plants through in vitro culture, Nobars [6] pointed out that this technique has been successfully used
to produce stress-tolerant plants from several species.
The accumulation of proline and soluble sugars as an osmotic tolerance mechanism has been
widely observed in many species. Proline may provide
osmoregulation and
stabilisation of
proteins and membrane during stress [9]. Soluble sugars, which accumulate in the vacuole, are a
major organic solute, involved in osmotic adjust- ment when plants are exposed to drought [9].
Sucrose, mannitol or sorbitol were studied by Sabili et al. [10] as osmotic stress agents on in
vitro-grown Chrysanthemum morifolium. Sucrose failed to elicit consistent osmotic stress symptoms
and enhanced both shoots and root growth. The osmotic potential of the tissue paralleled the in-
crease of mannitol or sorbitol concentrations on culture medium. The harmful effect of the osmotic
stress agent occurred in both shoot proliferation and rooting stages. Sorghum bicolor was tested in
vitro for drought stress by Dunca et al. [11]. Results showed that osmotic stress applied to in
vitro cultures reduced regeneration ability. How- ever, by screening the regenerated plants under
field conditions they obtained stress-tolerant re- generates with higher yield under stress conditions
than that of their parents.
Regeneration of plants displaying an increased tolerance to environmental stress is an important
goal for the biotechnological improvement of many plant species [6]. Therefore, the aim of this
investigation was to induce somaclonal variation in regenerated plants in order to select drought-
tolerant clones of T. minuta, which grows as a cash crop in some developing countries [2,3], using
a rapid in vitro regeneration protocol [12].
2. Materials and methods
2
.
1
. General materials and methods Sterile seedlings and cotyledon-derived shoot
clumps were obtained and maintained as reported previously [12]. Unless stated, 42 explants from
each treatment were cultured in seven Phytatrays II Sigma with 100 ml medium. Shoot and root
fresh and dry weights for plants developed in vitro were measured for 10 randomly selected plantlets
after gently removing the medium from the roots. The fresh weights for individual shoots and roots
were assessed immediately after blotting the plantlets on tissue, and the dry weights determined
after drying to a constant weight in an oven at 80°C and cooling to room temperature in a desic-
cator. Plants were adapted to greenhouse condi- tions of 25 – 31°C and 16-h photoperiod, as
reported previously [12]. The significance of differ- ences in shoot, leaf, stem, plant fresh and dry
weights, proline and soluble sugars content and water potential were tested by Analysis of Vari-
ance using Minitab for Windows Version 10.5 computer programme.
2
.
2
. Optimising mannitol concentration for osmotic stress
This preliminary experiment was carried out to optimise mannitol concentrations which could
then be used as a selective agent for osmotic stress. Shoot tips of 2-week-old sterile seedlings were
cultured on
1 2
-strength MS medium [13] containing 2 sucrose, 0.5 mg l
− 1
IAA shoot growth medium; SGM with 0, 20, 40, 60, 80 or 100 mM
mannitol. After 4 weeks, the number of segments which developed plantlets and the fresh and dry
weight of both shoots and roots were measured.
In order to study the ability of cotyledon ex- plants to regenerate shoots on medium containing
mannitol, 40 cotyledons from 1-week-old sterile seedlings were cultured onto eight Petri dishes
each with 25 ml MS medium containing 3 su- crose, 3 mg l
− 1
IAA, 10 mg l
− 1
BA callus growth medium; CGM with 0 or 60 mM mannitol. After
1 month, the developing shoot clumps were trans- ferred onto SGM to enhance adventitious shoot
growth.
2
.
3
. Shoot regeneration from cotyledon explants on medium containing mannitol
Cotyledons were cultured on CGM as stated previously. The developing shoot clumps were
subcultured onto CGM six times at monthly inter- vals Fig. 1, step 1. Following that, ca. 2-g pieces
of shoot clumps were subcultured onto fresh medium containing 0, 60 or 80 mM mannitol for 3
months at monthly intervals Fig. 1, step 2.
Pieces of shoot clumps ca. 2 g, previously cul- tured on CGM and CGM with 60 mM mannitol
were subcultured onto SGM containing 0 or 60 mM mannitol, respectively. In this trial, 72 shoot
clumps were cultured on 12 Phytatrays II for each treatment. Shoot clumps were subcultured twice
onto a fresh medium at monthly intervals Fig. 1, step 3. Developing shoots were micropropagated
as separate clones by single node culture on SGM for 2 months Fig. 1, step 4. Plants were hardened
off under greenhouse conditions Fig. 1, step 5.
For control plants, seeds were sown in the greenhouse. After 2 months, shoot tips from each
of 30 plants was excised. Segments were surface- sterilised with 10 sodium hypochlorite solution
BDH with a few drops of Tween 20 for 30 min, followed by several washes with sterile distilled
water. Nodes were cultured in SGM for 1 month before transferring to the greenhouse Fig. 1, step
4.
2
.
4
. In 6itro screening of clones for drought stress After growing the clones for 2 months in the
greenhouse, segments with the uppermost two identifiable nodes and the shoot tips were excised
and surface-sterilized. Single-node segments were cultured on SGM with 0 or 60 mM mannitol and
500 mg l
− 1
carbenicillin, to eliminate bacterial contamination Fig. 1, step 6. After 1 month the
percentage of nodes that developed into plantlets was assessed and shoot and root fresh and dry
weights were measured. The remaining plantlets that developed on medium without mannitol were
returned to the greenhouse Fig. 1, step 7.
Clones were grown in the greenhouse for 2 months before retesting for osmotic stress in vitro.
Two intermediate clones developed in non-stress conditions and two clones including a drought-tol-
erant clone and non-tolerant clone which devel- oped from callus cultured on mannitol-containing
medium were tested for osmotic stress in vitro. Control plants Fig. 1, step 4 were tested in the
same way as the clones. First and second upper- most nodes were surface-sterilised and cultured on
SGM containing 500 mg l
− 1
carbenicillin and 0, 30, 60 or 90 mM mannitol Fig. 1, step 8. After 1
month, shoot and root fresh and dry weights were assessed. The remaining plants grown on manni-
tol-free medium were micropropagated by shoot tips for 1 month and then transferred to the
greenhouse.
2
.
5
. Proline and soluble sugars determination Plants were grown in the greenhouse for 2
months then, first and second uppermost nodes of stress-selected clone, PM3, and non-stress-selected
clone, P4, were cultured as in Section 2.4 on SGM containing 0 or 30 mM mannitol. After 1 month
the new developing shoots were used to measure the proline content and soluble sugars. Samples of
1 g from each of 15 individual plantlets were used to measure proline content as described by Bates
et al. [14]. For soluble sugars, 15 individual plantlets from of the each clones were dried at
80°C, and 0.1 g from each individual plantlet was used to measure soluble sugars following the
method reported by Jermyn [15].
2
.
6
. In 6i6o screening of clones for drought stress After 2 months of growth in the greenhouse,
shoot tip segments of two clones, P4 selected on mannitol-free medium and PM3 selected on man-
nitol-containing medium, and control plants were cultured in vitro for a further month before being
returned to the greenhouse. Two weeks later
Fig. 1. Protocol designed to select drought-tolerant clones of T. minuta. CGM, callus growth medium = MS + 3 su-
crose + 10 mg l
− 1
BA + 3 mg l
− 1
IAA; SGM, shoot growth medium =
1 2
MS + 2 sucrose + 0.5 mg l
− 1
IAA.
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
