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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 b a College of Forestry and Horticulture, Sichuan Agricultural University, Ya’an, 625014 Sichuan, China b Zentrum Gru ¨ne Gentechnik, Staatliche Lehr- und Forschungsanstalt, D-67435 Neustadt an der Weinstraße, 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 l 1 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, ‘Scharo ¨’ 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: [email protected] (H. Tang). 0304-4238/00/$ – see front matter # 2000 Elsevier Science B.V. All rights reserved. PII:S0304-4238(99)00073-4

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Page 1: Somatic embryogenesis and organogenesis from immature ...directory.umm.ac.id/Data Elmu/jurnal/S/Scientia... · Somatic embryogenesis and organogenesis from immature embryo cotyledons

Somatic embryogenesis and organogenesis from

immature embryo cotyledons of three sour

cherry cultivars (Prunus cerasus L.)

Haoru Tanga,b,*, Zhenglong Rena, Gabi Krczalb

aCollege of Forestry and Horticulture, Sichuan Agricultural University,

Ya'an, 625014 Sichuan, ChinabZentrum 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: [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

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1. Introduction

There are more than 30 species of cherries, but only a few of them arecommercially cultivated. Sour cherry (Prunus cerasus L.), one of the economic-ally important species, offers dual market potential for both fresh fruits andprocessing, 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 inprogress by conventional means (Brown et al., 1996). It is, however, very limiteddue to the long generation cycles and highly heterozygous nature. Genetictransformation could provide a complementary approach to conventionalbreeding of sour cherry by the introduction of genes encoding desirable traits.

Somatic embryogenesis and organogenesis from in vitro cultures are aprerequisite of many plant genetic transformation techniques. In order to obtainstable and non-chimeric transgenic plants, there are two important issues thatmust be considered in the process of regenerating transgenic plants. The recipientcells must be accessible to the transformation vectors and transgenic plants mustoriginate from single cells (Dandekar et al., 1992). The protoplast-to-plant systemholds promise since every protoplast seems to be transformable. While thedelivery of DNA into protoplasts presents only a minor problem, establishingregeneration systems from protoplasts is a major problem. Despite numerousstudies defining protocols for plant regeneration from protoplasts in Rosaceae,including sour cherry, there are as yet no reports of protoplast-based genetransfers in this family (Ochatt and Patat-Ochatt, 1995). Furthermore, as soon as adifferent approach becomes accessible, the protoplast-to-plant transformationsystem will probably be avoided (Siemens and Schieder, 1996).

The somatic embryo-to-plant system provides an alternative approach. Somaticembryogenesis is a process in which somatic plant cells undergo differentiation toform embryos. Somatic embryos can be germinated to form plants and canmultiply to produce many more somatic embryos through a process referred to assecondary or repetitive embryogenesis. Repetitive somatic embryos have anunicellular origin in the epidermis (Polito et al., 1989), a most important propertyof somatic embryos that has been exploited for avoiding chimeric transformationof 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 apetiole-derived cell suspension in this species. Nevertheless, there have beenseveral reports on somatic embryogenesis from other Prunus species (Hammers-chlag et al., 1985; Druart, 1990; De March et al., 1993; Da Camara Machadoet al., 1995). Somatic embryo-to-plant transformation system has already beenexploited for cultivar improvement in some Prunus species (Da Camara Machadoet al., 1995; Druart et al., 1998; GutieÁrrez-Pesce et al., 1998). The aim of thisinvestigation was to get more information about somatic embryogenesis as well

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as organogenesis in sour cherry because this information was required for thisspecies 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 1998from the state virus-free stock plants orchard of the Landesanstalt fuÈr Pflanzenbauund Pflanzenschutz in Mainz, Germany. These cultivars were chosen for studybecause they are three of the most important ones commercially. The fruitscollected had developed to the stage between the gelatinous endosperm beginningto disappear and it having half disappeared. Maternal tree genotype effects werenot tested and fruits from the different trees of each cultivar were mixed randomlyas a group.

Before dissection, fruits were washed with tap water for 20±30 min andsurface-disinfected by immersion in 70% (v/v) ethanol/water solution for 30 sand 5% (w/v) Ca(ClO)2 fresh solution with two drops of Tween 20 per 100 ml for25 min, followed by three rinses in sterile deionized water. The fruits wereopened and the embryos were excised aseptically. The cotyledons served asexplants.

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 agarand 1 g lÿ1 casein hydrolysate. Medium was adjusted to pH 5.6 with 1 N NaOHor HCl prior to autoclaving at 1158C for 25 min. Ten different combinations ofplant growth regulators (Table 1) as filter-sterilised solutions were added tomedia after autoclaving and media were dispensed as 30 ml aliquots per94�16 mm Petri dish. Cotyledons were placed abaxial side down on the inductivemedia in Petri dishes. Dishes were sealed with `̀ Parafilm'' and put in continuousdarkness at 228C in a growth chamber. After 4 weeks on the inductive media, thecultures were transferred onto the basal MS medium. Transfers to fresh mediumwere made every 4 weeks.

2.3. Experimental treatments

The two cotyledons of one embryo were separated and the embryonic axis wasexcluded for investigating the potential for somatic embryogenesis in intact

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cotyledons. The intact cotyledons from all three cultivars were placed on the 10inductive media, 4±5 pairs of cotyledons in each Petri dish, 16±32 explants foreach treatment (Table 2).

In order to examine the potential of somatic embryogenesis from different partsof a cotyledon, the cotyledons from cultivar `Gerema' were trisectioned intodistal, median and proximal sections and put onto the same 10 inductive mediamentioned above, 5±6 trisections in each Petri dish, 16±21 explants for eachtreatment (Table 4).

In assessing the influence of the embryonic axis, the two cotyledons of oneembryo from cultivar `Schattenmorelle' were dissected into two kinds ofexplants, one with the embryonic axis attached to cotyledons and another withoutcotyledons attached, and placed onto 2, 3, 6, 7 and 8 inductive media mentionedabove, 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 connection with parenttissue may be termed somatic embryos. Using these criteria, we considered anexplant 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 cerasus L.) cotyledons

Medium Plant growth regulatorsa

Auxins (mg lÿ1) Cytokinins (mg lÿ1) A:Cb

2,4-D NAA IBA KT BAP

1 1 0 0 1 0 1 : 1

2 2 0 0 1 0 2 : 1

3 4 0 0 1 0 4 : 1

4 0 2 0.1 1 0 2 : 1

5 2 0 0 0 1 2 : 1

6 2 0 0.1 1 0 2 : 1

7 2 0 0 2 0 1 : 1

8 2 2 0 2 0 2 : 1

9 1 1 0 1 0 2 : 1

10 4 0 0 2 0 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: 3-

indolebutyric acid; KT: kinetin; NAA: a-naphthaleneacetic acid.b A:C�Auxin : Cytokinin.

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Table 2

Morphogenic responses of the intact cotyledons of sour cherry (Prunus cerasus L.) 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 wasobserved. Since the cotyledon responses to a given criterion were found to bemany and varied, we focused on embryo formation and on other morphogenicresponses as well and recorded them explant by explant after 3 months in culture.Since the somatic embryogenesis was the main event of concern in thisinvestigation, the embryogenic responses were analysed by using statisticalmethods. However, no significant differences were found among the experimentaltreatments due to a large proportion of irresponsive explants in each treatment.Therefore we described the experiments by using the percentage of embryogenicexplants and the numbers of somatic embryos per embryogenic explantthroughout the text followed.

3.1. Somatic embryogenesis and characterisation

Somatic embryos formed either individually or in groups, directly oncotyledons and mainly at the proximal ends (Fig. 1). The somatic embryossubsequently developed from globular stage to cotyledonary stage and they weredetached easily from the surrounding cells of their parental tissues. Somaticembryos showed great variability in their morphology. The typical somaticembryos 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 isolatedfrom the cotyledons and cultured on basal medium, occasionally secondarysomatic embryos formed at the radicle of primary somatic embryos. In a fewcases, secondary somatic embryos were found on abnormal ones (Fig. 3(C)). On

Fig. 1. Somatic embryos of sour cherry c.v. `Gerema' (Prunus cerasus L.) directly formed on

cotyledons.

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the basal medium in darkness, about 27% of well-developed somatic embryosgerminated, showing an elongated radicle and an emerging root. Aftertransferring these germinated somatic embryos onto fresh basal medium andexposing them to light (�45 mmol mÿ2 sÿ1 photosynthetic photon flux, 16 hphotoperiod), their cotyledons turned green and epicotyls appeared. One weeklater, shoot meristems began to grow. In some cases, the roots grew, but the shootmeristem failed to develop. A few plantlets were obtained (Fig. 4).

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.

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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).

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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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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 thecotyledons 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 thepresence of 0.1 mg lÿ1 IBA, the frequency of somatic embryogenesis wasenhanced and, at the same time, somatic embryos had fewer abnormalities andappeared earlier in comparison with those produced on media 3 and 7 (Tables 2and 3).

The percentage of explants show to be embryogenic in `Schattenmorelle' wasgreatest on medium 10, followed by media 9, 1, 3 and 7. Somatic embryos couldbe observed during the second subculture on cotyledons cultured on medium 10,with a high auxin concentration but moderate auxin/cytokinin ratio (2:1), but theyexhibited 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 somaticembryos was higher compared to those on medium 1 and 3 (Tables 2 and 3).

Somatic embryogenesis in `ScharoÈ' occurred only on the explants on medium2. Three typical somatic embryos per embryogenic cotyledon were producedduring the second subculture.

The average percentages of explants being embryogenic were similar for all thethree sections of cotyledons from `Gerema', but higher concentrations of plantgrowth regulators were needed for distal and median sections to produce somaticembryos than those for proximal sections (Table 4). Moreover, somatic embryosfrom distal sections on a higher auxin concentration medium (medium 10)showed more morphogenic variations than those on a moderate auxinconcentration medium (medium 6) in comparison with those from median andproximal sections. Medium 6, however, appeared to be optimum for all the threesections to undergo somatic embryogenesis.

The embryonic axis attachments to cotyledons from `Schattenmorelle' gave aninhibitory effect on somatic embryogenesis, as showed in Table 5. No somaticembryogenesis occurred on the cotyledons with embryonic axes while thosewithout embryonic axes were embryogenic in 3/5 experimental treatments.Likewise, the cotyledons without embryonic axes on media 6 and 7 producedmore typical somatic embryos when compared to those on medium 3, althoughtheir percentages of somatic embryogenesis on media 6 and 7 were similar to, orlower than, that on medium 3.

As for somatic embryogenesis, analyses of the responses of all the explants tothe experimental treatments demonstrated that somatic embryogenesis occurredprincipally when using 2 mg lÿ1 2,4-D plus 1 mg lÿ1 KT. Increasing the

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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

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.d Subculture 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 cerasus L.) 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 inducedsomatic embryogenesis, but it gave rise to abnormally developed embryos. Theaddition of 0.1 mg lÿ1 IBA to the inductive medium containing 2,4-D resulted inan increase of the incidence of somatic embryogenesis and a decrease ofabnormal somatic embryos. If the concentration of 2,4-D and NAA were equal,the incidence of somatic embryogenesis was reduced. No somatic embryogenesisoccurred when using NAA substituted for 2,4-D, even in the presence of0.1 mg lÿ1 IBA. The replacement of 1 mg lÿ1 KT by 1 mg lÿ1 BAP reducedsomatic embryogenesis.

3.2. Cotyledon-like structures

White cotyledon-like structures developed individually or in clusters, in somecases together with somatic embryos, on the surfaces of the explants (Fig. 5).Cotyledon-like structures initiated directly or indirectly on cotyledons and lookedlike somatic embryos at the beginning of their development. Unlike somaticembryos, they did not undergo the sequentially developmental stages andremained closely attached to their parental tissues. Sometimes they differentiatedcotyledon-like lobes, but no shoot meristems and radicles were found aftersectioning them under the dissection microscope. Additionally, they lookedunlike leaves due to their thickened shapes and without vein venation.

Cotyledon-like structures occurred on the intact cotyledons in all experimentaltreatments except medium 10, although there were some differences among thethree cultivars (Table 2). `Schattenmorelle' gave more cotyledon-like structuresthan `Gerema' and `ScharoÈ'. Medium 9 produced the highest percentage of

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 31 0 3.2 0 0 0 0 9.8 3.2 0 0

3 32 0 3.1 0 0 3.1 6.3 6.3 3.1 0 3.1

6 24 0 4.2 0 0 0 4.2 4.2 0 0 0

7 32 0 0 0 0 0 6.3 0 3.2 0 6.3

8 30 0 3.3 0 0 0 0 3.3 0 0 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.

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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 showeda higher percentage of explants having cotyledon-like structures than the distaland proximal sections (Table 4). The combination of 4 mg lÿ1 auxin, either 2,4-Dalone or 2,4-D plus NAA, with 2 mg lÿ1 KT (media 8 and 10) induced moreexplants to form cotyledon-like structures than the others. The proximal sectionswere less reactive than distal and median sections.

Cotyledons with and without embryonic axes reacted to the same experimentaltreatments in producing cotyledon-like structures but with different frequency(Table 5). On an average, cotyledons with embryonic axes gave 2.2% of explantsforming cotyledon-like structures whereas cotyledons without the embryonicaxes gave 5.0% cotyledon-like structures.

3.3. Other adventitious structures

Formation of other adventitious structures such as leaves, shoots and rootsfrequently occurred on the cotyledons. These adventitious structures appearedseparately, either one structure alone or two structures at different sites, on eachexplant. Leaves and shoots formed individually, whereas roots developed eitherindividually or, in most cases, in groups at the surfaces of explants.

Among the three cultivars tested, `ScharoÈ' showed little ability to formadventitious 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. cerasus L.) immature

cotyledons.

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formation between the two cultivars, `Gerema' showed higher root formationwhile `Schattenmorelle' gave higher leaf formation.

After trisectioning the cotyledons of `Gerema', the ability to form leaves andshoots reduced while that to form roots increased (Table 4). The distal andmedian sections induced more explants to form roots, but gave fewer explants toform leaves and shoots. The proximal sections induced fewer explants to formroots, 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 thefive experimental treatments (Table 5). Without the embryonic axes, thecotyledons formed shoots and roots but no leaves.

4. Discussion

This paper reports somatic embryogenesis and organogenesis of sour cherryfrom immature embryo cotyledons. The system described here presents someinformation on morphogenic responses to the different experimental treatments,which might suggest further investigation on the regeneration potential in sourcherry.

The mechanisms of differentiation between a state of permissive determinationleading to a particular morphogenic pattern (somatic embryogenesis) and anotherstate which leads to leaves, shoots, roots or callus are not well understood. In vitroculture conditions, including certain chemical compounds, plant growthregulators, play a major role in triggering the morphogenic progress andregulating the switch of a somatic cell from one pathway to another. Sincesomatic embryogenesis and organogenesis are two mutually exclusive processesof in vitro differentiation (Ammirato, 1985), their induction requires distinctlydifferent conditions. Our results with immature cotyledons of Prunus cerasus

showed that somatic embryogenesis and organogenesis responded to the sameinductive conditions. Similar results were also obtained in Prunus avium (DeMarch et al., 1993) and in Juglans nigra (Long et al., 1995). This phenomenonmight reflect the genotypic differences in the ability to activate key elements inthe morphogenic pathway.

Because of genotypic specificity, the requirements of different kinds of plantgrowth regulators and their proportion for morphogenesis vary from one speciesto another. In callus cultures of Petunia inflata and Petunia hybrida, the additionof 2,4-D led to embryogenesis, the use of IAA/BAP led to the development ofadventitious shoots with roots, and NAA/BAP to root development (Rao et al.,1973). In embryonic tissue cultures of apple, the interaction of NAA and BAPresulted in shoot formation, IAA and BAP in bud and root formation and 2,4-Dand BAP in proembryo and bud formation (Rubos and Pryke, 1984). Our results

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with Prunus cerasus immature cotyledons showed that somatic embryogenesisoccurred principally when using the combinations of 2,4-D and KT. Using NAAand 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) inPrunus avium immature cotyledon cultures.

Many studies concerned with in vitro morphogenesis underscore theimportance of auxin/cytokinin ratio in the culture medium. In Prunus cerasus,the auxin/cytokinin ratio is not only the factor to be considered. Somatic embryoswere obtained from the cotyledons cultured on the inductive media with eitherhigh or low auxin/cytokinin ratio, but they showed atypical development.Cotyledons cultured on media with moderate auxin/cytokinin ratio formedsomatic embryos earlier and produced more typical somatic embryos. Moreover,the addition of 0.1 mg lÿ1 IBA increased the incidence of somatic embryogenesisand 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 theembryogenic capacity in the callus of Prunus subhirtella autumno rosa. Theinteraction between two auxins and the competency to form somatic embryosremains to be understood.

It was widely found that when a complete organ was cut into pieces, the varioussegments differed in their morphogenic capability. The regenerative capacity ofcotyledon fragments was usually greater in proximal sections than in distalsections (Cheng, 1976; Mante et al., 1989; Schmidt and Kardel-Meisner, 1992).Our study showed that gradients in shoot and leaf formation were apparent whilethey were not apparent in root and somatic embryo formation. These might berelated to the development of endogenous gradients of hormones or to theinteraction and balance between the endogenous hormones and the exogenousplant growth regulators in forming morphogenic structures.

Kouider et al. (1984) found that if the embryonic axis was left in place, noadventitious structures, even no callus, formed on the cotyledons of apple,whereas intact cotyledons without embryonic axes and different excisions ofcotyledons produced morphogenic structures. Mante et al. (1989) reported thatformation of shoots in cotyledons from different Prunus species was entirelydependent upon the removal of the embryonic axis and the presence of theproximal region of cotyledon, but Schmidt and Kardel-Meisner (1992) couldregenerate shoots from different sections of cotyledons from five cultivars ofPrunus avium. Both the strongly inhibitory effects on morphogenesis of theembryonic axes attached to the cotyledons and the capability of cotyledon pieceswithout proximal regions differentiating into different morphogenic structureswere demonstrated in our study.

While somatic embryogenesis has many potential advantages for geneticimprovement, some limitations remain to be overcome before somatic

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embryogenic systems can be applied for operational production in sour cherry.Selection experiments using zygotic embryos or their components are of littlecommercial value for clonally propagated species, but they serve to highlight thepressing need for extending somatic embryogenesis to a wide range of explants.Further studies on selecting the most competent organs or tissues for somaticembryogenesis, testing the optimum conditions for inducing somatic embryos andincreasing somatic embryo production, may provide precise information for sourcherry somatic embryogenesis and, at the same time, provide repetitive somaticembryogenic lines for genetic manipulation experiments.

Acknowledgements

Thanks are due to the German Ministry for Science and Technology forfinancial support and the Landesanstalt fuÈr Pflanzenbau und Pflanzenschutz inMainz, Germany, for supplying plant materials. We would like to thank Mr.MoÈrbel for help in collecting plant materials and Dr. Martin for help in paperpreparation. Also Dr. Reustle for good suggests on experiments and Mr. Wahl forphotography.

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