Plant Cell Reports (1985) 4:289-292 Plant Cell Reports © Springer-Verlag 1985

Callus production from photoautotrophic soybean cell culture protoplasts

Vijay K. Chowhury i and J a c k M. W i d h o l m 2

1 Department of Genetics, Haryana Agricultural University, Hisar 125 004, India 2 Department of Agronomy, University of Illinois, 1102 S Goodwin Ave., Urbana, IL 61 801, USA

Received March 1, 1985 / Revised version received August 30, 1985 - Communicated by G. B. Collins

SUMMARY :

Protoplasts were prepared from a photoauto- trophic (PA) cell line of Glycine max (soybean). A yield of 75 to 90% after two to three hours diges- tion in a mixture of i% Cellulase RI0, 0.2% Pectolyase Y23 and 2% Driselase was obtained. Cell division and colony formation occurred from approxi- mately 18% of the plated protoplasts. The cultured protoplasts were as sensitive to the herbicide atrazine, a photosynthetic inhibitor, as the original PA cells under the same conditions. Proto- plasts and cells of a heterotrophic (HT) soybean culture were not as sensitive to atrazine. The isolated protoplasts retained the PA characteristics of the parental culture in the callus and cell sus- pension cultures obtained from the protoplasts. The chromosome numbers in the parental cell line and in cells derived from the isolated protoplasts (both PA and HT) were found to be largely (99%) the normal diploid number of 40.

Abbreviations:

INTRODUCTION:

BA, Benzylaminopurine 2,4-D, 2,4-dichlorophenoxyacetic acid HT, Heterotrophic MES, 2-(morpholino) ethane sulfonic

acid NAA, Naphthaleneacetic acid PA, Photoautotrophic PCM, Protoplast culture medium.

Glycine max (soybean) is one of the world's most important grain legumes. The development of a wide range of tissue culture and protoplast tech- niques may open up the possibility of genetically manipulating this species in vitro. The isolation and culture of protoplasts from soybean cell suspen- sions has been reported (Gamborg et al., 1969; Kao et al., 1971 and Miller et al., 1971). Such proto- plasts have been used for fusion (Gamborg et al., 1974; Wetter and Kao, 1980), in ultrastructural investigations (Fowke et al. 1976), in studies of DNA replication (Cress et al., 1978) and in studies of virus infection (Jarvis and Murakishi, 1980).

We report here the enzymatic isolation of protoplasts from a photoautotrophic cell line of Glycine max (Horn et al. 1983) and the regeneration

Offprint requests to: J. M. Widholm

of callus and cell suspension cultures from these with characteristics like those of the original cell strain. This protoplast system could be valuable in biochemical, fusion and genetic transformation studies.

MATERIALS AND METHODS:

Heterotrophic (HT) cell cultures

The callus cultures were established from the central part of the hypocotyl of aseptically germi- nated seedlings of Glycine max (A 3127, Asgrow) on B5 medium (Gamborg et al., 1968) supplemented with 2 g/i casein hydrolysate (enzymatic). These cultures were then maintained on L2 medium (Phillips & Collins, 1981) supplemented with 2,4-D (0.4 mg/l), NAA (4.65 mg/l) and kinetin (2.15 mg/l). Suspension cultures were initiated by transferring approximate- ly 500 mg friable callus into 50 ml B5 medium sup- plemented with 2 g/l casein hydrolysate. The cultures were shaken on a gyratory shaker (131 rpm) at 28 ± I°C and were subcultured every 8-10 d.

Photoautotrophic (PA) cell suspension cultures

The photoautotrophic (PA) cells were established using callus initiated from 'Corsoy' soybean cotyledons (Horn et al. 1983) and were main- tained in a liquid medium lacking sucrose (KT-0) (Horn et al. 1983) in a 5% CO 2 atmosphere under continuous light. The cells were subcultured at 7 d intervals.

Protoplast isolation

Five to seven day old fine suspensions of single cells and small aggregates, were collected by centrifugation at 400xg for I0 min. The pellet (300 mg fresh weight) was suspended in 10 ml of 1.0% Cellulase RIO (Yakult Pharmaceuticals Ind., Japan); 0.2% Pectolyase Y23 (Seishin Pharmaceuticals Inds. LTD., Japan) and 2.0% Driselase (Kyowa Hakko Kogyo Co. LTD, Japan) dissolved in IS-I protoplast wash medium (Gamborg et al., 1983) consisting of 0.7 mM KH2P04, 6.0 mM CaCI2-2H20 , 0.15 M sorbitol, 0.15 M mannitol, 0.i M glucose and 3 mM MES buffer at pH 5.6. The incubation was for 2 to 3 h in the dark on a rotary shaker (20 rpm) at 28 ± I°C. The protoplasts were harvested by centrifugation at lOOxg for I0 min. The pellet was resuspended in

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IS-I medium and the protoplasts were purified by

suspending in 20% sucrose in 35 ml Babcock bottles and centrifuging at 100xg for i0 min. Purified protoplasts were removed from the neck o~ the bottle and 2 ml with a protoplast density of i0 /ml were

cultured in 6 cm Petri dishes for 8 d under continuous light at 28 ± 1°C in liquid PCM: SL

mineral salts (Collins and Phillips, 1982), B5 vitamins (Gamborg, 1975), calcium and multivitamins (Gamborg et al. 1968) and the following in mg/l: casamino acids, 125; sodium citrate i0; L-glutamate,

I00; sodium pyruvate, 5; ribose, 125; sorbitol, 27300; sucrose, 150; m-inositol, 150; glucose, 36000; 2,4-D, 0.22; picloram, 0.06; BA, 0.i. The medium pH was adjusted to 5.7 and was filter sterilized. On day 8, 1.5 ml of fresh KT-I medium (Horn et al., 1983) supplemented with 15 g/l sorbitol, was added and after another 12 d, 2 ml of KT-I medium was added. Finally cell aggregates

which formed were cultured on KT-I medium solidified with 0.6% BRL (low melting point) agarose (Bethesda Research Laboratories, Gaithersburg, MD, USA) under

continuous light (fluorescent, 300 lux). Plating efficiencies were determined in experi-

ments where protoplasts were incubated for 5d in PCM

liquid medium and then were mixed with an equal volume of KT-I medium with 15 g/l sorbitol and 1.2%

BRL low melting point, Sea Prep or BRL agarose. Plating efficiency was the number of colonies formed

divided by the number of protoplasts cultured

originally.

Chlorophyll determination

Chlorophyll content was determined by grinding

tissue collected on Miracloth filters in a mortar and pestle with 80% acetone and 20% water. The

equation of Arnon (1949) was used.

Atrazine sensitivity

The isolated protoplasts and the normal cells

were cultured for I0 days in PCM medium containing different concentrations of atrazine ranging from 0.i BM to 100 ~M. Atrazine was dissolved in absolute ethanol before adding to the medium. Con- trols were run with ethanol alone. The percent viability as measured by the capability of the cells

and protoplasts to exclude phenosafranine dye (Widholm 1972) and the total chlorophyll content of cells and protoplasts were determined 10 d after

inoculation.

Cytological studies

Five day old suspensions were fixed in 3:1

ethanol and glacial acetic acid for 8 h and then washed 3-4 times in distilled water. Hydrolysis of cells was carried out in 0.2 N HCI for five min at 60°C. The hydrolyzed cells were washed and preserved in 70% ethanol at O°C until used. The slides were prepared according to the method

described by Paka and Widholm (1984) and 225 mitotic figures were examined of each line.

RESULTS AND DISCUSSION:

Experiments using the PA soybean cells 5d after subculturing showed that the enzyme mixture I, con- sisting of i% Cellulase RIO, 0.2% Pectolyase Y23 and 2% Driselase, was the most effective of the combi- nations used (Table i), releasing protoplasts from 85% of the cells. In other experiments, cells were harvested at 2 d intervals following inoculation from day 2 up to day 11. The 5 d cells released the highest number of protoplasts (75-90%) using the enzyme mixture i with the release complete, 2 to 3 h after the beginning of the incubation. Figure IA shows a typical protoplast preparation following purification.

These protoplasts began to divide after 3d in the protoplast culture medium (Figure IB) and many

showed sustained divisions to form cell clumps (Figure IC). These clumps could grow to form green colonies and callus when plated on solidified medium (Figure ID). Final plating efficiencies based on the original number of protoplasts cultured of ca. 18% were obtained on medium with Sea Prep and BRL

agarose while ca. 25% efficiency occurred with BRL low melting point agarose.

Freshly prepared protoplasts and complete cells of both the PA and HT soybean suspension cultures were incubated with different concentrations of atrazine to determine their sensitivity. It was felt that PA cells and protoplasts relied more on photosynthesis than did HT cells or protoplasts so would therefore be more sensitive to a photosynthetic inhibitor such as atrazine. As seen in Figure 2 the cells and protoplasts from the PA cultures were more easily killed by atrazine than were the HT ones in the i0 d incubation period. Atrazine also decreased the chlorophyll content of

Table I. Percent of protoplasts released from five d old photoautotrophic cells after a four h incubation in

different enzyme mixtures dissolved in IS-I medium.

Enzyme mixture

Enzyme i 2 3 4 5 6 7 8 9 10

................................. ~ .................................

Cellulase RI0 1.0 - 1.5 2.0 - 2.0 - 2.5 - Pectolyase Y23 0.2 0.5 - 0.4 - - 1.0 - 1.5 - Hemicellulase (ICN- 1.0 - - 2.5 - - 2.5 - 3.0

Pharmaceuticals Ohio Rhozyme HP 150 - 2.0 2.0 - 2.0 - 1.5 - -

Cellulysin . . . . . 1.5 - - - Driselase 2.0 - - 1.5 - 2.5 - 2.0 2.5 -

Macerozyme - - 1.0 - - 1.0 - - - Macerase - 1.0 - - 1.5 1.5 - 1.5 - 1.0

Meicelase - - - 4.0 - 1.5 . . . . - 2.5 - 3.5 4.0

Rohament P . . . .

% Release 85 2.5 30 20 25 0 35 0 52 0

291

100

Figure i. Photographs of freshly isolated PA protoplasts (A), PA protoplasts showing first cell division after 72h (B), cell-cluster after 15 d (C), callus formation from clusters on BRL low melting point agarose medium (D), The bars in A, B, and C equal 100 ~m, and in D equals I cm.

80

"-- / t

60 ._J

<~ 40 >

20

0 .1 1.0 10 S0 100 ATRAZINE (#M)

Figure 2. Effect of atrazine on the viability of PA cells (o), and protoplasts (o) and on HT cells (D) and protoplasts ([]) after a I0 d treatment.

the protoplasts and cells of the PA cultures with the protoplasts being somewhat more sensitive (Figure 3). Atrazine is known to inhibit electron transport at the reducing side of photosystem II (Vermaas and Govindjee 1981) so the bleaching seen would be a secondary effect. The HT cells and protoplasts contained little or no chlorophyll, as expected, since they were maintained on a sucrose containing medium. It should be noted, however, that even though these results indicate that photo- synthesis is important to the viability of the cells

60O

!°00 i

,oo o 1:o lo -go 16o

ATRAZ1"NE (#1"1 }

Figure 3. Effect of atrazine on the chlorophyll content of PA cells (e) and protoplasts (o) and on HT cells (m) and protoplasts (D) after a I0 d treatment.

and protoplasts of the PA culture, the medium used in this experiment with atrazine (PCM) did contain 3.6% glucose. The genotypes of the PA cells and protoplasts used in these experiments were not the same but in other experiments comparing 'Corsoy' PA and HT cell atrazine sensitivities (Horn and Widholm 1984), very large differences were seen with the PA cells being much more sensitive as also noted here.

The growing protoplasts did retain high chlorphyll levels after the 12 d incubation without

292

Table 2: Growth and chlorophyll content in the PA cells and in cultures derived from protoplasts of the PA cells in media with zero or 1% sucrose after 12 d incubation in continuous light (mean of five flasks with standard deviation).

Cultures Medium a Fresh weight b (gram)

Chlorophyll content (~g/g fresh wt.)

Normal PA cells PA Protoplast derived cells

Derived cells Normal PA cells PA Protoplast derived cells

KT-I 3.8 + 0.82 419 + 42 KT-I 3.71 ± 0.73 478 ± 53

KT-O 3.47 + 0.62 652 + 62 KT-O 3.75 ± 0.93 517 ± 71

a KT-I - i% Sucrose in ~he medium. KT-0 No sucrose + 5% CO 2 atmosphere.

b Increase in fresh weight over 500 mg initial weight.

atrazine (Figure 3). The chlorophyll level near 550 ~g/g fresh weight (Figure 3) is close to that of the PA cells analyzed in Table 2. The actual

chlorophyll levels in the PA cells used to prepare the protoplasts for the atrazine experiments (Figure 3) were not measured, however.

The dark green callus (Figure ID) which formed from the PA protoplasts was placed in liquid medium lacking sucrose under 5% CO 2 and continuous light (PA cell growth conditions) and growth began imme- diately. After the second transfer under these conditions, one typical protoplast-derived culture grew at the same rate as the original PA line in both 0 and 1% sucrose containing medium with doubling times near 4 d (Table 2). The chlorophyll contents were also similar.

Cytological observations of both the original HT and PA cultures and the protoplast derived cul- tures from each showed the chromosome number to be largely normal (2n = 40). Less than 1% of the cells showed variation from this number (aneuploidy and polyploidy).

Thus we have been able to prepare protoplasts in high yield from a rapidly-growing photoautotrophic soybean cell suspension culture. These protoplasts could find several uses including the isolation of organelles for biochemical and genetic studies. The protoplasts can reform cell walls and divide to form cultures similar to the original in growth rate and ehlorophyll levels. Even though the original cell line has been cultured for ca. five years now, the chromosome numbers of the PA cells and the cells grown from protoplasts prepared from the PA cells are still normal (2n = 4O).

ACKNOWLEDGEMENTS

This work was supported by funds from the Illinois Agricultural Experiment Station and from Agrigenetics Research Associates. The PA stock cultures were provided by Sue Rogers.

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