Plant Science 160 (2000) 15–26

Analysis of habituated embryogenic lines in Asparagus officinalisL.: growth characteristics, hormone content and ploidy level of

calli and regenerated plants

Anne Limanton-Grevet a,b,c, Bruno Sotta d, Spencer Brown e, Marc Jullien a,*a UMR INRA/INA P-G Biologie des Semences, INRA Versailles, Route de Saint-Cyr, 78026 Versailles Cedex, France

b Laboratoire ‘in 6itro’, J. Marionnet GFA, Route de Courmemin, 41230 Soings-en-Sologne, Francec Asparagus b6, Postbus 6219, 5960 AE Horst, France

d Laboratoire de Physiologie du De6eloppement des Plantes, UMR de Physiologie Cellulaire et Moleculaire des Plantes,Uni6ersite Pierre et Marie Curie (P VI), Tour 53, 4 place Jussieu, 75252 Paris Cedex 05, France

e Cytometrie, Institut des Sciences Vegetales, CNRS, 91198 Gif-sur-Y6ette, France

Received 23 March 2000; received in revised form 26 July 2000; accepted 31 July 2000

Abstract

Habituated asparagus embryogenic lines derived from eleven genotypes were maintained on hormone-free medium and grewactively through secondary embryogenesis. Secondary embryos were of single cell origin and emerged from the transversal divisionof some epidermal or subepidermal cotyledonary cells of primary embryos. The intensity of secondary embryogenesis was foundto be variable between embryogenic lines. Plants regenerated from three of these lines have been previously demonstrated to carrya mutation whose phenotype was the direct appearance of somatic embryos on apices or nodes cultured on hormone-free medium.Habituated lines of embryogenic calli and various tissues of embryogenic mutant and wild type plants were analysed for theirhormonal content in ABA, IAA, iP, Z and their metabolites ABA-GE, iPA, iMP, ZR. No significant difference was foundbetween different embryogenic lines, except the level of iPA, or between cladophyll or apex cultures of mutant and wild typeplants. Flow cytometry analyses indicated only 34% of the embryogenic lines were diploid, most of the others being tetraploid,but 62% of regenerated plants from these lines were diploid. This indicated the process of maturation and conversion selecteddiploid embryos in the embryogenic lines. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Asparagus officinalis L.; Somatic embryogenesis; Habituation; Mutation; Hormone; Flow cytometry; Ploidy level

www.elsevier.com/locate/plantsci

1. Introduction

Asparagus has a low multiplication rate usingconventional methods [1,2]. In vitro clonal propa-gation using shoot multiplication from culturedmeristems and shoot tips leads to higher efficiencybut is very labour intensive [3]. Somatic embryoscould be the most efficient method for clonalmicropropagation of plants [4] and thus could also

be useful for asexual multiplication of asparagus.Different procedures for somatic embryogenesishave been described in Asparagus officinalis fromdifferent types of explants such as hypocotyls [5],terminal buds [6–8], stems [9,10], crowns ofseedlings [10], cladophylls [11] and mesophyll cellculture [12]. In most cases, embryogenic calli wereinduced and maintained on a medium containinggrowth regulators, especially auxins and cy-tokinins. Then, they had to be transferred on amedium with reduced hormone concentration toexpress their embryogenic potential, allowing em-bryo development and plant conversion. Jullien

* Corresponding author. Tel.: +33-1-3083-3074/+33-1-4408-1831; fax: +33-1-3083-3099.

E-mail address: jullien@versailles.inra.fr (M. Jullien).

0168-9452/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0 1 68 -9452 (00 )00356 -3

A. Limanton-Gre6et et al. / Plant Science 160 (2000) 15–2616

[12] and Delbreil et al. [13] described the produc-tion of long-term habituated embryogenic lines (Hlines) growing through repeated secondary em-bryogenesis on hormone-free medium with a veryhigh potential for plant multiplication which canbe used for genetic transformation of asparagus[14]. Plants regenerated from H lines exhibited anincreased embryogenic capacity compared to thecontrol plants. For three of these lines, the highembryogenic capacity was transmitted to theprogeny, following a Mendelian pattern, providingevidence for a dominant monogenic mutation thatimproved somatic embryogenesis [15,16].

An essential aspect of in vitro plant recovery isthe conformity of regenerated plants. In the caseof somatic embryogenesis in asparagus, little at-tention was paid to somaclonal variations withonly a few exceptions concerning variations inploidy levels [17–20]. As synthetic auxins haveoften been considered as largely responsible forpolyploidisation [21], H lines maintained on hor-mone-free medium could give a material less sub-ject to ploidy level changes than embryogenic linesmaintained in the presence of auxin, as usuallyused for asparagus [8–10,17–20]. The indicationof the mutational origin of H lines cited above wasanother example of somaclonal variation andquestioned about the function of correspondinggenes. Genes implicated in the embryogenic capac-ity have been identified in alfalfa [22], in maize[23,24] and in Dactylis glomerata [25], but theirfunction has not been defined. A relation betweenembryogenic capacity and hormonal metabolismhas been found in alfalfa [26] and wheat [27] wherethe embryogenic capacity could be regulated bythe ratio of abscisic acid/IAA.

The aim of this study was to characterize severalH lines (including two previously demonstratedmutant lines) for future plant production. Growthmodalities of the lines were compared by weightmeasurements, sieving of the cultures for embryosize and cytological analysis of growing tissues.Second, the intensity of secondary embryogenesisof various lines was tentatively related to theirhormonal content and was compared to the hor-monal composition in vegetative tissue and culti-vated apices of the embryogenic mutant and wildtype plants. Third, the ploidy level and its stabilityin embryogenic calli and regenerated plants wereexamined by flow cytometry to define long termusable H lines.

2. Materials and methods

2.1. Plant material

The habituated embryogenic lines (H lines) usedwere derived from eleven genotypes: two femaleclones (CO1 and CO3) and four male clones (8,186, DDNO5 and JMal) provided by J. MarionnetGFA (Soings en Sologne, France); two femaleclones (A1 and A2), two male clones (A3 and A4)provided by Asparagus bv (Horst, Holland) and81A a F1 hybrid provided by INRA (Versailles,France). The H lines derived from these genotypesare indicated in the text and named as following:for instance A2L2 was the H line n° 2 obtainedfrom the genotype A2. The H lines 8L1 [15], A3L3and A4L1 [16] carry a dominant ‘high embryo-genic’ mutation that enables vegetative tissue todevelop somatic embryos when cultivated on hor-mone free medium.

2.2. Cultures of habituated embryogenic lines

Isolation of H lines was described previously[13,16]. Briefly, shoot apices dissected from adultplants were cultured 1 month on MSN (basalmedium) containing naphthaleneacetic acid(NAA) 10 mg l−1 then were subcultured on basalmedium for development of H lines. Basalmedium consisted of Murashige and Skoogmacronutrients [28], Nitsch micronutrients [29],Nitsch and Nitsch vitamins [30], 2% sucrose and0.7% agar (Biomar). The pH was adjusted to 5.7before autoclaving. Most of the H lines weremaintained 2 years by subculturing every monthon basal medium. For subcultures, embryogeniccallus (0.1 g) containing mainly elongated andmature embryos coming from a precedent culturewere plated on 3MM paper (Whatman) and cul-tured on 20 ml basal medium in a Petri dish. Forrecovery of H lines without an auxin treatment,shoot apices or nodes of diploid regenerated plantsA4L1 and 81AL2 were cultured on basal medium.All cultures were put in a growth chamber with 16h per day fluorescent light providing 40–70 mmolm−2 s−1 at 25°C and 70% relative humidity.

To determine the distribution of embryo devel-opment stages, the embryogenic calli were dissoci-ated in distilled water then sieved on variousmeshes (0.2, 0.4, 0.8 and 1.6 mm). The freshweight of each fraction was measured. The 0.2–0.4

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mm fraction contained globular embryos, the 0.4–0.8 fraction globular and bipolar embryos, the0.8–1.6 mm fraction bipolar elongated embryosand the 1.6 mm and over fraction mature embryoswith a chlorophyllous cotyledon. Four repetitionscorresponding to four Petri dishes were made.

2.3. Embryo con6ersion

For plant recovery, 0.1 g callus from H lineswas plated in Petri dishes on basal medium con-taining 36 g l−1 maltose and solidified with 10 gl−1 Phytagel [8]. After 1 month of culture, matureembryos were transferred to glass pots containing30 ml of germination medium: MSN solidifiedwith 2 g l−1 Phytagel. Later (1 month), plantletswere transferred to test tubes containing 20 ml ofthe same medium where they developed during 2months. Plantlets were then transferred to thegreenhouse.

2.4. Histological analysis

Embryos were fixed overnight in a 0.2% glu-taraldehyde, 0.4% formaldehyde solution, rinsedthree times in water and dehydrated in successiveethanol solutions from 10 to 100%. They wereembedded in Technovit 7100 and cut at 3–4 mm.Sections were stained with toluidine blue.

Scanning electron microscopy was carried outusing a Phillips 625M microscope. Samples werefixed by cryodesiccation in a Cryostans System CT1500 or dehydrated by critical point method afterglutaraldehyde fixation and ethanol dehydration.

2.5. Hormone analysis

2.5.1. Plant materialAll plants and calli used for hormone analyses

were diploid. The hormonal content of 8 H lines 1year old was analysed in callus samples collected 2weeks after subculture. Plants regenerated fromthe mutant H line A4L1 and wild type plants fromthe clone A4 were compared for their hormonalcontent in different tissues. Cladophylls of mutantand wild type plants were collected on stems at theend of growth. One to seven mutant and wild typeplants were analysed. Buds of young spears (15–20 cm high) collected on mutant or wild typeplants 1 year old, grown in greenhouses, weredissected and cultured on basal medium for 0, 7,14 or 25 days.

Samples were frozen in liquid nitrogen,lyophilised and stored 1 month at room tempera-ture in a desiccator. Before extraction each samplewas ground with a ball mill.

2.5.2. Extraction, purification and fractionationExtractions were performed at 4°C in darkness

for 60 h from about 40 mg of tissue powder in 5ml of 80% aqueous methanol supplemented withBHT (butylhydroxytoluen) 40 mg l−1 as antioxi-dant. 3H-ABA and 3H-IAA were added to theextracts to measure extraction efficiency. A prefil-ter (0.2 mm) connected to a Sep-Pak cartridge wasequilibrated with 10 ml of 80% aqueous methanolbefore sample loading. Eluates were reduced byrotary evaporation and taken up with 0.2% formicacid up to 500 ml and injected into a C18(Macheray-Nagel) liquid chromatography (HPLC)column. Elution was performed at 0.8 ml min−1

with a HPLC (System gold, Beckman) with a 0.2%formic acid/methanol gradient. Retention time ofABA (abscisic acid), ABA-GE (abscisic acid glu-cose ester), IAA (indoleacetic acid), iP (isopen-tenyladenine), iMP (isopentenyladenosinemonophosphate), iPA (isopentenyladenosine), Z(zeatin) and ZR (zeatin-9-riboside) were deter-mined by separate injection of pure compounds(Sigma) as standards. A total of 40 fractions of 0.8ml were collected. They were evaporated to dry-ness in a speed-vac concentrator, methylated with250 ml of diazomethane in ether, evaporated againto dryness and finally taken up with 1.5 ml dis-tilled water with 0.2 g l−1 NaN3 as preservative.Aliquots (50 ml) of fractions were submitted toscintillation counting in order to determine ABAand IAA recovery, or to enzyme-linked immuno-sorbent assay (ELISA).

2.5.3. ELISA procedureThe whole procedure was described by Julliard

et al. [31]. ABA and ABA-GE were measuredusing anti-ABA monoclonal antibodies (LPDP229), IAA using anti-IAA polyclonal antibodies(LPDP 47), iP, iPA and iMP using anti-iPA poly-clonal antibodies (LPDP 5), and ZR and Z usinganti-ZR polyclonal antibodies (LPDP 17). Microt-itration plates were coated with ABA, IAA, iPAor ZR conjugated to ovalbumin. After washingthe plates five times, 50 ml of the fractions or 50 mlof solutions containing different concentrations ofmethylated ABA, IAA, iPA, or ZR standard were

A. Limanton-Gre6et et al. / Plant Science 160 (2000) 15–2618

added followed by 50 ml of anti-hormone antibodysolution. Plates were incubated 2 h at 4°C indarkness. After washing, anti-hormone antibodiesbound to the plates were quantified by means ofan anti-mouse antibody for ABA (Sigma) andanti-rabbit antibodies for IAA, iPA and ZR linkedto a peroxidase system (Sigma). The peroxidasesubstrate (ABTS: 2,2%-azino-bis (3-ethylbenzthaz-oline-6-sulfonic acid)), diluted in a perboratebuffer, was added and optical density was mea-sured at 405 nm. The measures were repeated 5times for each sample.

2.5.4. Statistical analysisHormone levels in H lines and in explants from

embryogenic and wild type plants were analysedusing a Fisher test at P=0.05 or a Student test atP=0.05.

2.6. Ploidy le6el analysis through flow cytometry

Ploidy level analyses were conducted with Hlines calli and regenerated plants. Callus sampleswere generally taken from H lines 1 month aftersubculture, corresponding to the end of the growthphase. For asparagus plants, aerial parts includingstem fragments and cladophylls were used. About0.1 g of fresh matter was chopped with a razorblade in 600 ml Galbraith buffer [32] containing0.5% Triton X-100 and 0.01 M sodiummetabisulfite, sieved through a 30 mm filter. RNasewas added to 10 mg ml−1 and BET to 50 mg ml−1.Plants from the eight genotypes were used as

controls, and tomato plants (Lycopersicon esculen-tum) as a reference. For each sample, 5000 nucleiwere analysed on a cytometer (EPICS V fromCoultronics France) with an Argon laser 400 mW,514 nm [33]. Samples were considered as diploidwhen a peak of 2C nuclei was observed and astetraploid when the peak corresponding to 2Cnuclei was absent and a peak of 4C nuclei wasobserved.

3. Results

3.1. Growth of habituated embryogenic lines

From a total of 40 H line calli isolated fromeleven asparagus genotypes [15,16], eight linesfrom six different genotypes were chosen as repre-sentative of the variation in the intensity of sec-ondary embryogenesis. They grew actively during20–30 days of subculture (Fig. 1). The growth ratethen slowed and the stationary phase was reachedmore or less rapidly depending on the line. De-pending on the line, 2.3–4.8 g of fresh matter wereproduced in 7 weeks per Petri dish (correspondingto an increase of 2300–4800%). The line CO3L1exhibited a decrease of fresh weight after 35 days.

3.2. Intensity of secondary embryogenesis

H line calli were sieved through different meshesto evaluate the development of somatic embryos 2weeks after subculture. The distribution of embryostages in various H lines differed greatly. The over1.6 mm fraction containing mature embryos repre-sented 80–28% of the total fresh weight of the Hlines (Fig. 2). The extreme H lines were A3L3-JMalL3, which were rich in mature embryos andcontained few globular embryos, and DDNO5L2-CO3L1, which contained few mature embryos andnumerous globular and bipolar embryos. Otherlines were intermediary. Different lines derivedfrom the same genotypes (JMal and DDNO5)were similar.

3.3. Embryo setting in habituated embryogeniclines

Mature and elongated somatic embryos begin toproduce secondary embryos about 1 week afterthe subculture. The secondary embryos emerged

Fig. 1. Growth of habituated embryogenic callus lines onbasal medium. The values are mean of ten repetitions9stan-dard deviation. The inoculum was 0.1 g. FW. 186L1 is the Hline number 1 isolated from the genotype 186.

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Fig. 2. Development stages of somatic embryos in differenthabituated embryogenic lines. H lines grown on a Petri dishfor 2 weeks were sieved on different meshes (0.2, 0.4, 0.8, and1.6 mm). Four Petri dishes were analysed for each line. \1.6mm: mature embryos; 0.8–1.6 mm: bipolar embryos; 0.4–0.8mm: globular+bipolar embryos; 0.2–0.4 mm: globular em-bryos.

mature embryos, like A4L1 (Fig. 2), secondaryembryogenesis occasionally occurred on the epi-dermis (Fig. 3F).

3.4. Hormonal content of H lines calli andregenerated plants

The eight H lines examined above have beencultured on hormone-free medium for more than 1year. The hormonal content of these H line calliexhibiting contrasting intensities of secondary em-bryogenesis was examined 2 weeks after subcul-ture, during active growth (Table 1). The ABAlevel was highly variable from one line to anotherand ranged from 220 to 5850 pmol g dryweight−1. IAA levels were more homogenousfrom one line to another ranging from 207 to 1032pmol g dry weight−1. The cytokinins iP, iPA andiMP were in most of the samples below the detec-tion level. No significant difference in Z and ZRcontent was observed. CO3L1 was the only H linepresenting a significant difference in hormone(iPA) content with the other lines. This lineshowed extremely active secondary embryogenesisas illustrated in Fig. 3G and contained a very highproportion of globular embryos (Fig. 2).

The mutant H line A4L1 was selected from thegenotype A4. Analyses were conducted on adultmutant plants and adult plants of the male cloneA4 as the wild type control. Freshly harvestedcladophylls and meristems dissected from youngspears and cultured on basal medium for 0, 7, 14or 25 days were analysed. Hormone levels in thecladophylls varied widely between plants, but hor-mone levels in cladophylls from mutant and wildtype plants were not significantly different accord-ing to Fisher’s test (not shown). No significantdifferences in hormone content appeared betweenshoot meristems of wild-type and mutant plantscultivated on hormone-free medium for 0, 14 or 25days, (not shown) a time corresponding with theemergence of somatic embryos on the basal part ofthe cultured apices of the high embryogenic mu-tant [16].

3.5. Ploidy le6el of H lines and regenerated plants

We analysed 32 H lines coming from eightdifferent genotypes maintained on hormone-freemedium. The lines were about 12 months old. Fig.5 illustrates the distribution of fluorescence ofBET-stained nuclei in a diploid control plant (A)

from epidermal and subepidermal cotyledonarylayers, where large cells presented a transversaldivision (Fig. 3A, Fig. 4A), which appeared to bethe first step of secondary embryogenesis. Sec-ondary embryos were therefore of single cellorigin. A second division of one or of the two cellsled to the formation of a three-celled or a four-celled pro-embryo (Fig. 3B, Fig. 4A). After subse-quent divisions, globular embryos wererecognisable (Fig. 3D–E, Fig. 4B–C). Not all cellspresenting divisions produced a somatic embryo.In particular, numerous cells in deeper cotyle-donary cell layers presented transversal divisions(Fig. 3C), while globular embryos were only visi-ble in the superficial layers (Fig. 3E–F). The emer-gence of secondary embryos was responsible forthe disorganisation of the epidermis when sec-ondary embryogenesis was very intensive (Fig. 3G,Fig. 4D). Later, secondary globular embryos weredetached from the primary embryos (Fig. 3E, G).No tissue connection related secondary embryosto the original embryos (Fig. 3E). Secondary em-bryogenesis was not synchronised (Fig. 4C).

A great variation in intensity of secondary em-bryogenesis was observed between lines. H linescontaining numerous globular embryos and fewmature embryos, like CO3L1 (Fig. 2), exhibitedintensive secondary embryogenesis (Fig. 3G). In Hlines showing few globular embryos and many

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and in the calli of diploid H lines (B, C), and atetraploid H line (D). Among 32 lines, 35% werediploid, 59% tetraploid, and 3% hexaploid or ane-uploid (Table 2). To evaluate the possibility ofploidy level changes during subculture on hor-mone-free medium, ten diploid H lines maintainedon hormone-free medium were analysed at differ-ent ages during another year. After 9 months, onediploid line had become tetraploid and after 20months, two others had become tetraploid (that is

30% of tetraploid to compare to the 59% of te-traploid detected 1 year earlier).

A total of 45 plants regenerated from eight ofthe previously examined 1-year-old H lines wereanalysed and classified into three groups (Table 3)depending on the ploidy level of regeneratedplants. In group 1 the plants showed the sameploidy level as the corresponding H line. In partic-ular, the diploid H line 186L1 produced onlydiploid plants. In group 2 some of the plants had

Fig. 3. Cytological analysis of secondary embryogenesis. Longitudinal sections of somatic embryos from the H lines A4L1 (A–F)and CO3L1 (G) 7 (A–D) and 18 days (E–G) after subculture on basal medium. (A) First transversal division of a subepidermalcell in the cotyledon of a primary embryo (arrow). e: epidermis. Bar: 10 mm. (B) Four-celled pro-embryo that arise fromtransversal divisions of a subepidermal cell (arrow). e: epidermis. Bar: 10 mm. (C) Reactivated cells in the 7th–12th cell layers fromthe epidermis of the cotyledon (arrows). e epidermis. Bar: 10 mm. (D) Globular embryo emerging from the disrupted epidermisof a primary embryo (arrow). e: epidermis. Bar: 10 mm. (E) A group of globular embryos emerging from the primary embryo. Bar:10 mm. (F) Secondary embryos (arrows) emerging from the superficial layer of the cotyledon (cot) of a somatic embryo in A4L1H line. Bar: 0.1 mm. (G) A palisade of secondary embryos (arrow) that developed from the superficial layer of the cotyledon (cot)in CO3L1 line. Bar: 0.1 mm.

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Fig. 4. Scanning electron microscopy of secondary embryogenesis. (A) Two (arrow) and four celled proembryos (black and whitearrowheads) appeared on epidermis of a primary embryo from the H line A4L1 7 days after subculture. (B) Globular embryo (ge)emerging through the epidermis of a primary embryo from the H line A4L1, 7 days after subculture. (C) Intensive emergence ofsecondary embryos on a primary embryo from the H line CO3L1, 12 days after subculture. The development of secondaryembryos was nonsynchronous. Very young globular embryos (arrows), post globular embryos (arrowheads) and elongatedembryos (ee) can be distinguished. (D) Complete disorganisation of epidermis following proliferation of superficial cells of aprimary embryo from the embryogenic line CO3L1, 12 days after subculture. Clusters of globular somatic embryos (arrowheads)emerged through dislocated epidermis cells (arrows).

Table 1Hormone content in embryogenic lines derived from six asparagus genotypesa

ABA IAA iMP iP iPA Z ZREmbryogenic line ABA-GE

3165 950 0A3L3 065 1.5 37 25116 207 9A4L1 16 4 24 5

5850 407 10 091 0JMalL3 33 82487 414 11 0JMalL4 024 54 8

2015 332 50 360 0.5186L1 31 132700 338 93 0.5DDNO5L1 50 32 32761 442 67 027 2DDNO5L2 10 6

220CO3L1 103223 55 2 21b 20 1

a Two callus samples were analysed for each H line; values are given in pmol g−1 DW. ABA, abscisic acid; ABA-GE, abscisicacid glucose ester; IAA, indoleacetic acid; iP, isopentenyladenine; iMP, isopentenyladenosine monophosphate; iPA, isopenteny-ladenosine; Z, zeatin; and ZR, zeatin-9-riboside.

b A significant difference with the other embryogenic lines was detected at P=0.05.

a higher ploidy level than the corresponding Hline. Typically, the H line A4L1 was diploid, andthe plants regenerated were diploid or tetraploid.In group 3 some of the plants showed a lowerploidy level than the H line, for instance thetetraploid H line 81AL2. Existence of group 2 and3 suggests some H lines contained a mixture ofdiploid and tetraploid cells.

3.6. Ploidy le6el of H lines re-isolated from theregenerated plants without auxin treatment

As plants regenerated from H lines exhibited ahigh embryogenic ability, manifested by the re-ap-pearance of H lines from explants cultured onhormone-free medium [15,16], 23 new H lines wereobtained without growth regulators from cultured

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apices of diploid regenerated plants. We usedA4L1 and 81AL2 regenerants. A total of 91% ofthe new H lines were found to be diploid, and 9%were found to be tetraploid (Table 4). The propor-tion of diploid H lines obtained here was verymuch higher than after induction in presence ofauxin (34%) (Table 1). This confirmed the strongimplication of the auxin in polyploidisation. How-ever, it also proved that ploidy changes couldoccur in the absence of exogenous hormone, al-though they were less frequent.

4. Discussion

We examined the main characteristics of habitu-ated embryogenic lines isolated from eleven geno-types of asparagus. Cytological analyses done onsome of the H lines revealed that they grewthrough secondary embryogenesis. Secondary em-bryogenesis was direct and began with two succes-sive transversal divisions of an epidermal orsubepidermal cotyledonary cell leading to the for-mation of a linear four-celled proembryo (Figs. 3

Fig. 5. Typical DNA histograms of BET-stained nuclei extracted from embryogenic lines. Nuclei from a tomato leaf were usedas a DNA standard. (A) DNA histogram from cladophylls of a diploid A4 asparagus plant. (B) DNA histogram of the diploidH line 186L1. (C) DNA histogram of the diploid H line A4L1. (D) DNA histogram of the tetraploid H line 81AL2. 2C T, 4CT: peak of 2C and 4C tomato nuclei. 2C A, 4C A, 8C A: peak of 2C, 4C and 8C asparagus nuclei.

Table 2Ploidy level of embryogenic lines determined by flow cytometrya

Ploidy level Total

4n2n 6n Aneuploid

Number of lines 19 (59%) 1 (3%) 1 (3%) 3211 (34%)

a The H lines were 1 year old.

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Table 3Ploidy level of the plants regenerated from embryogenic linesa

Ploidy level of the embryogenic linesGroup Number of regenerated plantsEmbryogenic lines

2n 4n

2n 10 –1 186L12n 2A3L3 –

8L1 4n – 14n – 1A3L2

2n 72 10A4L1

3 CO1L1 4n 1 –4n 1 –A2L14n 781AL2 5

Total 28(62%) 17 (38%)

a H lines are classified in three groups. Group 1: regenerated plants showed the same ploidy level as the callus line. Group 2:some of the regenerated plants showed a higher ploidy level than the callus line. Group 3: some of the regenerated plants showeda smaller ploidy level than the callus line.

and 4). Similar linear four-celled proembryos werealso observed in Freesia refracta embryos culturesafter two successive periclinal divisions of subepi-dermal cells [34]. Globular embryos developed thenrapidly from the primary embryo, disrupting theepidermis. In some of the H lines, the emergence ofsecondary embryos was only occasional on thesurface of primary embryos (Fig. 3F). In other Hlines (CO3L1 for example), which exhibited anextreme intensity of secondary embryogenesis, sub-cultured embryos became rapidly covered by apalisade of globular embryos, resulting in the com-plete disorganisation of the epidermis (Fig. 3G, Fig.4D). In this case H lines presented a high ratio ofglobular and bipolar embryos to mature embryos(Fig. 2). These lines were generally difficult toconvert into whole plants contrary to the less activeH lines. Whatever the intensity of secondary em-bryogenesis of the lines, their growth kinetics wereabout the same but the extreme H line CO3L1displayed hastened senescence (Fig. 1).

Most of the habituated embryogenic lines heredepicted can regenerate whole plants through so-matic embryo conversion and the derived plantsshowed a high embryogenic character, i.e. apicescultures regenerated somatic embryos then H lineswithout any auxin treatment [15]. For the H lines8L1, A3L3 and A4L1, this character was governedby a dominant mutation [15,16]. Consequently,habituation which is a remarkable characteristic ofthe embryogenic lines described here is closelyrelated to the embryogenic mutation. Habituation

has been defined as a heritable loss in the require-ments of cultured plant cells for exogenous growthhormones, which can originate from epigenetic aswell as genetic changes [35]. It could be attributedto an increased biosynthesis of the growth sub-stances, a decrease in their rate of degradation, analtered sensitivity of the cells to the growth sub-stances or an interaction of some or all of these[35,36]. Comparing the hormonal composition of Hline calli, no correlation between intensity of sec-ondary embryogenesis and hormone levels could beestablished, except in term of cytokinin because thelevel of iPA was significantly increased in theextreme H line CO3L1. The ribosides of cytokininare very active in bioassays [37]. The abundance of

Table 4Ploidy level of habituated embryogenic lines reisolated fromdiploid high embryogenic plants, without hormone treatmenta

Number of embryogenic linesPlant

2n 4n

A4L1 n°1 6 1A4L1 n°2 3 181AL2 n°1 8 –81AL2 n°2 4 –

21 (91%)Total 2 (9%)

a Nodes or apices from diploid plants regenerated from theH lines A4L1 and 81AL2 were cultivated on basal hormone-free medium where they produced new embryogenic lineswhose ploidy level was examined. A4L1 n°1 is a diploidplants regenerated from the embryogenic line A4L1.

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iPA could therefore explain the very high mitoticactivity of line CO3L1 illustrated in Fig. 3G. Thehigh level of iPA was correlated with normal levelof cytokinin bases and ribotides (Table 1). Refer-ring to cytokinin pathways [37], this could indicatea normal interconversion metabolism and allowspostulating a defect in the oxidation side chaincleavage by cytokinin oxidase.

No significant differences in hormone contentbetween cladophylls of wild type and cladophyllsof mutant plants could be identified. This is notvery surprising, as mutant plants were morpholog-ically indistinguishable from wild type plant. Inthe same way, no significant hormonal differencesappeared between mutant and wild type meristemscultured for 0, 7, 14 or 25 days when somaticembryos began to appear on the A4L1 mutantexplants. Consequently, the expression of the mu-tation was not correlated with a notable modifica-tion of the hormonal economy of the explantedapices. Because of difficulties in embryo conver-sion the mutant status of the H line CO3L1 hasnot been confirmed even if probable. Because ofthe different hormonal status of this H line incomparison with the others we can infer that thehabituated and embryogenic character have prob-ably different origin (and could be controlled bydifferent mutations?) affecting either hormonalmetabolism or sensitivity. Rare attempts havebeen made to associate the embryogenic compe-tence with endogenous hormone levels. InMedicago falcata the embryogenic capacity waspositively correlated with IAA level and negativelywith ABA level but not with cytokinin level [26].In wheat, the embryogenic capacity was related toa low IAA and a low ABA content and increasedcytokinin level [27]. In Dactylis glomerata no sig-nificant differences were found in the endogenousIAA levels of embryogenic and non-embryogenicgenotypes but cytokinin decreased in the latter[38]. Therefore the relation between embryogeniccapacity and hormone content is very dependenton the species. In asparagus, the embryogenicmutation A4L1 seemed not to be related to hor-mone metabolism.

Habituated embryogenic lines isolated afterNAA treatment were only 34% diploid 1 yearlater, after 11 subcultures on basal medium, theothers being tetraploid. The polyploidisation pro-cess clearly slow down during the following yearof subculture. The ploidy level of plants regener-

ated from the 1 year old H lines was foundvariable and not always correlated with the ploidylevel of the corresponding H lines (Table 3). Incase of the diploid A4L1 line which regenerated ahigh proportion of tetraploid plant it is clear fromits DNA histogram of BET-stained nuclei (Fig.5C) that the tissues contained a significant ratio oftetraploid nuclei. That tetraploid lines regeneratedfrequently diploid plants (Table 3) was more sur-prising, because generally no peak correspondingto the 2C ploidy level could be detected on theDNA histogram of BET-stained nuclei of the lines(Fig. 5D). Probably a few diploid cells are presentin H lines which appeared favoured through theregeneration process as described before in em-bryogenic culture of peal millet [39]. However, theploidy level of regenerated plants was generallyunpredictable only considering the ploidy level ofthe corresponding H line, except for the H line186L1, which seemed homogeneously diploid. Oneway to stabilise the diploid level of H lines couldbe to reinitiate them from apices or nodes ofregenerated plants on auxin free medium. In thislast case, more than 90% of the new H lines werediploid (Table 4) in comparison with the 34%obtained after NAA induction of the H lines.Reports on ploidy level in asparagus plants regen-erated from somatic embryos are conflicting inliterature, varying from diploid [17,19], tetraploid[18] to a mixture of diploid and tetraploid plants[20]. All the regenerated plants studied came fromembryogenic lines generally obtained through avisual selection on an auxin-containing medium. Ithas been shown [16] this selection process allowedin fact the obtaining of H lines and of auxin-de-pendent embryogenic lines. The diversity of theploidy status of the H lines here presented proba-bly explained the diversity of results reported inliterature. Obtaining exclusively diploid plantsthrough somatic embryogenesis in asparagus needprobably the use of stable diploid embryogeniclines, which appear rather rare.

Acknowledgements

The authors thank Jean-Marie Pollien andKrystina Gofron for care of plants and Dr IanSmall for English correction. The work was sup-ported by the CIFRE (Convention Industrielle deFormation par la Recherche) grant n°542/96.

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