Plant Science 158 2000 61 – 70
Assessment of factors affecting adventitious shoot regeneration from in vitro cultured leaves of apricot
Olaya Pe´rez-Tornero, Jose´ Egea, Alicia Vanoostende, Lorenzo Burgos
Departamento de Mejora y Patologı´a Vegetal, Centro de Edafologı´a y Biologı´a, Aplicada del Segura, CSIC, P.O. Box
4195
,
30080
Murcia, Spain Received 20 April 2000; accepted 2 June 2000
Abstract
Relatively high percentages of adventitious shoot regeneration have consistently been obtained from leaves of some apricot cultivars. For the cultivar ‘Helena’, explants from the proliferation medium were more reactive than those from the elongation
medium. The best results were obtained with thidiazuron TDZ. When 6-benzylamino-purine BAP was used instead of TDZ, the regeneration percentages were very low. High a-naphthaleneacetic acid NAA concentration had an important effect upon the
decrease of the secretion of phenolic substances. Young expanding leaves with the adaxial side touching the culture medium, maintained for 2 or 3 weeks in darkness, produced the best results. There was a significant genotypic variability in adventitious
bud formation. Several caulinar meristems arose from very small areas of the leaf but only one meristem developed to form a shoot when those buds were transferred to elongation medium. However, the fact that several caulinar meristems exist in early
steps could be an advantage when genetically transforming these leaves, since a high number of cells would have the possibility of being transformed and producing adventitious transformed buds. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Prunus armeniaca; Leaf age; Tissue culture; Thidiazuron; Organogenesis; Woody plant www.elsevier.comlocateplantsci
1. Introduction
Adventitious regeneration is a key step in the application of genetic engineering techniques to
the breeding of plants. Fruit trees are among the most recalcitrant to produce adventitious shoots,
although reports on the successful regeneration and transformation of different species have ap-
peared in recent years [1 – 3]. Reports of in vitro adventitious shoot regeneration from mature ex-
plants of Prunus are few. Adventitious shoots have been obtained from leaves of Prunus domestica [4],
Prunus canescens [5], P. dulcis [6], P. persica [7] and P. serotina and P. a6ium [8]. In apricot
Prunus armeniaca L., shoot regeneration through adventitious induction has been achieved fre-
quently from juvenile explants or endosperm [9 – 11]. Because apricot is a highly heterozygous
species, the use of seed-derived material should be avoided whenever elite clones have already been
selected. Thus, it is important to establish effective protocols for induction of shoot regeneration from
mature apricot explants. Only one paper about regeneration in apricot from adult tissue has been
published to date [12]. The authors of that report found the results to be poorly reproducible.
The present study was designed to explore the conditions required for induction of adventitious
buds and regeneration of shoots from explants of mature apricot trees propagated in vitro.
2. Materials and methods
2
.
1
. Maintenance of shoot cultures This study has been carried out with the Ameri-
Corresponding author. Tel.: + 34-968-215717; fax: + 34-968- 266613.
E-mail address
:
burgosnatura.cebas.csic.es L. Burgos. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 3 0 3 - 4
can cultivar
‘Helena’ obtained
in the
apricot-breeding program at the Horticultural Crops Research Laboratory in Fresno, California
and kindly provided by Dr Craig A. Ledbetter. In vitro shoots were maintained by sub-culturing at
3-week intervals onto a shoot multiplication medium.
This medium
consisted of
QL macronutrients [13] and micronutrients, vitamins
and organic compounds as described previously [14], 3 sucrose and 0.6 agar Hispanlab, S.A.,
and was
supplemented with
3.1 m
M N
6
-benzylamino-purine BAP
and 0.2
m M
indole-3-butyric acid IBA. The pH of the medium was adjusted to 5.7 prior to autoclaving
at 121°C for 20 min. Cultures were maintained at 22 9 1°C under cool white fluorescent tubes 55
m mol m
− 2
s
− 1
with a 16-h photoperiod.
2
.
2
. General strategy for regeneration The first four apical expanding leaves were
harvested in sterile water from 3-week-old prolif- eration shoots. Each leaf was transversely cut
three or four times across the midrib without fully separating the segments. Leaves were cul-
tured with the adaxial side in contact with the regeneration medium, which consisted of a basal
medium with the same composition as the multi- plication medium, with the exception of the
growth regulators, unless stated otherwise. All media were autoclaved for 20 min at 121°C and
then dispensed aseptically, 25 ml per sterile plas- tic Petri dish 8.5 cm diameter × 1.5 cm depth.
For each treatment at least five Petri dishes were prepared, each containing seven leaves. Experi-
ments, where regeneration was achieved, were re- peated
at least
twice. After
explants were
positioned on the medium, the dishes were sealed with parafilm. All explants were incubated
in the dark at 22 9 1°C for 3 weeks before expo- sure to light with a 16-h photoperiod unless
stated otherwise. Following regeneration, shoots were transferred from the Petri plates to the me-
dia used for micropropagation.
2
.
3
. Effect of leaf origin, type of material, type and concentration of growth regulators
In this experiment, leaves from proliferated shoots and leaves and internodes from elongated
shoots were used. Shoots were elongated in a basal medium with the same composition as the
multiplication medium but supplemented with 0.9 mM BAP and 0.2 mM IBA. For the experi-
ments with internodes, two Petri dishes were pre- pared
with ten
internodes per
dish. The
regeneration medium was supplemented with cy- tokinin, as BAP 4.4, 13.2 or 22.0 mM or 1-
Phenyl-3-1,2,3-thiadiazol-5-yl urea
Thidiazuron, TDZ 2.3, 4.5, 9.0, 11.3 or 13.5 m
M, and auxin, as a-Naphthaleneacetic acid NAA; 0.5, 1.3, 2.7, 4.0 or 5.4 mM; Table 1.
2
.
4
. Effect of leaf age and position on the culture medium
Young leaves, corresponding to the first four apical expanded leaves and also the last four
older leaves, from the proliferating shoots were cultured onto the RM12, RM18 and RM21 me-
dia Table 1, with the adaxial or abaxial side touching the culture medium.
2
.
5
. Control of the secretion of phenolic substances Young expanding leaves were harvested in
sterile water with 0.1 ascorbic acid and cul- tured in different regeneration media. Also,
leaves were cultured in regeneration media sup- plemented with 0.01 citric acid and 0.01
ascorbic acid or 0.1 polyvinylpyrrolidone PVP, molecular weight 10 000. RM10, RM12 and
RM13 Table 1 were used in these experiments. Also, media RM19, RM20, RM21 and RM22
were tried, in which the NAA concentration was increased to 4.0 or 5.4 mM Table 1.
2
.
6
. Effect of light intensity Young expanding leaves were cultured in the
media RM12, RM15 and RM18 Table 1. After the culture in the dark, leaves were transferred
to 55 or 110 mmol m
− 2
s
− 1
light intensity.
2
.
7
. Effect of the dark period Young expanding leaves were harvested from
explants in proliferation and then cultured in RM12, RM18 and RM21 Table 1. Four dark
treatments were tried, where culture plates were first incubated in the dark for 1, 2, 3 or 4 weeks
and then transferred to the light.
2
.
8
. Effect of using an induction medium followed by an expression medium
Young expanding leaves were cultured in the media RM12, RM18 and RM21 Table 1. After 3
weeks in the dark, the explants were transferred to auxin-free media expression medium with 1.1
m M or 2.3 mM TDZ, when the induction medium
was RM12, and 2.3, 4.5 or 9.0 mM TDZ when the induction medium was RM18 or RM21. Also,
when RM18 was used as induction medium, ex- pression media with 2.2 or 4.4 mM BAP, and
without TDZ or NAA, were tried Table 1.
2
.
9
. Effect of different gelling agents Young expanding leaves were harvested from
Table 1 Combination of growth regulators and source of the plant material used in the regeneration media
a
Growth regulators mM Induction medium
Expression medium Source of material
BAP TDZ
NAA I.E.
L.E. L.P.
Culture media TDZ
BAP NAA
x x
x 0.5
4.4 RM 1
RM 2 x
4.4 x
x 1.3
4.4 x
RM 3 x
2.7 x
13.2 x
RM 4 x
x 0.5
x x
x RM 5
13.2 1.3
13.2 x
x x
2.7 RM 6
RM 7 x
22.0 x
x 0.5
x x
1.3 x
22.0 RM 8
RM 9 x
x x
2.7 22.0
x x
x RM 10
2.3 0.5
x x
x RM 11
2.3 1.3
x x
x RM 12
2.7 2.3
x RM 12
2.3 2.7
1.1 x
RM 12 2.3
2.3 2.7
RM 13 x
x x
0.5 4.5
x x
x RM 14
4.5 1.3
x x
x RM 15
4.5 2.7
0.5 x
x RM 16
9.0 x
x x
x 9.0
1.3 RM 17
x x
x RM 18
9.0 2.7
9.0 x
9.0 2.7
RM 18 4.5
x 9.0
2.7 RM 18
x 2.3
RM 18 2.7
9.0 x
RM 18 9.0
2.7 4.4
9.0 x
RM 18 2.7
2.2 RM 19
x 4.0
2.3 x
RM 20 2.3
5.4 x
9.0 4.0
RM 21 9.0
x 9.0
RM 21 4.0
9.0 x
4.0 4.5
RM 21 RM 21
x 4.0
9.0 2.3
x RM 22
9.0 5.4
x RM 23
4.4 2.3
2.7 x
RM 24 2.7
9.0 4.4
x 11.3
RM 25 4.0
4.0 13.5
x RM 26
a
I.E., internodes from explants in elongation; L.E., leaves from explants in elongation; L.P., leaves from explants in proliferation; x, treatments tested in this study.
explants in proliferation and cultured in a regener- ation medium with 0.6 agar Hispanlab, S.A.,
0.45 agargel or 0.25 gelrite Sigma. The rest of the medium components and culture conditions
were the same as for the above experiments, and the growth regulators of the media RM12, RM18
and RM21 Table 1 were used.
2
.
10
. Effect of the genotype Young expanding leaves were harvested from
explants in proliferation of the cultivars ‘Helena’, ‘Bu´lida’, ‘Canino’ and ‘Lorna’. Also, young ex-
panding leaves from explants in elongation of the cultivar ‘Canino’ were used. Growth regulators
used were those in the media RM12, RM18 and RM21 Table 1. The macronutrients used for the
last three cultivars were a WPM modification, as described previously [14]. The rest of the medium
components and culture conditions were the same as for the above experiments, except that 0.45
agargel was used as gelling agent.
2
.
11
. E6aluation criteria and statistical analysis For up to 8 weeks after the dark period the
number of leaves forming adventitious buds and the number of buds formed per leaf were recorded.
Data were analysed by a maximum likelihood analysis of variance and, when necessary, specific
contrasts of maximum likelihood were designed.
2
.
12
. Light microscopy Sections of young expanding leaves that stayed
for 0, 1, 2, 3 or 4 weeks in the regeneration medium were fixed in FAA 90 of 70 ethanol,
5 of 40 formaldehyde, 5 glacial acetic acid. After this, the leaves were placed in 70 ethanol
to remove the fixer, dehydrated using a tertiary- butyl alcohol series TBA and then embedded in
Paraplast. Serial sections 5 or 10 mm deep were mounted on slides impregnated with an adhesive
of gelatine, glycerine and 3 formaldehyde. To stain the samples, the Paraplast was eliminated
with xylene. The leaves were stained with a mix- ture of 0.71 Na
2
HPO
4
, 0.48 citric acid and 0.025 toluidine blue for 30 s, then they were
rinsed in water, dried and mounted with a syn- thetic mounting medium.
2
.
13
. Scanning electron microscopy Sections of young expanding leaves that were
kept for 4 or 5 weeks in the regeneration medium were fixed for 4 h at room temperature in a 0.1
M-phosphate buffer, pH 7.2, containing 3 glu- taraldehyde. Afterwards, samples were postfixed
with 1 osmium tetroxide in the same buffer, and dehydrated in an acetone series. Subsequently, the
samples were transferred into amyl acetate and processed using the critical point method BASES
UNION CT20. The specimens were sputtered with gold and observed using a Jeol-T300 scan-
ning electron microscope [15].
3. Results
