at 4 Ž for several months. Axillary buds were disinfected trimmed as described earlier (Sugiura et al., 1986).

2 538 R. Tao, J. Ito A. Sugiura stored in plastic bags at 4 Ž for several months. Axillary buds were disinfected trimmed as described earlier (Sugiura et al., 1986). The apical portions of the buds (2 mm long) were cultured on MS agar medium (Murashige Skoog, 1962) supplemented with 5 µm zeatin. The cultures were maintained at 28 Ž under 16 hr day -1 photoperiod (light intensity : 20 µmol 1 2 sec -1 m -2) by subculturing to the same fresh medium every five weeks. For the first experiment, effect of the number of subcultures on rooting was tested with the shoots subcultured from 3 to 38 times. After five weeks of subculture, basal ends of shoots were quick dipped in 1.25 mm 3-indolebutyric acid (IBA) 50% ethanol solution placed on 1/2 MS (1/2 N) agar medium (Tao Sugiura, 1992). Controls were shoot tip cultures from very young seedling plants of 'Jiro'. Seeds of 'Jiro' were sown in vermiculite incubated at 28 Ž under 16 hr day -1 photoperiod. After 30 days of germination, shoot tips were disinfected trimmed aseptically to about 3 mm. They were cultured in vitro as with the same procedures for the dormant buds of 'Jiro'. After three subcultures, shoots were treated with the IBA solution described above transferred on 1/2 MS (1/2 N) agar medium to induce rooting. For rooting, the shoots were incubated at 28 Ž in darkness during the first 10 days transferred to 16 hr day -1 photoperiod. Single shoots were each placed in a separate flask containing 30 ml of medium. There were 30 explants per treatment. For seedling plants, there were 10 clones derived from 10 different seeds three shoots for each clone were induced to root. Thus 30 shoots were tested as a whole. For the second experiment, growth behavior of micropropagated trees seedlings of 'Jiro' were compared. 'Jiro' shoots subcultured for 35 times on MS agar medium containing 5 µm zeatin, were treated with 1.25 mm IBA 50% ethanol solution placed on 1/2 MS (1/2 N) medium solidified with 0.2% Gellan Gum (Wako Pure Chemicals Co. Ltd., Tokyo, Japan). Cultures were incubated at 28 in darkness during the first 10 days then Ž transferred to 16 hr day -1 photoperiod. After 7 weeks of culture, rooted shoots were removed from the rooting medium planted in vermiculite in plastic culture vessels which had been autoclaved. They were cultured at 28 Ž under a 24 hr day -1 photoperiod. After new shoot growth occurred, the covers were gradually removed from the vessels. In early June (about 15 weeks after rooting treatment), the plantlets were transplanted to clay pots ( cm) filled with a mixture of s vermiculite (2 : 1, v/v) grown in a frame covered with plastic film that was open at the sides. The plants were fertilized with Hoagl's No. 2 solution every five days until late September. As controls, seeds of 'Jiro' were sown in clay pots in April grown as with the same procedure for the micropropagated plantlets. In winter, eight plants each for micropropagated seedling plants, were harvested by carefully washing the soil from the roots. Then the roots were separated from the shoots. The roots the shoots were oven-dried at 60 Ž for three days weighed. Additional four trees for each type were observed during the second growing season to determine if they had initiated flower buds in the first growing season. Throughout the experiments, all the media contained 3% sucrose. Agar (Agar powder for microbiological culture medium; Wako Pure Chemicals Co. Ltd., Tokyo, Japan) at 0.8% was used for agar media. The ph of the media was adjusted to 5.7 ~ 5.8 before autoclaving at 121 Ž for 15 min. Results Discussion The rooting ability of the shoots from adult plants increased with repeated subcultures (Fig. 1). The final rooting percentage was only 3.3% in the shoots after three subcultures increasing to 90% after 38 subcultures. If the commercially feasible rooting percentage is assumed to be more than 60%, it can be achieved after 25 subcultures. The shoots from juvenile persimmon seedlings, on the contrary, had 70% rooting after three subcultures. Since rooting ability is often considered as an important parameter for rejuvenation (Hackett, 1985; Pierik, 1990), the observations that increasing rooting ability was obtained with successive subcultures in the shoots from adult plants while the high rooting ability at an early stage of subculture in the shoots from juvenile seedlings could suggest that the shoots from adult plants were rejuvenated progressively with subcultures. However, growth characters other than rooting ability should be investigated because a strict correlation between rooting rejuvenation does not always

3 J. Japan. Soc. Hort. Sci. 63 (3) : exist (Ununger Ekberg, 1987). In terms of shoot dry weight, micropropagated trees grew more vigorously than seedlings (Table 1). However, the greater trunk diameter in the micropropagated plants did not result in greater tree height (Table 1). Judging from the trunk diameter, the tissue cultured trees reached grafting size in the first growing season, whereas one or more additional growing seasons would be necessary for the seedling trees. This feature might be advantageous for the use of micropropagated trees as rootstocks because of the fast initial growth rate. Since vegetative vigor generally declines with the age, more vigorous vegetative growth observed in the micropropagated trees could also indicate that they were rejuvenated during repeated subcultures in vitro. Further, as judged by root growth, the tissue cultured plants had significantly greater development than the seedling trees (Table 1). The micropropagated trees developed many lateral fine roots, whereas the seedling trees had one vigorously growing tap root from which only a few lateral roots elongated (Fig.2). Japanese persimmon is known to be particularly prone to transplantation shock (Kitagawa Glucina, ). The use of micropropagated trees 1984 would alleviate the transplantation shock because the vast root system would help sustain early plant establishment. Finally we investigated the flowering ability of the micropropagated plants to see if they were truly rejuvenated. As opposed to our presumption, two of the four micropropagated plants bore their first flowers in the second growing season, while no seedling tree bore flowers. No abnormality was observed in the flowers (Fig. 3). The term juvenility is defined as that physiological state of a seedling plant during which it cannot be induced to flower, rejuvenation is defined as a phase reversal to juvenile form (Hackett, 1985). It would Fig. 1. Effect of the number of subcultures on in vitro rooting of persimmon shoots from adult seedling plants of 'Jiro'. Thirty explants were used per treatment. Table 1. Comparison of the growth in micropropagated seedling plants of ' Jiro' after the first growing season.

540 R. Tao, J. Ito A. Sugiura have taken at least three years before flowering occurred if the micropropagated plants had reverted to the juvenile phase.

4 540 R. Tao, J. Ito A. Sugiura have taken at least three years before flowering occurred if the micropropagated plants had reverted to the juvenile phase. The early flowering along with some juvenile characteristics observed in the micropropagated plants can be explained only by assuming that they were partially rejuvenated. Thus we conclude that reinvigoration or partial rejuvenation (Pierik, 1990) occurred was responsible for the early flowering the juvenile characteristics observed in propagated plants from adult explants. determine whether the plants could be further with continuing subcultures to pletely to vigorated tion over stocks to plantation the microwe did not rejuvenated return com- the juvenile phase; still, the use of reinself-rooted trees has commercial applicathe use of grafted trees on seedling rootachieve precocity, alleviation of transshock, uniformity of growth. Acknowledgement We Davis, the thank for G. his C. Martin, critical 2. Difference in growth between (left) seedling (right) first growing season. plants micropropagated of 'Jiro' after the of California, correction of manuscript. Literature Fig. Univ. reading Cited Debergh, P. H. R. H. Zimmerman Micropropagation : technology application. Kluwer Academic Publ., Dordrecht. Economou, A. S. P. E. Read. production deciduous Microcutting from sequential reculturing of hardy azalea shoot tips. HortScience 21 : Hackett, W. P Juvenility, maturation, uvenation in woody plants. Horticultural 7 : Kitagawa, H. P. G. Glucina. ture ton. Murayama, in New Zeal Persimmon Sci. Inf. Publ. H., R. Tao, T. Tanaka In vitro shoot proliferation cul- Cent., Welling- A. Sugiura. rooting of several Japanese persimmon cultivars. Soc. Hort. Sci. 58 : (In Japanese glish summary). Murashige, T. F. Skoog for rapid growth bioassays rereviews j J. Japan. with En- A revised medium with tobacco tis- sue cultures. Physiol. Plant. 15 : Pierik, R. L. M Rejuvenation micropropagation. Newsletter Intl. Assoc. Plant Tissue Cult. 62 : Striskarajah, S., G. Mullins Y. Nair Fig. 3. First flowering of the micropropagated plants of 'Jiro' during the second growing season. duction of adventitious rooting in difficult-to-propagate cultivars of apple. Lett. 24 : 1-9. vitro Plant No. Inin Sci.

5 J. Japan. Soc. Hort. Sci. 63(3) : Sugiura, A., R. Tao, H. Murayama T. Tomana In vitro propagation of Japanese persimmon. HortScience 21 : Tao, R. A. Sugiura Micropropagation of Japanese persimmon (Diospyros kaki L.), p In : Y. P. S. Bajaj (ed.). Biotechnology in Agriculture Forestry, vol. 18. High-Tech Micropropagation 2. Springer-Verlag, Berlin, Heiderberg. Tetsumura, T., R. Tao A. Sugiura Effect of cytokinin types on the in vitro propagation of Japanese persimmon (Diospyros kaki Thunb.). Plant Tissue Culture Letters 8 : (In Japanese with English summary). Ununger, J. I. Ekberg Rejuvenation of juvenile growth characters in Picea abies Sc. J. For. Res. 2 :

Keywords: Agarwood, satalum, resin, micropropagation, tok

Keywords: Agarwood, satalum, resin, micropropagation, tok Application of Tissue Culture Techniques in Woody Species Conservation, Improvement and Development in Vietnam: Agarwood (Aquilaria crassna Pierre ex LeComte) via tip Culture Tran Van Minh Institute of