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
The first change, observed within 7 days on regeneration medium, was the enlargement of ex-
plants to twice their original size. Morphogenesis occurred mostly on the cut edges and midribs or in
association with vascular tissue. Most of the buds developed from calli, but sometimes appeared to
arise directly from petiole tissue. The segments closer to the petiole were more regenerable than
distal leaf segments. The first adventitious buds were observed 3 or 4 weeks after the beginning of
the experiment. Most regeneration occurred from the calli in contact with the medium. Generally,
the number of buds per regenerating explant was one although up to 3 or 4 buds per leaf have been
found.
Frequently, the
buds developed
leaf rosettes.
In general, explants from the proliferation medium were more reactive than those from the
elongation medium. The regeneration percentages were very low or zero when leaves or internodes
from explants in elongation were used.
The proportion of leaves that produced adventi- tious buds was significantly greater with TDZ than
with BAP. When BAP was used the average regen- eration percentage was 0.8, with a maximum of
3.6 of regenerating explants. When the results of using different concentrations of TDZ or NAA
were analysed, the regeneration percentages were significantly affected by the TDZ concentration
P B 0.05 but not by NAA concentration. The best results were obtained with a TDZ concentra-
tion of 9.0 mM Table 2. The NAA concentration had an important effect decreasing the secretion of
Table 2 Regeneration percentages obtained in media with different concentrations of TDZ and NAA
a
Growth regulators Culture media
Percentage of regenerating leaves Number of shoots per regenerating leaf
mean 9 S.E. mean 9 S.E.
mM NAA
TDZ 0.5
RM 10 3.6 9 3.5
2.3 1
RM 11 2.3
1.3 7.1 9 4.9
1 RM 12
2.3 2.7
15.9 9 4.6 1.3 9 0.2
4.0 7.1 9 4.9
2.3 1
RM 19 RM 20
5.4 2.3
14.3 9 6.6 1.5 9 0.3
0.5 3.6 9 3.5
4.5 1
RM 13 4.5
RM 14 1.3
3.6 9 3.5 1
4.5 RM 15
2.7 7.1 9 3.1
1 0.5
14.3 9 6.6 9.0
1 RM 16
1.3 10.7 9 5.9
RM 17 1
9.0 2.7
24.3 9 5.1 9.0
1.2 9 0.1 RM 18
9.0 RM 21
4.0 23.8 9 5.4
1.1 9 0.1 9.0
RM 22 5.4
10.7 9 5.9 1.3 9 0.3
4.0 10.7 9 5.9
11.3 1
RM 25 4.0
17.9 9 7.2 RM 26
1 13.5
a
Leaves were excised from proliferating shoots. Table 3
Effect of leaf age and position in the culture medium on the percentage of regenerating leaves Culture media
Position
a
Age Percentage of regenerating leaves
Number of shoots per regenerating leaf mean 9 S.E.
mean 9 S.E. Adaxial
14.3 9 6.6 Young
1 RM 12
Young RM 12
Abaxial 2.9 9 2.8
1 RM 12
Adaxial Old
8.6 9 4.7 1.3 9 0.3
Abaxial 2.9 9 2.9
Old 1
RM 12 Young
RM 18 Adaxial
22.9 9 7.1 1.1 9 0.1
Young RM 18
Abaxial 5.7 9 3.9
1 Adaxial
11.4 9 5.4 Old
1 RM 18
RM 18 Abaxial
Old –
Adaxial 23.8 9 5.4
Young 1.1 9 0.1
RM 21 Young
RM 21 Abaxial
– Adaxial
RM 21 2.9 9 2.9
Old 1
Abaxial Old
– RM 21
a
Abaxial and adaxial leaf side touching the culture medium.
phenolic substances. The best results were ob- tained when the NAA concentration was above
2.7 mM.
Young expanding leaves produced better results than adult leaves. Regeneration percentages in-
creased at least two-fold when young leaves were used Table 3. Also, the leaf position in the
culture medium was an important factor. The best results were obtained when the leaf adaxial side
was touching the culture medium, in this position the regeneration obtained was four- or five-fold
higher than when the leaf abaxial side was in contact with the medium Table 3.
The treatments that decreased the secretion of phenolic substances, and did not affect the regen-
eration percentages, were PVP and the increased NAA concentration in the regeneration medium.
The rest of the treatments either did not have any effect on the phenolic secretion or diminished re-
generation percentages.
The increase of light intensity produced a higher secretion of phenolic substances, but had no sig-
nificant effect on the regeneration percentages.
The dark period had a significant effect on the regeneration percentage P B 0.001. The regenera-
tion was zero or very low with a dark period of 1 or 4 weeks and the best results were obtained with
2 or 3 weeks Fig. 1, without significant differ- ences between these two periods in darkness.
The use of an expression medium different to the induction medium had no significant effect on
the regeneration percentages. Moreover, a consid- erable increase in the secretion of phenolic sub-
stances was observed data not shown.
When different types of gelling agents were used, the regeneration percentages were signifi-
cantly affected P B 0.01. The highest regenera-
Fig. 3. Regeneration percentages from leaves of the cultivars ‘Helena’, ‘Bu´lida’, ‘Canino’ and ‘Lorna’ cultured in the regen-
eration media RM 12
, RM 18
and RM 21
.
Fig. 1. Effect of the dark period on the regeneration percent- ages from ‘Helena’ leaves, obtained from explants in prolifer-
ation, when the regeneration media RM 12
, RM 18
and RM 21
were used.
tion percentages were obtained with agar and agargel as gelling agents Fig. 2. When agargel
was used, this clearer gel allowed a better visibility of the adventitious buds.
The genotype significantly affected P B 0.001 the regeneration percentages. While in ‘Canino’
the regeneration obtained was significantly higher than in ‘Helena’ P B 0.001, in ‘Lorna’ and ‘Bu´l-
ida’ zero or very low regeneration percentages were obtained Fig. 3. Leaves of ‘Bu´lida’ and
‘Lorna’ formed very few calli and died. In ‘Canino’, when leaves from explant in prolifera-
tion or elongation were used, there were no signifi- cant differences in the regeneration percentages,
although slightly better results were obtained with leaves from explants in elongation Table 4.
When examined with the light or scanning elec- tron microscope, it was seen that leaf rosettes
regenerated from the cultured leaves were formed by a group of several adventitious buds Fig. 4. In
the region where adventitious buds were produced several caulinar meristems alongside the surface
were observed. However, when the leaf rosettes were transferred to a culture medium for their
elongation and proliferation, only one shoot developed.
4. Discussion
