Planta (Berl.) 126, 105--110 (1975) 9 by Springer-Verlag 1975
Nutritional Requirements for Growth of Vicia hajastana Cells and Protoplasts at a Very Low Population Density in Liquid Media* K. N. K a o and M. R. Michayluk National Research Council of Canada, Prairie Regional Laboratory, Saska~oon, Saskatchewan S7N OW9, Canada Received 3 June; accepted 6 June 1975
Summary. When Vicia ha]astana Grossh. cells or protoplasts were cultured at a high population density (ca. 5000 cells or protoplasts/ml), they were able to grow in a mineral-salt solution supplemented with sucrose (or glucose), a few vitamins, and 2,4-diehlorophenoxyacetic acid. They were not able to survive when cultured at a low population density unless the medium was supplemented with zeatin, naphthalene-l-acetic acid, nucleic-acid bases, amino acids, other sugars, sugar alcohols, and organic acids. Vicia cells were able to grow at an initial population density of 25-50 eells/ml in this defined medium. The population density could be lowered to 1-2 cells/ml with good growth when the mineral-salt medium was enriched with organic acids, sugars, sugar alcohols, coconut water, and casamino acids. The protoplasts also grew best in a medium enriched with these supplements. Three individual protoplasts were isolated and each one was cultured in a separate dish containing 4 ml of this medium. Within 3040 days, each one had grown indefinitely and formed a mass of cells
(C,a. 107). Introduction A v e r y high initial populatioa density (ca. 10000 cells/ml) was reqtdred for g r o w t h of plant cells in a mineral-salt liquid-culture medium. The initial population density could be lowered to 1000-2000 cells/ml if conditioned medium, casein hydrolysate, or free amino acids were added to the culture medium (Stuart and Street, 1969, 1971). I n order t o isolate desirable types of cells such as m u t a n t s or heterokaryocytes, further reduction of the initial cell population density is desirable. Various organic compounds such as glucose, amino acids etc. can be absorbed and/or released b y plant cells because of the permeability of the cell m e m b r a n e (Stuart and Street, 1971; Sargent and King, 1974; Ruesink, 1973; Maretzki et al., 1974). The inability of the plant cells to grow at a v e r y low population density m a y be caused b y excessive diffusion of metabolic intermediates into the medium, resulting in their dilution in the cell t o a level below t h a t required for survival (Ham, 1973). If this is the case, the cells should be able to grow at a v e r y low initial population density in a m e d i u m enriched with the appropriate metabolic intermediates. The object of this s t u d y was to develop such a medium for Vicia hajastana Grossh. cells and protoplasts. * NRCC No. 14694
106
K . N . Kao and M. R. Michayluk Material and Methods
Vicia ha]astana Grossh. cell suspension cultures, 1-2 days old and grown in Medium 1 (Kao et al., 1974) were used as the source of cells. The suspension culture consisted of single ceils and clusters of up to 200 cells. When the population density was below 50 cells/ml, only cells and cell clusters which were able to pass through a 125-~m mesh-size stainless-steel filter were used. The average number of ceils per cluster in the filtrate was 10 cells, ranging from 2 to 20 cells. The cell numbers were determined in two ways: (1) by calculation from packed cell volume divided by average cell size; (2) by direct counting using an inverted microscope. Vicia protoplasts were obtained in the following way: 1 ml of 1-day-old suspension culture and 1 ml of an enzyme solution consisting of 7 mM CaCl~, 0.7 mM NaH~PO 4, 0.35 M sorbitol, 0.35 M manaitol and 3 mM MES [2-(N-morpholino)-ethanesulfonic acid] buffer, 2 % Onozuka cellulase P 1500 (Kinki Yakult Mfg. Co., Ltd., Nishinomiya, Japan), 2 % Rhozyme HP 150 (Rehm and Haas Co. Canada, Ltd., West Mill, Ontario and 1% Sigma pectinase (Sigma Chemical Comp., St. Louis, Mo. USA) (pH 5.6; KOH) were mi~xedin a 60 • 15 mm Petri dish (Falcon) and incubated for 6 h at 24 ~ At the end of the incubation period, the protopl~sts were passed through an 80 ~m filter. The filtrate was centrifuged at 50 • g for 6 min. The supernatant was removed with a Pasteur pipette, and the protoplasts were resnspended in 10 ml of protoplast culture medium (Tables i and 3B) and again centrifuged. After the fourth washing, the protoplasts were resnspended in the same protoplast culture medium at various cell densities (Kao et al., 1973). The mineral salts and various additives used for the media are listed in Table 1. All the media were filter-sterilized. The cells were cultured in plastic Petri dishes (Falcon or Kimble) in a thin layer of liquid (4 ml per 60 • 15 mm dish), incubated (24~ in diffuse co01-white fluorescent lamps (45 lux • 10 h) (40 W., Canadian General Electric, Winnipeg, Manitoba) inside a plastic box which served as a humidity chamber. The protoplasts were cultured either in a thin layer of liquid as were the cells, or in 100 ~1 drops as described previously (Ka0 et al., 1973). Unless otherwise stated, the experiments were repeated at least 3 times. Results
1. Cells (Table 2) A l t h o u g h Vicia cells c u l t u r e d at a high p o p u l a t i o n d e n s i t y were able to grow in a m i n e r a l - s a l t m e d i u m s u p p l e m e n t e d w i t h sucrose, glucose, v i t a m i n s a n d 2,4-dichlorophenoxyacetic acid (2,4-D), or 2,4=D plus z e a t i n a n d 1-naphthaleneacetic a c i d = N A A (Media 1 a n d 2, T a b l e 2B), t h e y were n o t able t o survive w h e n t h e y were c u l t u r e d a t a low p o p u l a t i o n d e n s i t y unless t h e m e d i u m (Medium 2) was s u p p l e m e n t e d w i t h a m i n o acids, nucleic acid bases, o t h e r sugars, sugar alcohols, a n d organic acids of t h e t r i c a r b o x y l i c - a c i d cycle (Medium 6). The m i n i m u m cell d e n s i t y r e q u i r e d for g r o w t h in this m e d i u m was 25-50 cells/ml. T h e population d e n s i t y could also be lowered c o n s i d e r a b l y when t h e m i n e r a l - s a l t m e d i u m (Medium 3) was s u p p l e m e n t e d w i t h B a c t o v i t a m i n - f l e e casamino acids (Difco Lab., D e t r o i t , Mich., U S A ) a n d coconut w a t e r (Medium 7). T h e n u m b e r of cells p e r ml r e q u i r e d for g r o w t h in this m e d i u m was 50-250. The p o p u l a t i o n d e n s i t y could be f u r t h e r lowered w h e n M e d i u m 7 was enriched w i t h sugars, sugar alcohols a n d organic acids (Medium 8). W e were able t o grow 1-2 cells/ml in t h i s m e d i u m . The t i m e r e q u i r e d t o g r o w from a few cells t o ca. 107 cells in 4 ml of this m e d i u m was b e t w e e n 20 a n d 30 days. Only slight gain was o b s e r v e d when o n l y free a m i n o acids a n d nucleic-acid bases were a d d e d t o M e d i u m 2 (Medium 5). This was also t r u e when o n l y organic acids or organic acids in c o m b i n a t i o n w i t h t h e sugars were used to enrich M e d i u m 2 (Media 3 a n d 4).
Growth of Cells and Protoplasts at Low Population Density
107
Table 1. Compounds used in components of various media
a) Mineral salt/or all media (mg/1)a NHtNO 3 KNO 3 CaC12. 2H~O MgSOa. 7H20 KH2PO 4 KC1 Sequestrene| 330Fe
600 1900 600 300 170 300 28
KI H3BOa MnSO 4. tt~O ZnS04" 7tt~O Na~Mo04.2H20 CuSO 4- 5H20 CoC12. 6H~O
Media 1 to 8
Media 1 p to 8 p
0.75 3.00 10.00 2.00 0.25 0.025 0.025
b) Sucrose and glucose (g/l) Sucrose Glucose
20 10
0.25 68.4
c) Vitamins/or all media (mg/1) Inositol Nicotinamide Pyridoxine ttCI Thiamine HCI D-Calcium pantothenate Folic acid p-Aminobenzoic acid
100 1 1 1 1 0.4 0.02
Biotin Choline chloride Riboflavin Ascorbic acid Vitamin A Vitamin Ds Vitamin B12
0.01 1.00 0.20 2.00 0.01 0.01 0.02
Media 1 and l p
Media 2 to 8
Media 2p to 8p
0.5 0 0
0.1 0.2 1.0
0.2 0.5 1.0
d) Hormones (rag/l) 2,4-D Zeatin NAA
e) Organic acids (rag/l) (adjusted to pH 5.5 with NH40H ) Sodium pyruvate Citric acid
20 40
Malie acid Fumarie acid
40 40
Rhamnose Cellobiose Sorbitol Mannitol
250 250 250 250
]) Other sugars and sugar alcohols (mg/1) Fructose Ribose Xylose Mannose
250 250 250 250
g) 21 L-amino acids (including glycine), glutamine and asparagine (mg/1) All were used at a concentration of 0.1 mg/l except the following: Glutamine 5.6 Glutamic acid Alanine 0.6 Cysteine
0.6 0.2
h) Nucleic-acid bases (mg/1) Adenine Guanine Thymine
0.10 0.03 0.03
Uracil Hypoxanthine Xanthine
0.03 0.03 0.03
i) Casein hydrolyzate and coconut water Vitamin-free easamino acid Coconut water b
250 (rag/l) 20 (ml/t)
]) pH 5.6 (NaOH) for all the media All media were filter sterilized a The composition of the mineral salts was modified from B5 (Gamborg et al., 1968). b From mature fruits; heated to 60 ~ for 30 min.
108
K. N. Kao and M. R. Miehayluk Table 2. Influence of initial cell density upon growth in different media
A. Growth Medium
1 2 3
4 5 6 7 8
+ + + + --
Initial cell density (No. of cells/ml) 5600
1200
250
100
50
-4-+ +-4-
~ -4--4-
•
-
-
-4--4-4--4+-4-
-4-+ +-4-4--4-
-4--4-4--4-4--4-
. . .
+-4-
-4--4-
-4--4-
-4-+
+
+-4-
++ ++
-4++
++
~ ++
+-4-
= = = =
25
5
_<2
-
.
.
. .
10
.
. .
. .
. .
i
-
-
+-4-
-
-
++
-
-
-
-
+
++
Growth occurred in all replicates. Growth occurred in most of the replicates. Growth occurred in some of the replicates. No growth in any of the replicates.
B. Oomponent8 o] media Medium
Components Mineral salts, Zeatin sucrose, and NAA glucose, vitamins and 2,4-D
Organic acids
2 3 4 5
-4"4-4-4-
-4-4-4-4-4-4-
. -}-4--
6 7 8
-4-
-}-
+ -4-
4- = Present;
- -
=
Other sugars
Free amino acids and nucleic acid bases
Casamino acids and
--4-4-
---4-
---
+
-4-4-
-f-
---
+
--
--
--
+
-4-
-4-
-4-
.
.
coconut
water
.
-
+
Absent.
T h e free a m i n o acids a n d n u c l e i c - a c i d bases i n excess q u a n t i t y were v e r y toxic t o p l a n t cells. W h e n t h e q u a n t i t y was i n c r e a s e d b y 5 t i m e s i n c o m p a r i s o n t o t h a t u s e d i n M e d i u m 6, t h e m i n i m u m n u m b e r of cells r e q u i r e d for g r o w t h i n c r e a s e d t o 2 5 0 - 5 0 0 cells/ml. T h e t o l e r a n c e of t h e V. ha]astana cells a t h i g h p o p u l a t i o n d e n s i t y t o v a r i o u s c o m p o u n d s was v e r y good; e.g. t h e y g r e w well i n a m e d i u m c o n t a i n i n g 500 mg/1 g l u t a m i n e ; h o w e v e r , t h e r e was n o g r o w t h w h e n t h e i n i t i a l p o p u l a t i o n d e n s i t y was l o w e r e d t o less t h a n 10,000 cells/ml. All t h e s e m e d i a c o n t a i n e n o u g h m i n e r a l salt t o s u p p o r t t h e g r o w t h of cells t o 5 • l 0 s cells p e r m l of m e d i u m . Media 1 a n d 2 a r e v e r y low i n N a +. H o w e v e r , a d d i t i o n of 50 ml/1 NaC1 t o M e d i u m 2 d i d n o t r e s u l t i n a n y s i g n i f i c a n t c h a n g e i n t h e m i n i m u m n u m b e r of cells r e q u i r e d for g r o w t h .
Growth of Cells and Protoplasts at Low Population Density
109
Table 3. Influence of initial proto1,1ast density upon growth in different media
A. Growth Medium
Initial proto1,last density (No. of proto1,1asts per ml) 5000
1000
-}-+ ++
. ++
41,
-5-}++
++ ++
51 )
.~- -4- a
_~_~a
•
.~-a
6p, 7p
++a
++a
++a
+a
-5+ ++
-5+ ++
-5+ ++
-}-+ ++
-?+ ++
ip 21, 31,
81,
500 .
.
250
+
. .
+-5 ++
++ ++
100
25
10
• -
--
--
_a
_a
_a
__a
+a
•
__
__
-}-+
+-5
•
+
+
+
. .
50
. .
< 1
. .
-{:h
5
.
m
m
m
+
++
+ + = Growth occurred in all replicates. + = Growth occurred in most of the replicates. • = Growth occured in some of the replicates. -- No growth in any of the replicates. a Two replicates.
B. Components o~ media Medium
Components Mineral salts, Zeatin sucrose, and NAA glucose, vitamins and 2,4-D
Organic acids
.
Casamino acids and coconut water
+ +
. +
+
+
+
--
--
--
4p
+ + + -4-
+ + + +
-~-+ --
+ + + --
-+ + --
----[-
+
+
+
+
-
+
.
.
Free amino acids and nucleic acid bases
11, 2p 31, 51, 6p 71, Sp
.
Other sugars
.
. .
.
+=Present;--=Absent.
2. Protoplasts (Table 3) The n u t r i t i o n a l r e q u i r e m e n t s for growth of Vicia protoplasts at a v e r y low initial p o p u l a t i o n d e n s i t y was v e r y similar to those for Vicia cells. The protoplasts required a m i n o acids, tricarboxylic-acid cycle organic acids, nucleic-acid bases, sugars a n d sugar alcohols, etc. for growth. However, the protoplasts h a d poorer growth i n the media c o n t a i n i n g free a m i n o acids a n d nucleic-acid bases t h a n the cells. T h e y grew best i n a m e d i u m enriched with organic acids, sugars, sugar alcohols, casamino acids a n d coconut water (Medium 8p). Three i n d i v i d u a l protoplasts were isolated a n d each one was cultured i n a separate dish c o n t a i n i n g 4 ml of this m e d i u m . W i t h i n 30-40 days, we observed t h a t each one h a d grown indefinitely a n d formed a mass of cells (ca. 107). The protoplasts grew b e t t e r in a t h i n
llO
K . N . Kao and M. R. Michayluk
l a y e r of m e d i u m t h a n in t h e drops. W e h a v e n e v e r failed t o grow p r o t o p l a s t s in M e d i u m 8p a t a n y of t h e p o p u l a t i o n densities using t h e t h i n - l a y e r m e t h o d .
Discussion I t is clearly d e m o n s t r a t e d in t h e p r e s e n t e x p e r i m e n t s t h a t Vicia cells were able t o grow a t a v e r y low initial p o p u l a t i o n d e n s i t y (25-50 cells/ml) in a defined m e d i u m (Medium 6). I t was also d e m o n s t r a t e d t h a t a single p r o t o p l a s t was able to form a cell wall a n d t h e n d i v i d e i n d e f i n i t e l y in a semi-defined m e d i u m (Medium 8p) w i t h o u t s u p p o r t from o t h e r p r o t o p l a s t s or cells. The i n a b i l i t y of cells or protoplasts to grow in a low p o p u l a t i o n d e n s i t y could be because of (1) diffusion of n e c e s s a r y m e t a b o l i t e s from t h e cell i n t o t h e m e d i u m to such a n e x t e n t t h a t t h e y could n o t survive, or (2) an excessive a m o u n t of certain c o m p o u n d s in t h e m e d i u m resulting in t o x i c i t y t o t h e cells. Our results i n d i c a t e t h e g r e a t i m p o r t a n c e of t h e former factor. The simplest w a y t o grow cells or p r o t o p l a s t s a t a v e r y low p o p u l a t i o n d e n s i t y is to s u p p l e m e n t a culture m e d i u m w i t h casamino acids a n d coconut w a t e r because these substances s u p p l y m a n y m e t a b o l i c i n t e r m e d i a t e s for g r o w t h a n d also possess d e t o x i f y i n g p r o p e r t i e s (Tulecke et al., 1961; Nolan, 1971; N o l a n a n d Nolan, 1972). References Gamborg, O.L., Miller, R.A., Ojima, K.: Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151-158 (1968) Ham, R.G.: Dilution plating and nutritional considerations. A. Animal cells. In: Tissue culture methods and applications, p. 254-261, P.F. Kruse, Jr., Patterson, M.K. Jr., eds. New York: Acad. Press 1973 Kao, K.N., Constabel, F., Michayluk, M.R., Gamborg, O.L.: Plant protoplast fusion and growth of intergeneric hybrid cells. Planta (Berl.) 120, 215-227 (1974) Kao, K.N., Gamborg, O.L., Michayluk, M.R., Keller, W.A., Miller, R.A.: The effects of sugars and inorganic salts on cell regeneration and sustained division in plant protoplasts. In: Protoplastes et fusion de cellules somatiqnes v~g6tales, J. Tempd., ed. Coll. Intern. Cent. Nat. Rech. Sci. No. 212, p. 207-213 (1973) Maretzki, A., Thorn, M., Nickell, L. G. : Utilization and metabolism of carbohydrates in cell and callus cultures. In: Tissue culture and plant science, p. 329-361, H.E. Street, ed. London: Acad. Press 1974 Nolan, R.A.: Amino acids and growth factors in vitamin-free casamino acids. Myeologia 63, 1231-1234 (1971) Nolan, R.A., Nolan, W. G. : Elemental analysis of vitamin-free casamino acids. Appl. Microbiol. 24, 290-291 (1972) Ruesink, A. : Surface membrane properties of isolated protoplasts. In: Protoplastes et fusion de eeltules somatiques v~g~tales, J. Temp~, ed. Colloq. Intern. Cent. Nat. Rech. Sci. No. 212, p. 41-49 (1973) Sargent, P.A., King, J. : Investigations of growth-promoting factors in conditioned soybean root cells and in the liquid medium in which they grow: ammonium, glutamine, and amino acids. Canad. J. Bot. 52, 1747-1755 (1974) Stuart, R., Street, H. E. : Studies on the growth in culture of plant cells. IV. The initiation of division in suspensions of stationary phase cells of Acer pseudoplatanus L. J. exp. Bot. 20, 556 571 (1969) Stuart, R., Street, H.E. : Studies on the growth in culture of plant cells. X. Further studies on the conditioning of culture media by suspensions of Acer pseudoplatanu8 L. cells. J. exp. Bot. 22, 96-106 (1971) Tuleehke, W., Weinstein, L. H., Rutner, A., Laurencot, H. J., Jr. : The biochemical composition ol coconut water (coconut milk) as related to its use in plant tissue culture. Contr. Boyce Thompson Inst. 21, 115-128 (1961)
