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Induction and selection of chloroplast-coded mutations in Nicotiana
[43]
I S O L A T I O N OF P L A S T I D M U T A T I O N S IN
Nicotiana
611
To obtain mtDNA restriction patterns, which will provide visible fragments after ethidium bromide staining, about 2/xg of digested mtDNA, per gel-slot, is required. Therefore the amount of leaf material should be increased to about 100 g (fresh weight). Furthermore, an additional purification of the mitochondria by a sucrose gradient step is needed. Thus the mitochondrial fraction is layered over a discontinuous sucrose gradient, the mitochondria are collected, from the 1.2/1.45 M sucrose interphase, into a 30-ml Corex tube and diluted with 3 volumes of Buffer D. The tube is then centrifuged (SS34, Sorvall) at 11,150 rpm for 20 rain to obtain a purified mitochondrial pellet. To isolate mtDNA from cell suspensions the above detailed procedure should be modified as follows: (1) 150 g (fresh weight) cells is used per extraction; (2) Buffer A is modified to contain 0.3 M mannitol (rather than 0.5 M sucrose), the EDTA is reduced to 3 mM, and the pH is adjusted to 8.0; (3) the cells are homogenized in a French Press at 3000 psi; and (4) the DNase is reduced to 100/zg ml -~ and incubation is at room temperature for 30 min. Acknowledgment The experimental work of the authors is supported by the Lea and Julia Forscheimer Fund for MolecularGenetics.
[43] I n d u c t i o n a n d S e l e c t i o n o f C h l o r o p l a s t - C o d e d M u t a t i o n s in Nicotiana
By ROBERT FLUHR and AGNES CSI~PLO The majority of characterized plastome mutants in higher plants arose as spontaneous variegations which occur at a frequency of approximately 1 in 50,000 plants. ~However, the spontaneous rate for specific chloroplast traits 2 has been estimated to be as low as 1 x 10 -9. In some cases, such as the isolation of a Solanum nigrum variant resistant to atrazine, naturally occurring nuclear mutator genes have been indicated as responsible for t H. Kutzelnigg and W. Stubbe, Sub cell. Biochem. 3, 73 (1974). 2 C. J. Arntzen and J. H. Deusing, in "Advances in Gene Technology, Molecular Genetics of Plants and Animals" (F. Ahmad, K. Dawney, J. Schultz, and R. W. Voellny, eds.), p. 1. Academic Press, New York, 1983.
METHODS IN ENZYMOLOGY. VOI.. 118
Copyrighl V 1986 by Academic Press, Inc All rights of reproduction in any lk~rm reserved
612
CELL CULTURE AND TRANSFORMATION
[43]
increasing these low frequencies, z,3 The polyploidic nature of the chloroplast genetic organization and the large number of chloroplasts per cell g have presented investigators with considerable difficulties in the isolation o f specific chloroplast mutants. The first such mutants isolated were with the help of cell culture techniques and included two streptomycin-resistant isolates in tobacco. 5'6 Chemically induced mutagenesis to facilitate the isolation of chloroplast mutants has been carried out in Nicotiana plumbaginifolia protoplasts 7 tobacco seeds 8 and seeds o f A n t i r r h i n u m majus and Lycopersicon esculentum. 9 In all these cases mutagenesis with N-nitroso-N-methylurea (NMU) or N-nitroso-N-ethylurea (NEU) was thought to increase the preponderance o f plastid mutations. N M U causes methylation o f nucleic acids at guanidine bases; in addition, nuclear chromosomal breakage has been reported to Occur.~° It is, therefore, important when using mutagens to prove the plastome origin of the induced mutant characters. This can be ascertained by showing a nonMendelian inheritance pattern 9 or following the linkage of chloroplast traits via protoplast fusion.l~ H e r e we describe selection techniques used to isolate general and specific plastid mutations via seeds and protoplasts o f tobacco. Isolation o f Chloroplast M u t a n t s via S e e d Mutagenesis: Procedure
A stock solution o f 100 m M N M U (Sigma) in 70% ethanol and 0.1% acetic acid is kept at - 2 0 ° and diluted 20-fold with water before use. T o b a c c o seeds are soaked in 5 m M N M U for 2 hr at room temperature, surface sterilized by immersion in 3% (w/v) sodium hypochlorite for 20 min, and rinsed thoroughly with sterile water. Seed germination and rooting are in Nitsch medium containing 1% agar and 20 g/liter sucrose 3 w. Stubbe and R. G. Herrmann, in "Methods in Chloroplast Molecular Biology (M. Edelman, R. B. Hallick, and N.-H. Chua, eds.), p. 149. Elsevier/North-Holland Biomedical Press, Amsterdam, 1982. 4 N . Steele-Scott and J. V. Possingham, J. Exp. Bot. 31, 1081 (1980). 5 p. Maliga, A. Sz-Breznovits, and L. Marton, Nature (London) 255, 401 (1975). 6 N. Umiel, Z. Pflanzenphysiol. 92, 295 (1979). 7 A. Cseplo and P. Maliga, Curt. Genet. 6, 105 (1982). s R. Fluhr, D. Aviv, E. Galun, and M. Edelman, Proc. Natl. Acad. Sci. U.S.A. 82, 1485 (1985). 9 R. Hagemann, in "Methods in Chloroplast Molecular Biology" (M. Edelman, R. B. Hallick, and N.-H. Chua, eds.), p. 119. Elsevier/North-Holland Biomedical Press, Amsterdam, 1982. 10L. Fishbein, W. G. Flamm, and H. L. Falk, "Chemical Mutagens: Environmental Effects on Biological Systems." Academic Press, New York, 1970. 11E. Galun and D. Aviv, this volume [42].
[43]
ISOLATION OF PLASTID MUTATIONS IN
TABLE
613
Nicotiana
l
PLANT AND PROTOPLAST GROWTH MEDIA COMPONENTS"
Supplemented MS medium h
K3'
Nitsch a
P"
1.
NHaNOs
1650
250
720
2.
KNO~
1900
2500
--
380
3.
CaCI2
333
680
166
333
4.
MgSO4
181
121
90
36
5.
KH2PO4
170
68
170
6.
Fe EDTA
7.
H3BO3
8.
MnSO4.
9.
36.7 H20
-65
36
330
36.7
6.2
3.0
--
6.2
16.9
10.0
--
16.9
Z n S O 4 • 7H_,O
8.6
2.0
--
8.6
10.
Ki
0.83
0.75
--
0.83
11.
Na2MoO4 • 2H20
2.5
I).25
--
2.5
12.
CuSO4" 5HzO
0.025
0.025
--
0.025
13.
CoCIz • 6 H z O
0.025
0.025
--
0.025
14.
(NH4)2SO4
--
134
--
--
15.
N a H 2 P O 4 • 2H,_O
--
150
--
--
16.
Thiamine-HCI
10
10
--
--
17.
Nicotinic acid
0.5
I
--
--
18.
Pyridoxine-HCI
0.5
I
--
--
19.
myo-lnositol
100
100
--
--
20.
D-Xylose
--
250
--
--
21.
Glycine
2
--
22.
Biotin
0.5
--
--
--
23.
Folic acid
0.5
--
--
--
2
media are adjusted to p H 5.6. Values in milligram/liter. from T. Murashige and F. S k o o g , Physiol. Plant. 15, ' P. M a l i g a , Plant Mol. Biol. Rep. 1, 137 11983). a j . p. Nitsch, Phytomorphology 19, 389 11969). " A. C s 6 p l 6 a n d P. M a l i g a , MoL Gen. Genet. 196, 407 (1984).
--
" All
~' Adapted
473 11962).
(Table 1). Larger seeds such as tomato may require 12 to 24 hr immersion or an increase in mutagen concentration. The use of NMU requires ext r e m e caution. NMU is a highly carcinogenic substance. Mutagenesis is carried out in a fume hood with gloved hands. All liquid water and materials are detoxified in a 20% solution of NaOH in the fume hood. R e s u l t s . The above conditions for mutagenesis were found to produce in tobacco seedlings, variegation patterns in 50% of the seedlings' firsttrue leaves, while their survival rate was close to 100% due to maintenance of the plantlets on nutrient media. Higher concentrations induced stunting and a degree of sterility. A drastic reduction in variegation frequency occurs below 0.5 mM NMU.
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CELL CULTURE AND TRANSFORMATION
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To test the extent of maternally inherited pigment mutations induced by NMU, 38 seedlings (M0, which exhibited variegated patterns ranging from white to light green, were chosen randomly for further genetic analysis. No defects such as aberrant leaves or growth patterns were noticed. Heredity characteristics were established by using plants as both pollinators and pollen acceptors in the case of self-fertile plants (N. tabacum SR1, N. tabacum var. Xanthi) and only as pollen acceptor in the case of cytoplasmic male sterile lines (N. tabacum, line 92). The tests for inheritance type are summarized in Table II. A positive case of maternal inheritance was scored, when a variegated plant, as pollen recipient, showed in some seed pods, mixtures of mutant, variegated, and normal progeny; while in the reciprocal cross all progeny were normal. Twelve plants of this type were isolated. A positive case of nuclear inheritance was scored when the mutant trait was detected in the reciprocal crosses; this ocTABLE I1 PIGMENTATION INHERITANCE OF VARIEGATED MUTANT N. tabacum VARIETIES OBTAINED BY MUTAGENESIS WITH N-NITROSO-N-METHYLU REA
Number of variegated plants
Pigmentation of leaf sectors
Inheritance
White Light green White White Light green
Ub U U Maternal Maternal
White Light green White Light green White
U U Nuclear Nuclear Maternal
White Light green
Nuclear Maternal
N. tabacum--line 92 a
10 1 1 3 3 N. tabacum--SR-1 c
7 l 2 2 5 N. tabacum vat. Xanthi
1 1
Line 92: plants contain N. tabacurn type nucleus with N. undulata cytoplasm [R. Fluhr, D. Aviv, E. Galun, and M. Edelman, Theor. Appl. Genet. 67, 491 (1984)]. b U, undetermined. c SR-I plants contain maternally inherited streptomycin resistance [P. Maliga, A. Sz-Breznovits, and L. Marton, Nature (London) 255, 401 (1975)]. a
[43]
ISOLATION OF PLASTID MUTATIONS IN Nicotiana
615
curred in 3 plants. The inheritance type was scored as undetermined when the mutant characteristics were not detected in the M2 progeny. This may have occurred if the initial percentage of mutant chloroplast population was low or at a selective disadvantage; thus, in the process of somatic sorting out, the mutant cytoplasmic characteristic was lost. In one case a plant was obtained which contained two different mutant types, light green and white, indicating the presence of a mixed population of mutants. It should be noted that all the plant species used in this experiment are amphidiploid (4c), i.e., a nuclear background which would repress the appearance of any but dominant nuclear mutations. The use of male sterile lines such as N. tabacum line 92 is especially advantageous as it forces outcrossing of the M1 plant which prevents recessive mutations from appearing in subsequent M2 progeny. Clonal mutant lines were established from seed pods which yielded exclusively (or a high percentage of) pure mutant seedlings. Alternatively, mutant sectors from the M~ plant can be used directly as explants for somatic propagation, a procedure that will be elaborated on in the next section. We have used this general procedure to select for mutations for which no obvious selection scheme was possible, for example, a nonMendelian mutant lacking ribulosebisphosphate carboxylase was isolated. 12This mutant was isolated in two stages. First, mutant plants were screened ( - 3 0 0 plants) visually for mutant sectors of slightly reduced chlorophyll levels; this is especially obvious when the mutation is in the initial variegated stage, against the background of normal green pigmentation. A reduction in chlorophyll content has been noted to occur in spontaneous ribulosebisphosphate carboxylase plastome mutants of Oenothera. j3 In the second stage, the less than normal green mutant leaf sectors ( - 4 0 plants) were screened directly by SDS-polyacrylamide gel electrophoresis for their soluble and membrane-bound proteins. 12 One maternally inherited mutant lacked large and small subunits of ribulosebisphosphate carboxylase while other soluble or membrane-bound polypeptides of this mutant remained unchanged.
Selection for Antibiotic Resistance in Mutagenized Seedlings: Principle Tobacco seeds are treated with mutagen and then plated in selective media. The mutagen dose is adjusted to yield a high percentage of variegated plants without affecting the level of germination. The concentration of the selective agent in the media is adjusted to interfere directly with 12 R. Fluhr, Ph.D. Dissertation, The Weizmann Institute of Science, Rehovot, Israel (1984). 13 U. W. Hallier, J. M. Schmitt, U. Heber, S. S. Chaianova, and A. D. Volodarsky, Biochim. Biophys. Acta 504, 67 (1978).
616
CELL CULTURE AND TRANSFORMATION
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,ee0s ot,e
~.
) Y In selective medium
Mature ,lant followinc selfing r/and outcross~ng
FIG. I. Schematic outline of selection for antibiotic-resistant chloroplast mutants. NMU treated seeds were germinated in heterotrophic media containing antibiotic (selective media). Plantlets with resistant sectors in the first true leaves were obtained directly (arrow I). Cotyledons with resistant islands were used directly as explants in selective regeneration media (arrow II). Mutant plants from both procedures were transferred to heterotrophic, nonselective media and finally potted to obtain the mature flowering plant used for seed tests.
chloroplast processes but cellular division is left largely uninhibited. This results in chlorotic plantlets with more or less normal morphology. Plants exhibiting resistant green sectors are then selected visually. This procedure is shown schematically in Fig. 1. It is important to appreciate that resistant chloroplasts are not necessarily at a direct selective advantage, as chloroplast reproductive functions are apparently nuclear coded. Thus, to achieve selection one must assay for chloroplast resistances under conditions that will be nonlethal to the large majority of cells containing sensitive chloroplasts. This is accomplished by plating the treated seedlings on selective nutrient agar, on which plantlets are screened for resistant sectors. These plants are then transferred to nonselective media to enable the plastids to continue sorting-out under nonselective conditions. The plants are either self- or cross-pollinated and then individual seed pods are tested for resistant progeny.
Selection for Antibiotic Resistance in Mutagenized Seedlings: Procedure Seeds are mutagenized as described above at N M U doses (5 mM) which causes variegation in approximately 50% of the seedlings first true
[43]
ISOLATION OF PLASTID MUTATIONS IN Nicotiana
617
leaves as discussed previously. The seeds are plated on Nitsch media (Table I) containing 1% agar, 20 g/liter sucrose, and supplemented with one of the following antibiotics: 1000 rag/liter streptomycin, 50 rag/liter spectinomycin, or 500 rag/liter lincomycin. The seeds are plated at a density of 40-50 seeds per 10 cm plate. These antibiotic doses produce control plantlets with bleached cotyledon and first-true leaves in most Nicotiana species. Results. Treated seedlings (after 2 weeks) had a green granulated appearance in about 30% of the cotyledons. Fully expanded cotyledons (4 weeks) showed distinct green cell clusters (Fig. 2A) in 90% of the plantlets. Root elongation was inhibited in all plantlets, similar to non-mutagenized plantlets. First-true leaves (4 to 8 weeks) were generally all bleached. A small percentage of seedlings which exhibited green variegation patterns (Fig. 2B) were transferred to Nitsch agar without antibiotics. Under these nonselective conditions all subsequent leaves were totally
Fie. 2. (A) Cotyledon containing resistant Islands. Seeds were mutagenized with 5 mM NMU. and plated on 1 g/liter streptomycin. Green islands are clusters of cells containing chlorophyll pigment. Explants of this type of cotyledon yield variegated plantlets when plated on selective regeneration media. (B) Resistant variegation in first-true leaf. Mutagenized seeds were plated on Nitsch media containing 500 rag/liter lincomycin. A sector of green tissue is apparent (see arrow). The white surrounding area is nonresistant leaf tissue. The other seedlings similarly treated (upper left corner) show no resistant sectors.
618
CELL CULTURE AND TRANSFORMATION
[43]
TABLE III ANTIBIOTIC-REsISTANT AREAS IN COTYLEDONS AND FIRST-TRUE LEAVES OF MUTAGENIZED N. tabacum LINE 92a MI cotyledons
Mj first-true leaves
Selective agent
Antibiotic concentration (mg/liter)
Exhibiting green islands (%)
Antibiotic concentration (mg/liter)
Exhibiting green sectors (%)
Streptomycin Spectinomycin Lincomycin
1000 50 50
90 60 60
1000 50 500
1.0 1.2 1.9
a One thousand mutagenized seedlings were analyzed with each selective agent. No green islands in cotyledons or sectors in leaves were detected in 1000 control seedlings grown in the presence of selective agents.
green. Rooted plantlets (7 to 10 weeks) were transferred to a greenhouse. Plants were self-pollinated or pollinated with normal N. tabacum pollen in the case of male-sterile plants, and the resulting progeny were tested for antibiotic resistance. Table III shows that typically 1 to 2% of mutagenized seedlings exhibit green resistant sectors. The progeny seedlings of these plants were as expected: either mixtures of pure resistant, variegated for resistance or pure sensitive phenotypes. J.3.8 Mutant clonal lines (M2) were established from pods which yielded pure resistant or nearly pure resistant progeny. In general the progeny (M3) of the M2 plants were pure for the resistant phenotype indicating complete sorting out of the chloroplast types. All mutants selected in this manner exhibited maternal inheritance. 8 Cotyledonary material with green islands (Fig. 2) was used directly as explant material for mutant selection via plant regeneration (Fig. 1). The cotyledons were excised and placed on supplemented MS media (Table I) including 1% agar, 3% sucrose, 2 mg/liter 6-benzylaminopurine, 0.8 rag/ liter indole acetic acid, and the appropriate level of antibiotic (Table III). No attempt was made to excise the green areas directly as they were too small. Plantlets regenerated in this manner appeared variegated on selective media while nonmutagenized cotyledons either grew into undifferentiated white calli or regenerated completely white ptantlets. The MI plantlets generally produced very highly mixed M2 progeny for the selected trait. Pods containing pure mutant seeds were not isolated, using this method.
[43]
ISOLATION OF PLASTID MUTATIONS IN N i c o t i a n a
619
Isolation of Chloroplast Mutants via Protoplast: Principle Protoplasts are mutagenized and plated into callus inducing media. After a short period of cellular division the microcolonies are challenged with a selective agent which inhibits greening, but not other cellular processes. Selection for resistant calli is carried out visually. In contrast to mutant selection at the whole plant level, the plating of microcalli on selective media involves sorting out a smaller population of chloroplasts, all originating from one cell. Thus, the resistant calli and resultant regenerated plants are more homogeneous for the mutant trait. This method has been successfully used for the large scale isolation of lincomycin-resistant mutants, 14and the selection of herbicide resistant mutants.~5 This method requires thorough knowledge of plant cell culture techniques and the use of plant species amenable to efficient protoplast isolation and subsequent plant regeneration.
Protoplast Isolation and Mutant Selection for Antibiotic and Herbicide Resistance: Procedure Leaf material, diploid N. plumbaginifolia or amphidiploid N. tabacum, is obtained from sterile plants maintained on MS salts (Table 1, items 1 to 13) with 3% sucrose and 1% agar. Protoplasts are isolated by shredding fully expanded leaves in a I0 cm tissue culture dish containing 10 ml of K3 media (Table I) with 0.4 M sucrose and 0.5% Driselase (Kyowa Hakko Kogyo Co., Tokyo, Japan). The area of shredded leaf material should nearly cover the dish's bottom surface. The plates are incubated 12 to 16 hr in the dark at 27 °. The protoplasts are carefully removed using a wide mouth pipette and filtered through a 63-/zm nylon net (Verseidag), and centrifuged at 300 g for 3 min. Intact protoplasts which float to the top are removed and diluted into 9 volumes of 150 mM NaC1, 125 mM CaCI2, 5 mM KCI, 5 mM glucose, pH 5.8, and centrifuged at 50 g for 2 rain. Protoplast pellets are gently resuspended in supplemented K3 media; supplemented K3 media is K3 media (Table I) containing 0.4 M glucose, 0.1 mg/liter 2,4-dichlorophenoxyacetic acid, 0.2 mg/ liter 6-benzylaminopurine, and 1 rag/liter a-naphthaleneacetic acid. The protoplasts are plated at a density of 105 protoplasts/ml in 5-cm plates, 5 ml in each dish and maintained at 28 ° and low light (100 lux). If mutagenesis is performed, filter-sterilized N E U is added to 0.1 to 0.3 mM. The mutagen is not washed out as it is inactivated within approximately 3 days. 14 A. Cs6pl6 and P. Maliga, Mol. Gen. Genet. 196, 407 (1984). ~5 A. Cs6pl6, in preparation.
620
CELL CULTURE AND TRANSFORMATION
[43]
T A B L E IV NUMBER OF RESISTANT CLONES ISOLATED FROM N. plumbaginifolia PROTOPLASTS Lincomycin resistance (1 g/liter)
Terbutryne resistance (10 -4 M )
Concentration o f mutagen (NEU) (raM)
N u m b e r of calli screened ( x 105)
Number of resistant calli isolated
Number of calli screened ( × 105)
0.0 0.1 0.3
0.58 1.20 0.40
6 70 32
1.5 2.0 --
Number of resistant calli isolated 1 --
The osmoticum in the medium is gradually lowered every 7 to 10 days by diluting the media in the following manner. First dilution, an equal volume of supplemented K3 media with 0.3 M glucose is added; second dilution, an equal volume of supplemented K3 media with 0.2 M glucose is added. The resultant microcolonies are plated into supplemented MS salts (Table I, components 1-19) containing 0.2 M glucose, I g/liter lincomycin, 1 mg/liter 6-benzylaminopurine, and 0.1 mg/liter a-naphthaleneacetic acid solidified with 0.7% agar. The plates are maintained at 28°, I000 lux. Resistant green colonies are isolated within 1 to 2 months and retested on the same media but with 0.1 M glucose (2% w/v). In the case of selection for herbicide resistance, a third dilution of the protoplasts in supplemented K3 media with 0.1 M glucose is carried out. The microcolonies are diluted into MS salts as above but with 0.015 M glucose (0.3% w/v) and 10-4 M terbutryne (instead of lincomycin). Resistant colonies are screened and retested in the same medium but with 0.015 M glucose. Calli which exhibit resistance through two subcultures on restrictive media are transferred for shoot induction to the same media (0.05 M sucrose) without antibiotic or herbicide, and the a-naphthaleneacetic acid is omitted. Plants are rooted on P media (Table I) and transferred to the greenhouse for genetic analysis. Results. Table IV summarizes the data for two experiments involving selection of resistance to the antibiotic lincomycin 7,~4,~6and the herbicide terbutryne.~5 Approximately 75% of the lincomycin-resistant calli regenerated normal looking plants. When plant leaves were retested on supplemented MS media containing antibiotics, 90% were truly lincomycin re16 A. Csrpl6, F. Nagy, and P. Maliga, MoL Gen. Genet. 198, 7 (1984).
[43]
ISOLATION OF PLASTID MUTATIONS IN
Nicotiana
621
FIG. 3. Lincomycin resistance test of a putative lincomycin-resistant plant regenerated from mutagenized protoplasts. Leaf sections were tested on callus forming media, as described in the text, containing 1000 rag/liter lincomycin. On this media the sensitive leaves (Np) form white calli, while the resistant leaf sections (LR415) form green calli.
sistant. On this media, resistant plants formed green calli while sensitive control plants formed white caUi (Fig. 3). All the plants which resulted from the 0.3 mM N E U treatment exhibited functional male sterility. A certain increase in the amount of mutant resistant calli isolated was seen using 0.1-0.3 mM NEU. It should be noted that in selection for nuclear mutations a 100-fold increase in isolation frequency was reported with 0.3 mM N E U when using haploid N. plumbaginifolia protoplasts. 17 In nearly all cases, seed progeny of resistant mutants were homogeneously mutant, probably as a result of the extended selection period at the undifferentiated callus stage. Hence, complete sorting out of the mutant chloroplasts was obtained in subculturing the green areas of resistant calli. In each instance the mutant phenotype exhibited maternal inheritance. Crucial to the successful use of the herbicide selection procedure was the reduction of the glucose concentration of the selective media to 0.015 M. At higher glucose concentration (0. I M), sensitive calli still maintained 17 L. Marton, T. M. Dung, R. R. Mendel, and P. Maliga, Mol. Gen. Genet. 59, 191 (1982).
622
CELL CULTURE AND TRANSFORMATION
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TABLE V EFFECTIVE SCREENING CONCENTRATIONS OF HERBICIDES IN N. plumbaginifolia CELL CULTURES°
Herbicide
Class
Effective screening concentration
T e r b u t r y n e atrazine Metribuzin Metobromuron Bromacil Bromoxynil
s-Triazine as-Triazine Urea type Uracil type Nitrile type
10 _4 10 -4 10 -4 10 _5 2.0 × l0 -6
a Media for selection was s u p p l e m e n t e d K3 with 0.015 M glucose.
their green color. Raising the herbicide concentration to higher levels in the presence of 0.1 M glucose only caused secondary inhibitory effects to become apparent. The reduced glucose media has been found to be effective in providing selective screening for additional herbicides shown in Table V. The terbutryne-resistant isolates showed maternal inheritance, were resistant to atrazine (10 -4 M), and exhibited photochemical properties similar to naturally occurring s-triazine-resistant plants. ~8,19
Concluding Remarks The direct isolation of chloroplast mutants through seed mutagenesis is applicable when the following conditions exist. (1) The plantlets respond to NMU treatment with a high percentage of variegation in the first true leaves. (2) The selection agent used is plastid specific and can be transported through the plants' vascular system. The use of mutagenized seedlings avoids the added variance caused by prolonged cell culture such as deformed plants or induced male sterility. The high frequency of green islands appearing in cotyledonary tissue (Table III) is surprising. Although mutations appearing in this tissue have no genetic continuity in the cells of the apical meristem, plants regenerated from them yield mutant seed. 8 Thus, the number of cells composing the cotyledon and the rate of plastid sorting-out in those cells potentiate the mutagen effect. This material shows great potential for the isolation of low frequency mutations. The protoplasts method for mutant isolation is less dependent upon J8 C. J. A r n t z e n , K. Pfister, and K. E. Steinback, in "Herbicide Resistance in P l a n t s " (H. M. L e B a r o n a n d J. Gressel, eds.), p. 185. Wiley, N e w York, 1982. ~9 S. D e m e t e r , I. V a s s , E. Hideg, and A. Sallai, Biochim. Biophys. Acta (in press).
[43]
I S O L A T I O N OF P L A S T I D M U T A T I O N S IN
Nicotiana
623
the application of chemical mutagens. As Table IV shows, the use of large scale selection at the microcolony level enables successful screening even without NMU. Additional mutations such as herbicide resistance have been recovered using this technique. In general, the resultant M~ plants regenerated from resistant calli are more homogeneous for the mutant trait.
I S O L A T I O N OF P L A S T I D M U T A T I O N S IN
Nicotiana
611
To obtain mtDNA restriction patterns, which will provide visible fragments after ethidium bromide staining, about 2/xg of digested mtDNA, per gel-slot, is required. Therefore the amount of leaf material should be increased to about 100 g (fresh weight). Furthermore, an additional purification of the mitochondria by a sucrose gradient step is needed. Thus the mitochondrial fraction is layered over a discontinuous sucrose gradient, the mitochondria are collected, from the 1.2/1.45 M sucrose interphase, into a 30-ml Corex tube and diluted with 3 volumes of Buffer D. The tube is then centrifuged (SS34, Sorvall) at 11,150 rpm for 20 rain to obtain a purified mitochondrial pellet. To isolate mtDNA from cell suspensions the above detailed procedure should be modified as follows: (1) 150 g (fresh weight) cells is used per extraction; (2) Buffer A is modified to contain 0.3 M mannitol (rather than 0.5 M sucrose), the EDTA is reduced to 3 mM, and the pH is adjusted to 8.0; (3) the cells are homogenized in a French Press at 3000 psi; and (4) the DNase is reduced to 100/zg ml -~ and incubation is at room temperature for 30 min. Acknowledgment The experimental work of the authors is supported by the Lea and Julia Forscheimer Fund for MolecularGenetics.
[43] I n d u c t i o n a n d S e l e c t i o n o f C h l o r o p l a s t - C o d e d M u t a t i o n s in Nicotiana
By ROBERT FLUHR and AGNES CSI~PLO The majority of characterized plastome mutants in higher plants arose as spontaneous variegations which occur at a frequency of approximately 1 in 50,000 plants. ~However, the spontaneous rate for specific chloroplast traits 2 has been estimated to be as low as 1 x 10 -9. In some cases, such as the isolation of a Solanum nigrum variant resistant to atrazine, naturally occurring nuclear mutator genes have been indicated as responsible for t H. Kutzelnigg and W. Stubbe, Sub cell. Biochem. 3, 73 (1974). 2 C. J. Arntzen and J. H. Deusing, in "Advances in Gene Technology, Molecular Genetics of Plants and Animals" (F. Ahmad, K. Dawney, J. Schultz, and R. W. Voellny, eds.), p. 1. Academic Press, New York, 1983.
METHODS IN ENZYMOLOGY. VOI.. 118
Copyrighl V 1986 by Academic Press, Inc All rights of reproduction in any lk~rm reserved
612
CELL CULTURE AND TRANSFORMATION
[43]
increasing these low frequencies, z,3 The polyploidic nature of the chloroplast genetic organization and the large number of chloroplasts per cell g have presented investigators with considerable difficulties in the isolation o f specific chloroplast mutants. The first such mutants isolated were with the help of cell culture techniques and included two streptomycin-resistant isolates in tobacco. 5'6 Chemically induced mutagenesis to facilitate the isolation of chloroplast mutants has been carried out in Nicotiana plumbaginifolia protoplasts 7 tobacco seeds 8 and seeds o f A n t i r r h i n u m majus and Lycopersicon esculentum. 9 In all these cases mutagenesis with N-nitroso-N-methylurea (NMU) or N-nitroso-N-ethylurea (NEU) was thought to increase the preponderance o f plastid mutations. N M U causes methylation o f nucleic acids at guanidine bases; in addition, nuclear chromosomal breakage has been reported to Occur.~° It is, therefore, important when using mutagens to prove the plastome origin of the induced mutant characters. This can be ascertained by showing a nonMendelian inheritance pattern 9 or following the linkage of chloroplast traits via protoplast fusion.l~ H e r e we describe selection techniques used to isolate general and specific plastid mutations via seeds and protoplasts o f tobacco. Isolation o f Chloroplast M u t a n t s via S e e d Mutagenesis: Procedure
A stock solution o f 100 m M N M U (Sigma) in 70% ethanol and 0.1% acetic acid is kept at - 2 0 ° and diluted 20-fold with water before use. T o b a c c o seeds are soaked in 5 m M N M U for 2 hr at room temperature, surface sterilized by immersion in 3% (w/v) sodium hypochlorite for 20 min, and rinsed thoroughly with sterile water. Seed germination and rooting are in Nitsch medium containing 1% agar and 20 g/liter sucrose 3 w. Stubbe and R. G. Herrmann, in "Methods in Chloroplast Molecular Biology (M. Edelman, R. B. Hallick, and N.-H. Chua, eds.), p. 149. Elsevier/North-Holland Biomedical Press, Amsterdam, 1982. 4 N . Steele-Scott and J. V. Possingham, J. Exp. Bot. 31, 1081 (1980). 5 p. Maliga, A. Sz-Breznovits, and L. Marton, Nature (London) 255, 401 (1975). 6 N. Umiel, Z. Pflanzenphysiol. 92, 295 (1979). 7 A. Cseplo and P. Maliga, Curt. Genet. 6, 105 (1982). s R. Fluhr, D. Aviv, E. Galun, and M. Edelman, Proc. Natl. Acad. Sci. U.S.A. 82, 1485 (1985). 9 R. Hagemann, in "Methods in Chloroplast Molecular Biology" (M. Edelman, R. B. Hallick, and N.-H. Chua, eds.), p. 119. Elsevier/North-Holland Biomedical Press, Amsterdam, 1982. 10L. Fishbein, W. G. Flamm, and H. L. Falk, "Chemical Mutagens: Environmental Effects on Biological Systems." Academic Press, New York, 1970. 11E. Galun and D. Aviv, this volume [42].
[43]
ISOLATION OF PLASTID MUTATIONS IN
TABLE
613
Nicotiana
l
PLANT AND PROTOPLAST GROWTH MEDIA COMPONENTS"
Supplemented MS medium h
K3'
Nitsch a
P"
1.
NHaNOs
1650
250
720
2.
KNO~
1900
2500
--
380
3.
CaCI2
333
680
166
333
4.
MgSO4
181
121
90
36
5.
KH2PO4
170
68
170
6.
Fe EDTA
7.
H3BO3
8.
MnSO4.
9.
36.7 H20
-65
36
330
36.7
6.2
3.0
--
6.2
16.9
10.0
--
16.9
Z n S O 4 • 7H_,O
8.6
2.0
--
8.6
10.
Ki
0.83
0.75
--
0.83
11.
Na2MoO4 • 2H20
2.5
I).25
--
2.5
12.
CuSO4" 5HzO
0.025
0.025
--
0.025
13.
CoCIz • 6 H z O
0.025
0.025
--
0.025
14.
(NH4)2SO4
--
134
--
--
15.
N a H 2 P O 4 • 2H,_O
--
150
--
--
16.
Thiamine-HCI
10
10
--
--
17.
Nicotinic acid
0.5
I
--
--
18.
Pyridoxine-HCI
0.5
I
--
--
19.
myo-lnositol
100
100
--
--
20.
D-Xylose
--
250
--
--
21.
Glycine
2
--
22.
Biotin
0.5
--
--
--
23.
Folic acid
0.5
--
--
--
2
media are adjusted to p H 5.6. Values in milligram/liter. from T. Murashige and F. S k o o g , Physiol. Plant. 15, ' P. M a l i g a , Plant Mol. Biol. Rep. 1, 137 11983). a j . p. Nitsch, Phytomorphology 19, 389 11969). " A. C s 6 p l 6 a n d P. M a l i g a , MoL Gen. Genet. 196, 407 (1984).
--
" All
~' Adapted
473 11962).
(Table 1). Larger seeds such as tomato may require 12 to 24 hr immersion or an increase in mutagen concentration. The use of NMU requires ext r e m e caution. NMU is a highly carcinogenic substance. Mutagenesis is carried out in a fume hood with gloved hands. All liquid water and materials are detoxified in a 20% solution of NaOH in the fume hood. R e s u l t s . The above conditions for mutagenesis were found to produce in tobacco seedlings, variegation patterns in 50% of the seedlings' firsttrue leaves, while their survival rate was close to 100% due to maintenance of the plantlets on nutrient media. Higher concentrations induced stunting and a degree of sterility. A drastic reduction in variegation frequency occurs below 0.5 mM NMU.
614
CELL CULTURE AND TRANSFORMATION
[43]
To test the extent of maternally inherited pigment mutations induced by NMU, 38 seedlings (M0, which exhibited variegated patterns ranging from white to light green, were chosen randomly for further genetic analysis. No defects such as aberrant leaves or growth patterns were noticed. Heredity characteristics were established by using plants as both pollinators and pollen acceptors in the case of self-fertile plants (N. tabacum SR1, N. tabacum var. Xanthi) and only as pollen acceptor in the case of cytoplasmic male sterile lines (N. tabacum, line 92). The tests for inheritance type are summarized in Table II. A positive case of maternal inheritance was scored, when a variegated plant, as pollen recipient, showed in some seed pods, mixtures of mutant, variegated, and normal progeny; while in the reciprocal cross all progeny were normal. Twelve plants of this type were isolated. A positive case of nuclear inheritance was scored when the mutant trait was detected in the reciprocal crosses; this ocTABLE I1 PIGMENTATION INHERITANCE OF VARIEGATED MUTANT N. tabacum VARIETIES OBTAINED BY MUTAGENESIS WITH N-NITROSO-N-METHYLU REA
Number of variegated plants
Pigmentation of leaf sectors
Inheritance
White Light green White White Light green
Ub U U Maternal Maternal
White Light green White Light green White
U U Nuclear Nuclear Maternal
White Light green
Nuclear Maternal
N. tabacum--line 92 a
10 1 1 3 3 N. tabacum--SR-1 c
7 l 2 2 5 N. tabacum vat. Xanthi
1 1
Line 92: plants contain N. tabacurn type nucleus with N. undulata cytoplasm [R. Fluhr, D. Aviv, E. Galun, and M. Edelman, Theor. Appl. Genet. 67, 491 (1984)]. b U, undetermined. c SR-I plants contain maternally inherited streptomycin resistance [P. Maliga, A. Sz-Breznovits, and L. Marton, Nature (London) 255, 401 (1975)]. a
[43]
ISOLATION OF PLASTID MUTATIONS IN Nicotiana
615
curred in 3 plants. The inheritance type was scored as undetermined when the mutant characteristics were not detected in the M2 progeny. This may have occurred if the initial percentage of mutant chloroplast population was low or at a selective disadvantage; thus, in the process of somatic sorting out, the mutant cytoplasmic characteristic was lost. In one case a plant was obtained which contained two different mutant types, light green and white, indicating the presence of a mixed population of mutants. It should be noted that all the plant species used in this experiment are amphidiploid (4c), i.e., a nuclear background which would repress the appearance of any but dominant nuclear mutations. The use of male sterile lines such as N. tabacum line 92 is especially advantageous as it forces outcrossing of the M1 plant which prevents recessive mutations from appearing in subsequent M2 progeny. Clonal mutant lines were established from seed pods which yielded exclusively (or a high percentage of) pure mutant seedlings. Alternatively, mutant sectors from the M~ plant can be used directly as explants for somatic propagation, a procedure that will be elaborated on in the next section. We have used this general procedure to select for mutations for which no obvious selection scheme was possible, for example, a nonMendelian mutant lacking ribulosebisphosphate carboxylase was isolated. 12This mutant was isolated in two stages. First, mutant plants were screened ( - 3 0 0 plants) visually for mutant sectors of slightly reduced chlorophyll levels; this is especially obvious when the mutation is in the initial variegated stage, against the background of normal green pigmentation. A reduction in chlorophyll content has been noted to occur in spontaneous ribulosebisphosphate carboxylase plastome mutants of Oenothera. j3 In the second stage, the less than normal green mutant leaf sectors ( - 4 0 plants) were screened directly by SDS-polyacrylamide gel electrophoresis for their soluble and membrane-bound proteins. 12 One maternally inherited mutant lacked large and small subunits of ribulosebisphosphate carboxylase while other soluble or membrane-bound polypeptides of this mutant remained unchanged.
Selection for Antibiotic Resistance in Mutagenized Seedlings: Principle Tobacco seeds are treated with mutagen and then plated in selective media. The mutagen dose is adjusted to yield a high percentage of variegated plants without affecting the level of germination. The concentration of the selective agent in the media is adjusted to interfere directly with 12 R. Fluhr, Ph.D. Dissertation, The Weizmann Institute of Science, Rehovot, Israel (1984). 13 U. W. Hallier, J. M. Schmitt, U. Heber, S. S. Chaianova, and A. D. Volodarsky, Biochim. Biophys. Acta 504, 67 (1978).
616
CELL CULTURE AND TRANSFORMATION
[43]
,ee0s ot,e
~.
) Y In selective medium
Mature ,lant followinc selfing r/and outcross~ng
FIG. I. Schematic outline of selection for antibiotic-resistant chloroplast mutants. NMU treated seeds were germinated in heterotrophic media containing antibiotic (selective media). Plantlets with resistant sectors in the first true leaves were obtained directly (arrow I). Cotyledons with resistant islands were used directly as explants in selective regeneration media (arrow II). Mutant plants from both procedures were transferred to heterotrophic, nonselective media and finally potted to obtain the mature flowering plant used for seed tests.
chloroplast processes but cellular division is left largely uninhibited. This results in chlorotic plantlets with more or less normal morphology. Plants exhibiting resistant green sectors are then selected visually. This procedure is shown schematically in Fig. 1. It is important to appreciate that resistant chloroplasts are not necessarily at a direct selective advantage, as chloroplast reproductive functions are apparently nuclear coded. Thus, to achieve selection one must assay for chloroplast resistances under conditions that will be nonlethal to the large majority of cells containing sensitive chloroplasts. This is accomplished by plating the treated seedlings on selective nutrient agar, on which plantlets are screened for resistant sectors. These plants are then transferred to nonselective media to enable the plastids to continue sorting-out under nonselective conditions. The plants are either self- or cross-pollinated and then individual seed pods are tested for resistant progeny.
Selection for Antibiotic Resistance in Mutagenized Seedlings: Procedure Seeds are mutagenized as described above at N M U doses (5 mM) which causes variegation in approximately 50% of the seedlings first true
[43]
ISOLATION OF PLASTID MUTATIONS IN Nicotiana
617
leaves as discussed previously. The seeds are plated on Nitsch media (Table I) containing 1% agar, 20 g/liter sucrose, and supplemented with one of the following antibiotics: 1000 rag/liter streptomycin, 50 rag/liter spectinomycin, or 500 rag/liter lincomycin. The seeds are plated at a density of 40-50 seeds per 10 cm plate. These antibiotic doses produce control plantlets with bleached cotyledon and first-true leaves in most Nicotiana species. Results. Treated seedlings (after 2 weeks) had a green granulated appearance in about 30% of the cotyledons. Fully expanded cotyledons (4 weeks) showed distinct green cell clusters (Fig. 2A) in 90% of the plantlets. Root elongation was inhibited in all plantlets, similar to non-mutagenized plantlets. First-true leaves (4 to 8 weeks) were generally all bleached. A small percentage of seedlings which exhibited green variegation patterns (Fig. 2B) were transferred to Nitsch agar without antibiotics. Under these nonselective conditions all subsequent leaves were totally
Fie. 2. (A) Cotyledon containing resistant Islands. Seeds were mutagenized with 5 mM NMU. and plated on 1 g/liter streptomycin. Green islands are clusters of cells containing chlorophyll pigment. Explants of this type of cotyledon yield variegated plantlets when plated on selective regeneration media. (B) Resistant variegation in first-true leaf. Mutagenized seeds were plated on Nitsch media containing 500 rag/liter lincomycin. A sector of green tissue is apparent (see arrow). The white surrounding area is nonresistant leaf tissue. The other seedlings similarly treated (upper left corner) show no resistant sectors.
618
CELL CULTURE AND TRANSFORMATION
[43]
TABLE III ANTIBIOTIC-REsISTANT AREAS IN COTYLEDONS AND FIRST-TRUE LEAVES OF MUTAGENIZED N. tabacum LINE 92a MI cotyledons
Mj first-true leaves
Selective agent
Antibiotic concentration (mg/liter)
Exhibiting green islands (%)
Antibiotic concentration (mg/liter)
Exhibiting green sectors (%)
Streptomycin Spectinomycin Lincomycin
1000 50 50
90 60 60
1000 50 500
1.0 1.2 1.9
a One thousand mutagenized seedlings were analyzed with each selective agent. No green islands in cotyledons or sectors in leaves were detected in 1000 control seedlings grown in the presence of selective agents.
green. Rooted plantlets (7 to 10 weeks) were transferred to a greenhouse. Plants were self-pollinated or pollinated with normal N. tabacum pollen in the case of male-sterile plants, and the resulting progeny were tested for antibiotic resistance. Table III shows that typically 1 to 2% of mutagenized seedlings exhibit green resistant sectors. The progeny seedlings of these plants were as expected: either mixtures of pure resistant, variegated for resistance or pure sensitive phenotypes. J.3.8 Mutant clonal lines (M2) were established from pods which yielded pure resistant or nearly pure resistant progeny. In general the progeny (M3) of the M2 plants were pure for the resistant phenotype indicating complete sorting out of the chloroplast types. All mutants selected in this manner exhibited maternal inheritance. 8 Cotyledonary material with green islands (Fig. 2) was used directly as explant material for mutant selection via plant regeneration (Fig. 1). The cotyledons were excised and placed on supplemented MS media (Table I) including 1% agar, 3% sucrose, 2 mg/liter 6-benzylaminopurine, 0.8 rag/ liter indole acetic acid, and the appropriate level of antibiotic (Table III). No attempt was made to excise the green areas directly as they were too small. Plantlets regenerated in this manner appeared variegated on selective media while nonmutagenized cotyledons either grew into undifferentiated white calli or regenerated completely white ptantlets. The MI plantlets generally produced very highly mixed M2 progeny for the selected trait. Pods containing pure mutant seeds were not isolated, using this method.
[43]
ISOLATION OF PLASTID MUTATIONS IN N i c o t i a n a
619
Isolation of Chloroplast Mutants via Protoplast: Principle Protoplasts are mutagenized and plated into callus inducing media. After a short period of cellular division the microcolonies are challenged with a selective agent which inhibits greening, but not other cellular processes. Selection for resistant calli is carried out visually. In contrast to mutant selection at the whole plant level, the plating of microcalli on selective media involves sorting out a smaller population of chloroplasts, all originating from one cell. Thus, the resistant calli and resultant regenerated plants are more homogeneous for the mutant trait. This method has been successfully used for the large scale isolation of lincomycin-resistant mutants, 14and the selection of herbicide resistant mutants.~5 This method requires thorough knowledge of plant cell culture techniques and the use of plant species amenable to efficient protoplast isolation and subsequent plant regeneration.
Protoplast Isolation and Mutant Selection for Antibiotic and Herbicide Resistance: Procedure Leaf material, diploid N. plumbaginifolia or amphidiploid N. tabacum, is obtained from sterile plants maintained on MS salts (Table 1, items 1 to 13) with 3% sucrose and 1% agar. Protoplasts are isolated by shredding fully expanded leaves in a I0 cm tissue culture dish containing 10 ml of K3 media (Table I) with 0.4 M sucrose and 0.5% Driselase (Kyowa Hakko Kogyo Co., Tokyo, Japan). The area of shredded leaf material should nearly cover the dish's bottom surface. The plates are incubated 12 to 16 hr in the dark at 27 °. The protoplasts are carefully removed using a wide mouth pipette and filtered through a 63-/zm nylon net (Verseidag), and centrifuged at 300 g for 3 min. Intact protoplasts which float to the top are removed and diluted into 9 volumes of 150 mM NaC1, 125 mM CaCI2, 5 mM KCI, 5 mM glucose, pH 5.8, and centrifuged at 50 g for 2 rain. Protoplast pellets are gently resuspended in supplemented K3 media; supplemented K3 media is K3 media (Table I) containing 0.4 M glucose, 0.1 mg/liter 2,4-dichlorophenoxyacetic acid, 0.2 mg/ liter 6-benzylaminopurine, and 1 rag/liter a-naphthaleneacetic acid. The protoplasts are plated at a density of 105 protoplasts/ml in 5-cm plates, 5 ml in each dish and maintained at 28 ° and low light (100 lux). If mutagenesis is performed, filter-sterilized N E U is added to 0.1 to 0.3 mM. The mutagen is not washed out as it is inactivated within approximately 3 days. 14 A. Cs6pl6 and P. Maliga, Mol. Gen. Genet. 196, 407 (1984). ~5 A. Cs6pl6, in preparation.
620
CELL CULTURE AND TRANSFORMATION
[43]
T A B L E IV NUMBER OF RESISTANT CLONES ISOLATED FROM N. plumbaginifolia PROTOPLASTS Lincomycin resistance (1 g/liter)
Terbutryne resistance (10 -4 M )
Concentration o f mutagen (NEU) (raM)
N u m b e r of calli screened ( x 105)
Number of resistant calli isolated
Number of calli screened ( × 105)
0.0 0.1 0.3
0.58 1.20 0.40
6 70 32
1.5 2.0 --
Number of resistant calli isolated 1 --
The osmoticum in the medium is gradually lowered every 7 to 10 days by diluting the media in the following manner. First dilution, an equal volume of supplemented K3 media with 0.3 M glucose is added; second dilution, an equal volume of supplemented K3 media with 0.2 M glucose is added. The resultant microcolonies are plated into supplemented MS salts (Table I, components 1-19) containing 0.2 M glucose, I g/liter lincomycin, 1 mg/liter 6-benzylaminopurine, and 0.1 mg/liter a-naphthaleneacetic acid solidified with 0.7% agar. The plates are maintained at 28°, I000 lux. Resistant green colonies are isolated within 1 to 2 months and retested on the same media but with 0.1 M glucose (2% w/v). In the case of selection for herbicide resistance, a third dilution of the protoplasts in supplemented K3 media with 0.1 M glucose is carried out. The microcolonies are diluted into MS salts as above but with 0.015 M glucose (0.3% w/v) and 10-4 M terbutryne (instead of lincomycin). Resistant colonies are screened and retested in the same medium but with 0.015 M glucose. Calli which exhibit resistance through two subcultures on restrictive media are transferred for shoot induction to the same media (0.05 M sucrose) without antibiotic or herbicide, and the a-naphthaleneacetic acid is omitted. Plants are rooted on P media (Table I) and transferred to the greenhouse for genetic analysis. Results. Table IV summarizes the data for two experiments involving selection of resistance to the antibiotic lincomycin 7,~4,~6and the herbicide terbutryne.~5 Approximately 75% of the lincomycin-resistant calli regenerated normal looking plants. When plant leaves were retested on supplemented MS media containing antibiotics, 90% were truly lincomycin re16 A. Csrpl6, F. Nagy, and P. Maliga, MoL Gen. Genet. 198, 7 (1984).
[43]
ISOLATION OF PLASTID MUTATIONS IN
Nicotiana
621
FIG. 3. Lincomycin resistance test of a putative lincomycin-resistant plant regenerated from mutagenized protoplasts. Leaf sections were tested on callus forming media, as described in the text, containing 1000 rag/liter lincomycin. On this media the sensitive leaves (Np) form white calli, while the resistant leaf sections (LR415) form green calli.
sistant. On this media, resistant plants formed green calli while sensitive control plants formed white caUi (Fig. 3). All the plants which resulted from the 0.3 mM N E U treatment exhibited functional male sterility. A certain increase in the amount of mutant resistant calli isolated was seen using 0.1-0.3 mM NEU. It should be noted that in selection for nuclear mutations a 100-fold increase in isolation frequency was reported with 0.3 mM N E U when using haploid N. plumbaginifolia protoplasts. 17 In nearly all cases, seed progeny of resistant mutants were homogeneously mutant, probably as a result of the extended selection period at the undifferentiated callus stage. Hence, complete sorting out of the mutant chloroplasts was obtained in subculturing the green areas of resistant calli. In each instance the mutant phenotype exhibited maternal inheritance. Crucial to the successful use of the herbicide selection procedure was the reduction of the glucose concentration of the selective media to 0.015 M. At higher glucose concentration (0. I M), sensitive calli still maintained 17 L. Marton, T. M. Dung, R. R. Mendel, and P. Maliga, Mol. Gen. Genet. 59, 191 (1982).
622
CELL CULTURE AND TRANSFORMATION
[43]
TABLE V EFFECTIVE SCREENING CONCENTRATIONS OF HERBICIDES IN N. plumbaginifolia CELL CULTURES°
Herbicide
Class
Effective screening concentration
T e r b u t r y n e atrazine Metribuzin Metobromuron Bromacil Bromoxynil
s-Triazine as-Triazine Urea type Uracil type Nitrile type
10 _4 10 -4 10 -4 10 _5 2.0 × l0 -6
a Media for selection was s u p p l e m e n t e d K3 with 0.015 M glucose.
their green color. Raising the herbicide concentration to higher levels in the presence of 0.1 M glucose only caused secondary inhibitory effects to become apparent. The reduced glucose media has been found to be effective in providing selective screening for additional herbicides shown in Table V. The terbutryne-resistant isolates showed maternal inheritance, were resistant to atrazine (10 -4 M), and exhibited photochemical properties similar to naturally occurring s-triazine-resistant plants. ~8,19
Concluding Remarks The direct isolation of chloroplast mutants through seed mutagenesis is applicable when the following conditions exist. (1) The plantlets respond to NMU treatment with a high percentage of variegation in the first true leaves. (2) The selection agent used is plastid specific and can be transported through the plants' vascular system. The use of mutagenized seedlings avoids the added variance caused by prolonged cell culture such as deformed plants or induced male sterility. The high frequency of green islands appearing in cotyledonary tissue (Table III) is surprising. Although mutations appearing in this tissue have no genetic continuity in the cells of the apical meristem, plants regenerated from them yield mutant seed. 8 Thus, the number of cells composing the cotyledon and the rate of plastid sorting-out in those cells potentiate the mutagen effect. This material shows great potential for the isolation of low frequency mutations. The protoplasts method for mutant isolation is less dependent upon J8 C. J. A r n t z e n , K. Pfister, and K. E. Steinback, in "Herbicide Resistance in P l a n t s " (H. M. L e B a r o n a n d J. Gressel, eds.), p. 185. Wiley, N e w York, 1982. ~9 S. D e m e t e r , I. V a s s , E. Hideg, and A. Sallai, Biochim. Biophys. Acta (in press).
[43]
I S O L A T I O N OF P L A S T I D M U T A T I O N S IN
Nicotiana
623
the application of chemical mutagens. As Table IV shows, the use of large scale selection at the microcolony level enables successful screening even without NMU. Additional mutations such as herbicide resistance have been recovered using this technique. In general, the resultant M~ plants regenerated from resistant calli are more homogeneous for the mutant trait.