GENTIANA HETEROCARYONS PRODUCTION WITH APPLICATION

OF ECM 2001(BTX) PULSE GENERATORA. Fiuk and J. J. Rybczyński

Botanical Garden – Center for Biological Diversity Conservation of the Polish Academy of Sciences, Prawdziwka 2 str, 02-973 Warsaw, Poland

This work was financed by MS&I; Grant 3 P04C 037 23

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).

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 1:1. 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.

Conclusions:

1) 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

DC Voltage (V/cm) 1000 800 600 400

Amount of burst protoplasts (%) 28.16 22.26 12.68 8.51

Source of mesophyll

protoplats

DC Voltage (V/cm)

800 600 400

G. tibetica 6.38 ± 1.72 5.16 ± 1.61 4.67 ± 0.73

G. cruciata 7.13 ± 1.16 6.41 ± 0.46 4.96 ±0.83

G. pannonica 8.66 ± 1.07 7.94 ± 2.60 6.80 ± 0.50

Source

of mesophyll protoplats

DC Voltage V/cm

800 600 400

Heterocarions Homocarions Heterocarions Homocarions Heterocarions Homocarions

G. tibetica 2.90 ± 0.57 3.48 ± 2.29 2.86 ± 1.13 2.30 ± 0.65 2.24 ± 1.11 2.42 ± 0.37

G. cruciata 2.48 ± 0.87 3.87 ± 0.41 2.59 ± 0.22 3.82 ± 0.68 1.98 ± 0.54 3.0 ± 1.08

G. pannonica 4.76 ± 0.79 3.89 ± 0.56 4.21 ± 0.81 3.73 ± 1.89 4.55 ± 0.86 2.91 ± 0.38

Parameters G. kurroo + G. tibetica G. kurroo + G. cruciata G. kurroo + G. pannonica

AC

Voltage

(Vol/cm)

Duration (s)

Voltage after fusion

(Vol/cm)

Voltage

(Vol/cm)

Duration (s)

Voltage after fusion

(Vol/cm)

Voltage

(Vol/cm)

Duration (s)

Voltage after fusion

(Vol/cm)

20 02 0 20 02 0 20 02 0

DC

Duration (µs)

Voltage

(Vol/cm)

Number of pulse

Duration (µs)

Voltage

(Vol/cm)

Number of pulse

Duration (µs)

Voltage

(Vol/cm)

Number of pulse

30 600 2 30 600 2 30 400 2

Fig. 4. Optimal fusion condisions for particular species combination.

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. 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.

Fig. 1. The influence of DC voltage for share of burst protoplast after fusion of G. kurroo and G. cruciata (%).

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

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

A CB

A CB