Abstract:
The present study reports an efficient protocol for
isolation and regeneration of
protoplasts from callus of
Fritillaria imperialis
L. There is no published method
recommended for protoplast isolation and regenerati
on from
Fritillaria imperialis
L. A
range of factors, which influence the success of is
olation and regeneration of
F. imperialis
protoplasts, were investigated. From the results ob
tained, callus Fresh Weight (FW) of 0.4
g produced the highest number of viable protoplasts
, which was 1.12×10
5
protoplasts g
-1
FW. The highest amount of viable protoplasts (1.01×
10
5
protoplasts g
-1
FW) was obtained
when the mannitol concentration was maintained at 9
% (w/v). The best treatment for
isolation of
F. imperialis
protoplast (1.37×10
5
protoplasts g
-1
FW) was treatment with 2%
cellulase and 0.1% pectinase with 9% mannitol for 8
h. For enhancement of the
protoplasts division and the percentage of colony f
ormation, different concentrations
from Casein Hydrolysate (CH), 2,4-Dichlorophenoxyac
etic acid (2,4-D) and Benzyl-
Adenine (BA) were used. The results revealed that c
ell wall and colony formation were
better in liquid medium than those on semi-solid me
dium. The highest plating efficiency
(1.26×10
6
per g FW) and highest callus formation were obtain
ed using the medium
containing 0.5 mg L
–1
2,4-D, 1 mg L
–1
BA, and 200 mg L
–1
CH. Micro-calli were formed
after one month of culture. Many plantlets were for
med on the calli after transfer of the
proliferated calli to regeneration medium. The high
est plantlet regeneration (100%) was
obtained using the medium containing 0.5
mg L
–1
(Naphthalene
Acetic Acid) NAA and 1.5
mg L
–1
BA.
Keywords:
Callus formation, Medium, Protoplast culture, Viabi
lity.
