Alan AR et al., Plant Cell Reports, 26, 1345-1355 (2007)
Summary
An approach of combining flow cytometry
(FCM) analysis with morphological and chemical profiling
was used to assess the genetic stability and bioactive
compound diversity in a Scutellaria baicalensis Georgi
(Huang-qin) germplasm collection that was clonally
maintained in in vitro for a period of over 6 years. Based
on the FCM analysis of nuclei samples from young shoots,
the nuclear DNA content of S. baicalensis was calculated
as 0.84 pg/2C. FCM analysis showed no significant variation
in the nuclear DNA contents and ploidy levels in the
long-term in vitro maintained germplasm lines. Germplasm
lines, acclimatized to ex vitro conditions, exhibited distinctive
plant growth and bioactive compound production
capacities. The high level of genetic stability observed in
in vitro maintained S. baicalensis lines opens up a variety
of opportunities such as allowing long-term aseptic preservation
and easy distribution of well-characterized germplasm
lines of this medicinal plant species. This study
represents a novel approach for continuous maintenance,
monitoring, and production of medicinal plant tissues with
specific chemistry.
Objectives
An approach of combining flow cytometry
(FCM) analysis with morphological and chemical profiling
was used to assess the genetic stability and bioactive
compound diversity in a Scutellaria baicalensis Georgi
germplasm collection that was clonally
maintained in in vitro for a period of over 6 years.
Materials
Seeds of Scutellaria baicalensis, a diploid (2n = 2x = 18) species
were obtained from Richter’s Herb
Specialists (Goodwood, ON, Canada) and used as source
material for all experiments.
Explant
Seeds
Initial culture
In vitro-propagated control plants: Ten Scutellaria baicalensis seeds were surface sterilized by
immersion in a 70% ethanol solution for 5 seconds and sterilization
solution (20% commercial bleach with 0.1% Tween-
20) for 20 min. Sterilized seeds were rinsed three times
with sterile distilled water and placed in Magenta boxes
containing 65 ml of MSO medium (pH 5.75) comprised of
Murashige and Skoog (MS) salts, Gamborg B5 (B5) vitamins, 2.5 g/l gelrite (Sigma Chemical
Co., St. Louis, MO, USA) and 30 g/l sucrose. Seeds germinated
within 10 days and developed into plants with
well established roots and shoots 2 months after the germination.
Shoot multiplication
In vitro-propagated control plants: Nine nodal explants prepared from each plant
were sub-cultured in the Magenta boxes with fresh MSO
medium and grown for another 2 months. Cultures were
maintained in a culture room set at 16 h light (25–40 μmol m–2 s–1) at 28 ℃ and 8 h dark at 24 ℃.
Germplasm lines: The collection of in vitro-maintained S. baicalensis lines
was established from intact seedlings in the
presence of thidiazuron after a
limited exposure to the chemical mutagen ethylnitrosourea. The resulting germplasm collection was maintained
via aseptic clonal propagation of nodal cuttings, which
were sub-cultured at 2-month intervals on a growth regulator
free culture medium (MSO). The maintenance was
performed by placing nine nodal explants in each Magenta
box containing 65 ml of MSO and each line was maintained
in three boxes (total of 27 nodal explants). All
cultures were maintained in a culture room with 16 h light
(25–40 μmol m–2 s–1) at 22 ℃ and 8 h dark at 17 ℃. In general, the in vitro-propagated plantlet produces
about two roots and two shoots by the end of a 2-
month period. Plants of long-term maintained germplasm
lines retained their capacity to produce healthy roots and
shoots, but growth performances of lines were not similar. In vitro maintained S. baicalensis plants showed
tendency toward multiple shoot production (〜3 shoots per
plant) although the nodal explants had only two preexisting
buds.
Rooting
Nodal explants
were sub-cultured in the Magenta boxes with fresh MSO
medium. Roots
formed on the plantlets after a period of 4–6 weeks.
Acclimation
Planting
The flats were transferred to the greenhouse at
the end of the fourth week where 15–25 plants from each
line were transplanted into 6-inch standard pots filled with
a soil-less mixture of equal volumes of perlite and vermiculite.
Cultivation conditions
Potted plants were grown under typical greenhouse
conditions.