Somatic embryogenesis and organogenesis from
immature embryo cotyledons of three sour
cherry cultivars (
Prunus cerasus
L.)
Haoru Tang
a,b,*, Zhenglong Ren
a, Gabi Krczal
ba
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
Scientia Horticulturae 83 (2000) 109±126
* Corresponding author. Tel.: +49-6321-671487; fax: +49-6321-671222. E-mail address:htang.slfa-nw@agrarinfo.rpl.de (H. Tang).
0304-4238/00/$ ± see front matter#2000 Elsevier Science B.V. All rights reserved.
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 economic-ally 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).
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)2fresh 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 micro-elements 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
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 organiza-tion with a shoot and root meristem and lacking vascular connecorganiza-tion 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 1
Media used to induce somatic embryogenesis in sour cherry (Prunus cerasusL.) cotyledons
Medium Plant growth regulatorsa
Auxins (mg lÿ1) Cytokinins (mg lÿ1) A:Cb
Basal medium was MS supplemented with 30 g lÿ1
sucrose and 1 g lÿ1
casein hydrolysate and
solidified with 7 g lÿ1agar (pH 5.6).
a
Abbreviations: BAP: 6-benzylaminopurine; 2,4-D: 2,4-dichlorophenoxyacetic acid; IBA:
3-indolebutyric acid; KT: kinetin; NAA:a-naphthaleneacetic acid.
b
Table 2
Morphogenic responses of the intact cotyledons of sour cherry (Prunus cerasusL.) on different media (Table 1)
Md No. of explants and percentage of explants being morphogenic
Gerema ScharoÈ Schattenmorelle
Ep SE CL S L R Ep SE CL S L R Ep SE CL S L R
1 16 0 6.3 0 6.3 12.5 22 0 4.5 0 0 0 26 3.8 7.7 3.8 0 0
2 18 0 5.6 5.6 5.6 44.4 22 4.5 9.1 0 0 0 24 0 8.3 4.2 0 0
3 20 5.0 5.0 0 0 25.0 24 0 11.5 0 0 0 26 3.8 7.7 0 3.8 0
4 20 0 0 10.0 0 40.0 26 0 0 0 0 0 28 0 3.6 25.0 7.1 42.9
5 20a ± ± ± ± ± 24
0 4.2 0 0 0 22 0 9.1 4.5 0 0
6 20 10.0 5.0 0 0 0 24 0 0 0 0 0 24 0 8.3 8.3 0 4.2
7 20 5.0 10.0 10.0 0 25.0 24 0 11.5 0 0 0 28 3.6 3.6 0 3.6 3.6
8 21 0 5.6 5.6 0 27.8 26 0 0 0 0 0 22 0 9.1 0 4.5 50.0
9 21 0 9.5 9.5 0 28.6 26 0 13.3 0 0 0 24 4.2 12.5 3.1 0 25.0
10 18 0 0 4.8 0 14.3 30 0 0 0 0 0 32 6.3 0 0 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.
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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
Fig. 1. Somatic embryos of sour cherry c.v. `Gerema' (Prunus cerasusL.) directly formed on
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 (45mmol mÿ2sÿ1 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).
Fig. 2. Typical somatic embryos of sour cherry (P. cerasusL.). 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.
Fig. 3. (Continued).
Fig. 4. A plantlet derived from a somatic embryo of sour cherry c.v. `Schattenmorelle' (P. cerasus
L.) immature cotyledons.
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
Table 3
Comparison of somatic embryo production in immature cotyledons of sour cherry (Prunus cerasusL.) and the influence of media on the appearance
and the developmental types of somatic embryos
Mediuma Gerema Schattenmorelle
Epb %c Sub.d No. of somatic embryos per
embryogenic cotyledon
Epb %c Sub.d No. of somatic embryos per
embryogenic cotyledon
Total Typical Atypical Total Typical Atypical
1 16 0 ± 0 0 0 26 3.8 2nd 3 2 1
3 20 5.0 2nd 3 2 1 26 3.8 2nd 3 2 1
6 20 10.0 1st 5 5 0 24 0 ± 0 0 0
7 20 5.0 2nd 3 2 1 28 3.6 1st 4 3 1
9 21 0 ± 0 0 0 24 4.2 3rd 4 2 2
10 18 0 ± 0 0 0 32 6.3 2nd 4 2 2
Average 19.4 2.1 ± 2.8 2.3 0.5 25.6 2.3 ± 3.0 1.8 1.2
a
Media as in Table 1.
b
No. of explants.
c
Percentage of explants showing to be embryogenic.
dSubculture of embryos appearing.
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Table 4
Morphogenic responses of different parts of the cotyledon (distal, median or proximal) of sour cherry (Prunus cerasusL.) c.v. `Gerema' to different
media (Table 1)
Md Ep Percentage of explants being morphogenic
Distal section Median section Proximal section
SE CL S L R SE CL S L R SE CL S L R
1 16 0 6.3 0 0 50.0 0 6.3 0 0 31.3 0 6.3 0 6.3 12.5
2 18 0 0 0 0 60.1 0 0 0 0 44.4 5.6 5.6 5.6 5.6 44.4
3 20 0 5.0 0 0 25.0 5.0 15.0 0 0 30.0 0 5.0 0 0 25.0
4 20 0 0 0 0 40.0 0 0 5.0 0 40.0 0 0 10.0 0 40.0
5 20 0 0 0 0 25.0 0 0 0 0 25.0 0 0 0 0 12.5
6 20 10.0 0 0 0 5.0 10.0 0 0 0 25.0 10.0 0 0 0 40.0
7 20 0 5.0 0 0 30.0 5.0 5.0 0 0 20.0 5.0 10.0 10.0 0 25.0
8 21 0 12.5 0 0 56.3 0 23.8 4.7 0 44.4 0 5.6 5.6 0 27.8
9 21 0 9.5 0 0 3.8 0 14.3 0 0 47.6 0 0 9.5 0 28.6
10 18 11.1 0 0 0 11.1 0 11.1 0 0 9.5 0 11.1 4.8 0 14.3
Av 19.4 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.
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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 lÿ1IBA 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
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
Table 5
Morphogenic responses in cotyledons of sour cherry (Prunus cerasusL.) 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
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.
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
Fig. 5. Cotyledon-like structures of sour cherry c.v. `Schattenmorelle' (P. cerasusL.) immature
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 inJuglans 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 ofPetunia inflataandPetunia 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
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 aviumimmature 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.
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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