Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol83.Issue2.Febr2000:
Scientia Horticulturae 83 (2000) 109±126
Somatic embryogenesis and organogenesis from
immature embryo cotyledons of three sour
cherry cultivars (Prunus cerasus L.)
Haoru Tanga,b,*, Zhenglong Rena, Gabi Krczalb
a
College of Forestry and Horticulture, Sichuan Agricultural University,
Ya'an, 625014 Sichuan, China
b
Zentrum GruÈne Gentechnik, Staatliche Lehr- und Forschungsanstalt,
D-67435 Neustadt an der Weinstraûe, Germany
Accepted 5 May 1999
Abstract
Immature cotyledons of open-pollinated fruits from three sour cherry cultivars (Prunus cerasus
L.) were excised and cultured on Murashige and Skoog medium supplemented with various
combinations of auxin and cytokinin to induce somatic embryogenesis. Somatic embryogenesis
occurred principally when using the combinations of 2,4-dichlorophenoxyacetic acid plus kinetin.
Using a-naphthaleneacetic acid or 6-benzylaminopurine reduced the incidence of somatic
embryogenesis. Conversely, formation of cotyledon-like structures, leaves, shoots and roots was
enhanced. The addition of 0.1 mg lÿ1 3-indolebutyric acid to the inductive medium was beneficial
to the induction of somatic embryogenesis. In a few cases, secondary somatic embryos formed and
well-developed somatic embryos germinated. Of the three cultivars tested, `ScharoÈ' was less
responsive than `Gerema' and `Schattenmorelle' when cultured under equivalent conditions. After
trisectioning the cotyledons of cultivar `Gerema', morphogenic gradients were apparent in shoot
and leaf formation but not in root and somatic embryo formation. The embryonic axes attached to
the cotyledons of cultivar `Schattenmorelle' had an inhibitory effect on morphogenesis. # 2000
Elsevier Science B.V. All rights reserved.
Keywords: Cotyledon-like structures; Embryo culture; Embryonic axis; Morphogenic gradient;
Trisections of cotyledon
* Corresponding author. Tel.: +49-6321-671487; fax: +49-6321-671222.
E-mail address: [email protected] (H. Tang).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 7 3 - 4
110
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
1. Introduction
There are more than 30 species of cherries, but only a few of them are
commercially cultivated. Sour cherry (Prunus cerasus L.), one of the economically important species, offers dual market potential for both fresh fruits and
processing, as well as for ornamental, rootstock and timber uses (Brown et al.,
1996). To meet the need for high quality cultivars, sour cherry improvement is in
progress by conventional means (Brown et al., 1996). It is, however, very limited
due to the long generation cycles and highly heterozygous nature. Genetic
transformation could provide a complementary approach to conventional
breeding of sour cherry by the introduction of genes encoding desirable traits.
Somatic embryogenesis and organogenesis from in vitro cultures are a
prerequisite of many plant genetic transformation techniques. In order to obtain
stable and non-chimeric transgenic plants, there are two important issues that
must be considered in the process of regenerating transgenic plants. The recipient
cells must be accessible to the transformation vectors and transgenic plants must
originate from single cells (Dandekar et al., 1992). The protoplast-to-plant system
holds promise since every protoplast seems to be transformable. While the
delivery of DNA into protoplasts presents only a minor problem, establishing
regeneration systems from protoplasts is a major problem. Despite numerous
studies defining protocols for plant regeneration from protoplasts in Rosaceae,
including sour cherry, there are as yet no reports of protoplast-based gene
transfers in this family (Ochatt and Patat-Ochatt, 1995). Furthermore, as soon as a
different approach becomes accessible, the protoplast-to-plant transformation
system will probably be avoided (Siemens and Schieder, 1996).
The somatic embryo-to-plant system provides an alternative approach. Somatic
embryogenesis is a process in which somatic plant cells undergo differentiation to
form embryos. Somatic embryos can be germinated to form plants and can
multiply to produce many more somatic embryos through a process referred to as
secondary or repetitive embryogenesis. Repetitive somatic embryos have an
unicellular origin in the epidermis (Polito et al., 1989), a most important property
of somatic embryos that has been exploited for avoiding chimeric transformation
of many woody plants (Dandekar et al., 1992).
There are, however, few publications on somatic embryogenesis in Prunus
cerasus. So far only Durzan (1985) reported somatic embryogenesis from a
petiole-derived cell suspension in this species. Nevertheless, there have been
several reports on somatic embryogenesis from other Prunus species (Hammerschlag et al., 1985; Druart, 1990; De March et al., 1993; Da Camara Machado
et al., 1995). Somatic embryo-to-plant transformation system has already been
exploited for cultivar improvement in some Prunus species (Da Camara Machado
et al., 1995; Druart et al., 1998; GutieÁrrez-Pesce et al., 1998). The aim of this
investigation was to get more information about somatic embryogenesis as well
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
111
as organogenesis in sour cherry because this information was required for this
species prior to transformation experiments.
2. Materials and methods
2.1. Plant materials
Open-pollinated fruits were collected from sour cherry (Prunus cerasus L.)
cultivars `Gerema', `ScharoÈ' and `Schattenmorelle' virus-free trees in May 1998
from the state virus-free stock plants orchard of the Landesanstalt fuÈr Pflanzenbau
und Pflanzenschutz in Mainz, Germany. These cultivars were chosen for study
because they are three of the most important ones commercially. The fruits
collected had developed to the stage between the gelatinous endosperm beginning
to disappear and it having half disappeared. Maternal tree genotype effects were
not tested and fruits from the different trees of each cultivar were mixed randomly
as a group.
Before dissection, fruits were washed with tap water for 20±30 min and
surface-disinfected by immersion in 70% (v/v) ethanol/water solution for 30 s
and 5% (w/v) Ca(ClO)2 fresh solution with two drops of Tween 20 per 100 ml for
25 min, followed by three rinses in sterile deionized water. The fruits were
opened and the embryos were excised aseptically. The cotyledons served as
explants.
2.2. Culture conditions
The basal medium consisted of Murashige and Skoog (MS) macro- and microelements and vitamins, supplemented with 30 g lÿ1 sucrose, 7 g lÿ1 Sigma agar
and 1 g lÿ1 casein hydrolysate. Medium was adjusted to pH 5.6 with 1 N NaOH
or HCl prior to autoclaving at 1158C for 25 min. Ten different combinations of
plant growth regulators (Table 1) as filter-sterilised solutions were added to
media after autoclaving and media were dispensed as 30 ml aliquots per
9416 mm Petri dish. Cotyledons were placed abaxial side down on the inductive
media in Petri dishes. Dishes were sealed with ``Parafilm'' and put in continuous
darkness at 228C in a growth chamber. After 4 weeks on the inductive media, the
cultures were transferred onto the basal MS medium. Transfers to fresh medium
were made every 4 weeks.
2.3. Experimental treatments
The two cotyledons of one embryo were separated and the embryonic axis was
excluded for investigating the potential for somatic embryogenesis in intact
112
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Table 1
Media used to induce somatic embryogenesis in sour cherry (Prunus cerasus L.) cotyledons
Medium
Plant growth regulatorsa
Auxins (mg lÿ1)
1
2
3
4
5
6
7
8
9
10
Cytokinins (mg lÿ1)
2,4-D
NAA
IBA
KT
BAP
1
2
4
0
2
2
2
2
1
4
0
0
0
2
0
0
0
2
1
0
0
0
0
0.1
0
0.1
0
0
0
0
1
1
1
1
0
1
2
2
1
2
0
0
0
0
1
0
0
0
0
0
A:Cb
1:1
2:1
4:1
2:1
2:1
2:1
1:1
2:1
2:1
2:1
Basal medium was MS supplemented with 30 g lÿ1 sucrose and 1 g lÿ1 casein hydrolysate and
solidified with 7 g lÿ1 agar (pH 5.6).
a
Abbreviations: BAP: 6-benzylaminopurine; 2,4-D: 2,4-dichlorophenoxyacetic acid; IBA: 3indolebutyric acid; KT: kinetin; NAA: a-naphthaleneacetic acid.
b
A:CAuxin : Cytokinin.
cotyledons. The intact cotyledons from all three cultivars were placed on the 10
inductive media, 4±5 pairs of cotyledons in each Petri dish, 16±32 explants for
each treatment (Table 2).
In order to examine the potential of somatic embryogenesis from different parts
of a cotyledon, the cotyledons from cultivar `Gerema' were trisectioned into
distal, median and proximal sections and put onto the same 10 inductive media
mentioned above, 5±6 trisections in each Petri dish, 16±21 explants for each
treatment (Table 4).
In assessing the influence of the embryonic axis, the two cotyledons of one
embryo from cultivar `Schattenmorelle' were dissected into two kinds of
explants, one with the embryonic axis attached to cotyledons and another without
cotyledons attached, and placed onto 2, 3, 6, 7 and 8 inductive media mentioned
above, 4±5 pairs per Petri dish, 24±32 explants each treatment (Table 5).
3. Results
According to Reinert (1973), adventitious structures having bipolar organization with a shoot and root meristem and lacking vascular connection with parent
tissue may be termed somatic embryos. Using these criteria, we considered an
explant to be embryogenic when at least one somatic embryo which had a well-
Table 2
Morphogenic responses of the intact cotyledons of sour cherry (Prunus cerasus L.) on different media (Table 1)
No. of explants and percentage of explants being morphogenic
ScharoÈ
Gerema
Schattenmorelle
Ep
SE
CL
S
L
R
Ep
SE
CL
S
L
R
Ep
SE
CL
S
L
R
1
2
3
4
5
6
7
8
9
10
16
18
20
20
20a
20
20
21
21
18
0
0
5.0
0
±
10.0
5.0
0
0
0
6.3
5.6
5.0
0
±
5.0
10.0
5.6
9.5
0
0
5.6
0
10.0
±
0
10.0
5.6
9.5
4.8
6.3
5.6
0
0
±
0
0
0
0
0
12.5
44.4
25.0
40.0
±
0
25.0
27.8
28.6
14.3
22
22
24
26
24
24
24
26
26
30
0
4.5
0
0
0
0
0
0
0
0
4.5
9.1
11.5
0
4.2
0
11.5
0
13.3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
26
24
26
28
22
24
28
22
24
32
3.8
0
3.8
0
0
0
3.6
0
4.2
6.3
7.7
8.3
7.7
3.6
9.1
8.3
3.6
9.1
12.5
0
3.8
4.2
0
25.0
4.5
8.3
0
0
3.1
0
0
0
3.8
7.1
0
0
3.6
4.5
0
0
0
0
0
42.9
0
4.2
3.6
50.0
25.0
6.3
Av
19.4
2.1
4.6
4.6
1.0
21.6
24.8
0.04
5.0
0
0
0
25.6
2.3
6.6
5.0
1.9
12.9
Corresponding letter(s): Md: media as in Table 1; Ep: no. of explants; SE: somatic embryos; CL: cotyledon-like structures; S: shoots; L: leaves; R:
roots; Av: average.
a
Cultures were lost due to contamination.
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Md
113
114
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Fig. 1. Somatic embryos of sour cherry c.v. `Gerema' (Prunus cerasus L.) directly formed on
cotyledons.
defined hypocotyl region and one or more distinct or fused cotyledons was
observed. Since the cotyledon responses to a given criterion were found to be
many and varied, we focused on embryo formation and on other morphogenic
responses as well and recorded them explant by explant after 3 months in culture.
Since the somatic embryogenesis was the main event of concern in this
investigation, the embryogenic responses were analysed by using statistical
methods. However, no significant differences were found among the experimental
treatments due to a large proportion of irresponsive explants in each treatment.
Therefore we described the experiments by using the percentage of embryogenic
explants and the numbers of somatic embryos per embryogenic explant
throughout the text followed.
3.1. Somatic embryogenesis and characterisation
Somatic embryos formed either individually or in groups, directly on
cotyledons and mainly at the proximal ends (Fig. 1). The somatic embryos
subsequently developed from globular stage to cotyledonary stage and they were
detached easily from the surrounding cells of their parental tissues. Somatic
embryos showed great variability in their morphology. The typical somatic
embryos had both a shoot and a root pole with two distinct cotyledons (Fig. 2),
while the atypical ones had fused or thickened cotyledons, aberrant apex,
branched apices or twin embryos (Fig. 3). When somatic embryos were isolated
from the cotyledons and cultured on basal medium, occasionally secondary
somatic embryos formed at the radicle of primary somatic embryos. In a few
cases, secondary somatic embryos were found on abnormal ones (Fig. 3(C)). On
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
115
Fig. 2. Typical somatic embryos of sour cherry (P. cerasus L.). Somatic embryos from cotyledons
of c.v. `Gerema' (A) and c.v. `Schattenmorelle' (B) showed both a shoot and a root pole with two
distinct cotyledons.
the basal medium in darkness, about 27% of well-developed somatic embryos
germinated, showing an elongated radicle and an emerging root. After
transferring these germinated somatic embryos onto fresh basal medium and
exposing them to light (45 mmol mÿ2 sÿ1 photosynthetic photon flux, 16 h
photoperiod), their cotyledons turned green and epicotyls appeared. One week
later, shoot meristems began to grow. In some cases, the roots grew, but the shoot
meristem failed to develop. A few plantlets were obtained (Fig. 4).
116
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Fig. 3. Atypical somatic embryos from immature cotyledons of sour cherry (P. cerasus L.). Twin
somatic embryos (A), thickened somatic embryos (B) and ``trumpet-like'' somatic embryo, from
which a secondary somatic embryo formed (C).
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
117
Fig. 3. (Continued ).
Fig. 4. A plantlet derived from a somatic embryo of sour cherry c.v. `Schattenmorelle' (P. cerasus
L.) immature cotyledons.
118
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Regardless of the experimental treatments, `Gerema' and `Schattenmorelle',
respectively exhibited 2.7% and 2.3% of explants to be embryogenic while
`ScharoÈ' showed very few (0.04%) (Table 2). Somatic embryogenesis in
`Gerema' was equal on media 3 and 7, with a high auxin/cytokinin ratio (4:1)
and a low auxin/cytokinin ratio (1:1), respectively. It did not occur on the
cotyledons on media with moderate auxin/cytokinin ratios (2:1) unless 0.1 mg lÿ1
IBA was added to the medium containing 2,4-D but not NAA (medium 6). In the
presence of 0.1 mg lÿ1 IBA, the frequency of somatic embryogenesis was
enhanced and, at the same time, somatic embryos had fewer abnormalities and
appeared earlier in comparison with those produced on media 3 and 7 (Tables 2
and 3).
The percentage of explants show to be embryogenic in `Schattenmorelle' was
greatest on medium 10, followed by media 9, 1, 3 and 7. Somatic embryos could
be observed during the second subculture on cotyledons cultured on medium 10,
with a high auxin concentration but moderate auxin/cytokinin ratio (2:1), but they
exhibited much more abnormalities than those from other media (Table 3).
Cotyledons on medium 7, with a high cytokinin concentration but a low auxin/
cytokinin ratio (1:1), formed somatic embryos earlier and rate of typical somatic
embryos was higher compared to those on medium 1 and 3 (Tables 2 and 3).
Somatic embryogenesis in `ScharoÈ' occurred only on the explants on medium
2. Three typical somatic embryos per embryogenic cotyledon were produced
during the second subculture.
The average percentages of explants being embryogenic were similar for all the
three sections of cotyledons from `Gerema', but higher concentrations of plant
growth regulators were needed for distal and median sections to produce somatic
embryos than those for proximal sections (Table 4). Moreover, somatic embryos
from distal sections on a higher auxin concentration medium (medium 10)
showed more morphogenic variations than those on a moderate auxin
concentration medium (medium 6) in comparison with those from median and
proximal sections. Medium 6, however, appeared to be optimum for all the three
sections to undergo somatic embryogenesis.
The embryonic axis attachments to cotyledons from `Schattenmorelle' gave an
inhibitory effect on somatic embryogenesis, as showed in Table 5. No somatic
embryogenesis occurred on the cotyledons with embryonic axes while those
without embryonic axes were embryogenic in 3/5 experimental treatments.
Likewise, the cotyledons without embryonic axes on media 6 and 7 produced
more typical somatic embryos when compared to those on medium 3, although
their percentages of somatic embryogenesis on media 6 and 7 were similar to, or
lower than, that on medium 3.
As for somatic embryogenesis, analyses of the responses of all the explants to
the experimental treatments demonstrated that somatic embryogenesis occurred
principally when using 2 mg lÿ1 2,4-D plus 1 mg lÿ1 KT. Increasing the
Table 3
Comparison of somatic embryo production in immature cotyledons of sour cherry (Prunus cerasus L.) and the influence of media on the appearance
and the developmental types of somatic embryos
Gerema
Ep
b
1
3
6
7
9
10
16
20
20
20
21
18
Average
19.4
a
Schattenmorelle
%
c
0
5.0
10.0
5.0
0
0
2.1
d
Sub.
No. of somatic embryos per
embryogenic cotyledon
%c
Total
Typical
Atypical
±
2nd
1st
2nd
±
±
0
3
5
3
0
0
0
2
5
2
0
0
0
1
0
1
0
0
26
26
24
28
24
32
3.8
3.8
0
3.6
4.2
6.3
±
2.8
2.3
0.5
25.6
2.3
Media as in Table 1.
No. of explants.
c
Percentage of explants showing to be embryogenic.
d
Subculture of embryos appearing.
b
Epb
Sub.d
No. of somatic embryos per
embryogenic cotyledon
Total
Typical
Atypical
2nd
2nd
±
1st
3rd
2nd
3
3
0
4
4
4
2
2
0
3
2
2
1
1
0
1
2
2
±
3.0
1.8
1.2
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Mediuma
119
120
Table 4
Morphogenic responses of different parts of the cotyledon (distal, median or proximal) of sour cherry (Prunus cerasus L.) c.v. `Gerema' to different
media (Table 1)
Ep
Percentage of explants being morphogenic
Distal section
1
2
3
4
5
6
7
8
9
10
16
18
20
20
20
20
20
21
21
18
Av
19.4
Median section
Proximal section
SE
CL
S
L
R
SE
CL
S
L
R
SE
CL
S
L
R
0
0
0
0
0
10.0
0
0
0
11.1
6.3
0
5.0
0
0
0
5.0
12.5
9.5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
50.0
60.1
25.0
40.0
25.0
5.0
30.0
56.3
3.8
11.1
0
0
5.0
0
0
10.0
5.0
0
0
0
6.3
0
15.0
0
0
0
5.0
23.8
14.3
11.1
0
0
0
5.0
0
0
0
4.7
0
0
0
0
0
0
0
0
0
0
0
0
31.3
44.4
30.0
40.0
25.0
25.0
20.0
44.4
47.6
9.5
0
5.6
0
0
0
10.0
5.0
0
0
0
6.3
5.6
5.0
0
0
0
10.0
5.6
0
11.1
0
5.6
0
10.0
0
0
10.0
5.6
9.5
4.8
6.3
5.6
0
0
0
0
0
0
0
0
12.5
44.4
25.0
40.0
12.5
40.0
25.0
27.8
28.6
14.3
2.1
3.9
0
0
30.1
2.1
7.2
1.0
0
32.0
2.1
4.4
4.7
1.0
27.3
Corresponding letter(s): Md: media as in Table 1; Ep: no of explants; SE: somatic embryos; CL: cotyledon-like structures; S: shoots; L: leaves; R:
roots; Av: average.
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Md
121
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Table 5
Morphogenic responses in cotyledons of sour cherry (Prunus cerasus L.) c.v. `Schattenmorelle' as
affected by the embryonic axis attachments to cotyledons on different media (Table 1)
Md
Ep
Percentage of explants being morphogenic
Cotyledons with embryonic axis
Cotyledons without embryonic axis
SE
CL
S
L
R
SE
CL
S
L
R
2
3
6
7
8
31
32
24
32
30
0
0
0
0
0
3.2
3.1
4.2
0
3.3
0
0
0
0
0
0
0
0
0
0
0
3.1
0
0
0
0
6.3
4.2
6.3
0
9.8
6.3
4.2
0
3.3
3.2
3.1
0
3.2
0
0
0
0
0
0
0
3.1
0
6.3
26.7
Av
29.8
0
3.4
0
0
0.7
4.0
5.4
2.0
0
7.4
Corresponding letter(s): Md: media as in Table 1; Ep: no. of explants; SE: somatic embryos; CL:
cotyledon-like structures; S: shoots; L: leaves; R: roots; Av: average.
concentrations of 2,4-D from 2 to 4 mg lÿ1 or KT from 1 to 2 mg lÿ1 induced
somatic embryogenesis, but it gave rise to abnormally developed embryos. The
addition of 0.1 mg lÿ1 IBA to the inductive medium containing 2,4-D resulted in
an increase of the incidence of somatic embryogenesis and a decrease of
abnormal somatic embryos. If the concentration of 2,4-D and NAA were equal,
the incidence of somatic embryogenesis was reduced. No somatic embryogenesis
occurred when using NAA substituted for 2,4-D, even in the presence of
0.1 mg lÿ1 IBA. The replacement of 1 mg lÿ1 KT by 1 mg lÿ1 BAP reduced
somatic embryogenesis.
3.2. Cotyledon-like structures
White cotyledon-like structures developed individually or in clusters, in some
cases together with somatic embryos, on the surfaces of the explants (Fig. 5).
Cotyledon-like structures initiated directly or indirectly on cotyledons and looked
like somatic embryos at the beginning of their development. Unlike somatic
embryos, they did not undergo the sequentially developmental stages and
remained closely attached to their parental tissues. Sometimes they differentiated
cotyledon-like lobes, but no shoot meristems and radicles were found after
sectioning them under the dissection microscope. Additionally, they looked
unlike leaves due to their thickened shapes and without vein venation.
Cotyledon-like structures occurred on the intact cotyledons in all experimental
treatments except medium 10, although there were some differences among the
three cultivars (Table 2). `Schattenmorelle' gave more cotyledon-like structures
than `Gerema' and `ScharoÈ'. Medium 9 produced the highest percentage of
122
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Fig. 5. Cotyledon-like structures of sour cherry c.v. `Schattenmorelle' (P. cerasus L.) immature
cotyledons.
explants with cotyledon-like structures, followed by media 7 and 1 for `Gerema',
7 and 3 for `ScharoÈ', 5 and 8 for `Schattenmorelle'.
Of the three sections of cotyledons from `Gerema', the median sections showed
a higher percentage of explants having cotyledon-like structures than the distal
and proximal sections (Table 4). The combination of 4 mg lÿ1 auxin, either 2,4-D
alone or 2,4-D plus NAA, with 2 mg lÿ1 KT (media 8 and 10) induced more
explants to form cotyledon-like structures than the others. The proximal sections
were less reactive than distal and median sections.
Cotyledons with and without embryonic axes reacted to the same experimental
treatments in producing cotyledon-like structures but with different frequency
(Table 5). On an average, cotyledons with embryonic axes gave 2.2% of explants
forming cotyledon-like structures whereas cotyledons without the embryonic
axes gave 5.0% cotyledon-like structures.
3.3. Other adventitious structures
Formation of other adventitious structures such as leaves, shoots and roots
frequently occurred on the cotyledons. These adventitious structures appeared
separately, either one structure alone or two structures at different sites, on each
explant. Leaves and shoots formed individually, whereas roots developed either
individually or, in most cases, in groups at the surfaces of explants.
Among the three cultivars tested, `ScharoÈ' showed little ability to form
adventitious structures in the given experimental treatments, but `Gerema' and
`Schattenmorelle' did (Table 2). Although there was no big difference in shoot
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
123
formation between the two cultivars, `Gerema' showed higher root formation
while `Schattenmorelle' gave higher leaf formation.
After trisectioning the cotyledons of `Gerema', the ability to form leaves and
shoots reduced while that to form roots increased (Table 4). The distal and
median sections induced more explants to form roots, but gave fewer explants to
form leaves and shoots. The proximal sections induced fewer explants to form
roots, but gave more explants to form shoots.
If the embryonic axes were left in place, the cotyledons from `Schattenmorelle'
showed little ability to form roots and no ability to form leaves and shoots in the
five experimental treatments (Table 5). Without the embryonic axes, the
cotyledons formed shoots and roots but no leaves.
4. Discussion
This paper reports somatic embryogenesis and organogenesis of sour cherry
from immature embryo cotyledons. The system described here presents some
information on morphogenic responses to the different experimental treatments,
which might suggest further investigation on the regeneration potential in sour
cherry.
The mechanisms of differentiation between a state of permissive determination
leading to a particular morphogenic pattern (somatic embryogenesis) and another
state which leads to leaves, shoots, roots or callus are not well understood. In vitro
culture conditions, including certain chemical compounds, plant growth
regulators, play a major role in triggering the morphogenic progress and
regulating the switch of a somatic cell from one pathway to another. Since
somatic embryogenesis and organogenesis are two mutually exclusive processes
of in vitro differentiation (Ammirato, 1985), their induction requires distinctly
different conditions. Our results with immature cotyledons of Prunus cerasus
showed that somatic embryogenesis and organogenesis responded to the same
inductive conditions. Similar results were also obtained in Prunus avium (De
March et al., 1993) and in Juglans nigra (Long et al., 1995). This phenomenon
might reflect the genotypic differences in the ability to activate key elements in
the morphogenic pathway.
Because of genotypic specificity, the requirements of different kinds of plant
growth regulators and their proportion for morphogenesis vary from one species
to another. In callus cultures of Petunia inflata and Petunia hybrida, the addition
of 2,4-D led to embryogenesis, the use of IAA/BAP led to the development of
adventitious shoots with roots, and NAA/BAP to root development (Rao et al.,
1973). In embryonic tissue cultures of apple, the interaction of NAA and BAP
resulted in shoot formation, IAA and BAP in bud and root formation and 2,4-D
and BAP in proembryo and bud formation (Rubos and Pryke, 1984). Our results
124
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
with Prunus cerasus immature cotyledons showed that somatic embryogenesis
occurred principally when using the combinations of 2,4-D and KT. Using NAA
and BAP instead reduced the incidence of somatic embryogenesis. Conversely,
formation of cotyledon-like structures, leaves, shoots and roots was enhanced.
These results parallel the observations achieved by De March et al. (1993) in
Prunus avium immature cotyledon cultures.
Many studies concerned with in vitro morphogenesis underscore the
importance of auxin/cytokinin ratio in the culture medium. In Prunus cerasus,
the auxin/cytokinin ratio is not only the factor to be considered. Somatic embryos
were obtained from the cotyledons cultured on the inductive media with either
high or low auxin/cytokinin ratio, but they showed atypical development.
Cotyledons cultured on media with moderate auxin/cytokinin ratio formed
somatic embryos earlier and produced more typical somatic embryos. Moreover,
the addition of 0.1 mg lÿ1 IBA increased the incidence of somatic embryogenesis
and the production of typical somatic embryos. Da Camara Machado et al. (1995)
even found that the addition of 0.06 mg lÿ1 IBA was necessary to induce the
embryogenic capacity in the callus of Prunus subhirtella autumno rosa. The
interaction between two auxins and the competency to form somatic embryos
remains to be understood.
It was widely found that when a complete organ was cut into pieces, the various
segments differed in their morphogenic capability. The regenerative capacity of
cotyledon fragments was usually greater in proximal sections than in distal
sections (Cheng, 1976; Mante et al., 1989; Schmidt and Kardel-Meisner, 1992).
Our study showed that gradients in shoot and leaf formation were apparent while
they were not apparent in root and somatic embryo formation. These might be
related to the development of endogenous gradients of hormones or to the
interaction and balance between the endogenous hormones and the exogenous
plant growth regulators in forming morphogenic structures.
Kouider et al. (1984) found that if the embryonic axis was left in place, no
adventitious structures, even no callus, formed on the cotyledons of apple,
whereas intact cotyledons without embryonic axes and different excisions of
cotyledons produced morphogenic structures. Mante et al. (1989) reported that
formation of shoots in cotyledons from different Prunus species was entirely
dependent upon the removal of the embryonic axis and the presence of the
proximal region of cotyledon, but Schmidt and Kardel-Meisner (1992) could
regenerate shoots from different sections of cotyledons from five cultivars of
Prunus avium. Both the strongly inhibitory effects on morphogenesis of the
embryonic axes attached to the cotyledons and the capability of cotyledon pieces
without proximal regions differentiating into different morphogenic structures
were demonstrated in our study.
While somatic embryogenesis has many potential advantages for genetic
improvement, some limitations remain to be overcome before somatic
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
125
embryogenic systems can be applied for operational production in sour cherry.
Selection experiments using zygotic embryos or their components are of little
commercial value for clonally propagated species, but they serve to highlight the
pressing need for extending somatic embryogenesis to a wide range of explants.
Further studies on selecting the most competent organs or tissues for somatic
embryogenesis, testing the optimum conditions for inducing somatic embryos and
increasing somatic embryo production, may provide precise information for sour
cherry somatic embryogenesis and, at the same time, provide repetitive somatic
embryogenic lines for genetic manipulation experiments.
Acknowledgements
Thanks are due to the German Ministry for Science and Technology for
financial support and the Landesanstalt fuÈr Pflanzenbau und Pflanzenschutz in
Mainz, Germany, for supplying plant materials. We would like to thank Mr.
MoÈrbel for help in collecting plant materials and Dr. Martin for help in paper
preparation. Also Dr. Reustle for good suggests on experiments and Mr. Wahl for
photography.
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Somatic embryogenesis and organogenesis from
immature embryo cotyledons of three sour
cherry cultivars (Prunus cerasus L.)
Haoru Tanga,b,*, Zhenglong Rena, Gabi Krczalb
a
College of Forestry and Horticulture, Sichuan Agricultural University,
Ya'an, 625014 Sichuan, China
b
Zentrum GruÈne Gentechnik, Staatliche Lehr- und Forschungsanstalt,
D-67435 Neustadt an der Weinstraûe, Germany
Accepted 5 May 1999
Abstract
Immature cotyledons of open-pollinated fruits from three sour cherry cultivars (Prunus cerasus
L.) were excised and cultured on Murashige and Skoog medium supplemented with various
combinations of auxin and cytokinin to induce somatic embryogenesis. Somatic embryogenesis
occurred principally when using the combinations of 2,4-dichlorophenoxyacetic acid plus kinetin.
Using a-naphthaleneacetic acid or 6-benzylaminopurine reduced the incidence of somatic
embryogenesis. Conversely, formation of cotyledon-like structures, leaves, shoots and roots was
enhanced. The addition of 0.1 mg lÿ1 3-indolebutyric acid to the inductive medium was beneficial
to the induction of somatic embryogenesis. In a few cases, secondary somatic embryos formed and
well-developed somatic embryos germinated. Of the three cultivars tested, `ScharoÈ' was less
responsive than `Gerema' and `Schattenmorelle' when cultured under equivalent conditions. After
trisectioning the cotyledons of cultivar `Gerema', morphogenic gradients were apparent in shoot
and leaf formation but not in root and somatic embryo formation. The embryonic axes attached to
the cotyledons of cultivar `Schattenmorelle' had an inhibitory effect on morphogenesis. # 2000
Elsevier Science B.V. All rights reserved.
Keywords: Cotyledon-like structures; Embryo culture; Embryonic axis; Morphogenic gradient;
Trisections of cotyledon
* Corresponding author. Tel.: +49-6321-671487; fax: +49-6321-671222.
E-mail address: [email protected] (H. Tang).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 7 3 - 4
110
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
1. Introduction
There are more than 30 species of cherries, but only a few of them are
commercially cultivated. Sour cherry (Prunus cerasus L.), one of the economically important species, offers dual market potential for both fresh fruits and
processing, as well as for ornamental, rootstock and timber uses (Brown et al.,
1996). To meet the need for high quality cultivars, sour cherry improvement is in
progress by conventional means (Brown et al., 1996). It is, however, very limited
due to the long generation cycles and highly heterozygous nature. Genetic
transformation could provide a complementary approach to conventional
breeding of sour cherry by the introduction of genes encoding desirable traits.
Somatic embryogenesis and organogenesis from in vitro cultures are a
prerequisite of many plant genetic transformation techniques. In order to obtain
stable and non-chimeric transgenic plants, there are two important issues that
must be considered in the process of regenerating transgenic plants. The recipient
cells must be accessible to the transformation vectors and transgenic plants must
originate from single cells (Dandekar et al., 1992). The protoplast-to-plant system
holds promise since every protoplast seems to be transformable. While the
delivery of DNA into protoplasts presents only a minor problem, establishing
regeneration systems from protoplasts is a major problem. Despite numerous
studies defining protocols for plant regeneration from protoplasts in Rosaceae,
including sour cherry, there are as yet no reports of protoplast-based gene
transfers in this family (Ochatt and Patat-Ochatt, 1995). Furthermore, as soon as a
different approach becomes accessible, the protoplast-to-plant transformation
system will probably be avoided (Siemens and Schieder, 1996).
The somatic embryo-to-plant system provides an alternative approach. Somatic
embryogenesis is a process in which somatic plant cells undergo differentiation to
form embryos. Somatic embryos can be germinated to form plants and can
multiply to produce many more somatic embryos through a process referred to as
secondary or repetitive embryogenesis. Repetitive somatic embryos have an
unicellular origin in the epidermis (Polito et al., 1989), a most important property
of somatic embryos that has been exploited for avoiding chimeric transformation
of many woody plants (Dandekar et al., 1992).
There are, however, few publications on somatic embryogenesis in Prunus
cerasus. So far only Durzan (1985) reported somatic embryogenesis from a
petiole-derived cell suspension in this species. Nevertheless, there have been
several reports on somatic embryogenesis from other Prunus species (Hammerschlag et al., 1985; Druart, 1990; De March et al., 1993; Da Camara Machado
et al., 1995). Somatic embryo-to-plant transformation system has already been
exploited for cultivar improvement in some Prunus species (Da Camara Machado
et al., 1995; Druart et al., 1998; GutieÁrrez-Pesce et al., 1998). The aim of this
investigation was to get more information about somatic embryogenesis as well
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
111
as organogenesis in sour cherry because this information was required for this
species prior to transformation experiments.
2. Materials and methods
2.1. Plant materials
Open-pollinated fruits were collected from sour cherry (Prunus cerasus L.)
cultivars `Gerema', `ScharoÈ' and `Schattenmorelle' virus-free trees in May 1998
from the state virus-free stock plants orchard of the Landesanstalt fuÈr Pflanzenbau
und Pflanzenschutz in Mainz, Germany. These cultivars were chosen for study
because they are three of the most important ones commercially. The fruits
collected had developed to the stage between the gelatinous endosperm beginning
to disappear and it having half disappeared. Maternal tree genotype effects were
not tested and fruits from the different trees of each cultivar were mixed randomly
as a group.
Before dissection, fruits were washed with tap water for 20±30 min and
surface-disinfected by immersion in 70% (v/v) ethanol/water solution for 30 s
and 5% (w/v) Ca(ClO)2 fresh solution with two drops of Tween 20 per 100 ml for
25 min, followed by three rinses in sterile deionized water. The fruits were
opened and the embryos were excised aseptically. The cotyledons served as
explants.
2.2. Culture conditions
The basal medium consisted of Murashige and Skoog (MS) macro- and microelements and vitamins, supplemented with 30 g lÿ1 sucrose, 7 g lÿ1 Sigma agar
and 1 g lÿ1 casein hydrolysate. Medium was adjusted to pH 5.6 with 1 N NaOH
or HCl prior to autoclaving at 1158C for 25 min. Ten different combinations of
plant growth regulators (Table 1) as filter-sterilised solutions were added to
media after autoclaving and media were dispensed as 30 ml aliquots per
9416 mm Petri dish. Cotyledons were placed abaxial side down on the inductive
media in Petri dishes. Dishes were sealed with ``Parafilm'' and put in continuous
darkness at 228C in a growth chamber. After 4 weeks on the inductive media, the
cultures were transferred onto the basal MS medium. Transfers to fresh medium
were made every 4 weeks.
2.3. Experimental treatments
The two cotyledons of one embryo were separated and the embryonic axis was
excluded for investigating the potential for somatic embryogenesis in intact
112
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Table 1
Media used to induce somatic embryogenesis in sour cherry (Prunus cerasus L.) cotyledons
Medium
Plant growth regulatorsa
Auxins (mg lÿ1)
1
2
3
4
5
6
7
8
9
10
Cytokinins (mg lÿ1)
2,4-D
NAA
IBA
KT
BAP
1
2
4
0
2
2
2
2
1
4
0
0
0
2
0
0
0
2
1
0
0
0
0
0.1
0
0.1
0
0
0
0
1
1
1
1
0
1
2
2
1
2
0
0
0
0
1
0
0
0
0
0
A:Cb
1:1
2:1
4:1
2:1
2:1
2:1
1:1
2:1
2:1
2:1
Basal medium was MS supplemented with 30 g lÿ1 sucrose and 1 g lÿ1 casein hydrolysate and
solidified with 7 g lÿ1 agar (pH 5.6).
a
Abbreviations: BAP: 6-benzylaminopurine; 2,4-D: 2,4-dichlorophenoxyacetic acid; IBA: 3indolebutyric acid; KT: kinetin; NAA: a-naphthaleneacetic acid.
b
A:CAuxin : Cytokinin.
cotyledons. The intact cotyledons from all three cultivars were placed on the 10
inductive media, 4±5 pairs of cotyledons in each Petri dish, 16±32 explants for
each treatment (Table 2).
In order to examine the potential of somatic embryogenesis from different parts
of a cotyledon, the cotyledons from cultivar `Gerema' were trisectioned into
distal, median and proximal sections and put onto the same 10 inductive media
mentioned above, 5±6 trisections in each Petri dish, 16±21 explants for each
treatment (Table 4).
In assessing the influence of the embryonic axis, the two cotyledons of one
embryo from cultivar `Schattenmorelle' were dissected into two kinds of
explants, one with the embryonic axis attached to cotyledons and another without
cotyledons attached, and placed onto 2, 3, 6, 7 and 8 inductive media mentioned
above, 4±5 pairs per Petri dish, 24±32 explants each treatment (Table 5).
3. Results
According to Reinert (1973), adventitious structures having bipolar organization with a shoot and root meristem and lacking vascular connection with parent
tissue may be termed somatic embryos. Using these criteria, we considered an
explant to be embryogenic when at least one somatic embryo which had a well-
Table 2
Morphogenic responses of the intact cotyledons of sour cherry (Prunus cerasus L.) on different media (Table 1)
No. of explants and percentage of explants being morphogenic
ScharoÈ
Gerema
Schattenmorelle
Ep
SE
CL
S
L
R
Ep
SE
CL
S
L
R
Ep
SE
CL
S
L
R
1
2
3
4
5
6
7
8
9
10
16
18
20
20
20a
20
20
21
21
18
0
0
5.0
0
±
10.0
5.0
0
0
0
6.3
5.6
5.0
0
±
5.0
10.0
5.6
9.5
0
0
5.6
0
10.0
±
0
10.0
5.6
9.5
4.8
6.3
5.6
0
0
±
0
0
0
0
0
12.5
44.4
25.0
40.0
±
0
25.0
27.8
28.6
14.3
22
22
24
26
24
24
24
26
26
30
0
4.5
0
0
0
0
0
0
0
0
4.5
9.1
11.5
0
4.2
0
11.5
0
13.3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
26
24
26
28
22
24
28
22
24
32
3.8
0
3.8
0
0
0
3.6
0
4.2
6.3
7.7
8.3
7.7
3.6
9.1
8.3
3.6
9.1
12.5
0
3.8
4.2
0
25.0
4.5
8.3
0
0
3.1
0
0
0
3.8
7.1
0
0
3.6
4.5
0
0
0
0
0
42.9
0
4.2
3.6
50.0
25.0
6.3
Av
19.4
2.1
4.6
4.6
1.0
21.6
24.8
0.04
5.0
0
0
0
25.6
2.3
6.6
5.0
1.9
12.9
Corresponding letter(s): Md: media as in Table 1; Ep: no. of explants; SE: somatic embryos; CL: cotyledon-like structures; S: shoots; L: leaves; R:
roots; Av: average.
a
Cultures were lost due to contamination.
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Md
113
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H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Fig. 1. Somatic embryos of sour cherry c.v. `Gerema' (Prunus cerasus L.) directly formed on
cotyledons.
defined hypocotyl region and one or more distinct or fused cotyledons was
observed. Since the cotyledon responses to a given criterion were found to be
many and varied, we focused on embryo formation and on other morphogenic
responses as well and recorded them explant by explant after 3 months in culture.
Since the somatic embryogenesis was the main event of concern in this
investigation, the embryogenic responses were analysed by using statistical
methods. However, no significant differences were found among the experimental
treatments due to a large proportion of irresponsive explants in each treatment.
Therefore we described the experiments by using the percentage of embryogenic
explants and the numbers of somatic embryos per embryogenic explant
throughout the text followed.
3.1. Somatic embryogenesis and characterisation
Somatic embryos formed either individually or in groups, directly on
cotyledons and mainly at the proximal ends (Fig. 1). The somatic embryos
subsequently developed from globular stage to cotyledonary stage and they were
detached easily from the surrounding cells of their parental tissues. Somatic
embryos showed great variability in their morphology. The typical somatic
embryos had both a shoot and a root pole with two distinct cotyledons (Fig. 2),
while the atypical ones had fused or thickened cotyledons, aberrant apex,
branched apices or twin embryos (Fig. 3). When somatic embryos were isolated
from the cotyledons and cultured on basal medium, occasionally secondary
somatic embryos formed at the radicle of primary somatic embryos. In a few
cases, secondary somatic embryos were found on abnormal ones (Fig. 3(C)). On
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
115
Fig. 2. Typical somatic embryos of sour cherry (P. cerasus L.). Somatic embryos from cotyledons
of c.v. `Gerema' (A) and c.v. `Schattenmorelle' (B) showed both a shoot and a root pole with two
distinct cotyledons.
the basal medium in darkness, about 27% of well-developed somatic embryos
germinated, showing an elongated radicle and an emerging root. After
transferring these germinated somatic embryos onto fresh basal medium and
exposing them to light (45 mmol mÿ2 sÿ1 photosynthetic photon flux, 16 h
photoperiod), their cotyledons turned green and epicotyls appeared. One week
later, shoot meristems began to grow. In some cases, the roots grew, but the shoot
meristem failed to develop. A few plantlets were obtained (Fig. 4).
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H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Fig. 3. Atypical somatic embryos from immature cotyledons of sour cherry (P. cerasus L.). Twin
somatic embryos (A), thickened somatic embryos (B) and ``trumpet-like'' somatic embryo, from
which a secondary somatic embryo formed (C).
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
117
Fig. 3. (Continued ).
Fig. 4. A plantlet derived from a somatic embryo of sour cherry c.v. `Schattenmorelle' (P. cerasus
L.) immature cotyledons.
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H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Regardless of the experimental treatments, `Gerema' and `Schattenmorelle',
respectively exhibited 2.7% and 2.3% of explants to be embryogenic while
`ScharoÈ' showed very few (0.04%) (Table 2). Somatic embryogenesis in
`Gerema' was equal on media 3 and 7, with a high auxin/cytokinin ratio (4:1)
and a low auxin/cytokinin ratio (1:1), respectively. It did not occur on the
cotyledons on media with moderate auxin/cytokinin ratios (2:1) unless 0.1 mg lÿ1
IBA was added to the medium containing 2,4-D but not NAA (medium 6). In the
presence of 0.1 mg lÿ1 IBA, the frequency of somatic embryogenesis was
enhanced and, at the same time, somatic embryos had fewer abnormalities and
appeared earlier in comparison with those produced on media 3 and 7 (Tables 2
and 3).
The percentage of explants show to be embryogenic in `Schattenmorelle' was
greatest on medium 10, followed by media 9, 1, 3 and 7. Somatic embryos could
be observed during the second subculture on cotyledons cultured on medium 10,
with a high auxin concentration but moderate auxin/cytokinin ratio (2:1), but they
exhibited much more abnormalities than those from other media (Table 3).
Cotyledons on medium 7, with a high cytokinin concentration but a low auxin/
cytokinin ratio (1:1), formed somatic embryos earlier and rate of typical somatic
embryos was higher compared to those on medium 1 and 3 (Tables 2 and 3).
Somatic embryogenesis in `ScharoÈ' occurred only on the explants on medium
2. Three typical somatic embryos per embryogenic cotyledon were produced
during the second subculture.
The average percentages of explants being embryogenic were similar for all the
three sections of cotyledons from `Gerema', but higher concentrations of plant
growth regulators were needed for distal and median sections to produce somatic
embryos than those for proximal sections (Table 4). Moreover, somatic embryos
from distal sections on a higher auxin concentration medium (medium 10)
showed more morphogenic variations than those on a moderate auxin
concentration medium (medium 6) in comparison with those from median and
proximal sections. Medium 6, however, appeared to be optimum for all the three
sections to undergo somatic embryogenesis.
The embryonic axis attachments to cotyledons from `Schattenmorelle' gave an
inhibitory effect on somatic embryogenesis, as showed in Table 5. No somatic
embryogenesis occurred on the cotyledons with embryonic axes while those
without embryonic axes were embryogenic in 3/5 experimental treatments.
Likewise, the cotyledons without embryonic axes on media 6 and 7 produced
more typical somatic embryos when compared to those on medium 3, although
their percentages of somatic embryogenesis on media 6 and 7 were similar to, or
lower than, that on medium 3.
As for somatic embryogenesis, analyses of the responses of all the explants to
the experimental treatments demonstrated that somatic embryogenesis occurred
principally when using 2 mg lÿ1 2,4-D plus 1 mg lÿ1 KT. Increasing the
Table 3
Comparison of somatic embryo production in immature cotyledons of sour cherry (Prunus cerasus L.) and the influence of media on the appearance
and the developmental types of somatic embryos
Gerema
Ep
b
1
3
6
7
9
10
16
20
20
20
21
18
Average
19.4
a
Schattenmorelle
%
c
0
5.0
10.0
5.0
0
0
2.1
d
Sub.
No. of somatic embryos per
embryogenic cotyledon
%c
Total
Typical
Atypical
±
2nd
1st
2nd
±
±
0
3
5
3
0
0
0
2
5
2
0
0
0
1
0
1
0
0
26
26
24
28
24
32
3.8
3.8
0
3.6
4.2
6.3
±
2.8
2.3
0.5
25.6
2.3
Media as in Table 1.
No. of explants.
c
Percentage of explants showing to be embryogenic.
d
Subculture of embryos appearing.
b
Epb
Sub.d
No. of somatic embryos per
embryogenic cotyledon
Total
Typical
Atypical
2nd
2nd
±
1st
3rd
2nd
3
3
0
4
4
4
2
2
0
3
2
2
1
1
0
1
2
2
±
3.0
1.8
1.2
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Mediuma
119
120
Table 4
Morphogenic responses of different parts of the cotyledon (distal, median or proximal) of sour cherry (Prunus cerasus L.) c.v. `Gerema' to different
media (Table 1)
Ep
Percentage of explants being morphogenic
Distal section
1
2
3
4
5
6
7
8
9
10
16
18
20
20
20
20
20
21
21
18
Av
19.4
Median section
Proximal section
SE
CL
S
L
R
SE
CL
S
L
R
SE
CL
S
L
R
0
0
0
0
0
10.0
0
0
0
11.1
6.3
0
5.0
0
0
0
5.0
12.5
9.5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
50.0
60.1
25.0
40.0
25.0
5.0
30.0
56.3
3.8
11.1
0
0
5.0
0
0
10.0
5.0
0
0
0
6.3
0
15.0
0
0
0
5.0
23.8
14.3
11.1
0
0
0
5.0
0
0
0
4.7
0
0
0
0
0
0
0
0
0
0
0
0
31.3
44.4
30.0
40.0
25.0
25.0
20.0
44.4
47.6
9.5
0
5.6
0
0
0
10.0
5.0
0
0
0
6.3
5.6
5.0
0
0
0
10.0
5.6
0
11.1
0
5.6
0
10.0
0
0
10.0
5.6
9.5
4.8
6.3
5.6
0
0
0
0
0
0
0
0
12.5
44.4
25.0
40.0
12.5
40.0
25.0
27.8
28.6
14.3
2.1
3.9
0
0
30.1
2.1
7.2
1.0
0
32.0
2.1
4.4
4.7
1.0
27.3
Corresponding letter(s): Md: media as in Table 1; Ep: no of explants; SE: somatic embryos; CL: cotyledon-like structures; S: shoots; L: leaves; R:
roots; Av: average.
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Md
121
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Table 5
Morphogenic responses in cotyledons of sour cherry (Prunus cerasus L.) c.v. `Schattenmorelle' as
affected by the embryonic axis attachments to cotyledons on different media (Table 1)
Md
Ep
Percentage of explants being morphogenic
Cotyledons with embryonic axis
Cotyledons without embryonic axis
SE
CL
S
L
R
SE
CL
S
L
R
2
3
6
7
8
31
32
24
32
30
0
0
0
0
0
3.2
3.1
4.2
0
3.3
0
0
0
0
0
0
0
0
0
0
0
3.1
0
0
0
0
6.3
4.2
6.3
0
9.8
6.3
4.2
0
3.3
3.2
3.1
0
3.2
0
0
0
0
0
0
0
3.1
0
6.3
26.7
Av
29.8
0
3.4
0
0
0.7
4.0
5.4
2.0
0
7.4
Corresponding letter(s): Md: media as in Table 1; Ep: no. of explants; SE: somatic embryos; CL:
cotyledon-like structures; S: shoots; L: leaves; R: roots; Av: average.
concentrations of 2,4-D from 2 to 4 mg lÿ1 or KT from 1 to 2 mg lÿ1 induced
somatic embryogenesis, but it gave rise to abnormally developed embryos. The
addition of 0.1 mg lÿ1 IBA to the inductive medium containing 2,4-D resulted in
an increase of the incidence of somatic embryogenesis and a decrease of
abnormal somatic embryos. If the concentration of 2,4-D and NAA were equal,
the incidence of somatic embryogenesis was reduced. No somatic embryogenesis
occurred when using NAA substituted for 2,4-D, even in the presence of
0.1 mg lÿ1 IBA. The replacement of 1 mg lÿ1 KT by 1 mg lÿ1 BAP reduced
somatic embryogenesis.
3.2. Cotyledon-like structures
White cotyledon-like structures developed individually or in clusters, in some
cases together with somatic embryos, on the surfaces of the explants (Fig. 5).
Cotyledon-like structures initiated directly or indirectly on cotyledons and looked
like somatic embryos at the beginning of their development. Unlike somatic
embryos, they did not undergo the sequentially developmental stages and
remained closely attached to their parental tissues. Sometimes they differentiated
cotyledon-like lobes, but no shoot meristems and radicles were found after
sectioning them under the dissection microscope. Additionally, they looked
unlike leaves due to their thickened shapes and without vein venation.
Cotyledon-like structures occurred on the intact cotyledons in all experimental
treatments except medium 10, although there were some differences among the
three cultivars (Table 2). `Schattenmorelle' gave more cotyledon-like structures
than `Gerema' and `ScharoÈ'. Medium 9 produced the highest percentage of
122
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
Fig. 5. Cotyledon-like structures of sour cherry c.v. `Schattenmorelle' (P. cerasus L.) immature
cotyledons.
explants with cotyledon-like structures, followed by media 7 and 1 for `Gerema',
7 and 3 for `ScharoÈ', 5 and 8 for `Schattenmorelle'.
Of the three sections of cotyledons from `Gerema', the median sections showed
a higher percentage of explants having cotyledon-like structures than the distal
and proximal sections (Table 4). The combination of 4 mg lÿ1 auxin, either 2,4-D
alone or 2,4-D plus NAA, with 2 mg lÿ1 KT (media 8 and 10) induced more
explants to form cotyledon-like structures than the others. The proximal sections
were less reactive than distal and median sections.
Cotyledons with and without embryonic axes reacted to the same experimental
treatments in producing cotyledon-like structures but with different frequency
(Table 5). On an average, cotyledons with embryonic axes gave 2.2% of explants
forming cotyledon-like structures whereas cotyledons without the embryonic
axes gave 5.0% cotyledon-like structures.
3.3. Other adventitious structures
Formation of other adventitious structures such as leaves, shoots and roots
frequently occurred on the cotyledons. These adventitious structures appeared
separately, either one structure alone or two structures at different sites, on each
explant. Leaves and shoots formed individually, whereas roots developed either
individually or, in most cases, in groups at the surfaces of explants.
Among the three cultivars tested, `ScharoÈ' showed little ability to form
adventitious structures in the given experimental treatments, but `Gerema' and
`Schattenmorelle' did (Table 2). Although there was no big difference in shoot
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
123
formation between the two cultivars, `Gerema' showed higher root formation
while `Schattenmorelle' gave higher leaf formation.
After trisectioning the cotyledons of `Gerema', the ability to form leaves and
shoots reduced while that to form roots increased (Table 4). The distal and
median sections induced more explants to form roots, but gave fewer explants to
form leaves and shoots. The proximal sections induced fewer explants to form
roots, but gave more explants to form shoots.
If the embryonic axes were left in place, the cotyledons from `Schattenmorelle'
showed little ability to form roots and no ability to form leaves and shoots in the
five experimental treatments (Table 5). Without the embryonic axes, the
cotyledons formed shoots and roots but no leaves.
4. Discussion
This paper reports somatic embryogenesis and organogenesis of sour cherry
from immature embryo cotyledons. The system described here presents some
information on morphogenic responses to the different experimental treatments,
which might suggest further investigation on the regeneration potential in sour
cherry.
The mechanisms of differentiation between a state of permissive determination
leading to a particular morphogenic pattern (somatic embryogenesis) and another
state which leads to leaves, shoots, roots or callus are not well understood. In vitro
culture conditions, including certain chemical compounds, plant growth
regulators, play a major role in triggering the morphogenic progress and
regulating the switch of a somatic cell from one pathway to another. Since
somatic embryogenesis and organogenesis are two mutually exclusive processes
of in vitro differentiation (Ammirato, 1985), their induction requires distinctly
different conditions. Our results with immature cotyledons of Prunus cerasus
showed that somatic embryogenesis and organogenesis responded to the same
inductive conditions. Similar results were also obtained in Prunus avium (De
March et al., 1993) and in Juglans nigra (Long et al., 1995). This phenomenon
might reflect the genotypic differences in the ability to activate key elements in
the morphogenic pathway.
Because of genotypic specificity, the requirements of different kinds of plant
growth regulators and their proportion for morphogenesis vary from one species
to another. In callus cultures of Petunia inflata and Petunia hybrida, the addition
of 2,4-D led to embryogenesis, the use of IAA/BAP led to the development of
adventitious shoots with roots, and NAA/BAP to root development (Rao et al.,
1973). In embryonic tissue cultures of apple, the interaction of NAA and BAP
resulted in shoot formation, IAA and BAP in bud and root formation and 2,4-D
and BAP in proembryo and bud formation (Rubos and Pryke, 1984). Our results
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H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
with Prunus cerasus immature cotyledons showed that somatic embryogenesis
occurred principally when using the combinations of 2,4-D and KT. Using NAA
and BAP instead reduced the incidence of somatic embryogenesis. Conversely,
formation of cotyledon-like structures, leaves, shoots and roots was enhanced.
These results parallel the observations achieved by De March et al. (1993) in
Prunus avium immature cotyledon cultures.
Many studies concerned with in vitro morphogenesis underscore the
importance of auxin/cytokinin ratio in the culture medium. In Prunus cerasus,
the auxin/cytokinin ratio is not only the factor to be considered. Somatic embryos
were obtained from the cotyledons cultured on the inductive media with either
high or low auxin/cytokinin ratio, but they showed atypical development.
Cotyledons cultured on media with moderate auxin/cytokinin ratio formed
somatic embryos earlier and produced more typical somatic embryos. Moreover,
the addition of 0.1 mg lÿ1 IBA increased the incidence of somatic embryogenesis
and the production of typical somatic embryos. Da Camara Machado et al. (1995)
even found that the addition of 0.06 mg lÿ1 IBA was necessary to induce the
embryogenic capacity in the callus of Prunus subhirtella autumno rosa. The
interaction between two auxins and the competency to form somatic embryos
remains to be understood.
It was widely found that when a complete organ was cut into pieces, the various
segments differed in their morphogenic capability. The regenerative capacity of
cotyledon fragments was usually greater in proximal sections than in distal
sections (Cheng, 1976; Mante et al., 1989; Schmidt and Kardel-Meisner, 1992).
Our study showed that gradients in shoot and leaf formation were apparent while
they were not apparent in root and somatic embryo formation. These might be
related to the development of endogenous gradients of hormones or to the
interaction and balance between the endogenous hormones and the exogenous
plant growth regulators in forming morphogenic structures.
Kouider et al. (1984) found that if the embryonic axis was left in place, no
adventitious structures, even no callus, formed on the cotyledons of apple,
whereas intact cotyledons without embryonic axes and different excisions of
cotyledons produced morphogenic structures. Mante et al. (1989) reported that
formation of shoots in cotyledons from different Prunus species was entirely
dependent upon the removal of the embryonic axis and the presence of the
proximal region of cotyledon, but Schmidt and Kardel-Meisner (1992) could
regenerate shoots from different sections of cotyledons from five cultivars of
Prunus avium. Both the strongly inhibitory effects on morphogenesis of the
embryonic axes attached to the cotyledons and the capability of cotyledon pieces
without proximal regions differentiating into different morphogenic structures
were demonstrated in our study.
While somatic embryogenesis has many potential advantages for genetic
improvement, some limitations remain to be overcome before somatic
H. Tang et al. / Scientia Horticulturae 83 (2000) 109±126
125
embryogenic systems can be applied for operational production in sour cherry.
Selection experiments using zygotic embryos or their components are of little
commercial value for clonally propagated species, but they serve to highlight the
pressing need for extending somatic embryogenesis to a wide range of explants.
Further studies on selecting the most competent organs or tissues for somatic
embryogenesis, testing the optimum conditions for inducing somatic embryos and
increasing somatic embryo production, may provide precise information for sour
cherry somatic embryogenesis and, at the same time, provide repetitive somatic
embryogenic lines for genetic manipulation experiments.
Acknowledgements
Thanks are due to the German Ministry for Science and Technology for
financial support and the Landesanstalt fuÈr Pflanzenbau und Pflanzenschutz in
Mainz, Germany, for supplying plant materials. We would like to thank Mr.
MoÈrbel for help in collecting plant materials and Dr. Martin for help in paper
preparation. Also Dr. Reustle for good suggests on experiments and Mr. Wahl for
photography.
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