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Bioscience Methods 2012, Vol.3, No.3, 21
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26
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22
aflatoins which were produced by aspergillus flavus
and harmful to human beings and livestock. When
extracts and vitamin C were used together, it appeared
stronger oxidation resistance (Djenane
et al., 2003).
Furthermore,
R. officinalis
L. can be used for soil and
water conservation, city landscaping and family potted
(as mozzie buster). Therefore, it’s a kind of economic
plant that has prosperous exploiting and utilizing
value (Li et al
.
, 2006).
With the increasing demand of
R. officinalis
L. on
food processing
、
medical, beauty and health care, this
not only enhances the
R. officinalis
L processing
industry, but also the rise of effective and high quality
cultivation, therefore, it requires higher quality
breeding and cultivation of rosemary.
Usually, there are three propagation methods: seed
breeding, and cutting propagation. However, seed
germination rate is quite low (about 13%) that it’s not
easy to survival by cutting propagation. Although
laying propagation has high survival rate, there are no
enough plantlets for large area cultivating (Chen and
Kang, 2009).
Despite of controversial breeding, some significant
biotechnologies which are beneficial to improvement of
economic production and food crops, such as genetic
engineering, cell engineering, haploid induction and
somatic mutation, largely depended on the techniques
that explants can regenerate and become intact plants
(
Vijendra
et al., 2005). We adapt
in vitro
culture
technique
s,
select the rosemary as the explants in
this research, induces the leaves to dedifferentiation
and become callus, later becomes adventitious buds
and finally grow to intact plant. This not only offers
the callus and intact plants to rosemary improvement
by modern biotechnologies, but also quickly obtain
large amount of rosemary materials that the genetic
traits are similar for large-scale cultivation. Meanwhile,
it establishes the rosemary callus cultivation system
in order to produce secondary metabolism product
by cell engineering techniques.
1 Results and Analysis
1.1 Screening the optimal sterilization reagents
The treatment of different sterilizing reagents showed
that 0.2% HgCl
2
(w/v, 5 min) was the optimal
treatment (Table 1). From the table 1, we knew that,
with 75% (v/v) ethanol, the contamination rate of
explants was low as well as the survival rate and
sometimes all of them were dead. It was estimated that
when the alcohol-soluble substances of
R. officinalis
L
may be dissolved, thus, the leaves were dead.
Therefore, the explants sterilization cannot use 75%
(v/v) ethanol, which will result in large amount of
explants dead.
1.2 Callus induction of
R. officinalis
L. leaves
The mix proportion test of different concentration of
sucrose and hormone indicated that MS medium with
6
-
BA 0.5 mg/L, NAA 0.5 mg/L, and 50 g/L sucrose
was proved to be the optimal medium for the
production of calli, which was able to start to
dedifferentiation after 6 days. There are more
explants that was successful to form callus (88%).
However, it needed 9 days that the MS 30 g/L sucrose
medium with start to dedifferentiation. From the
table 2, we knew that the speed of callus
dedifferentiation was much faster in 50 g/L sucrose
medium than that in 30 g/L sucrose medium and the
growth status of callus was better (Table 2; Figure 1).
1.3 Regeneration of
R. officinalis
L. callus
After the dedifferentiation, the leaves became callus,
some callus were used to subculture for proliferation
subculture
，
the other callus removed to dedifferentiation
medium. The adjustment of cytokinins and auxin ratio
Table 1 Effect of different sterilizing reagents on
Rosmarinus officinalis
L. explants
Treatment
Sterilizing reagents
Holding time
Observed results
No. of explants Rate of contamination Survival rate Death rate
1
75% ethanol+0.2% HgCl
2
30 s+5 min
40
25% (75% sterile)
10%
90%
2
75% ethanol+2% NaClO
30 s+15 min 40
50% (50% sterile)
15%
85%
3
2% NaClO
15 min
40
80% (20% sterile)
65%
35%
4
0.2% HgCl
2
5 min
40
7.5% (92.5% sterile)
95%
5%
Note: Death rate = No. of
R. officinalis
L. leaf sterile (but dead) / No. of sterile×100%