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GENTIANA HETEROCARYONS PRODUCTION WITH
APPLICATION OF ECM 2001(BTX) PULSE GENERATOR
A. Fiuk and J. J. Rybczynski Botanical Garden
Center for Biological Diversity Conservation of
the Polish Academy of Sciences, Prawdziwka 2 str,
02-973 Warsaw, Poland
Introduction Since ninetieth we have been working
on Gentiana genus biotechnology exploring high
level of morphogenic potential at multicellular
and single cell explants. The somatic
hybrydization which employs protoplasts opens
avenue to combine the genomic value of nucleus
and cytoplasm of both parents the process of
electrofusion as the method of hybridization of
somatic cells consist on two consecutive stages
protoplast pearl chains formation controlled by
Alternating Current (AC) and direct fusion
stimulated by Direct Current (DC).
Fot. 1. Succesive stage of protoplast fusion
A) green leaf mesophyll protoplasts of G.
cruciata and cell suspension protoplasts of G.
kurroo before the fusion
B) first stage - protoplast pearl chain
formation C) second stage -
fusion
Aim The aim of the presentation was to work out
condition for high yield production of
heterocaryons of Gentiana spp. with the
application of electrofusion (EF) protoplasts.
Material and Methods Experiments were carried out
with four species of gentian. Two types of
protoplasts were fused. White protoplasts were
isolated from three years old embryogenic
suspension of Gentiana kurroo. For protoplast
isolation cell suspensions were digested with 1.5
Cellulase RS (Yakult Honsha Co., LTD), 1.5
Macerozyme R10 (Yakult Honsha Co., LTD), 0.5
Driselase (Fluka AG), 0.25 Hemicellulase
(Sigma), 0.04 Pectolyase Y23 (Seishin
Pharmaceutical Co., LTD), 0.976 MES and 9.0
mannitol diluted in CPW solution during 12 hrs
into the dark. Green mesophyll protoplasts were
isolated from cotyledons and first leaves of
seedling G. cruciata, G. pannonica and G.
tibetica with application of 1.0 Cellulase and
0.5 Macerozyme R10 (the rest components and
conditions were the same like in case of cell
suspension). After three times washing in CPW
with 9.0 mannitol density of green and white
protoplasts was established on the level 5x104
and 1x105 respectively. Fusion partners were
mixed in fusion buffer in ratio 11. Fusion
buffer includes 1.0 mM CaCl2 . 5 H2O 5.0 mM MES
9.0 mannitol with adjused pH 7.2.
Electrofusion was done with the help of ECM 2001
(BXT) Pulse Generator and application of special
type of Microslide chamber. Following parameters
were tested AC-field at 10, 15, 20 , 25 and 30
V/cm for 2 s and DC-firld for obtaining fusion at
400, 600, 800 and 1000 V/cm with two pulses of 30
µs. Electrofusion conditions were work out on the
basis of observation directly after fusion and 24
hrs later with computer program Analysis.
Results At the presence of AC 10 and 15 V/cm
chains formation was to slow. AC 25 and 30 V/cm
were caused protoplast burning or large amount of
multiple fusion. The best AC for all species
combination was 20 V/cm. At the presence of DC
1000 V/cm 28.1 of fused protoplasts burst
directly after fusion (Fig. 1) and almost all 24
h after fusion. The fusion efficiency varied
from 4,67 to 8, 66 at DC 800, 600 and 400 V/cm
with two pulses of 30 µs (Fig. 2). Lesser values
of DC were caused reduction of fusants yield.
Source of green protoplast was influenced on
fusion frequency. The highest fusion efficiency
was achieved when white protoplasts of G.
kurroo and green protoplasts of G. pannonica
were partners. Share of hetero- and homocaryons
were similar (Fig. 3). Optimal fusion conditions
were established on the basis of microscopic
observation after 24 h (Fig. 4). Differences
were observed only in the scope of DC voltage.
The best DC voltage for G. kurroo G. tibetica
or G. cruciata was 600 V/cm and for G. kurroo
G. pannonica 400 V/cm. Succesive fusion stages
were showed on figures XY.
Fot. 2. Distribution of chloroplasts (Ch) and
amyloplasts (Am) in the heterocaryon after
fusion A) closely located
to plasmolemma B) chloroplasts
distribution in cytoplasm
C) chroloplasts surrounding nucleus
Fig. 3. The influence of DC voltage for hetero-
and homocaryons creating () during fusion of
suspension delivered protoplast of G. kurroo
with mesophyl protoplsts of three gentiana
species.
Fig. 1. The influence of DC voltage for share of
burst protoplast after fusion of G. kurroo and G.
cruciata ().

• Conclusions
• Heterocaryon production was correlated to the
  quality of protoplasts
• 2) Using electrifield the production of
  heterocaryons was species dependent
• 3) The best results were achieved with the
  application of voltage 400 600 Vol/cm DC

Fig. 4. Optimal fusion condisions for particular
species combination.
Fig. 2. The influence of DC voltage for fusion
efficiency () of suspension delivered protoplast
of G. kurroo with mesophyl protoplsts of G.
tibetica, G. cruciata and G. pannonica.
This work was financed by MSI Grant 3 P04C 037
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