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Bioscience Methods 2012, Vol.3, No.3, 21
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receptor which makes use of enhancing the
rosemary quality by modern biotechnologies, but also
quickly produced large amount of plants with similar
genetic traits. This became the theoretical basis for
rosemary
in vitro
experiment. Simultaneously, this also
provided the theoretical basis that extract the effective
ingredient from callus directly.
The results also showed that 75% ethanol (30~40 s)
with other sterilizing reagents could result in large
explants death, which suggested that there were
abundant ethanol-soluble components in
R. officinalis
L.
leaf. If utilizing ethanol sterilizing reagents, the
content will be dissolved and the cell die. Therefore,
the 75% ethanol mixed sterilizing reagents is not
suitable for this research. Meanwhile, MS medium
with higher sucrose concentration (50 g/L) will
promote rosemary leaves to dedifferentiation. This
suggested that the rosemary leaves contain lots of
essential oil, higher carbon source ratio that is
beneficial to enhance the growth of rosemary with
Alcohol-soluble substances and lipid. All of results
provided the theory for the plant
in vitro
culture.
3 Materials and Methods
3.1 Materials
Leaf explants from spring shoots of
R. officinalis
L.;
Basal medium of Murashige and Skoog (MS)
(Murashige and Skoog, 1962) with 3%~5% (w/v)
sucrose, 0.7% Agar, pH 5.8 and different concentration
of plant growth regulators; sterilizing these mediums
under 121
℃
, 0.1-0.5 kPa 20 min; distilled water; 75%
(v/v) ethanol; 0.2% HgCl
2
(w/v); 2% sodium
hypochlorite (NaClO, v/v).
3.2 Comparative study of the sterilization effects of
the explants primary cultivation
R. officinalis
L. leaves and spring stems (3~5 cm)
bought from market, washed by running water for
0.5~1 hours, sterilized by different sterilizing
reagents (Table 1), washed by sterile still water for
3~5 times, and cultured. The temperature is (25±2)
℃
with 70%~80% relative humidity. The light intensity
is 2 500 Lux with 14 hours in the daytime and 10
hours at night. After cultured 3 d, 6 d, the status of
explants were observed and then record the data every
10 days. or without pre-sterilized in 70% (v/v) ethanol
for 30~40 s, and then in commercial bleach (2% sodium
hypochlorite) for 15 min, or 0.2% HgCl
2
(w/v) for 5
min (Table 1), and then washed 3 to 5 times in
distilled water. Leaves were excised from young stems
and placed on solid agar sterile medium for callus
induction. They maintained in the culturing room at
(25±2)
℃
and in a 14 h light (2 500 Lux), 8 h dark
photoperiod.
3.3 Callus induction of
R. officinalis
To investigate the callus induction medium, this
research designed mixture of 30 g/L source, 50 g/L
source with cytokinins and auxin, separately (Table 2).
Note: callus induction (%) = No. of calli from leaf /
No. of cultured leaf × 100%; Start-up period (unit; d):
The days are from the cultivation to 50% leaf blades
that began to differentiate and form calli.
3.4 Regenerating of
R. officinalis
L. calli
When calli expanded to 1.5~2.0 cm
2
, some of them
continued to subculture in the original medium, while
the other induce to dediffireciate to roots with
different cell division concentration. This research has
designed various cytokinins and auxin ratio to
investigate the callus dedifferentiation conditions.
Note: budding rate (%) =No. of budding calli (piece of)
/ No. of culturing calli (piece of)×100%.
3.5 Propagation of
R. officinalis
L. adventitious
buds
Adjusting the auxin concentration in medium, the
rosemary
adventitious buds
is predicted to induce the
lateral buds propagation. The auxin compose and
densities is in
Table 4. Note: Proliferation rate = No.
of shoots / No. of transferring shoots×100%.
3.6 The rooting and the formation of complete
plants
In the process of plant tissue culture, medium
inducing shoots would inhibit the root growth, and
vice versa. Therefore, The adventitious buds that
coming from callus didn’t have root. It needed to
reduce cytokinins and auxin concentration to grow
into an intact plantlet. Select the adventitious buds
with 2~5 cm in length to rooting and set up three
different NAA concentration; When roots came into
3~6 with about 2~4 cm in length, they could transfer
into the soil to grow up.
Author Contributions
YMD and ZNL conceived the overall study, performed the