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Colorimetric estimation of leaf-protoplast potential-to-divide by use of 2,3,5-triphenyl tetrazolium chloride
J. Plant Physiol. Vol.
141. pp. 111-114 {1992}
Colorimetric Estimation of Leaf-Protoplast Potential-toDivide by Use of 2,3,5-Triphenyl Tetrazolium Chloride MASAMI WATANABE, YUKIO WATANABE,
and NORITSUGU
SHIMADA
Laboratory of Plant Nutrition, Faculty of Horticulture, Chiba University, 648 Matsudo, Chiba 271, Japan Received February 2,1992 . Accepted May 10, 1992
Summary
Purified protoplasts from Petunia hybrida reduced 2,3,5-triphenyl tetrazolium chloride (TTC), whereas cellular or subcellular debris gave a negative TTC test. The TTC reduction was decreased with increasing concentration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and iodoacetate (IA). Consequently it could be correlated with plating efficiency. The most formazan was produced by the protoplasts from top-developing leaves. FDA staining could not detect the difference in viability of protoplasts isolated from different growth stages of leaves. The TTC reduction test described here offers a colorimetric estimation for the potential of protoplasts to form colonies.
Key words: Petunia hybrida, plating efficiency, protoplast, 7TC, viability. Abbreviations: BA = benzyladenine; 2,4-D = 2,4-dichlorophenoxyacetic acid; FDA = fluorescein diacetate; IA = iodoacetate; MES = 2-(N-morpholino) ethanesulfonic acid; TTC = 2,3,5-triphenyl tetrazolium chloride; MNNG = N-methyl-N'-nitro-N-nitrosoguanidine; MS = Murashige and Skoog.
Introduction
One definition of plant protoplast viability is the capacity of an isolated protoplast to divide, form a callus, and ultimately give rise to a plantlet. The process of protoplast isolation affects the state of viability (Watts et ai., 1974); a proportion of isolated protoplasts will not survive subsequent culture. This proportion depends upon the potential of viability. Therefore, protoplasts should be monitored for viability at an early stage. A number of tests have been applied to check protoplast viability in order to determine what portion of damaged protoplasts appears during isolation or in culture. Intactness of the plasmalemma can be measured by dye exclusion. Ability of tht plasmalemma to block the entry of a molecule of dye into the cell is normally measured with Evans blue (Kanai and Edwards, 1973; Glimelius et ai., 1974). In this test, dead or dying protoplasts stain blue, while those with an intact plasmalemma remain unstained. Percentage viability can then be assessed by counting. If the vacuole is disrupted, the protoplast will die. © 1992 by Gustav Fischer Verlag, Stuttgart
Intactness of the tonoplast can be assayed by use of neutral red (Basham and Bateman, 1975). The dye traverses the intact plasmalemma and is concentrated in the vacuoles. Dead or damaged cells will stain completely. Fluorescein diacetate (FDA) staining depends on plasmalemma integrity and an associated esterase activity (Larkin, 1976). The esterase cleaves the acetate from the FDA as it passes into the protoplasts, and the cells fluoresce on irradiation under ultraviolet light. In contrast to the dye exclusion methods, only viable protoplasts will fluoresce. In this paper we report the use of triphenyl tetrazolium chloride (TTC) as a protoplast viability test. It has formerly been used in conjunction with seed germinability (Porter et ai., 1947), cold injury of tissue (Purcell and Young, 1963), and cells in suspension culture (Kuriyama et aI., 1989). In contrast to FDA staining which depends on plasmalemma integrity and its associated esterase activity, TTC reduction is caused by dehydrogenase activity and may be a more direct estimate of protoplast viability. However, TTC reduction tests reported previously cannot be directly applied to
112
MASAMI WATANABE, YUKIO WATANABE, and NORITSUGU SHIMADA
leaf protoplasts because of endogeneous pigment interference. The present work provides a qualitative assessment of viability in batches of protoplasts.
Materials and Methods
Plant materials Seeds of Petunia hybrida cv. Recovery White were commercially obtained (Sakata seed Co. Ltd.).
Protoplast isolation Leaf protoplasts were prepared from leaves of 8 to 10-week-old plants grown in a growth chamber under a 14-h photoperiod with a photon fluence density of 50 Ilmol m- 2 S-I of fluorescent tubes and at 25/20°C for the light/dark period. Leaf blades were sterilized with 70 % ethanol for 10 s followed by treatment in 0.6 % final concentration of sodium hydrochlorite for 5 min which, in turn, was followed by several washes in sterile water. Lower epidermis from leaves of P. hybrida were peeled away and incubated for 12 h in an enzyme solution containing 0.1 % Cellulase Y-C (Seishin Pharmaceutical Co. Ltd.), 0.02 % Pectolyase Y-23 (Seishin Pharmaceutical Co. Ltd.), 5 mM MES (pH 5.8), and 0.6 M sorbitol. Isolated protoplasts were sieved through nylon mesh (82Ilm) and purified by flotation on 0.7 M sucrose.
1TC reduction test Leaf protoplasts, in 10-mL screw cap centrifuge tubes, were suspended in 3 mL of 0.05 M K 2HP0 4-KH 2P0 4-O.6 M sorbitol buffer (PH 7.4) containing 0.05 % of TTC % (w/v) and incubated for 16 h at 30°C in the dark. The TTC solution was filter-sterilised. All procedures described above were conducted aseptically. TTC reduction was determined as follows: Protoplasts were collected by centrifugation at 180 x g for 10 min. Water insoluble formazan was extracted with 5mL of 95% (v/v) ethanol in a boiling water bath for 5 min (Steponkus and Lanphear, 1967). The extract was cooled and diluted to 10 mL with 95 % ethanol. The absorbance was recorded at 520 nm. A wavelength still in the region of high absorption by reduced TTC but with minimum absorption by tissue pigments, namely 520 nm, was chosen for determination of the TTC reduction.
MNNG and lA treatment Leaf protoplast of P. hybrida were treated with 25, 50 and 100mgL- 1 final concentration of MNNG and 0.01, 0.1, 1 and 10 mM of IA for 1 h at 25°C in light. The MNNG solutions were discarded and the protoplasts washed three times with 10mL 0.6M sorbitol. Protoplasts for TTC reduction test were transferred to centrifuge tubes and cultured in 1 mL of MS liquid medium supplemented with 1.0 mgL- 1 2,4-D, 0.5 mgL -I BA, 2 % sucrose, 1 % glucose and 0.6 M sorbitol (pH 5.8) at 25°C for 5 d in the dark.
FDA staining FDA was added to the protoplast suspensions to give a final concentration of 0.01 %. After 5 min at room temperature, the protoplasts were examined for fluorescence using an Olympus FLM fluorescent microscope.
Chlorophyll determination Protoplasts were collected by centrifugation at 180 x g for 10 min and suspended in 80 % acetone with agitation. The solution was made up to 10 mL with 80 % acetone and centrifuged at 1600 x g for 5 min. The absorbance of the supernatant was recorded at 645 and 663 nm. Chlorophyll content was calculated according to Arnon (1949).
Protein assay The protoplast suspensions were centrifuged as above and suspended in a lysis buffer containing 50 mM Tris-HCI (PH 8.0), 10 mM EDTA-2Na. Protein content was determined by a Bio-Rad microprotein assay (Bio-Rad Co. Ltd.), using BSA as a standard.
Results
Steponkus and Lanphear (1967) working with leaf discs of an ivy showed that sodium hydrosulfate reduced formazan has a )" max at about 490 nm, a wavelength at which there is interference from endogenous plant pigments. They used 530 nm, a wavelength still in the region of high absorption by the reduced TTC, but minimum absorption by tissue pigments, for determination of formazan. In this work, tissue pigment interference was minimized by subtracting absorbance at 520 nm of 95 % ethanol extract of protoplasts incubated in an assay medium without TTC. As a result, the reduced water-insoluble formazan can be quantitatively recovered from the protoplasts. A result of the TTC reduction test for leaf protoplasts and a fraction of cellular or subcellular debris is presented in Fig. 1. Formazan can be detected in 1.0 , - " ' - - - - - - - - - - - - - - - - - - ,
w
U Z
o¢
'" o 0:
UJ
'" o¢
a 4
aa
5
aa
WAVEL E NGTH
Protoplast plating Leaf protoplasts of P. hybrida were cultured in 9-cm petri dishes at a cell density of 8.0-9.8 x 105 per dish in MS medium. They were maintained in the dark at 25°C. After 2 months the number of small calluses were determined.
6
aa
(nm)
Fig. 1: Absorption spectra of 95 % (v/v) ethanol extract of A) petunia leaf protoplasts incubated in the 0.05 % TTC assay medium containing 0.05M K-Pi buffer (PH 7.4) and 0.6M sorbitol, B) a fraction of cellular or subcellular debris incubated in the 0.05 % TTC assay medium.
Protoplast dividing potentiallity 0.0 4
I
aa
113
100
0.0 8
. ~
0.0 2
5
a
"
60
w
Cl.
a:
a
5a
a
Cl.
O'----'----'---------''---~--___+_----'
a
MNNG(ppm)
0.0 I
O. I
I
a
a:
20
a
I A(mM)
Fig. 2: Relationship between MNNG concentration and plating efficiency or TTC reduction: absorbance at 520 nm ( 0); relative plating efficiency (RPE) (.). Protoplasts were plated at a cell density of 8.0 x 105 per dish. Each point represents the average of two replicates.
leaf protoplasts, while the spectrum of debris fraction shows that there is no formazan.
Estimation
~
0.0 4
100
-; w
0/plating efficiency by TTC reduction test
Petunia leaf protoplasts were treated with MNNG, an alkylating agent, and lA, a metabolic inhibitor, to determine the relationship between plating efficiency and TTC reduction. Division and callus from leaf protoplasts of P. hybrida is easily induced. Fig. 2 shows that TTC reduction decreased as MNNG concentration increased, while plating efficiency decreased as the MNNG concentration reached 25 mgL -1. The TTC reduction was determined 5 dafter MNNG treatment. Plating efficiency of the control was 0.8 %. Fig. 3 also shows that the TTC reduction decreased as IA concentration increased, and TTC reduction was correlated with the plating efficiency of P. hybrida leaf protoplasts. Plating efficiency of the control was 0.7 %. The TTC reduction was determined immediately after protoplast isolation. Since protoplast viability affects plating efficiency, TTC reduction can provide an estimate of protoplast viability potential to form colonies at an early stage of culture. Comparative tests for estimation of viability potential of petunia leaf protoplasts with the TTC reduction test and FDA staining.
Fig. 3: Relationship between IA concentration and plating efficiency or TTC reduction: absorbance at 520nm (0); relative plating efficiency (RPE) (.). Protoplasts were plated at a cell density of 9.8 x 105 per dish. Each point represents the average of two replicates.
P. hybrida leaves were harvested from the apex of the plant just prior to flowering. Leaves were classified as A, B, C, according to their growth stages. Each group consisted of 2 or 3 leaves. Younger leaves (group A) contained less chlorophyll per protein than older leaves (group C). Protoplasts of younger leaves produced 2.8 to 3.5 times more formazan than older leaves. Therefore, protoplasts isolated from younger leaves just prior to flowering maintained a high metabolic activity. FDA staining could not detect the difference of activity between protoplasts isolated from leaves of different ages. Furthermore, FDA assessment was not correlated with plating efficiency.
Discussion
The TTC test has been used for examining seed viability and cold injury. Steponkus and Lanphear (1967) developed a refinement of the TTC test for determining cold injury by using leaf discs and stem segments of ivy. In our work, the TTC test was modified to evaluate leaf protoplast viability. Kun and Abood (1949) found that tetrazolium is an indicator of succinic dehydrogenase activity of a tissue homogenate. However, Roberts (1951) found that no single reductase
Table 1: Difference in the activity between protoplasts isolated from different growth stages of leaves. Stage
Leaf Area (cm2)
FW (g)
ChI/Protein
Formazan (Ilg)a
FDA Staining (%)b
Plating Efficiency (%)C
A B C
14.2 26.1 30.5
0.47 1.09 1.47
0.547 0.733 0.800
0.690 0.194 0.241
95 98 98
0.7
a TTC reduction was expressed as formazan which protoplasts produced per Ilg chlorophyll. Number of protoplasts counted: 300-400. Plating efficiency was determined using two replicates.
b C
o o
114
MASAMI WATANABE, YUKIO WATANABE, and NORITSUGU SHIMADA
system was responsible for the characteristic reduction in plant tissues; rather, they postulated that a general redox potential level, maintained by several physiologically active systems, brings about the reduction of tetrazolium. The TTC test is a two step reaction: substrate + NAD(P) + TTC (colorless) ~ Product + NAD(P)H + TTC (colorless) !. Product + NAD(P) + Formazan (colored), where A = a dehydrogenase, and B = a tetrazole reductase (Gahan, 1989). In this experiment, as the substrate and cofactor were omitted from the reaction medium, a formazan was produced presumably through the presence of endogenous substrate and cofactor. Differences in the amount of reduced formazan may be due to limitation of substrate and cofactor and hence represent metabolic activity potential at the time of the test. In fact, cellular or subcellular debris resulting in damaged plasmalemma gave a negative TTC test (Fig. 1). Relative plating efficiency of P. hybrida leaf protoplasts was decreased markedly to 8%, when 25mgL -1 of MNNG in MS medium was used, whereas absorbance of the reduced formazan extracted from 5-d cultured protoplasts was decreased slightly from 0.039 to 0.029. The difference may be due to their respective metabolic activity potentials at the particular time their TTC reduction was determined. Protoplasts treated with MNNG survive for more than 5 days; the 5-d cultured protoplasts have some dehydrogenase activities and hence they can reduce TTC. MNNG is an alkylating agent and affects DNA but not dehydrogenase-related metabolism. Therefore, the major difference in TTC reduction appears more than 5 dafter MNNG treatment. In the case of lA, the difference in TTC reduction emerged immediately after the IA treatment. Consequently, the TTC reduction test gives some information about metabolic activity potential, especially with respect to dehydrogenases. Robertson and Earle (1987) reported a procedure for the use of fluorescein-labelled tetrazolium as stains to detect photosynthetic activity in leaf protoplasts. Their methods and FDA staining are very fast but require a fluorescence microscope. The TTC reduction test described here focuses on a spectrophotometric method for viability potential in batches of protoplasts. This test could also be extended to a microscopic method for monitoring of each protoplast viability; the number of these stained protoplasts could be counted, because reduced formazan accumulates in small vacuoles of active protoplasts. A disadvantage of the TTC reduction test is that it is time-consuming; on the other hand it increases the possibility of predicting viability potential. Except for a limited number of plants, regeneration of plantlets from leaf protoplasts is still difficult. Division in isolated leaf protoplasts depends on the physiological state of the donor plant and the specific leaf position. The method
described here could specify the leaf positions for isolation of active protoplasts to form colonies. Acknowledgements
We thank Prof. G. Tamura for the use of a HITACHI U-3200 spectrophotometer and Dr. K. Ishikawa for the use of an Olympus FLM fluorescence microscope.
References ARNON, D. L.: Copper enzymes in isolated chloroplasts. Phenoloxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (1949). BASHASM, H. G. and D. F. BATEMAN: Relationship of cell death in plant tissue treated with a homogeneous endopectate lyase to cell wall degradation. Physiol. Plant Pathol. 5, 249-262 (1975). GAHAN, P. B.: Viability of plant protoplasts. In: BAJAJI, Y. P. S. (ed.): Biotechnology in agriculture and forestry, Vol. 8, 34-49. Springer-Verlag, Berlin (1989). GLIMELIUS, K., A. WALLIN, and T. ERIKSSON: Agglutinating effects of concanavalin A on isolated protoplasts of Daucus carota. Physiol. Plant. 31, 225-230 (1974). KANA!, R. and G. E. EDWARDS: Purification of enzymatically isolated mesophyll protoplasts from C), C 4 and crassulacean acid metabolism plants using an aqueous dextran-polyethylene glycol twophase system. Plant Physiol. 52, 484-490 (1973). KUN, E. and G. L. ABOOD: Colorimetric estimation of succinic dehydrogenase by triphenyltetrazolium chloride. Science 109, 144-146 (1949). KURIYAMA, A., K. WATANABE, S. UENO, and H. MITSUDA: Inhibitory effect of ammonium ion on recovery of cryopreserved rice cells. Plant Science 64, 231-235 (1989). LARKIN, P. J.: Purification and viability determinations of plant protoplasts. Planta 128, 213-216 (1976). PORTER, R. H., M. DURRELL, and J. H. ROMM: Use of 2,3,5-triphenyltetrazolium chloride as a measure of seed germinability. Plant Physiol. 22, 149-159 (1947). PURCELL, E. A. and H. R. YOUNG: The use of tetrazolium in assessing freeze damage in citrus trees. Am. Soc. Hor. Sci. 83, 352-358 (1963). ROBERTS, W. L.: Survey of factors responsible for reduction of 2,3,5triphenyltetrazolium chloride in plant meristems. Science 113, 692-693 (1951). ROBERTSON, D. and D. E. EARLE: Nitro-blue tetrazolium: A specific stain for photosynthetic activity in protoplasts. Plant Cell Reports 6, 70 -73 (1987). STEPONKUS, L. P. and O. F. LANPHEAR: Refinement of the triphenyl tetrazolium chloride method of determining cold injury. Plant Physiol. 42, 1423 -1426 (1967). WATTS, J. W., F. MOTOYOSHI, and J. W. KRUNG: Problems associated with the production of stable protoplasts of cells of tobacco mesophyll. Ann. Bot. 38, 667 -671 (1974).
141. pp. 111-114 {1992}
Colorimetric Estimation of Leaf-Protoplast Potential-toDivide by Use of 2,3,5-Triphenyl Tetrazolium Chloride MASAMI WATANABE, YUKIO WATANABE,
and NORITSUGU
SHIMADA
Laboratory of Plant Nutrition, Faculty of Horticulture, Chiba University, 648 Matsudo, Chiba 271, Japan Received February 2,1992 . Accepted May 10, 1992
Summary
Purified protoplasts from Petunia hybrida reduced 2,3,5-triphenyl tetrazolium chloride (TTC), whereas cellular or subcellular debris gave a negative TTC test. The TTC reduction was decreased with increasing concentration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and iodoacetate (IA). Consequently it could be correlated with plating efficiency. The most formazan was produced by the protoplasts from top-developing leaves. FDA staining could not detect the difference in viability of protoplasts isolated from different growth stages of leaves. The TTC reduction test described here offers a colorimetric estimation for the potential of protoplasts to form colonies.
Key words: Petunia hybrida, plating efficiency, protoplast, 7TC, viability. Abbreviations: BA = benzyladenine; 2,4-D = 2,4-dichlorophenoxyacetic acid; FDA = fluorescein diacetate; IA = iodoacetate; MES = 2-(N-morpholino) ethanesulfonic acid; TTC = 2,3,5-triphenyl tetrazolium chloride; MNNG = N-methyl-N'-nitro-N-nitrosoguanidine; MS = Murashige and Skoog.
Introduction
One definition of plant protoplast viability is the capacity of an isolated protoplast to divide, form a callus, and ultimately give rise to a plantlet. The process of protoplast isolation affects the state of viability (Watts et ai., 1974); a proportion of isolated protoplasts will not survive subsequent culture. This proportion depends upon the potential of viability. Therefore, protoplasts should be monitored for viability at an early stage. A number of tests have been applied to check protoplast viability in order to determine what portion of damaged protoplasts appears during isolation or in culture. Intactness of the plasmalemma can be measured by dye exclusion. Ability of tht plasmalemma to block the entry of a molecule of dye into the cell is normally measured with Evans blue (Kanai and Edwards, 1973; Glimelius et ai., 1974). In this test, dead or dying protoplasts stain blue, while those with an intact plasmalemma remain unstained. Percentage viability can then be assessed by counting. If the vacuole is disrupted, the protoplast will die. © 1992 by Gustav Fischer Verlag, Stuttgart
Intactness of the tonoplast can be assayed by use of neutral red (Basham and Bateman, 1975). The dye traverses the intact plasmalemma and is concentrated in the vacuoles. Dead or damaged cells will stain completely. Fluorescein diacetate (FDA) staining depends on plasmalemma integrity and an associated esterase activity (Larkin, 1976). The esterase cleaves the acetate from the FDA as it passes into the protoplasts, and the cells fluoresce on irradiation under ultraviolet light. In contrast to the dye exclusion methods, only viable protoplasts will fluoresce. In this paper we report the use of triphenyl tetrazolium chloride (TTC) as a protoplast viability test. It has formerly been used in conjunction with seed germinability (Porter et ai., 1947), cold injury of tissue (Purcell and Young, 1963), and cells in suspension culture (Kuriyama et aI., 1989). In contrast to FDA staining which depends on plasmalemma integrity and its associated esterase activity, TTC reduction is caused by dehydrogenase activity and may be a more direct estimate of protoplast viability. However, TTC reduction tests reported previously cannot be directly applied to
112
MASAMI WATANABE, YUKIO WATANABE, and NORITSUGU SHIMADA
leaf protoplasts because of endogeneous pigment interference. The present work provides a qualitative assessment of viability in batches of protoplasts.
Materials and Methods
Plant materials Seeds of Petunia hybrida cv. Recovery White were commercially obtained (Sakata seed Co. Ltd.).
Protoplast isolation Leaf protoplasts were prepared from leaves of 8 to 10-week-old plants grown in a growth chamber under a 14-h photoperiod with a photon fluence density of 50 Ilmol m- 2 S-I of fluorescent tubes and at 25/20°C for the light/dark period. Leaf blades were sterilized with 70 % ethanol for 10 s followed by treatment in 0.6 % final concentration of sodium hydrochlorite for 5 min which, in turn, was followed by several washes in sterile water. Lower epidermis from leaves of P. hybrida were peeled away and incubated for 12 h in an enzyme solution containing 0.1 % Cellulase Y-C (Seishin Pharmaceutical Co. Ltd.), 0.02 % Pectolyase Y-23 (Seishin Pharmaceutical Co. Ltd.), 5 mM MES (pH 5.8), and 0.6 M sorbitol. Isolated protoplasts were sieved through nylon mesh (82Ilm) and purified by flotation on 0.7 M sucrose.
1TC reduction test Leaf protoplasts, in 10-mL screw cap centrifuge tubes, were suspended in 3 mL of 0.05 M K 2HP0 4-KH 2P0 4-O.6 M sorbitol buffer (PH 7.4) containing 0.05 % of TTC % (w/v) and incubated for 16 h at 30°C in the dark. The TTC solution was filter-sterilised. All procedures described above were conducted aseptically. TTC reduction was determined as follows: Protoplasts were collected by centrifugation at 180 x g for 10 min. Water insoluble formazan was extracted with 5mL of 95% (v/v) ethanol in a boiling water bath for 5 min (Steponkus and Lanphear, 1967). The extract was cooled and diluted to 10 mL with 95 % ethanol. The absorbance was recorded at 520 nm. A wavelength still in the region of high absorption by reduced TTC but with minimum absorption by tissue pigments, namely 520 nm, was chosen for determination of the TTC reduction.
MNNG and lA treatment Leaf protoplast of P. hybrida were treated with 25, 50 and 100mgL- 1 final concentration of MNNG and 0.01, 0.1, 1 and 10 mM of IA for 1 h at 25°C in light. The MNNG solutions were discarded and the protoplasts washed three times with 10mL 0.6M sorbitol. Protoplasts for TTC reduction test were transferred to centrifuge tubes and cultured in 1 mL of MS liquid medium supplemented with 1.0 mgL- 1 2,4-D, 0.5 mgL -I BA, 2 % sucrose, 1 % glucose and 0.6 M sorbitol (pH 5.8) at 25°C for 5 d in the dark.
FDA staining FDA was added to the protoplast suspensions to give a final concentration of 0.01 %. After 5 min at room temperature, the protoplasts were examined for fluorescence using an Olympus FLM fluorescent microscope.
Chlorophyll determination Protoplasts were collected by centrifugation at 180 x g for 10 min and suspended in 80 % acetone with agitation. The solution was made up to 10 mL with 80 % acetone and centrifuged at 1600 x g for 5 min. The absorbance of the supernatant was recorded at 645 and 663 nm. Chlorophyll content was calculated according to Arnon (1949).
Protein assay The protoplast suspensions were centrifuged as above and suspended in a lysis buffer containing 50 mM Tris-HCI (PH 8.0), 10 mM EDTA-2Na. Protein content was determined by a Bio-Rad microprotein assay (Bio-Rad Co. Ltd.), using BSA as a standard.
Results
Steponkus and Lanphear (1967) working with leaf discs of an ivy showed that sodium hydrosulfate reduced formazan has a )" max at about 490 nm, a wavelength at which there is interference from endogenous plant pigments. They used 530 nm, a wavelength still in the region of high absorption by the reduced TTC, but minimum absorption by tissue pigments, for determination of formazan. In this work, tissue pigment interference was minimized by subtracting absorbance at 520 nm of 95 % ethanol extract of protoplasts incubated in an assay medium without TTC. As a result, the reduced water-insoluble formazan can be quantitatively recovered from the protoplasts. A result of the TTC reduction test for leaf protoplasts and a fraction of cellular or subcellular debris is presented in Fig. 1. Formazan can be detected in 1.0 , - " ' - - - - - - - - - - - - - - - - - - ,
w
U Z
o¢
'" o 0:
UJ
'" o¢
a 4
aa
5
aa
WAVEL E NGTH
Protoplast plating Leaf protoplasts of P. hybrida were cultured in 9-cm petri dishes at a cell density of 8.0-9.8 x 105 per dish in MS medium. They were maintained in the dark at 25°C. After 2 months the number of small calluses were determined.
6
aa
(nm)
Fig. 1: Absorption spectra of 95 % (v/v) ethanol extract of A) petunia leaf protoplasts incubated in the 0.05 % TTC assay medium containing 0.05M K-Pi buffer (PH 7.4) and 0.6M sorbitol, B) a fraction of cellular or subcellular debris incubated in the 0.05 % TTC assay medium.
Protoplast dividing potentiallity 0.0 4
I
aa
113
100
0.0 8
. ~
0.0 2
5
a
"
60
w
Cl.
a:
a
5a
a
Cl.
O'----'----'---------''---~--___+_----'
a
MNNG(ppm)
0.0 I
O. I
I
a
a:
20
a
I A(mM)
Fig. 2: Relationship between MNNG concentration and plating efficiency or TTC reduction: absorbance at 520 nm ( 0); relative plating efficiency (RPE) (.). Protoplasts were plated at a cell density of 8.0 x 105 per dish. Each point represents the average of two replicates.
leaf protoplasts, while the spectrum of debris fraction shows that there is no formazan.
Estimation
~
0.0 4
100
-; w
0/plating efficiency by TTC reduction test
Petunia leaf protoplasts were treated with MNNG, an alkylating agent, and lA, a metabolic inhibitor, to determine the relationship between plating efficiency and TTC reduction. Division and callus from leaf protoplasts of P. hybrida is easily induced. Fig. 2 shows that TTC reduction decreased as MNNG concentration increased, while plating efficiency decreased as the MNNG concentration reached 25 mgL -1. The TTC reduction was determined 5 dafter MNNG treatment. Plating efficiency of the control was 0.8 %. Fig. 3 also shows that the TTC reduction decreased as IA concentration increased, and TTC reduction was correlated with the plating efficiency of P. hybrida leaf protoplasts. Plating efficiency of the control was 0.7 %. The TTC reduction was determined immediately after protoplast isolation. Since protoplast viability affects plating efficiency, TTC reduction can provide an estimate of protoplast viability potential to form colonies at an early stage of culture. Comparative tests for estimation of viability potential of petunia leaf protoplasts with the TTC reduction test and FDA staining.
Fig. 3: Relationship between IA concentration and plating efficiency or TTC reduction: absorbance at 520nm (0); relative plating efficiency (RPE) (.). Protoplasts were plated at a cell density of 9.8 x 105 per dish. Each point represents the average of two replicates.
P. hybrida leaves were harvested from the apex of the plant just prior to flowering. Leaves were classified as A, B, C, according to their growth stages. Each group consisted of 2 or 3 leaves. Younger leaves (group A) contained less chlorophyll per protein than older leaves (group C). Protoplasts of younger leaves produced 2.8 to 3.5 times more formazan than older leaves. Therefore, protoplasts isolated from younger leaves just prior to flowering maintained a high metabolic activity. FDA staining could not detect the difference of activity between protoplasts isolated from leaves of different ages. Furthermore, FDA assessment was not correlated with plating efficiency.
Discussion
The TTC test has been used for examining seed viability and cold injury. Steponkus and Lanphear (1967) developed a refinement of the TTC test for determining cold injury by using leaf discs and stem segments of ivy. In our work, the TTC test was modified to evaluate leaf protoplast viability. Kun and Abood (1949) found that tetrazolium is an indicator of succinic dehydrogenase activity of a tissue homogenate. However, Roberts (1951) found that no single reductase
Table 1: Difference in the activity between protoplasts isolated from different growth stages of leaves. Stage
Leaf Area (cm2)
FW (g)
ChI/Protein
Formazan (Ilg)a
FDA Staining (%)b
Plating Efficiency (%)C
A B C
14.2 26.1 30.5
0.47 1.09 1.47
0.547 0.733 0.800
0.690 0.194 0.241
95 98 98
0.7
a TTC reduction was expressed as formazan which protoplasts produced per Ilg chlorophyll. Number of protoplasts counted: 300-400. Plating efficiency was determined using two replicates.
b C
o o
114
MASAMI WATANABE, YUKIO WATANABE, and NORITSUGU SHIMADA
system was responsible for the characteristic reduction in plant tissues; rather, they postulated that a general redox potential level, maintained by several physiologically active systems, brings about the reduction of tetrazolium. The TTC test is a two step reaction: substrate + NAD(P) + TTC (colorless) ~ Product + NAD(P)H + TTC (colorless) !. Product + NAD(P) + Formazan (colored), where A = a dehydrogenase, and B = a tetrazole reductase (Gahan, 1989). In this experiment, as the substrate and cofactor were omitted from the reaction medium, a formazan was produced presumably through the presence of endogenous substrate and cofactor. Differences in the amount of reduced formazan may be due to limitation of substrate and cofactor and hence represent metabolic activity potential at the time of the test. In fact, cellular or subcellular debris resulting in damaged plasmalemma gave a negative TTC test (Fig. 1). Relative plating efficiency of P. hybrida leaf protoplasts was decreased markedly to 8%, when 25mgL -1 of MNNG in MS medium was used, whereas absorbance of the reduced formazan extracted from 5-d cultured protoplasts was decreased slightly from 0.039 to 0.029. The difference may be due to their respective metabolic activity potentials at the particular time their TTC reduction was determined. Protoplasts treated with MNNG survive for more than 5 days; the 5-d cultured protoplasts have some dehydrogenase activities and hence they can reduce TTC. MNNG is an alkylating agent and affects DNA but not dehydrogenase-related metabolism. Therefore, the major difference in TTC reduction appears more than 5 dafter MNNG treatment. In the case of lA, the difference in TTC reduction emerged immediately after the IA treatment. Consequently, the TTC reduction test gives some information about metabolic activity potential, especially with respect to dehydrogenases. Robertson and Earle (1987) reported a procedure for the use of fluorescein-labelled tetrazolium as stains to detect photosynthetic activity in leaf protoplasts. Their methods and FDA staining are very fast but require a fluorescence microscope. The TTC reduction test described here focuses on a spectrophotometric method for viability potential in batches of protoplasts. This test could also be extended to a microscopic method for monitoring of each protoplast viability; the number of these stained protoplasts could be counted, because reduced formazan accumulates in small vacuoles of active protoplasts. A disadvantage of the TTC reduction test is that it is time-consuming; on the other hand it increases the possibility of predicting viability potential. Except for a limited number of plants, regeneration of plantlets from leaf protoplasts is still difficult. Division in isolated leaf protoplasts depends on the physiological state of the donor plant and the specific leaf position. The method
described here could specify the leaf positions for isolation of active protoplasts to form colonies. Acknowledgements
We thank Prof. G. Tamura for the use of a HITACHI U-3200 spectrophotometer and Dr. K. Ishikawa for the use of an Olympus FLM fluorescence microscope.
References ARNON, D. L.: Copper enzymes in isolated chloroplasts. Phenoloxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (1949). BASHASM, H. G. and D. F. BATEMAN: Relationship of cell death in plant tissue treated with a homogeneous endopectate lyase to cell wall degradation. Physiol. Plant Pathol. 5, 249-262 (1975). GAHAN, P. B.: Viability of plant protoplasts. In: BAJAJI, Y. P. S. (ed.): Biotechnology in agriculture and forestry, Vol. 8, 34-49. Springer-Verlag, Berlin (1989). GLIMELIUS, K., A. WALLIN, and T. ERIKSSON: Agglutinating effects of concanavalin A on isolated protoplasts of Daucus carota. Physiol. Plant. 31, 225-230 (1974). KANA!, R. and G. E. EDWARDS: Purification of enzymatically isolated mesophyll protoplasts from C), C 4 and crassulacean acid metabolism plants using an aqueous dextran-polyethylene glycol twophase system. Plant Physiol. 52, 484-490 (1973). KUN, E. and G. L. ABOOD: Colorimetric estimation of succinic dehydrogenase by triphenyltetrazolium chloride. Science 109, 144-146 (1949). KURIYAMA, A., K. WATANABE, S. UENO, and H. MITSUDA: Inhibitory effect of ammonium ion on recovery of cryopreserved rice cells. Plant Science 64, 231-235 (1989). LARKIN, P. J.: Purification and viability determinations of plant protoplasts. Planta 128, 213-216 (1976). PORTER, R. H., M. DURRELL, and J. H. ROMM: Use of 2,3,5-triphenyltetrazolium chloride as a measure of seed germinability. Plant Physiol. 22, 149-159 (1947). PURCELL, E. A. and H. R. YOUNG: The use of tetrazolium in assessing freeze damage in citrus trees. Am. Soc. Hor. Sci. 83, 352-358 (1963). ROBERTS, W. L.: Survey of factors responsible for reduction of 2,3,5triphenyltetrazolium chloride in plant meristems. Science 113, 692-693 (1951). ROBERTSON, D. and D. E. EARLE: Nitro-blue tetrazolium: A specific stain for photosynthetic activity in protoplasts. Plant Cell Reports 6, 70 -73 (1987). STEPONKUS, L. P. and O. F. LANPHEAR: Refinement of the triphenyl tetrazolium chloride method of determining cold injury. Plant Physiol. 42, 1423 -1426 (1967). WATTS, J. W., F. MOTOYOSHI, and J. W. KRUNG: Problems associated with the production of stable protoplasts of cells of tobacco mesophyll. Ann. Bot. 38, 667 -671 (1974).