Fig. 2. Effect of mannitol concentrations on shoot fresh A and dry B and root fresh C and dry D weights on plants
developed from shoot tip segments of T. minuta after 1 month of culture. The bars show LSD for all pair comparisons at
P = 0.05.
Drought stressed plants were watered to 40 FC. After 2 months, plant heights were measured and
then plants were cut at the soil surface. WP was measured for individual plants using 10-cm long
shoot tips immediately after cutting. Individual leaf and stem fresh weights were assessed immedi-
ately and the dry weights were assessed after air drying for 2 weeks. The relative growth rates
RGR for plant fresh and dry weights were calcu- lated by the formula:
RGR g g
− 1
day
− 1
= log
e
W 2 − log
e
W 1T2 − T1 where W represent weight and T indicates harvest-
ing time [18].
3. Results
3
.
1
. Optimising mannitol concentration for osmotic stress
All segments cultured on medium with mannitol remained green, even those that did not show any
further growth. Fig. 2 shows that mannitol had an affect on plantlets grown from cultured segments.
On medium with 80 and 100 mM mannitol some segments developed only shoots that were too
small to measure. Only 68, 45, and 33 of shoot tips cultured on medium containing 40, 60 and 80
mM
mannitol, respectively,
developed into
plantlets. Plantlets developing on medium contain- ing 60 mM mannitol had shoot fresh and dry
weights of 53 and 52, and root fresh and dry weights of 71 and 59, respectively, of those of
plantlets developed on mannitol-free medium. The equivalent values for plantlets grown on medium
with 80 mM mannitol were 30, 38, 55 and 42.
All cotyledons cultured in CGM containing 60 mM mannitol became swollen and necrotic and
did not survive. However, cotyledons cultured in CGM without mannitol increased in size after 1
week in culture and callus was initiated from the basal end before developing adventitious shoots.
When 1-month-old shoot clumps were transferred from CGM to SGM for a month, an average of 20
shoots explant
− 1
was regenerated. Media with 60 and 80 mM mannitol were therefore used to apply
osmotic stress in vitro and to assess the drought stress of micropropagated plants in vitro.
plants were transplanted into 12 cm diameter plas- tic pots filled with 650 g air-dried clay soil. One
week later, 10 plants were randomly selected from each clone and control plants. Individual plant
heights were assessed and water potential WP measured using the pressure-chamber technique
[16]. Individual shoot fresh weights were assessed immediately. Shoot dry weights were measured
after air drying for 2 weeks.
Plants were arranged in a randomised block design with three blocks, each block with 18 plants
from each clone and the control plants. Within each block, half of the plants of each type were
well-watered to 100 field capacity FC and half exposed to drought conditions at 40 FC. Plants
were watered every 3 days. Field capacity of the soil in pots was assessed before the treatment as
reported by Tuomela [17]. Plants grown in soil with 40 FC were irrigated for the first time 6
days after starting water stress treatment. Before watering, three pots from each treatment in each
block were randomly selected and weighed. The amount of water required to restore the soil to
100 FC was added to the non-stressed group.
3
.
2
. Shoot regeneration from cotyledon explants on medium containing mannitol
After culture for 6 months on CGM without mannitol to encourage somaclonal variation,
shoot clumps were transferred to CGM containing 0, 60 or 80 mM mannitol Fig. 1, step 2. Two
weeks after transfer of shoot clumps onto CGM containing 60 mM mannitol, vitrified swollen
shoots developed but died at this stage although the differentiated clumps remained green. No
shoots elongated from shoot clumps cultured on the CGM. After a month, a little decrease in
growth was observed but all shoot clumps cultured in medium containing 60 or 80 mM mannitol
survived with some necrotic tissue forming espe- cially on medium containing 80 mM mannitol.
After subculturing onto fresh CGM for another month, shoot clumps cultured in medium contain-
ing 60 mM mannitol were healthy but 80 of shoot clumps cultured on medium containing 80
mM mannitol became necrotic and died. In the second subculture, all shoot clumps cultured onto
medium containing 80 mM mannitol died. After 3 months, 36 and 12 shoot clumps out of 72 clumps
cultured on CGM containing 0 and 60 mM man- nitol, respectively, survived. These surviving shoot
clumps were transferred onto SGM with 0 or 60 mM mannitol for shoot clumps previously cul-
tured on CGM with 0 or 60 mM mannitol, respec- tively, to stimulate shoot growth Fig. 1, step 3.
Some of the shoot clumps cultured onto SGM became necrotic and stopped growth regardless of
the medium used to maintain the shoot clumps during the previous 9 months. Many of the shoots
developed from shoot clumps cultured on medium containing mannitol died at an early stage after
reaching approximately 0.5 cm in length. The number of shoots developed from clumps at this
stage was 12 from five shoot clumps in mannitol- free medium and four shoots from two shoot
clumps in medium containing mannitol. Shoots developed on mannitol-free medium were named
as P1 – P12, and those developed in stressed medium named as PM1 – PM4. Two shoots from
those developed from non-stressed shoot clumps were discarded because of contamination.
Developing shoots were excised and microprop- agated as clones on SGM Fig. 1, step 4. Shoot
tip segments continued growth as a main stem but the two axillary buds on nodal segments developed
into shoots and all axillary buds on developing shoots sprouted. After 2 months of micropropaga-
tion all shoots became weak, developing small narrow leaves and long nodes. To achieve normal
growth, plants were transferred in vivo to a green- house Fig. 1, step 5. All transferred plantlets
survived and developed normal shoots within a month.
3
.
3
. In 6itro screening of clones for drought tolerance
Nodes from greenhouse-grown plants were ex- cised from each clone and cultured on SGM con-
taining 0 or 60 mM mannitol Fig. 1, step 6. The percentage of surviving explants which developed
new shoots varied from 75 to 100 in mannitol- free medium and from 30 to 100 in medium
containing mannitol. However, although the per- centage survival of segments on medium contain-
ing mannitol for clones P2, P6, P8, PM1 and PM2 was 100, the developing shoots and roots were
too small to obtain freshdry weight measurements Fig. 3.
There were significant differences among clones in both shoot fresh and dry weights and in the
response of these parameters to mannitol Figs. 4 and 5. There were also significant differences
among clones in root fresh and dry weights data not presented. Plantlets from clone PM3 cultured
on medium containing mannitol gave shoot fresh and dry weights of 298 and 27 mg
− 1
, respectively. These weights did not differ from those for
plantlets grown on mannitol-free medium. Plantlets from four of the clones developed on
medium without mannitol were transferred to the greenhouse for 2 months Fig. 1, step 7. These
clones and control plants were tested for drought tolerance by culturing nodal explants on SGM
containing 0, 30, 60 or 90 mM mannitol Fig. 1, step 8. Percentage survival was not a good indica-
tor of osmotic stress as most cultured nodes, even in medium containing the highest mannitol con-
centration, remained green while showing no fur- ther growth. Fig. 6 shows that shoot fresh and dry
weights were significantly decreased by all manni- tol concentrations for all plants except for PM3
clone, which previously showed a high tolerance to drought. There was no difference in biomass when
the drought-tolerant clone PM3 was grown on medium containing 0 or 30 mM mannitol. Plants
of two clones, one from the non-stress selected shoot clumps and one from stress-selected shoot
clumps were very sensitive to osmotic stress based on shoot fresh and dry weight measurements. A
drought-sensitive regenerated clone P4, from non- stress-selected shoot clumps, the drought tolerant
clone PM3 which developed from stress-selected shoot clumps, and the control plants grown on
SGM without mannitol, were all cloned from shoot tips and returned to the greenhouse to test
their response to drought stress in vivo.
3
.
4
. Proline and soluble sugars content Neither the proline content nor soluble sugars
were significantly increased in either clones when 30 mM mannitol was added to the growth
medium P = 0.11. Overall plantlets of clone PM3 exhibited a significantly higher proline content
P B 0.001 than plantlets of P4, but a significant difference between clones could not be shown for
the 0 and 30 mM mannitol individually. Overall PM3 plantlets grown in medium containing 0 or
Fig. 3. T. minuta nodal segments of from left to right PM3 on SGM, PM3 on SGM containing 60 mM mannitol and P10, P7, P3 and PM1 on SGM containing 60 mM mannitol, after 1 month in culture. The scale is in centimeters.
Fig. 4. Shoot fresh weight for T. minuta clones after 1 month of growth on medium containing mannitol. Clones have been arranged in descending order by weight of plants grown on medium with mannitol as a percentage of control plant weight. These
values are shown for each clone. The bar shows the LSD for all pair comparisons at P = 0.05. Clones P1 – P10 were developed on mannitol-free CGM and clones PM1 – PM4 were developed on CGM with mannitol.
Fig. 5. Shoot dry weights for T. minuta clones after 1 month of growth on medium containing mannitol. Clones have been arranged in descending order by weight of plants grown on medium with mannitol as a percentage of control plant weight. These
values are shown for each clone. The bar shows the LSD for all pair comparisons at P = 0.05. Clones P1 – P10 were developed on mannitol-free CGM and clones PM1 – PM4 were developed on CGM with mannitol.
30 mM mannitol had significantly higher soluble sugars content than P4 plantlets Fig. 7.
3
.
5
. In 6i6o testing of clones for drought tolerance When plants were exposed to the drought stress
in the greenhouse the bottom leaves of the stressed plant, especially the control and the non-stress-se-
lected clone P4, started to dry out after a month of drought stress. Before starting drought stress, con-
trol, non-stress-selected clone P4 and the stress-se- lected clone PM3 had a WP of − 0.121 to − 0.125
MPa, with no significant difference among them. Two months after drought stress, plants of the
stress-selected clone PM3 had a WP of − 0.178 MPa, significantly lower than the WP of plants of
the non-stress-selected clone P4 or the control plants grown in soil at 40 FC. In soil at 100 FC
there was no significant difference between control, non-stress selected P4 and stress-selected PM3
plants Table 1.
Plant height and plant fresh and dry weights did not differ significantly between the clones or be-
tween the clones and the control before water stress was applied or after 2 months growth in soil at 100
FC. Plant height and fresh and dry weights were significantly reduced by drought P B 0.001 for all
of these parameters. Plants of the stress-selected clone PM3 grown in soil with 40 FC had a plant
height and plant fresh and dry weight significantly greater than that of control or non-stress selected
P4 plants Table 1. Fresh and dry weights of stress-selected plants PM3 grown in soil at 40 FC
were 38 and 41, respectively of the unstressed control plants, whereas the equivalent values for
control plants were 19 and 22, and for the non-stress-selected clone P4 were 22 and 19.
Fig. 6. Shoot fresh A and dry B weights for T. minuta clones after 1 month of growth on medium containing manni-
tol. The bar shows the LSD for all pair comparisons at P = 0.05. Missing bars represent zero value. Clones P1 and P4
were developed on mannitol-free CGM and clones PM2 and PM4 were developed on CGM containing mannitol.
Fig. 7. Proline A and soluble sugars content B of T. minuta clones after 1 month of growth on medium containing 0 or 30
mM mannitol. The bar shows the LSD for all pair compari- sons at P = 0.05.
stress-selected clone PM3 had RGR fresh wt. which was significantly higher than for the non-
stress-selected P4 clone 152 and control plants 142. Also, stress-selected plants had RGR dry
wt. which was significantly higher than for P4 129 and control plants 143 Table 1.
4. Discussion
