Plant Science Letters, 10 (1977) 171--179 ©Elsevier/North-Holland Scientific Publishers, Ltd.
SELECTION AND DIFFERENTIATION OF AMINOPTERIN RESISTANT CELLS O F D A T U R A I N N O X I A
IRIS A. MASTRANGELO* and HAROLD H. SMITH Biology Department, Brookhaven National Laboratory, Upton, N.Y. 11973 (U.S.A.) (Received February 19th, 1977) (Accepted May 25th, 1977)
Primary cultured pith cells of Datura innoxia were subjected to a stepwise selection o f colonies resistant to the folic acid analog, aminopterin. The first t w o screenings were done at a concentration of 1 • I 0 -s M, followed by 2 • 10 -s M and 4 • 1"0-s M. The frequency of resistant colonies ranged from 5.4 • 10 -6 to 10 -7 during these steps. Resistance was maintained in cells through two subcultures (3 months) without aminopterin. Independently isolated resistant lines differed with respect to growth and ability to differentiate. Plants have been obtained from five resistant lines. One of these plants is fertile and has produced seeds.
The potential of using protoplast fusion techniques to bring together genetically diverse cell types, and thus to generate new genotypes, depends on reliable m e t h o d s for selecting the hybrid cells from the parental cells. Combined resistance to various drugs has proved to be a useful means for selecting hybrid products following fusion of cultured mammalian cells. It is important to have a stable, resistant phenotype, i.e. one with a genetic basis. Resistance of various cell t y p e s to methotrexate and aminopterin, which are analogs of folic acid, has been shown to involve genetic differences in dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP ÷ oxidoreductase, E.C. 22.214.171.124). Dihydrofolate reductase (H2FR) catalyzes the reduction of dihydrofolate to tetrahydrofolate which is an essential coenzyme for the synthesis of purines, * Guest Cytogeneticist. Abbreviations: BAP, benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid; I-I2FR, dihydrofolate reductase; IAA, indoleacetic acid; 2- IP, N 6-dimethylallyladenine; LM, liquid medium; SM, standard medium.
172 amino acids and t h y m i d i n e . Methotrexate and aminopterin inhibit this reaction by combining almost irreversibly with H2FR . Point mutations in the gene coding for H2FR confer resistance to methotrexate and aminopterin in Diplococcus pneumoniae [ 1 ]. Codominant mutations in Chinese hamster ovary cells that result in increased production of H2FR or reduced permeability of the cell membrane to antifolates also confer resistance [4,10]. Elevated levels of H2FR m R N A are found in Syrian hamster cells that are stably resistant [ 11 ]. Aminopterin resistant plant cells offer valuable experimental materials for studies of somatic cell genetics and gene regulation. In addition, they would be available for somatic fusion experiments with tobacco, soybean, sycamore and carrot cells, resistant to antibiotics , amino acid analogues  and base analogues [14--16] that have also been selected in culture. MATERIALS AND METHODS
Culture and plating o f Datura innoxia One-mm stem cross sections from haploid D. innoxia (n = 12), surface sterilized with 10% Chlorox, were placed on solid Murashige and Skoog medium  supplemented with 5.7 • 10 -6 M indoleacetic acid (IAA), 4.5 • 10 -6 M 2,4dichlorophenoxyacetic acid (2,4-D) and 4.9 • 1'0 -s M N6-dimethylallyladenine (2-IP). The pH was adjusted to 5.7. This is the standard medium (SM) used. Hormones were dissolved in 0.2 N NaOH. The 2,4-D was added to the medium prior to autoclaving; the other two hormones were filter sterilized. All cultures were maintained at 25 °C with 16 h of light (600 f.c.) and 8 h of darkness. In 3--4 weeks friable callus and stem sections were transferred to liquid triedium (LM) which is the same as SM but w i t h o u t agar. The liquid cultures were maintained on a gyratory shaker for 1--2 weeks, then filtered through several layers o f cheese cloth in order to remove larger cell aggregates and the stem sections. The frequency distribution of clumps with different cell numbers in the filtrate is shown in Table I where a unit is a single cell or cell clump and a viable unit is one t h a t contains at least one live cell. Viability of cells was microscopically determined by exclusion of 0.025% Evan's blue diluted in Murashige and Skoog salts and by observation of protoplasmic streaming . Cells were plated at final concentrations of 2--3 • 10 3 viable units/ml in SM or SM supplemented with aminopterin in 20 × 100 mm Petri plates. Aminopterin (Nutritional Biochemicals) was dissolved immediately prior to use at alkaline pH, which was then adjusted to pH 6.0, and filter sterilized. A m i n o p t e r i n dose-response curve The killing curve for aminopterin was determined by inhibition of colony formation by concentrations o f 10 -9 M to 4 • 1"0-s M. Cell suspension filtrates were plated in SM supplemented with aminopterin and solidified with 0.9% Bacto-Agar. Colonies which were 1 mm or larger were scored 4 weeks after plating. The frequency of colony formation in the test plates is expressed as a measure of
173 survival relative to the n u m b e r of control colonies (Fig. 1). Survival was 100% in 10 -9 M aminopterin.
Selection o f aminopterin resistant cells Wild type suspension cell populations were screened for aminopterin resistant cells by plating in SM and 1 • 1'0 -s M aminopterin. At this dose, the frequency of colony formation was 1 • 1"0-s (Fig. 1). A multiple selection step procedure was used because not all colonies initially f o r m e d in 1 • 10 -s M aminopterin continued to proliferate at t h a t drug concentration, while at higher initial drug levels t o o few cells survived to form colonies. The multiple step selection procedure invol~ cd: (1) Plating wild type suspension cells in SM with 1 • 1'0 -s M aminopterin. This is the 1st screen. (2) Overlaying plates containing colonies with fresh SM with 1 • 1'0 -s M aminopterin, or subculturing 1st screen survivors o n t o SM with 1. 10 -s M aminopterin. This subculture is the 2nd selection step. (3) Transplanting colonies that continue to proliferate o n t o SM with 2 • I 0 -s M and then 4 • 1'0-s M aminopterin. These subcultures are the 3rd and 4th selection steps. The increased levels o f the drug were used to distinguish differences in the drug resistant phenotype.
Regeneration o f aminopterin resistant plants Seven cell lines t h a t maintained aminopterin resistance through at least eight subcultures on 1 • l'0-s M aminopterin were induced to undergo morphogenesis by culturing t h e m on three differentiating media with and w i t h o u t 1 • 10 -s M aminopterin. The three media differed from SM by the following h o r m o n e substitutions: 1 • I 0 -6 M benzylaminopurine (BAP), 3 • 10 -6 M BAP and 22.9 • 1=0-6 M IAA + 4.6 • 1'0 -6 M kinetin, respectively. T w e n t y callus samples (50--75 mg) from each line were cultured on the six media. Unselected wild type D innoxia callus tissue served as the control. RESULTS
Plating procedures Table I presents the frequency distribution of groups of different cell numbers in suspension filtrates. The data are expressed as the range in percentage for eight experiments. The mean percent of viable units within each of these groups for all experiments is also given. Single cell units were observed most frequently, but viable single cells comprised only 6.7% of all units counted. The viable units in individual experiments ranged from 27 to 32%. The total viable units for all groups was 30.6% of which 22% (6.7/30.6) were single cells. Not all viable cell units divided to form visible colonies in 4 weeks. The plating efficiency, expressed in terms o f percentage o f viable cell units which form 1 m m or larger colonies in 4 weeks, ranged between 21 and 30%.
Aminopterin dose response curve D. innoxia tolerates aminopterin over a relatively wide concentration range, 10 -8 --10 -6 M, but there is a logarithmic relationship between killing and con-
174 TABLE I DISTRIBUTION OF CELLS ACCORDING TO NUMBER OF CELLS/UNIT AND VIABLE CELLS/UNIT IN FILTRATES FROM SUSPENSION CULTURES This table summarizes cell counts and viabilitj~ counts from eight experiments in which the total units counted was 1273. No. of cells/unit (%) 1
Range of cells/unit in individual experiments Viable units/total
Viable units in individual experiments
centration in the range of 10 -6 --10 -s M. The limited survival (1 • I0 -s) in 1 • 1'0-s M indicated that this was aneffective selective dose with which to screen for aminopterin resistant cells in D. innoxia (Fig. 1).
Frequency of aminopterin resistant cells T h e results o f t h e m u l t i p l e step selections are s u m m a r i z e d in T a b l e II. T h e f r e q u e n c y o f colonies resistant t o a m i n o p t e r i n d u r i n g t h e various selection steps r a n g e d f r o m 5.4 • 10 -6 t o 10 -7, T h e s e are f r e q u e n c i e s o f resistant c o l o n i e s relative t o t h e original t o t a l n u m b e r o f wild t y p e cells. F r o m wild t y p e c u l t u r e d cells, 74 colonies w e r e p i c k e d which survived o n 1 • 1'0 -s M a m i n o p t e r i n . On r e t e s t i n g b y s u b c u l t u r i n g t h e m again o n I • 10-s M a m i n o p t e r i n , 54% c o n t i n u e d t o p r o l i f e r a t e ( 4 0 / 7 4 ) . T h e colonies surviving the s e c o n d s u b c u l t u r e c o n t i n u e d t o grow d u r i n g s u b s e q u e n t subcultures on 1 • 1'0 -s M a m i n o p t e r i n . T h e s e 4 0 colonies d i f f e r e d in t h e i r ability t o survive a t greater c o n c e n t r a t i o n s o f t h e drug. In 2 • 10 -s M a m i n o p t e r i n , 1 4 / 4 0 o f t h e s e c o n d s u b c u l t u r e resistant colonies grew while o n l y f o u r o f t h o s e , i.e. 4 / 4 0 , c o u l d also p r o l i f e r a t e at 4 • 1'0 -s M a m i n o p t e r i n . T h e s e f o u r resistant colonies had b e e n isolated originally f r o m wild t y p e cultures b y first screening w i t h 1 • 10 -s M a m i n o p t e r i n and t h e y r e p r e s e n t 10 -7 o f t h e original cell p o p u l a t i o n . T h e f r e q u e n c y o f resistance t o 2 - 1"0-s M a n d 4 • 1'0 -s M a m i n o p t e r i n a m o n g 2nd s u b c u l t u r e resistant colonies is 35 and 10% respectively. T h e d e c r e a s e in colonies b e t w e e n t h e first a n d s e c o n d screen at 1 • 10 -s M a m i n o p t e r i n p r o b a b l y reflects statistical f l u c t u a t i o n s in c o l o n y f o r m a t i o n at this d r u g c o n c e n t r a t i o n w h i c h p e r m i t s s o m e n o n - r e s i s t a n t colonies t o survive.
O3 .J / LIJ (J
g W Q. >.. I---
i u_ 0 _J
u. 0 >0 Z W -"1
Fig. 1. D o s e response curve o f w i l d t y p e liquid s u s p e n s i o n cells to aminopterin. Survival w a s scored by ability to f o r m 1 - m m colonies. Survival w a s 100% in 1 0 -9 M aminopterin.
T A B L E II F R E Q U E N C Y O F D. INNOXIA CELLS R E S I S T A N T TO A M I N O P T E R I N Colonies > 1 m m were scored in the 1st screen. T h e s e c o l o n i e s were transplanted for the 2nd subculture. F o r t h e 3rd and 4 t h subculture inocula weighing a b o u t 5 0 mg w e r e t a k e n f r o m surviving colonies. S e l e c t i o n step
Aminopterin ( . 1 0 -s M)
Frequency of resistant colonies
Number of resistant colonies
1st Screen 2nd Subculture 3rd S u b c u l t u r e 4th S u b c u l t u r e
1 1 2 4
5.4 • 10 -~ 10 -6 3.5 • 10 -~ 10 -~
74 40 14 4
The different resistance to increased concentration o f aminopterin may indicate t h a t there are a n u m b e r o f mechanisms involved in increasing resistance or that the level o f resistance depends on the accumulation o f numbers of multiple alleles of a gene responsible for resistance [ 5].
Growth characteristics o f aminopterin resistant lines The increase in fresh weight o f three aminopterin resistant lines was compared (Table HI) after 49 or 58 days and again at 103 days of culture on SM and SM with 1 ° 1'0 -s M aminopterin. Line AR-54 grew more slowly than AR-36 and AR-33; the latter two lines increased 17.9- and 16-fold in weight, respectively, when cultured on SM for 49 days, while AR-54 multiplied its weight only 5.7 times when cultured 58 days on the same medium. Cells t h a t have been cultured continuously on SM with 1 • 10-s M aminopterin are given the postscript A. Lines AR-33A and AR-54A grew somewhat slower on aminopterin-containing medium than on SM (14.7- vs. 18.1-fold and 8.2- vs. l l . 9 - f o l d increase, respectively). Line AR-36A grew at nearly the same rate on both media (15.8- vs. 17.9-fold yield). Resistant lines from other cell types have different growth rates in the presence o f antifolates. These differences were matched by distinctive increases in H2FR production or particular levels of decreased affinity for m e t h o t r e x a t e T A B L E III INCREASE
IN F R E S H
D. I N N O X I A T h e m e a n fresh w e i g h t o f callus is c o m p a r e d t o its initial w e i g h t a f t e r g r o w t h o n SM and SM w i t h 1 • 10 -s M a m i n o p t e r i n . T h e m e a n w e i g h t and standard error o f the i n o c u l a w a s 143 ± 13 rag. The m e a n w e i g h t and standard e r r o r w e r e c a l c u l a t e d for 25 replicate s a m p l e s following culture.
AR-36 AR-36A* AR-33 AR-33A* AR-54 AR-54A* Wild t y p e
Days of growth
49 103 49 49 103 103 58 103 103 49
Standard m e d i u m (SM) rag/callus Final w t . Initial w t .
S M with 1 • 10 -s M rag/callus
Final w t . Initial w t .
2 5 4 0 ± 233 2328 ± 4 6 9
2788 +- 351
2258 ± 289
2288 ± 296 2 5 8 9 ± 303
1560 -+ 417 2100 ± 251
808 ± 74 1705 ± 337
5.7 11.9 19.5
1171 -+ 180 No g r o w t h
2793 ± 254
* T h e p o s t s c r i p t A indicates that t h e s e cells w e r e c o n t i n u o u s l y c u l t u r e d o n SM w i t h aminopterin.
177 and aminopterin
to indicate whether aminopterin
Data are not yet available for the
the observed differences
rate are related to the
Stability of the aminopterin resistant phenotype Table
III also demonstrates
AR-36, when grown in the absence of aminopterin. Cells grown for at least two s u b c u l t u r e s ( 3 m o n t h s ) o n S M w e r e c u l t u r e d a g a i n o n S M w i t h 1 • 1 0 -s M aminopterin.
to the yield following continuous
culture on amino-
pterin, AR-33 grew well (10.0- vs. 14.7-fold increase) and AR-36 grew exceedingly well (19.5-vs. 15.8-fold increase). Maintaining resistance in the absence o f s e l e c t i v e p r e s s u r e is c o n s i d e r e d conferred
the resistant phenotype
to be a strong indication
that a genetic change
Regeneration of aminopterin resistant plants The seven lines that were selected to undergo
T A B L E IV
SHOOT FORMATION AND GROWTH BY SEVEN AMINOPTERIN LINES O F D. I N N O X I A O N D I F F E R E N T I A T I N G M E D I A
T w e n t y i n o c u l a w e i g h i n g 5 0 - - 7 5 mg e a c h w e r e c u l t u r e d o n t h r e e m e d i a w i t h or w i t h o u t a m i n o p t e r i n (AM) as i n d i c a t e d . C o n c e n t r a t i o n o f a m i n o p t e r i n was 1 • 10 -s M. The n u m b e r o f i n o c u l a w h i c h d e v e l o p e d s h o o t s o r c o n t i n u e d t o p r o l i f e r a t e a f t e r culturing f o r 9 w e e k s is given.
AR-IA AR-2A AR-3A AR-33A AR-36A AR-38A AR-45A Callus (Control)
Shoots Growth Shoots Growth Shoots Growth Shoots Growth Shoots Growth Shoots Growth Shoots Growth Shoots Growth
1 • 10 -6 M BAP
3 • 10 -6 M BAP
22.9 • 1 0 - ' M I A A + 4.6 • 1 0 - ' M k i n e t i n
5 9 8 17 9 9 -7 8 19 8 15 12 19 10 20
--4 7 ---1 ----.10 10 ---
5 8 17 18 1 1 6 17 -5 -3 15 15 18 20
12 18 1 20 8 12 -17 3 14 13 20 10 18 16 20
----1 1 ---
178 the ability to differentiate shoots in the absence of aminopterin in at least one of the differentiating media (Table IV). When aminopterin was in the medium only two of the seven lines developed shoots. The aminopterin resistant phenotype was maintained in these two lines during the switch from undifferentiated callus growth to shoot Organization and formation. Unselected calli (control) neither grew nor differentiated shoots in aminopterin supplemented differentiating media but grew well and differentiated on all three media without added aminopterin. Roots were not induced by any of the three media. Healthy shoot systems were put through numerous root inducing regimens, so that whole, rooted plants were regenerated in four of the seven lines, two of which had developed shoots in aminopterin supplemented media. AR-36A and AR-38A have produced flowering haploid plants which are ready to be diploidized by colchicine treatment. Flowering plants have been obtained from three additional resistant lines. One of these has produced seed. T h e inheritance of aminopterin resistance in the progeny of these plants can now be tested. DISCUSSION D. innoxia is less sensitive to aminopterin than CHO cells  and baby hamster kidney cells  ; hence, various cell types display different sensitivities to aminopterin and other folic acid analogs. Vicia faba root tip cells, for example, cease dividing at higher concentrations of aminopterin than do some mammalian cells . Since these folic acid analogs affect a number of synthetic pathways when blocking formation of tetrahydrofolate by H2FR, sensitivity of a particular cell type may be a reflection of the pool size of various intermediates and of folate and dihydrofolate. Three of the seven cell lines in Table IV (AR-IA, AR-3A and AR-36A) had diminished ability, compared to control callus, to grow and form shoots on at least two of the three differentiating media. None of these lines formed shoots on aminopterin supplemented media. The two lines that did differentiate in aminopterin supplemented media (AR-2A and AR-45A) proliferated and differentiated well in its absence. The biochemical alterations resulting in aminopterin resistance may have some effect on purine synthesis. The availability of cytokinins, which have a purine backbone, would be expected to affect the capacity to differentiate. The multiple step method of selecting mutants is time consuming, involves lengthy manipulations and may encourage the selection of multiple site mutants. Furthermore, cells in culture are known to be able to gradually acclimate or habituate to the drug so that physiologic variants are selected rather than genetic mutants. It is significant, therefore, that D. innoxia cells do maintain their resistance in the absence of aminopterin. In addition, the genetic basis of the resistant phenotype can now be established by analyzing the offspring of the fertile plant that was regenerated from
179 a m i n o p t e r i n resistant callus. A d d i t i o n a l studies are also u n d e r w a y t o c o m p a r e t h e kinetics o f H 2 F R f r o m wild t y p e a n d a m i n o p t e r i n resistant cells. ACKNOWLEDGEMENT
Research carried out at Brookhaven National Laboratory, under the auspices of the United States Energy Research and Development Administration. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
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