Bulletin de la Société Botanique de France.

Lettres
Botaniques

ISSN: 0181-1797 (Print) (Online) Journal homepage: www.tandfonline.com/journals/tabg19

In vitro culture of the olive tree (Olea europaea L.):

present aspects and prospects

Luis A. Cañas

To cite this article: Luis A. Cañas (1988) In vitro culture of the olive tree (Olea europaea L.):
present aspects and prospects, Bulletin de la Société Botanique de France. Lettres Botaniques,
135:3, 263-277, DOI: 10.1080/01811797.1988.10824802

To link to this article: https://doi.org/10.1080/01811797.1988.10824802

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BuU. Soc. bot. Fr., 135, Lettres bot., 1988 (3), 263-277.

In vitro culture of the olive tree (Olea europaea L.)

present aspects and prospects ( * l

by Luis A. CANAS

Laboratoire de Biologie cellulaire, Faculté de Pharmacie

4 avenue de l'Obseroatoire, F-75270 Paris Cedex 06

Summary.- Short- and long-term objectives for research on tissue culture of the olive tree
(Olea europaea L) are described by the author in this paper. Severa! procedures to establish in vitro
cultures of the olive were used with interesting results in plant regeneration, from different expiant
sources and cultivars. In vitro culture methods, as vegetative propagation from olive naked embryos,
«meristem>> cultures (axillary bud stimulation and shoot proliferation), shoot rooting, callus culture,
plant regeneration, isolation, culture and division of olive protoplasts are described in a first section.
Future prospects for genetic improvement of this tree, a crop species of high economie relevance in
the Mediterranean countries, are discussed in the second section of this paper.

Résumé.- Les objectifs à court et à long terme dégagés par la recherche sur la culture in
vitro de l'olivier (Olea europaea L) sont décrits par l'auteur dans cet article. Quelques méthodes de
micropropagation in vitro à partir de différents ex plants et cultivars d'olivier ont été testés avec, dans
certains cas, des résultats encourageants. Dans la première partie sont décrites différentes méthodes
telles que la propagation végétative à partir d'embryons isolés, de culture de «méristèmes» (stimu-
lation de bourgeons axillaires et prolifération de tiges). enracinement de tiges, culture de cals, régéné-
ration de plantes, isolement, culture et division de protoplastes. Dans la deuxième partie, sont abor-
dées les perspectives de l'amélioration génétique de l'olivier, espèce dont l'importance économique
pour certains pays du Bassin Méditerranéen est considérable.

Key words : Olea europaea L., in vitro propagation, cali us culture, plant regeneration, proto-
piast culture.

Abbreviations.- IBA : indol-3-butyric acid ; 2ip : 2-isopentenyladenine ; GA3 : gibberel-

lic acid ; lAA : indole-3-acetic acid ; BN : Bourgin and Nitsch medium, 1967 ; NAA : naphthalene
acetic acid ; 2,4-D : 2.4-dichlorophenoxyacetic acid ; FDA : fluorescein diacetate ; GSH : reduced
gluthation.

(*)Conférence prononcée le 23 octobre 1987.

264 LETTRES BOTANIQUES

INTRODUCTION

The olive tree (Olea europaea L.) is a crop species of economie relevance within
the Mediterranean Basin. V arieties of the olive are cultivated mainly for oil (93 %) and
also for table olives (both black and green). There are very few dual-purpose varieties
but nonè of the existing ones are at ali satisfactory. Olive oil ranks six th in world pro-
duction of fluid vegetable fats, preceded by soya, peanut, sunflower, colza and cotton
seed oils. However, it is still first for its taste, its nutritional qualities and its use in diet
therapy. The 97 % of world olive production is supplied by the countries of the Medi-
terranean Basin (FAO Production Yearbook, 1981).
The classification of the family Oleaceae, to which Olea europaea L. belongs, is
not at ali clear. This species belongs to one of the 30 species of the genos Olea and is
known to have a large numher of varieties with a genome of 2n = 46 chromosomes.
The olive longevity is due to the presence of excrescences ( «<vuli») at the base
of the trunk which are rich in adventitious buds capable of giving rise to new branches
(suckers) and roots with a high degree of juvenility. So, the olive has been propagated
vegetatively by rooted suckers or «ovuli» for thousands of years and later by grafting
and cutting.
Modern genetic methods to improve the olive tree have been neglected due to
the seant information on its physiology and genetics, that, in turn, are difficult to
study due to the slow development of the plant and its long juvenile phase (10-15
years). Up to now, both clonai and mass selection of progeny have not given satisfacto-
ry results. Unfortunately, very little work has been done in the area of in vitro micro-
propagation of this tree (recalcitrant species), but recently sorne methods to obtain olive
plantlets from different explants have been devised and a new culture medium (OM)
formulated by comparing data from analysis of the main mineral elements found in
olive shoot-tips and embryos (for review see Rugini, 1986).
The existing olive cultivars ali have low productivity and the difficulties so far
encountered in an attempt to improve them with traditional methods of cross-breeding
necessitate research on alternative solutions to this problem. Several breeding program-
mes are currently interested in the development of a methodology for producing highly
productive hybrids through genetic engineering or protoplast fusion, drought and salt
resistant plants by stress-selection, olive plants with higher photosynthetic rates, homo-
zygous plants from anthers culture, new olive cultivars through induced mutation,
cryopreservation of germplasm from the numerous existing cultivars, production of new
cultivars via somatic embryogenesis and production of disease-free plants by thermothe-
rapy or in vitro culture of olive shoot-tips.
At present, we are interested in studying the molecular biology of the olive and
its genetic transformation, via T-DNA from Agrobacterium. Therefore, the in vitro
multiplication methods and the isolation of olive protoplasts are essential for our pur-
pose. ln fact, the absence of cell walls around protoplasts provides an opportunity to
introduce foreign genes into the plants by direct transfer methods, moreover protoplasts
are an useful tool for the isolation and purification of nuclear DNA and cytoplasmic
organelles such as chloroplasts and mitocondria.
 L. CANAS 265

I.- PRESENT ASPECTS

Vegetative propagation from in vitro cultured mature embry os

Germination of olive seed is, under field conditions, a slow and rare event,
impractical for the propagation of the plant. It has been suggested that the presence of
inhibitors in the endosperm of the seed prevents the germination of the embryo until
their inactivation by the low winter temperatures (Diamantoglou and Mitrakos, 1979).
Istanbouli and Neville (1977) found emhryonal dormancy in olive, which disappeared
gradually during seed conservation in a dry place. They further noted that olive emhryos
germinated hetter in the light than in the darkness at 18-20°C.
As olive has a long germination period, the in vitro germination of isolated em-
hryos should provide a method to obtain plantlets within a reasonahle time, thus
being a valuahle tool for the establishment of in vitro cultures of the olive suitable for
further manipulations in genetic improvement programmes.
At our lahoratory, concerning in vitro culture of olive embryos, peeled seeds
(without pericarp) of several Spanish cultivars (Bical, Cornezuelo, Gordal, Lechin,
Manzanillo, Marteno and Picual) and French cultivars (Picholine, Tanche, Cailletier and
Lucques) were surface-sterilized for 2 min. treatment with 70 % ethanol followed by
stirring in 20 % solution of commercial hleach (1.2 %active sodium hypochlorite) plus
sorne drops of Tween 80 during 15 min. After rinsing three times with sterile distilled
water, they are placed on a double layer of sterile filter paper inside a lOOmm glass
petridish. Five ml of sterile distilled water were added, the rims were sealed with pa-
raffin film and the dishes were placed overnight at 23 ± l°C in darkness. Then the
embryo was removed from endosperm by a razor blade onder sterile conditions in a
laminar flow hood. Isolated embryos (fig. 2A) were placed on top of germination
medium (OMg = l/20M proliferation medium without phytohormones) and cultured
in a growth chamber with cool white fluorescent tubes at 16 h light per day (2.5klx)
and 23 ± l°C of temperature (Canas et al., 1987).
The first visible sings of germination in OMg medium were the divergence and
greening of the cotyledons simultaneously to the elongation of the rootlet after one
week in culture (fig. lA). Shoots with visible buds and leaves were apparent at day 30
(fig. lB). Between days 45 and 50, depending on the individual embryo, plantlets
were already grown, their stems measuring around 3cm and their roots from 10 to
l2cm. Germination of embryos occurs at a higher frequency when fruits of the same
season are used as source. A yield of 40 % viable plantlets, depending on the cultivar,
was obtained from these embryos as middle term.
After two months of aseptic culture, seedlings were transplanted to soil condi-
tions (fig. lC) onder controlled environmental conditions in a transparent plastic
chamber, because they were greatly sensitive to dehydration. Plant survival was about
70-80% when olive plantlets were transferred to pots in a greenhouse.

«Meristem» cultures: axillary bud stimulation and shoot proliferation

In the olive, various attemps to culture meristems from field-grown or green-
bouse plants were unsuccessful, due to rapid oxidation after collection. Neverthe-
less, meristem culture was possible hy collecting the meristems from stabilized shoot
in vitro. Meristem culture is often used to obtain disease-free plants.
Rapid hud sprou t, node formation and shoot elongation with very small basal
callus were obtained when uninodal leafy explants (fig. lF) were soaked for 10 min.
266 LETTRES BOTANIQUES

in a filter-sterilized solution of GA 3 (50-l00mg.r 1 ) before planting in OM prolifera·

tion medium (table l) supplemented with 4mg.r 1 zeatin or 2iP.
Uninodalleafy explants were obtained from in vitro cultured seedlings or from
semiaseptic cultured wvuli». For this purpose, surface sterilized wvuli» (50-lOOg/
fresh wt), collected from December to March, were cultured for l month in a tray with
moist vermiculite (65 % relative humidity) at 25 ± l°C and l6h light (21Wm"2) per
day (fig. ID and E). Moreover, olive as strong apical dominance and from one unino·
dai expiant it is possible to obtain only two shoots (fJg. IF).

Root induction and elongation

Bi- or trinodal explants, obtained from stimulation of axillary buds, were used
for rooting phase.
Root induction of excised shoots was performed in 160 x 25mm glass tubes
with OMr medium (table l) supplemented with lmg.P IBA or NAA. When the first
roots began to appear in the base of the explants, they were transferred to a root elon·
gation medium (OMe, table l) in which the auxin was removed and replaced by zeatin
riboside or 2iP. The aerial part of the plantlet was kept under l6h light (21 Wm" 2 ) per
day at 23 ± l°C, while the developing radicular system was maintained completely in
darkness, by covering the surface of the medium with a sterile black cardboard mask
(previously boiled to avoid bleeding of the pigment) with a central hole to insert the
expiant, and by wrapping the outside in aluminium foi! up to the medium leve!.
For rooting to excised shoots, IBA was more active (90 % of rooting) followed
by NAA (70 %) and lAA (20 %) both in Spanish and French cultivars tested (Canas
et al., 1987), white in the case of ltalian olive cultivars, NAA was more active (80% of
rooting) followed by IBA (50%) and lAA (20 %) both in woody explants, embryogenie
material and mature shoots (Rugini, 1986).
When the excised shoots were transferred to OMr medium, long and thick roots
developed resulting in plantlets (fig. IG). The first roots began to appear on the base of
the explants after 8-10 days of culture. At this date, the plantlets were transferred to
a root elongation medium (OMe) that allowed rapid root elongation and subsequently
rapid growth restart, which is also inhibited by prolongated contact of the explants
with auxins. The use of a black cardboard mask and aluminium foil, for maintenance
of clark conditions in the developing radicular system, give a better result than the active
charcoal, which produces growth inhibition by adsorption of the hormones present in
the culture medium and this hecomes inactive. Dark was not necessary for root induc-
tion phase.
The rooting percentage of sorne olive cultivars may he increased hy inoculation
of the hase of the shoots with Agrobacterium rhizogenes (strains A4 and 1855) followed
by culture on a half-strengh OM hormone free medium. After 10-15 days, roots hegan
to appear in more than 50 % of the explants, but induced roots were always agropine-
negative and their genetic transformation has not been confirmed up to now.

Callus culture and regeneration to plants

Multiple shoot cultures have heen used most extensively for tissue culture pro·
pagation of woody plants, but up the date differentiation from callus is only possible
with a few numher of woody species. On the other band, transfer of in vitro regenerated
plants of Gimnosperm tree species to soit, is quite often unsuccessful, as sometimes
there is not viable vascular connection between roots and shoots. Also, somatic
 L. CANAS 267

1
Table 1.- Composition of olive media* (in mg.f )

Compounds OM! Or.lcli OMr O~le OMp

KN0 1100 950 550 275 950

3
NH N0 412 720 206 103 720
4 3
Ca(N0 J .4H 0
3 2 2
600 --- 300 150 ---
CaCl<!.2H 0 440 166 220 110 166
2
KC1 500 --- 250 125 ---
MgS0 .7H 0 1500 185 750 375 185
4 2
KH P0 340 68 170 85 68
2 4
FeS0 .7H o 27.8 27.8 13.9 6.95 27.8
4 2
Na EDTA 37.5 37.5 18.75 9.38 37.5
2
MnS0 .4H 0 22.3 22.3 11.15 5.58 22.3
4 2
H ao 12.4 12.4 6.2 3.1 12.4
3 3
znso .7H o 14.3 14.3 7.15 3.58 14.3
4 2
Na Mo0 .2H 0 0.25 0.25 0.13 0.06 0.25
2 4 2
CuSO .5H o 0.25 0.25 0.13 0.06 0.25
4 2
CoC1 .6H 0 0.025 0.025 0.013 0.006 0.025
2 2
KI 0.83 0.83 0.42 0.208 0.83
Myo-inositol 100 100 --- 50 100
Glycine 2 2 --- 1 2
Thiamine.HCl 0.5 0.5 --- 0.25 0.5
Pyridoxin.HCl 0.5 0.5 --- 0.25 0.5
Nicotinic a cid 5 5 --- 2.5 5
Biotin 0.05 0.05 --- 0.05 0.05
Folie acid 0.5 0.5 --- 0.25 0.5

Glutamine 2194 --- --- 550 500

Case in hydroly_sate --- 1000 --- --- ---
Zeatin 4 0.2-0.5 --- 1-2 0.5
II3A --- 5 1 --- ---
Suc rose 30000 20000 20000 20000 2000
Agar 6000 6000 7000 6000 ---
pH 5.8 5.8 5.8 5.8 5,7
(•)~ Olive proliferation medium. Rugini,1984.
( !> l~ Gr·l wi th BN macroelements,

*All media used in our experiments were adjusted to pH 5.8- 5.7 before auto-
claving at 115°C for 20min and thermolabile substances were added filtere~
268 LETTRES BOTANIQUES

embryogenesis in woody plants is not yet far enough advanced to guarantee genetic
stability of regenerated plants.
CaHus culture, cell suspensions and regeneration to whole plants are necessary
for genetic improvement via genetic engineering, protoplast fusion or selection for
resistance. ln the case of the olive tree, there is little literature on in vitro culture of
calli obtained from different expiant sources and cultivars (for review, see Rugini,
1986), and there is not a reference up to date about the use of cotyledonar pieces for
this purpose. only from apical twigs callus, shoot and root differentiation were obtai-
ned, but complete plant regeneration was not reported (Wang et al., 1979). Olive caHus
goes through three developemental stages : activation, division and formation, each
characterized by changes in ceU morphology, cell division, growth and relative RNA
acumulation according to these authors.
Callus initiation and maintenance, using both embryonic and mature material,
were achieved by Rugini in BN medium supplemented with 2,4-D (O.l-5mg.r 1 ) alone
or in combination with zeatin riboside (O.l-0.5mg.r 1 ), in the dark or in the light.
GSH (500mg.r 1 ) was added in the medium to prevent cali us browning produced by
the auxin, white was not necessary when NAA or IBA were used as auxins. In view of
callus initiation from cotyledon fragments, we have observed that the addition to OMc
medium (table 1) of a high concentration of auxin (5mg.r 1 IBA) and a low concentra-
tion of cytokinin (0.2-0.5mg.r 1 2iP or zeatin riboside), stimulate ceU division and rapid
callus development, but after 3 weeks of culture, the callus tissue grew poorly and its
1
growth was very much improved when subcultured on OMc medium with l-2mg.r
2
IBA and the same concentration of 2iP or zeatin riboside, in the light (1.5Wm" , 16h
per day) at 23 ± l°C.
In order to direct further developemnt and obtain calli bearing morphogenesis,
cotyledon-derived calli were transferred to OMc medium with different levels IBA al one
or in combination with 2iP (table 2). On OMc medium, lmg.r 1 of IBA alone induced
the maximum frequency of root differentiation (fig. 2B) and shoot induction was
greater when this medium was supplemented with 4mg.r 1 2iP (fig. 2C). A low concen-
tration of 2,4-D (O.lmg.r 1 ) plus zeatin rihoside (O.lmg.r 1 ), also stimulated shoot
formation occasionally in cotyledons cultured in the dark. Both shoot and root diffe-
rentiation from callus were obtained with different combinations of IBA and 2iP
(table 2).
In general, shoot generation percentage was higher in calli from cotyledon seg-
ments proximal to the embryo axis than the distal ones (table 3) both in cv. Tanche and
Picual. This result suggests that a gradient of regeneration potential exists from the
proximal to the distal region of the olive cotyledon. Whole plantlets were obtained
when the regenerated shoots were stimulated to produce adventitious roots on OMr me-
dium with lmg.r 1 IBA or NAA. After root elongation on OMe medium without auxin,
plantlets were transferred to peat and soil conditions with a great rate of plant survival
(75-80 %) (fig. 2D, E and F).
The phenotype variation among plants regenerated from callus culture was
reproted in several species which showed genetic variability after regeneration. ln our
case (Canas and Benbadis, 1988), results from morphometric measurements indicate
that there is a certain variability in the regenerated plants when compared with
seedlings. ln the olive tree, the normal phyHotaxis is opposite decussate, but on vigo-
rous suckers, more complicated phyllotaxis may occur (for instance, verticils of 3
 L. CANAS 269

Table 2.- Response of olive cotyledon calli to different levels of 2iP and/or IBA on OMc medium

Levels of hormone cal li!

% of total forming:
-1
tested in mg.l

cv. Tanche cv. Picual

Cal lus Roots Shoots Callus Roots Shoots
0.5 2iP 89 -- 11 91 -- 9
1.0 2iP 90 -- 10 90 -- 10
2.0 2iP 88 -- 12 91 -- 9
4.0 2iP 84 -- 16 86 -- 14

0.5 IBA 48 52 -- 62 38 --
1.0 IBA 33 67 -- 48 52 --
2.0 IBA 49 51 -- 57 43 --
4.0 IBA 64 36 -- 60 40 --
1.0 IBA + 2.0 2iP 90 -- 10 92 -- 8
1.0 IBA + 1.0 2iP 83 13 4 80 14 6
2.0 IBA + 1.0 2jP 87 10 3 85 13 2

(! ) = 90-100 call i for each treatment.

Table 3.- Percentage distribution of total morphogenetic calli in proximal and distal parts to em-
bryo axes

Levels of hormone % of calli forming shoots.

-1
tested in mg.l cv. Tanche cv. Picual

Proximal Distal Proximal Distal

----
0.5 2iP 54.5 45.5 55.5 44.5
1.0 2iP 50 50 50 50
2.0 2iP 58.3 41.7 55.5 44.5
4.0 2iP 75 25 78.5 21.5

1.0 IBA + 2.0 2iP 70 30 62.5 37.5

1.0 IBA + 1.0 2iP 50 50 66.6 33.4
2.0 IBA + 1.0 2iP 33.3 66.7 50 50
270 LETTRES BOTANIQUES

or 4 Ieaves) in which sorne leaves are double (Espagnac and Neville, 1969). The presence
or absence of these rare characters or anomalies in olive regenerated plants were chosen
to measure carly plant variability and after comparison and quantification by the simili-
tude coefficient of Sokal and Michener (Hideux, 1977), our results show that the cv.
Tanche has a greater variability than cv. Picual (from 0 to 1 and from 0.33 to 1 respecti-
vely). However, morphological changes frequently occur in seedlings and shoots from
trunk and big branch buds of plants in the field. They are probably associated with juve-
nility, so that they disappear in the adult plants. Since it is impossible to observe our
future adult plants, we can only conclude that this kind of morphological changes tends
to increase under caHus culture conditions (fig. 2D and E).
For this reason, a complete study of ploidy levels in regenerated plants is now in
progiess to determine if there is a relationship hetween morphometric variability and
ploidy levels, and to check if aneuploidy is present in these plants. Unfortunately, up
to date, there are not reports concerning chromosome analysis from olive plants regene-
rated in vitro, therefore it will be necessary to improve this technique in the case of
Olea europaea L. because this species has a genome with a large number of chromo-
somes (2n = 46).
On the other hand, sorne approaches for production and maintenance of olive
cell suspension cultures have been achieved by Rugini, using micropropagated leaves
and stems of several Italian cultivars, in an autophyton with BN medium supplemented
with zeatin riboside (O.lmg.r 1 ) plus 2,4-D (1mg.r 1 ) or IBA (5mg.r 1 ), in the light or
in the clark. When the auxin NAA (l-5mg.r 1 ) is used, the cells are smaller and overfed
but soon they break up. Preliminary studies have shown that this phenomenon is avoi-
ded by substituting BN medium with MS medium supplemented with more calcium,
magnesium and sulfate, and drastic reduction of NH~ (Rugini, 1986).

Transfer of plants to peat and soil conditions

Olive plantlets obtained from isolated emhryos, axillary buds or callus culture
were more sensitive to dehydration as compared to other woody species, when trans-
planted from in vitro to soil conditions.
Plantlets is plastic pots containing a 1 : 1 mixture of sand and peatmoss were pla-
ced in a transparent plastic chanher under controlled environmental conditions for one
month. Surviving plants (75-80 %) were then transferred to soil in a greenhouse and a
solution of GA 3 (300mg.r 1 ) was sprayed on the leaves to remove initial dormancy and
stimulate growth restart.
At present, several plants from in vitro cultures of cv.Tanche and cv.Picual are
ready for transplanting in the field, but the plants still retain their characteristic juvenile
appearance.

Isolation, culture and division of olive protoplasts. Developement of protoplast-derived

callus
Many reports exist on plant regeneration from protoplasts of herbaceous plants.
However, relatively few attempts have been made on the culture of protoplasts from
woody species (the fruit trees in particular) and most of these studies are preliminary.
the only arborescent species, in which plant regeneration from protoplasts have heen
reported, are Citrus, Santalum, Broussonetia and Pyrus (V ardy et al., 1982 ; Rao and
Ozias--Akins, 1985; Oka and Ohyama, 1985; Ochatt and Caso, 1986).
 L CANAS 271

Viable olive protoplasts from in vitro cultured leaves and cotyledons were obtai-
ned after overnight incubation in an enzyme solution containing 1-1.5 % driselase,
3.5mM CaCh and 0.5M sucrose, pH adjusted to 5.6. this combined method allowed
high yield of purified protoplasts, which floated and formed a dark green band at the
meniscus after centrifugation at 80xg for 5 min. Results on protoplast yield, viability,
cell wall regeneration and cell division are summarized in table 4.

Table 4.- Comparison of yield, viability, cell wall regeneration and cell division of protoplasts
from leaf mesophyll and green cotyledon of Olea europaea L. cv.Tanche.

Leaf mesophyll Green cotyledon

6 6
Yield (ppts.g/fresh wt) 2.3-2.7 x 10 1.4-1.6 x 10
Viability! (%) 78-83 87-91
Cell wall regen. (%) 46-51 61-68
Cell division~(%) 5.2-6. 3 8.7-9.8

(i) After 48h in culture by FDA method.

4
(9) Protopla~ts density: 3x10 ppts.ml-
1

Size of freshly isolated protoplasts ranged from 40 to 60 f.J.m in cotyledon proto-

plasts and from 30 to 45 f.J.m in leaf protoplasts. The cotyledon protoplasts varied consi-
derably with respect to their number of chloroplasts, from none (white protoplasts), to
very high density (green protoplasts), reflecting the variety in cell origin (fig. 3A).
Protoplast variability was measured after 48h in culture medium by the FDA
method, moreover olive protoplasts showed an endogenous fluorescence when were
observed under UV light (chloroplasts exibited red chlorophyll autofluorescence and
cytoplasm blue fluorescence, both in leaf and cotyledon protoplasts).
Purified protoplasts of various plating densities (104 to 10 ) were cultured in a
thin-layer of OMp culture medium (table 1) supplemented with 0.5mg.r 1 2,4-D, 1mg.r 1
NAA, 91 g.r 1 mannitol and 0.03 mM ornithine.
Cell wall regeneration was proved by Calcofluor White staining and by the chan-
ge of round protoplasts to oval or elongated cells (fig. 3B) after 4 days of culture in
darkness. Cell wall was synthesized in a large number of leaf and cotyledon protoplasts
(table 4), but the regenerated cells failed to divide when the plating density was below
2.5 x 104 ppts.ml- 1 • The results, obtained from 6 experiments, demonstrated that the
optimum plating density for cell division and proliferation was 3 x 104 ppts.mr 1 •
After cell wall regeneration, olive cells were cultured at low light intensities (1.5 Wm- 2 ,
16h per day) at 23 ± l°C and two weeks later, the culture medium was gradually dilu-
ted. Cell divisions were detected after 15 days of culture, both in leaf and cotyledon
protoplasts (fig. 3C), while cell clusters were obvious at approximately 4 weeks (fig. 3D).
Microcolonies from leaf protoplasts stopped their growth in OMp diluted medium
(OMp without auxins and mannitol and supplemented only with 0.2mg.r 1 zeatin) when
272 LETTRES BOTANIQUES

they were formed by 40-50 cells. However, cell colonies from cotyledon protoplasts
developed visible micocalli (1.5-2mm in diameter) after 6 weeks of culture. It is possible
that this difference hetween leaf and cotyledon protoplasts-derived cells arise from the
difference observed in viahility, cell wall regeneration and cell division at the heginning
of their culture or from the culture medium used, which is unfavourahle for leaf micro-
colony development.
When the microcalli were transferred to OMc agarose medium (OMp hasewithout
mannitol, supplemented with 0.5mg.l" 1 zeatin, 2.5mg.l" 1 IBA or NAA and solidified
with 0.6 % (w/v) Sea Plaque Agarose LMT) approximately 60-70% of transferred mi-
crocalli were able to survive and two kinds of active growth calli were induced : white
friable calli (12 % of total formed calli) and green compact calli (fig. 3E). White friable
callus also grew rapidly on OMc medium without phytohormones (Habituated callus).
Green calli grew more slowly than white calli on OMc agarose medium with
phytohormones and showed several organized structures after cytohistological studies.
For this purpose, the calli were fixed in AFA (absolut ethanol : formaldehyde : acetic
acid, 6 : 3 : 1), whased in 60 % ethanol and dehydrated in ethanol-xylene series. Small
fragments were included into paraffin, sectioned on a microtome and stained with Sa-
franin-Orange G.
Green calli showed superficial proliferation and unorganized parenchima-like
cells, when subcultured on OMc medium supplemented only with 0.1mg.r 1 zeatin
riboside and 0.1mg.r 1 2,4-D (callus maintenance medium). When green calli were
transferred to OMc agarose with 1mg.r 1 zeatin alone, in order to direct further deve-
lopment and induce morphogenesis, they developed sorne protuberances with white
globular shapes in their surface, which remind certain emhryoid or shoot primordia
forms. In addition, histological studies showed epiderm formation (fig. 3F) in the outer
region of the callus and sorne scattered tracheids or vascular bundles (fig. 3G) were
differentiated in the inner region. At the same time, vascular tissue nodules with tra-
cheids surrounded by cambium-like cells (fig. 3H) were observed in the same region.
These meristematic nodules were also detected in green calli cultured on OMc medium
with 1mg.r 1 NAA alone in a greater proportion, and they have been reported to be the
origin of root primordia in olive apical twigs-derived calli (Wang et al., 1979). In our
case, organogenesis from this type of nodules was not observed up to date. Also, no
results were obtained when the same conditions used for plant regeneration from coty-
ledon-derived calli were tested.
Having established a standard procedure for isolation, culture and division of
olive protoplasts (Canas et al., 1987), we are now engaged in a project to define the
factors governing the development of cell colonies, and the conditions under which
plant regeneration from protoplast-derived callus would be possible.

II.- CONCLUSIONS AND PERSPECTIVES

Genetic improvement of the olive trec (Olea europaeaL.) by traditional methods

has still not given satisfactory results, possihly due to the low productive capacity of the
existent cultivars characterized by a low photosynthetic efficiency.
The use of tissue culture methods could open new prospects for its improve-
ment, but until now research has yielded only preliminary results. This is partly due to
the fact that olive is a woody species in which morphogenesis, a condition necessary for
 L. CANAS 273

genetic improvement programmes, is still difficult to induce, except in certain geneti-

cally predisposed lines, and almost invariably not beyond the juvenile phase. This factor
makes difficult to obtain plants with specifie resistance and haploid plants (Rugini,
1986).
Good prospects for improvement of the species are more likely through the sti-
mulation of axillary huds. The use of ionizing radiations, during the proliferation phase,
could he a promising means, both for the induction of mutations and for isolation
of mu tated parts.
On the other hand, the olive is attacked by numerous parasites, as fungi (Cyclo-
conium oleaginum, Gloesporum olivarum, etc.), bacteria (Pseudomonas syringae pv.
savastanoi, which produces «olive knots» on the branches), virus (Nepovirus), nematho-
des (Meloidogyne incognito and javanica, Paratylenchus vandenbrandei, peraticus and
vulnus, etc. which are virus carried in sorne cases) and insects (Dacus oleae, Saissetia
oleae and Prais oleae ). Therefore, another objective is the possibility of obtaining
disease-free plant in the near future, using the apical meristems from in vitro-cultured
shoots in combination with thermotherapy. In this way, wa are now interested in the
development of basic molecular genetics and other biotechnological techniques to mani-
pulate this plant. This is a long-term project in which several groups are cooperating.
The specifie lines of this project are :
• development of genetic vectors able to transfer genes into Pseudomonas sy-
ringae pv. savastanoi, combining the ability of this bacterium to infect easily the olive
tree and the DNA transfer properties of Agrobacterium. As a first step the expression of
sorne Agrobacterium vir functions in P. savastanoi will be studied. ln addition, a bacte-
riophage (Nu) able to lysogenize P. savastanoi has been studied (Canas and Santaolalla,
1986) ; its use as a vector is precluded by its loss of infectivity under laboratory condi-
tions.
• cloning and analysis of markers selectable both in plants and hacteria (iaaMH
genes from P. savastanoi, Cornai and Kosuge, 1980).
• in vitro culture of the olive trec (micropropagation, protoplast culture and
plant regeneration from protoplast-derived callus.
• genomic genotheques, as weil as eDNA ones from fruits, isolated at different
stages of ripening, are being constructed to identify genes that are expressed of the fruit
drastically change and are mainly directed towards the synthesis of fatty acids. A detai-
led knowledge of the expression of such genes is likely to be crucial for future hiotech-
nological manipulations of this trec.
Procedures to transfer genes into the plant wiU have to be developed, as infec-
tion with Agrobacterium, has been found not to transform this plant in a stable fashion.
Systems to detect carly expres..'>ion of desired markers should finally be sought as the
olive trec develops very slowly and has a long juvenile phase.
Finally, the creation of a germplasm hank of shoot apices which are the ideal
material for cryopreservation, should be achieved with a large number of interesting
cultivars to use in future hreeding programmes and avoid their extinction.

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CANAS L.A. and M. SANTAOLALLA, 1986.- Pseudomonas syringae pv. savastanoi lipopolysaccha·
ride as receptor of a new bacteriophage. Can. J. M icrobiol., 32, 73-78.
274 LETTRES BOTANIQUES

CANAS LA., L CARRAMOLINO and M. VICENTE, 1987.- Vegetative propagation of the olive
tree from in vitro cultured embryos. Plant Sei., 50, 85-90.
CANAS LA., A.M. WYSSMANN and M.C. BENBADIS, 1987.- Isolation, culture and division of
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OCHATT S.J. and S.H. CASO, 1986.- Shoot regeneration from leaf mesophyll protoplasts of wild
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Fig. 1.- ln vitro propagation of the olive tree (0/ea europaea L.). A/ Germination of an olive em-
bryo after 1 week in OMg culture medium; c :green cotyledon, r : rootlet. BI Seedling after
1 month in culture ; c : cotyledons, r : secondary roots, h : hypocotyl, 1 : leaf, a : shoot
apices. Cl Plantlet derived from a germinated embryo after transferring to soil conditions.
Dl Semiaseptic culture of olive «Ovulb) on moist vermiculite ; arrows-stimulation of super-
ficial buds after 2 weeks of latence. E/ Developed shoots from <<OVuli» after 1 month in
culture. FI In vitro stimulation of axillary buds and shoot proliferation on OM culture
medium with 4mg.l" 1 of zeatin. G/ Plantlet of cv. Tanche obtained from in vitro rooted
shoot.
L. CANAS 275

Figure 1
 276 LETTRES BOTANIQUES

Fig. 2- In t1itro plant regeneration from cotyledon-derived calli. Al lsolated mature embryos. BI
Root regeneration from cotyledon-derived callus on OMc medium with 1 mg.r 1 of lB A. C.
Shoot regeneration on OMc medium with 4 mg.r 1 of 2iP. Dl Regenerated plant from cotyle-
don callus with «triple whorh• (verticils with 3 leaves), the normal phyllotaxis in the olive
tree is opposite decussate. El Rooted plant showinq double leaves artd dichotomy. FI Surviving
olive plants after transferring to peat and soil conditions in a greenhouse.
-------- . ·--·-----------------·-··------------·------......-------------------------------·-------------
Fig. 3.- Culture and division of cotyledon·derived protoplasts from Olea europoeo L. cv. Tanche.
O.Velopment of regenerated calli. Al Freshly isolated protopl•ts from cotyledon tissue. ar·
rows : white protoplasts (without chloroplastsl, bar : 4Qi.tm. BI Cell wall regeneration and elon.
gation, bar : 4'~· Cl First division, bar: 4~m. Dl Ce)l colony after 1 month in OMp culture
medium, bar : m, arrows : cell division. El Active callus growth from cotyledon-derived
protopl•ts on 0 c medium, Wc : white callus, Ge :green callus. FI Epiderr, formation in the
surface of green calli after 1 month in culture on OMc medium with 1 mg.r of zeatin (x 1601.
G/ Vascular tissue bundles inside the green callus lx 1601. H/ Meristematic nodule with tracheids
surrounded by cambium-li lee cells lx 300). Il Development of meristematic nodules with cam.
bium activitY lx 160).
L. CANAS 277

Figure 3