In Vitro Cell. Dev. Biol.ÐPlant 37:274±279, March±April 2001 DOI: 10.

1079/IVP2000147
q 2001 Society for In Vitro Biology
1054-5476/01 $10.0010.00

EFFICIENT AND SIMPLE PLANT REGENERATION VIA ORGANOGENESIS FROM LEAF SEGMENT
CULTURES OF PERSIMMON (DIOSPYROS KAKI THUNB.)

J. Y. CHOI1, H. J. KIM1, C. H. LEE2, J. M. BAE3, Y. S. CHUNG4, J. S. SHIN3, and N. I. HYUNG1*

1
Department of Horticultural Science, Sangmyung University, Chonan 330-180, Korea
2
Department of Horticultural Science, Korea University, Seoul 136-701, Korea
3
Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea
4
Division of Bioresources, Faculty of Life Sciences and Resources, Donga University, Pusan 604-714, Korea

(Received 15 March 2000; accepted 14 November 2000; editor F. Englemann)

Summary
An efficient and simple plant regeneration system via organogenesis from leaf segments of persimmon (Diospyros kaki
Thunb.) cultivars `Fuyu' and `Nishimurawase' has been developed. The regeneration capacity was influenced by the
culture vessels, gelling agents, plant growth regulators, and light conditions. Leaf explants taken from in vitro shoots were
cultured on a modified Murashige and Skoog medium (MS12N) for 16 wk without transfer to fresh medium. Adventitious
shoots appeared after 4 and 8 wk in culture of `Nishimurawase' and `Fuyu' tissues, respectively. The culture of leaf
explants in Erlenmeyer flasks with medium containing 4 g l21 agar enhanced shoot formation in comparison to media with
increased agar concentrations. Optimal shoot regeneration was obtained with 5 mg l21 (22.8 mM) zeatin and 0.1 mg l21
(0.05 mM) indole-3-butyric acid (IBA) for `Nishimurawase', and 10 mg l21 (45.6 mM) zeatin and 0.1 mg l21 (0.05 mM)
IBA for `Fuyu'. Shoot regeneration frequencies in both cultivars were 100%, and shoot numbers per explant reached up to
9.2 for `Nishimurawase' and 2.2 for `Fuyu'. Dark incubation during the first 4±5 wk was the most effective condition to
successfully influence shoot regeneration in both cultivars. While dark incubation was essential for adventitious shoot
formation by `Fuyu', it was only slightly beneficial to `Nishimurawase'. More than 80% of the regenerated shoots rooted
within 4 wk on hormone-free MS12N medium after having been dipped for 30 s in 250 mg l21 (1.1 mM) IBA solution.
Key words: adventitious shoot formation; culture vessel; plant growth regulators; light condition; dark culture.

Introduction
Persimmons (Diospyros kaki Thunb.) are an important fruit crop
in North-Eastern Asia, including Korea, Japan and China. However,
conventional breeding of persimmons is difficult due to their long
generation time, large plant sizes, and genetic heterozygosity.
Moreover, cross-breeding is hindered by the limited number of
cultivars, which carry male and/or hermaphrodite flowers.
Recent developments in biotechnology opened up several new
ways for persimmon breeding through the use of transformation in
which heterologous genes can be introduced into existing cultivars
(Tao et al., 1997). In many instances, however, the lack of an
efficient regeneration system is the limitation to the use of gene
transfer technologies for perennial fruit crops (Schuerman and
Dandekar, 1993). An efficient plant regeneration system is,
therefore, essential for transformation and propagation as well as
for in vitro breeding through selection of somaclonal variation and
somatic hybridization.
For persimmon, plant regeneration via organogenesis has been
achieved using hypocotyls of seeds (Yokoyama and Takeuchi, 1976;
*Author to whom correspondence should be addressed: Email nihyung@
smuc.ac.kr
Yamada et al., 1987) and primordial leaves of adult trees (Tao et al.,
1988). The method of regeneration and transformation using
hypocotyls cannot be used to introduce a gene directly into existing
cultivars, because the hypocotyls are of cross-bred origin, although
regeneration by this method (Nakamura et al., 1998) is fast and
efficient. The method of Tao et al. (1988) using leaf explants
requires a long time of culture and three or four steps: callus
induction from explants, adventitious shoot regeneration from the
callus, regenerated shoot elongation, and rooting. For future genetic
transformations, therefore, a regeneration system from vegetative
tissue, such as a leaf, without separate steps would be very
desirable. Moreover, it has been demonstrated that the formation of
adventitious shoots is dependent upon the genotype of the
persimmon; only eight out of 16 cultivars formed adventitious
buds within the frequency range of 2% to 72%, and the
commercially most important cultivar `Fuyu' failed to regenerate
and proved to be recalcitrant (Tao and Sugiura, 1992a).
In this study, we developed an efficient and simple system for
plant regeneration using the leaf segments excised from in vitro
shoot cultures of the persimmon cultivars `Fuyu' and `Nishimur-
awase'. Several culture conditions, such as culture vessels, gelling
agents, growth regulators, and light conditions which might
influence adventitious shoot formation from leaf explants, were
investigated.

274
 PLANT REGENERATION IN PERSIMMON 275

Fig. 1. Plant regeneration via organogenesis from in vitro cultured leaf explants of persimmon cvs `Fuyu' (A, C) and `Nishimurawase'
(B, D±F). The leaf explants produced callus and adventitious shoots mainly at the proximal end after 16 wk (A±D) on MS12N medium
supplemented with 5 mg l21 zeatin and 0.1 mg l21 IBA. The explants marked with an arrow in (A) and (B) were transferred to fresh
culture vessels for the sake of taking the photos in (C) and (D), respectively. The adventitious shoots produced roots after dipping them
into 250 mg l21 IBA solution and culturing on basal MS12N medium without plant growth regulator (E). After rooting, the regenerated
plantlets were acclimatized (F). Bars ˆ 10 mm:

Materials and Methods
Shoot culture. Axillary shoots of the persimmon (Diospyros kaki Thunb.)
cultivars `Fuyu' and `Nishimurawase' were cultured in vitro. MS basal
medium (Murashige and Skoog, 1962) supplemented with 2 mg l21
(8.9 mM) N6-benzyladenine (BA) was used for `Nishimurawase', and a
modified MS medium with half the normal NH4NO3 and KNO3 concentration
(MS12N; Sugiura et al., 1986) supplemented with 2 mg l21 (9.1 mM) zeatin
was used for `Fuyu'. The medium containing 30 g l21 sucrose and 8 g l21
agar (Sigma) was adjusted to pH 5.8 prior to the addition the agar and
autoclaved at 1218C for 15 min. The cultures were grown at 24±268C under
24-h continuous illumination provided by cool-white fluorescent lamps
(40 mmol m22 s21) and subcultured every 4 wk.
Adventitious shoot regeneration. Actively growing leaves from the shoots
were excised after 3 wk in subculture and cut transversely across the midrib
into segments. The leaf segments were placed with the adaxial surface in
contact with the medium. The cultures were maintained for 8 or 16 wk
without transfer to fresh medium. The culture conditions were the same as
those of the shoot cultures described above.
The percentage of shoot-forming explants and the shoot number per
regenerating explant were recorded. To examine the effects of gelling agents,
growth regulators, and light conditions on adventitious shoot formation, five
Erlenmeyer flasks (100 ml) containing five explants each were used.
Effect of culture vessel and light/dark incubation. The effects of two
different culture vessels, Petri dish …100  10 mm† and Erlenmeyer flask
(100 ml), as well as the effect of light/dark incubation, on adventitious shoot
276 CHOI ET AL.

regeneration were tested in `Nishimurawase'. After leaves were explanted

onto the medium, Petri dishes and Erlenmeyer flasks were sealed with
Parafilm (Whatman) and aluminum foil, respectively. The culture vessels
were placed in the dark (dark incubation) or under continuous illumination
(light incubation) for the entire culture period of 8 wk. The basal shoot
regeneration medium was composed of MS12N medium supplemented with
3 mg l21 (13.7 mM) zeatin, 0.1 mg l21 (0.5 mM) indole-3-butyric acid
(IBA), 30 g l21 sucrose, and 8 g l21 agar.
Effect of gelling agent. To compare gelling agents, 4, 6 and 8 g l21 agar
(Sigma) or 2.5 g l21 Phytagel (Sigma) were added to the basal shoot
regeneration medium of MS12N supplemented with 3 mg l21 (13.7 mM)
zeatin, 0.1 mg l21 (0.5 mM) IBA, and 30 g l21 sucrose. The cultures of
`Nishimurawase' were then maintained on those media in the dark for 8 wk.
Effect of zeatin and IBA, and light/dark treatment. The combined effect
of growth regulators and light/dark treatment was investigated in `Fuyu' and
`Nishimurawase'. MS12N medium was supplemented with various combina-
tions of zeatin (1, 3, 5, 10 mg l21; 4.6±45.6 mM) and IBA (0.01, 0.1,
1 mg l21; 0.05±4.9 mM). The cultures were maintained under light
provided by cool-white fluorescent lamps (40 mmol m22 s21) for the entire
16 wk of culture (light treatment) or in the dark for the initial 8 wk followed
by incubation under 24 h of continuous illumination (dark treatment).
Effect of time in the dark. Since the above experiment indicated that the
dark treatment improved the efficiency of adventitious shoot formation over
that of the light treatment, we tried to determine the optimal time in the dark
for `Fuyu' and `Nishimurawase'. After initial culturing in the dark for 0± Fig. 2. Effect of culture vessel and light/dark incubation on
adventitious shoot generation from in vitro-cultured leaf segments of
8 wk, the cultures were transferred into light for the balance of 16 wk of
persimmon cv. `Nishimurawase' after 8 wk in culture. Data represent
total culture time. The medium used for shoot induction was MS12N medium
containing 5 mg l21 (22.8 mM) zeatin and 0.1 mg l21 (0.5 mM) IBA for percentages or means ^ SE of five replicates.
both cultivars.
Rooting and acclimatization. Regenerated shoots were cut to 15±20 mm
lengths at the distal end. The proximal ends of the shoots were dipped into
250 mg l21 (1.1 mM) IBA solution for 30 s, and then transferred to the
rooting medium, consisting of MS12N with 30 g l21 sucrose and 8 g l21 agar
(Tao and Sugiura, 1992b). The cultures were incubated in the dark for 10 d
and then switched to light. After 4 wk in culture the rooted shoots (plantlets)
were washed with tap water and planted in pots containing autoclaved
artificial soil (1 part vermiculite:1 part perlite). After the plants successfully
acclimated, they were transplanted into pots containing soil and transferred
to a greenhouse.

Results

Shoot regeneration from leaf explant. Calluses formed after 2±

3 wk in culture at the proximal ends of leaf segments of persimmon
`Fuyu' and `Nishimurawase'. These calluses contained a large
amount of dark brown substances, most likely polyphenolic
compounds which were secreted into the culture medium (Fig. 1A,
B). After 4 or 8 wk, respectively, adventitious shoots began to grow
from the calluses of `Nishimurawase' and `Fuyu'. Occasionally,
shoots regenerated directly from the leaf blade in both cultivars.
The regenerated shoots then continued to gradually elongate in the
original medium without transfer to fresh medium (Fig. 1A±D). Fig. 3. Effects of gelling agents on adventitious shoot generation from in
Effect of culture vessel and light/dark incubation. The formation vitro-cultured leaf segments of persimmon cv. `Nishimurawase' after 8 wk in
of adventitious shoots on `Nishimurawase' tissue was greatly culture. Data represent percentages or means ^ SE of five replicates.
affected by the culture vessels used. In Erlenmeyer flasks, shoots
formed under both light and dark conditions. In contrast, shoot
regeneration in Petri dishes occurred at a lower frequency and only medium containing 4 g l21 agar than Phytagel. In comparing agar
under illumination, none formed upon dark treatment. The concentrations, the regeneration of shoots gradually increased in
Erlenmeyer flask culture with dark incubation produced the highest frequency as the agar concentration was reduced down to 4 g l21.
shoot regeneration, 60% (Fig. 2). Therefore, only Erlenmeyer flasks Effect of zeatin and IBA in combination with light/dark
were subsequently used as the culture vessel. treatment. The adventitious shoot formation from leaf explants of
Effect of gelling agent. The effect of gelling agents, agar and `Nishimurawase' was slightly influenced by light/dark treatment
Phytagel, on the adventitious shoot regeneration is shown in Fig. 3. depending on different combinations of zeatin and IBA concentra-
Shoot regeneration frequency was not greatly different among four tions. The optimal requirement for IBA in the light was generally
treatments, all of which showed higher than 40% regeneration. higher than that in the dark. On the other hand, high concentrations
However, the number of shoots per explant was higher in the (.3 mg l21) of zeatin appeared to effectively promote shoot
 PLANT REGENERATION IN PERSIMMON 277

TABLE 1

EFFECT OF PLANT GROWTH REGULATOR AND LIGHT/DARK TREATMENT ON SHOOT REGENERATION FROM LEAF SEGMENT EXPLANTS OF
PERSIMMON CV. `NISHIMURAWASE' AFTER 16 WK CULTURE ON MS12N MEDIUM

Plant growth regulator Light treatment Dark treatmenta

Shoot regeneration Shoot number per explant Shoot regeneration Shoot number per explant
Zeatin (mg l21) IBA (mg l21) (%)b …mean ^ SE†b (%)b …mean ^ SE†b
1.0 0.01 8.0 2.0 28.0 3.0 ^ 0.6
0.1 20.0 2.0 ^ 0.6 60.0 2.6 ^ 0.5
1.0 28.0 1.0 0.0 ±
3.0 0.01 100.0 7.0 ^ 1.1 92.0 8.2 ^ 1.4
0.1 92.0 4.1 ^ 0.8 80.0 7.1 ^ 1.3
1.0 48.0 2.4 ^ 0.4 0.0 ±
5.0 0.01 80.0 7.0 ^ 1.0 80.0 6.2 ^ 0.8
0.1 88.0 9.1 ^ 1.0 100.0 9.2 ^ 0.8
1.0 100.0 5.1 ^ 0.6 28.0 1.0
10.0 0.01 72.0 6.7 ^ 1.0 88.0 7.4 ^ 0.5
0.1 92.0 7.9 ^ 0.8 92.0 10.1 ^ 1.3
1.0 80.0 7.3 ^ 0.9 88.0 5.9 ^ 0.7

a
Leaf segment explants were cultured in the dark for 8 wk initially and then cultured under continuous light.
b
Data represent percentages or means ^ SE of five replicates.

TABLE 2

EFFECT OF PLANT GROWTH REGULATOR AND LIGHT/DARK TREATMENT ON SHOOT REGENERATION FROM LEAF SEGMENT EXPLANTS OF
PERSIMMON CV. `FUYU' AFTER 16 WK CULTURE ON MS12N MEDIUM

Plant growth regulator Light treatment Dark treatmenta

Shoot regeneration Shoot number per explant Shoot regeneration Shoot number per explant
Zeatin (mg l21) IBA (mg l21) (%)b (mean^SE)b (%)b (mean^SE)b
1.0 0.01 8 1.0 32 1.0
0.1 20 1.0 0 ±
1.0 0 ± 0 ±
3.0 0.01 20 1.0 80 1.8 ^ 0.3
0.1 32 1.0 88 1.7 ^ 0.2
1.0 12 1.0 0 ±
5.0 0.01 0 ± 52 3.0 ^ 1.0
0.1 20 1.5 ^ 0.5 80 2.0 ^ 0.5
1.0 8 2.0 60 1.8 ^ 0.3
10.0 0.01 32 1.7 ^ 0.3 96 3.1 ^ 0.6
0.1 40 2.0 ^ 0.8 100 2.2 ^ 0.2
1.0 32 2.3 ^ 0.7 12 1.0

a
Leaf segment explants were cultured in the dark for 8 wk initially and then cultured under continuous light.
b
Data represent percentages or means ^ SE of five replicates.

regeneration. The most effective combination for shoot regeneration
in `Nishimurawase' was either 3 mg l21 zeatin with 0.01 mg l21
IBA in light culture, or 5 mg l21 zeatin with 0.1 mg l21 IBA in
dark culture. Under those conditions, all explants produced
adventitious shoots in the light or dark with 7.0 or 9.2 shoots per
explants, respectively (Table 1).
In `Fuyu', shoot regeneration was not observed within 8 wk of
culture in the dark, but it was initiated in 1±2 wk after transfer of
the culture to light (total 9±10 wk). After an additional 8 wk of
culture in the light, another transfer into the dark further enhanced
the shoot regeneration as compared to continuous illumination.
The optimal concentrations of the growth regulators were
combinations of 10 mg l21 zeatin with 0.1 mg l21 IBA in both
light and dark treatment. Under these conditions, 40% and 100% of
regeneration were observed under light and dark treatment,
respectively. The number of shoots was 2.0 and 2.2 shoots per
explant, respectively (Table 2).
Effect of time in the dark. In the previous experiments we had
seen that dark culture was quite effective in generating adventitious
shoots. Therefore, the optimal period of time for which the leaf
cultures should be kept in the dark prior to transfer to light was
subsequently investigated.
The regeneration of adventitious shoots for `Nishimurawase' was
apparent after 3 wk in culture, and the numbers of shoots gradually
increased, reaching almost maximum levels after 8 wk. Dark
treatment slightly enhanced shoot regeneration in `Nishimurawase',
as shown in the previous experiment. We determined that 5 wk of
dark treatment produced the maximal response: 100% regeneration
and 4.6 shoots per explant (Table 3).
In `Fuyu', adventitious shoots started to emerge after 8 wk of
278 CHOI ET AL.

TABLE 3 Adventitious shoot regeneration from leaf explants was highly

influenced by the choice of culture vessel (Fig. 2). Erlenmeyer
EFFECT OF DARK PERIOD ON THE SHOOT REGENERATION FROM
LEAF SEGMENT EXPLANTS OF PERSIMMON CVS `NISHIMURAWASE'
flasks showed more efficient shoot regeneration than Petri dishes.
AND `FUYU'a Changes in gas composition in the culture vessels may be the key
element that can depress or raise the regeneration efficiency, since
Time (wk) `Nishimurawase' `Fuyu' an Erlenmeyer flask covered with aluminum foil holds more than
Dark Light 8 wk 16 wk 8 wk 16 wk the total volume and facilitates better gas exchange than a Petri
dish sealed with Parafilm. It is known that both carbon dioxide and
Shoot regeneration (%)b
0 16 68 76 0 12 ethylene accumulate and oxygen is reduced in sealed culture
1 15 80 92 4 28 vessels during in vitro culture, mainly due to gases emitted during
2 14 96 96 16 44 the manipulating process of explant inoculation and culture
3 13 80 88 16 44 (Buddendorf-Joosten and Woltering, 1994). To prevent ethylene
4 12 96 96 16 60
5 11 96 100 20 68 accumulation, which inhibits plant tissue regeneration, several
6 10 84 92 12 32 inhibitors of ethylene biosynthesis, such as AgNO3 and sodium
7 9 80 96 4 56 thiosulfate, or air permeable seals, such as Micropore (3M) paper
8 8 72 88 4 44 tape, have been used (Kuvshinov et al., 1999). These items can also
Shoot number per explantb
be used as alternative methods to control gas composition in
0 16 3.5 ^ 0.7 3.7 ^ 0.3 ± 1.0
1 15 5.2 ^ 0.8 5.2 ^ 0.6 1.0 1.6 ^ 0.4 Erlenmeyer flasks for persimmon culture.
2 14 5.0 ^ 0.4 6.5 ^ 0.3 1.5 ^ 0.4 1.5 ^ 0.3 In this experiment, two different gelling agents, agar and
3 13 4.4 ^ 0.8 5.1 ^ 0.5 1.8 ^ 0.5 1.9 ^ 0.6 Phytagel, were tested. Generally, agar is the most common gelling
4 12 4.0 ^ 0.6 4.2 ^ 0.3 1.5 ^ 0.4 1.7 ^ 0.4 agent utilized in plant regenerations. However, it has been reported
5 11 3.1 ^ 0.4 4.6 ^ 1.0 1.2 ^ 0.3 1.9 ^ 0.4
6 10 3.5 ^ 0.6 4.8 ^ 0.6 1.0 1.4 ^ 0.2 that Gelite (same as Phytagel) was superior to agar in promoting
7 9 2.6 ^ 0.7 4.0 ^ 0.5 1.0 1.2 ^ 0.1 adventitious shoot development in apple (Welander and Mahes-
8 8 2.3 ^ 0.4 3.3 ^ 0.6 1.0 1.6 ^ 0.4 waran, 1992) and carnation (Miller et al., 1991) cultures. For
persimmons, the shoot regeneration efficiency revealed not much
a
Explants were cultured on MS12N medium supplemented with 5 mg l21 difference between these two gelling agents, although the number of
zeatin and 0.1 mg l21 IBA.
b shoots was slightly higher using agar rather than Phytagel. Shoot
Data represent percentages or means ^ SE of five replicates.
regeneration increased as the agar concentration was reduced to
4 g l21, which is in agreement with observations made by Welander
culture, and the numbers increased up to 16 wk of culture. `Fuyu' and Maheswaran (1992). These investigators suggested that gelling
essentially required dark treatment for successful regeneration, agents might affect osmotic potentials and nutrient diffusion rates of
since under continuous light regeneration was extremely poor. At different media formulations.
least 2 wk of dark treatment was indispensable to give a sufficient By testing combinations of different concentrations of zeatin and
response. However, the most effective regeneration was obtained IBA, we found that high cytokinin and low auxin promoted
with 5 wk of dark treatment. Under these conditions, 68% adventitious shoot formation on the leaf explants. Tao and Sugiura
regeneration and 1.9 shoots per explant were achieved (Table 3). (1992a) and Tao et al. (1988) made similar observations with callus
Rooting and acclimatization. Regenerated shoots were cut and cultures derived from leaf explants of the `Jiro' persimmon. The
transferred to shoot culture medium for elongation and multi- most effective combinations were of 5 mg l21 zeatin and 0.1 mg l21
plication, before they were transplanted for rooting. The shoots were IBA for `Nishimurawase' and 10 mg l21 zeatin and 0.1 mg l21 IBA
dipped into 250 mg l21 1-naphthaleneacetic acid (NAA) solution for `Fuyu' cultured commonly with dark treatment. These zeatin
for 30 s and then cultured in hormone-free MS12N medium. requirements are higher than those previously used in the leaf
`Nishimurawase' and `Fuyu' rooted after 2 and 4 wk, respectively. explant experiments on 16 persimmon cultivars by Tao and Sugiura
Rooting percentages of over 80% were observed in both cultivars. (1992a), who cited maximum regeneration frequencies of 72% using
The rooted plantlets were then planted in pots containing artificial 2.2 mg l21 zeatin and 0.3 mg l21 IAA. However, our combinations
soil (1 part vermiculite: 1 part perlite) and allowed to acclimate. for `Nishimurawase' and `Fuyu' yielded shoot generation frequen-
Successfully acclimated plants were transferred to the greenhouse. cies of 100% (Tables 1 and 2). As we had expected, the optimal IBA
requirement under light conditions was slightly higher than that
Discussion required in the dark. This is probably due to the degradation of
auxin by light (Tao et al., 1988).
Plant regeneration from leaf explants of the persimmon varieties Almost all previous persimmon studies recommend culturing
`Nishimurawase' and `Fuyu' was achieved in vitro. In this simple under light (12-h photoperiod) for adventitious shoot formation (Tao
procedure, leaf explants were maintained in batch culture without and Sugiura, 1992b; Tao et al., 1988). Our results showed that light
transfer to fresh medium. This method made possible elongated inhibition of morphogenesis on the leaf segments was severe for
shoot production from leaf segments in only one step through the `Fuyu' and moderate for `Nishimurawase'. However, placing the
sequential processes of callus and shoot formation on the explants cultures in the dark for the first 4±5 wk of culture produced the
and the elongation of regenerated shoots. In comparison to the highest number of adventitious shoots in both cultivars. Suppression
method of Tao and Sugiura (1992a), which uses three separate steps of organogenesis by light has been observed also in apple (Korban
in the production of elongated shoots from leaf explants, our method et al., 1992). The inhibition in `Fuyu' indicates that initial dark
is very simple. treatment is critical for shoot regeneration in this recalcitrant
 PLANT REGENERATION IN PERSIMMON
persimmon variety. Therefore, it may be that the requirement of
dark incubation is in proportion to the level of recalcitrancy of
cultivars when it comes to adventitious shoot formation of
persimmons. Leaf cultures of `Fuyu' should be maintained in the
dark for at least the initial 4 wk and transferred to light for
morphogenesis and subsequent development of shoots. It is
noteworthy that the continuous dark treatment appeared to delay
the development of shoots.
The two cultivars in this experiment exhibited several different
characteristics during the process of shoot regeneration. The shoot
regeneration capacity was higher in `Nishimurawase' than in `Fuyu'.
`Nishimurawase' showed faster shoot regeneration, higher regenera-
tion percentage, and comparable shoot numbers per explant than
`Fuyu'. In addition, `Fuyu' was more sensitive to light and required
higher concentrations of zeatin for shoot regeneration than
`Nishimurawase'. These differences may explain why `Fuyu' was
considered a recalcitrant cultivar by Tao and Sugiura (1992a).
In the present experiment, the frequencies of adventitious shoot
regeneration by `Nishimurawase' and `Fuyu' reached up to 100%.
The optimal conditions for `Nishimurawase' used in this experiment
can also be applied to `Jiro' which yielded 100% shoot regeneration
under such conditions (data not shown). Furthermore, the
regenerated shoots rooted successfully with over 80% efficiency,
and the plantlets acclimatized and developed in the greenhouse
with normal phenotypes. In this present protocol we could produce
as many as 8.3 and 1.8 plantlets per leaf segment explant in 6 mo.
in `Nishimurawase' and `Fuyu', respectively. We hope that this
protocol will help in future genetic transformation, selection of
somaclonal variation and somatic hybridization and that it can be
applied for other recalcitrant cultivars of persimmon.
Acknowledgment
This research was supported by a grant from the Korea Science and
Engineering Foundation (project No. 95-04-02-10-01-3).
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