CALLUS CULTURE TECHNOLOGY OF SPRING SOFT WHEAT STRESS TOLERANT VARIETIES SELECTION - Biochemistry and Biotechnology: Research and Development - page 52

M ATERIALS AND M ETHODS

12 varieties of spring soft wheat, differed in adaptive rate and vegetation period, and 16
regenerants, which had been created on selective (salt (RS), acidity (RA) and control (RC)
media, were involved in research. Isolated plant tissues culture methods [[6]] , especially
barley tissue ones [[7]] were used for technology development and stress-tolerant regenerants
creation processes. Selection was divided into three steps: callus induction, its proliferation
and plant regeneration. Immature embryos taken at 20-21 or 27-28 days after the beginning of
the earing were used as explants. Cultivation was undertaken on modified Murashige-Skoog
medium (MS). 2,4-dichlorphenoxyacetic acid (2,4-D) was used as callus formation inductor.
Its level in induction and proliferation media had being optimized while investigation.
Expanded calluses were passaged to regeneration media, containing different concentrations
of IAA and kinetin or BAP, from proliferation one. Selective conditions, low acidity, high
concentration of NaCl for salinity simulation, of polyethylene glycol for drought simulation,
were created for tolerant cell lines selection at the two last steps. MS medium (pH 5.6-5.7)
was used as control. Levels of hormones and stress agents are described further. Laboratory
test of regenerants and their parental varieties salt tolerance was realized by roll method [[8]] .
Weight and length of plantlet organs in salt solution in comparison with clear water were
measured. Acid tolerance was tested by modified method using coefficient of tested plants
organ growth function reduction, which development is described further.

R ESULTS

Hormone Media Composition Optimization and Detection of Explants
Parameters Suitable for Technology

There are great variety of in vitro stress-tolerant wheat and barley selection protocols,
described in literature and differed in medium composition and cultivation conditions,
because of genotype great influence on callus culture growth and development [[9], [10], [2],
[7]] . Different levels (0,5; 1; 2; 3 mg/l) of 2,4-D was used for callus induction. 100% level of
callus formation was detected on the medium with 3 mg/l 2,4-D. But no regenerants formed
at the further steps of selection process after cultivation under these conditions. Calluses were
passaged to proliferation medium with halved 2,4-D level in comparison with induction
medium after 30 days of cultivation. The most active callus proliferation was detected on the
medium contained 0,5 mg/l 2,4-D. So far big calluses could be divided into parts to increase
the quantity of samples.
As the result the 2,4-D concentration of 1 and 0,5 mg/l in induction and proliferation
medium respectively were chosen for further research in spite of the fact that maximum level
of callusogenesis was detected at the 0,25 mg/l of 2,4-D (Table 1). But our latest
investigations cleared out that the most suitable for maintenance regeneration activity during
further cultivation is proliferation medium with 2.4-D level the same as in callus induction
one - 1 mg/l (Figure 1). Vegetative period of parental varieties also had a great influence on
callus induction. Early cultivars (Tajozhnaja, Novosibirskaja 15) had significant larger
percentage of formatted calluses, than late cultivars (Minusa, KS-1607) independently of 2.4-