The Components of Plant Tissue Culture Media II - Horticultural ...

138 The Components of Plant Tissue Culture Media II
plantlets was inversely proportional to the sucrose
level.
The dry weight of Lilium auratum bulbs and roots
on MS medium increased as the sucrose
concentration was augmented to 90 g/l but decreased
dramatically with 150 g/l because growth was
inhibited. The fresh weight/dry weight ratio of both
kinds of tissue once again declined progressively as
sucrose was added to the medium (0 - 150 g/l). In a
medium containing 30 mg/l sucrose, the number and
fresh weight of bulblets increased as MS salt strength
was raised from one eighth to two times its normal
concentration. An interaction between salt and
sucrose concentrations was demonstrated in that
optimum dry weight of bulbs could be obtained in
either single strength MS + 120 g/l sucrose
(osmolality, without sucrose inversion = ca. 492
mOsm/kg; Ψs = ca. -1.22 MPa), or double strength
MS + 60 g/l sucrose (osmolality, without sucrose
inversion = ca. 378 mOsm/kg; Ψs = ca. -0.94 MPa)
(Takayama and Misawa, 1979).
4.3.4. Morphogenesis
The osmotic effect of sucrose in culture solutions
was well demonstrated by a series of experiments on
tobacco callus by Brown et al., (1979): rates of callus
growth and shoot regeneration which were optimal on
culture media containing 3 per cent sucrose, could be
maintained when the sugar was replaced partially by
osmotically equivalent levels of mannitol. The
optimal (medium plus sucrose) here was between
-0.4 and -0.6 MPa, and increasing sucrose levels
above 3 per cent brought a progressive decrease in
shoot regeneration. Similar results were obtained by
Barg and Umiel (1977), but when they kept the
osmotic potential of the culture solution roughly
constant by additions of mannitol, the sucrose
concentrations optimal for tobacco callus growth or
morphogenesis were not the same (Fig. 4.2)
Brown and Thorpe (1980) subsequently found
that callus of Nicotiana capable of forming shoots,
had a water potential (Ψ) of -0.8 MPa, while nonshoot-forming
callus had a Ψ of -0.4 MPa. The two
relationships were:
Shoot forming callus
Ψcell = Ψs +Ψp + Ψm
–0.8 =–1 + 0.4 + 0 MPa
Non-shoot-forming callus
Ψcell = Ψs +Ψp + Ψm
–0.4 = –0 + 0.3 + 0 MPa
Correct water potential. An optimum rate of
growth and adventitious shoot formation of wheat
callus occurred on MS medium containing 2%
sucrose. Only a small number of shoots were
produced on the medium supplemented with 1%
sucrose, but if mannitol was added so that the total
Ψcell = Ψs was the same as when 2% sucrose was
present, the formation of adventitious shoots was
stimulated (Galiba and Erdei, 1986). Very similar
results were obtained by Lapeña et al. (1988). A
small number of shoot buds were produced from
Digitalis obscura hypocotyls on MS medium
containing 1% sucrose: more than twice as many if
the medium contained 2% sucrose (total Ψs given as
-0.336 MPa), or 1% sucrose plus mannitol to again
give a total Ψs equal to -0.336 MPa.
Water potential can modify commitment.
Morphogenesis can also be regulated by altering the
water potential of media. Shepard and Totten (1977)
found that very small (ca. 1-2 mm) calluses formed
from potato mesophyll protoplasts were unable to
survive in 1 or 2% sucrose, and the base of larger (5-
10 mm) ones turned brown, while the upper portions
turned green but formed roots and no shoots. The
calli became fully green only on 0.2-0.5% sucrose.
At these levels shoots were formed in the presence of
0.2-0.3 M mannitol. When the level of mannitol was
reduced to 0.05 M, the proportion of calli
differentiating shoots fell from 61% to 2%. The
possibility of an osmotic affect was suggested
because equimolar concentrations of myo-inositol
were just as effective in promoting shoot
regeneration.
Another way to modify morphogenesis is to
increase the ionic concentration of the medium. Pith
phloem callus of tobacco proliferates on Zapata et al.
(1983) MY1 medium supplemented with 10 –5 M IAA
and 2.5 x 10 –6 M kinetin, but forms shoots on
Murashige et al. (1972) medium containing 10 –5 M
IAA and 10 –5 M kinetin. These two media contain
very similar macronutrients (total ionic concentrations,
respectively 96 and 101mM), yet adding 0.5-
1.0% sodium sulphate (additional osmolality 89-130
mOsm/kg) decreased the shoot formation of callus
grown on the shoot-forming medium, but increased it
on the medium which previously only supported
callus proliferation (Pua et al., 1985a). Callus
cultured in the presence of sodium sulphate retained
its shoot-producing capacity over a long period,
although the effect was not permanent (Pua et al.,
1985b; Chandler et al., 1987). In these experiments
shoot formation was also enhanced by sodium
chloride and mannitol.
Increasing the level of sucrose from 1 to 3 per
cent in MS medium containing 0.3 mg/l IAA,