Primisulfuron herbicide-resistant tobacco plants: mutant selection in vitro by adventitious shoot formation from culturéd leaf discs

Plant Science, 79 ( 1991 ) 77-85 Elsevier Scientific Publishers Ireland Ltd.

77

Primisulfuron herbicide-resistant tobacco plants: mutant selection in vitro by adventitious shoot formation from cultur6d leaf discs C h r i s t i a n T. H a r m s , J o s e p h J. D i M a i o , S u s a n M. J a y n e , L a u r a A. M i d d l e s t e a d t , D a v i d V. N e g r o t t o , H o p e T h o m p s o n - T a y l o r a n d Alice L. M o n t o y a # C1BA-GE1G Y Corporation, Agricultural Biotechnology Research. P.O. Box 12257, Research Triangle Park. N C 27709 ( U. S. A. ) (Received April 16th, 1991; revision received June 24th, 1991 ; accepted June 27th, 1991 )

A simple procedure has been developed for the rapid and direct selection of herbicide-resistant mutant plants. The procedure uses adventitious shoot formation from suitable explants, such as leaf discs, on a shoot-inducing culture medium containing a toxic herbicide concentration. Resistant green shoots were thus isolated from tobacco (Nicotiana tabacum L.) leaf explants cultured on medium containing 100 ~g 1-~ primisulfuron, a new sulfonylurea herbicide. Resistant shoots were recovered from both haploid and diploid explants after UV mutagenesis, as well as without mutagenic treatment. Three mutant plants of separate origin were further analyzed biochemically and genetically. Their acetohydroxyacid synthase (AHAS) enzyme activity was less inhibited by sulfonylurea herbicides than that of unselected, sensitive wild type plants. The extent of inhibition of the A H A S enzyme a m o n g the three mutants was different for different sulfonylurea and imidazolinone herbicides suggesting different sites were affected by each mutation. Herbicide tolerance was scored for germinating seedling populations and was found to be inherited as a single dominant nuclear gene. Adventitious shoot formation from cultured leaf discs was used to determine the cross tolerance of mutant plants to various herbicidal A H A S inhibitors. The usefulness of this rapid and direct scheme for mutant selection based on adventitious shoot formation or embryogenesis is discussed. Key words: in vitro selection; mutant; herbicide resistance; sulfonylurea; acetohydroxyacid synthase; adventitious bud regeneration

Introduction

The value of plant mutants for basic scientific and applied studies has long been recognized [1]. In vitro cell selection has become a powerful tool to generate novel mutants (variants) in an increasing number of plant species [2]. The selection of herbicide-resistant mutants has been particularly successful, due in part to the simplicity of phenotype selection and in part due to the potential for application as genetic markers and as valuable new traits for crops. The case of sulfonylurea herbicide resistance has been exceedingly well studied genetically, biochemically Correspondence to." C.T. Harms, C I B A - G E I G Y Corporation, Agricultural Biotechnology Research, P.O. Box 12257, Research Triangle Park, NC 27709, U.S.A. 1"Deceased.

and on a molecular level [3-8]. Cell cultures and plants resistant to sulfonylurea and imidazolinone herbicides have been obtained in several plant species using a variety of in vitro selection schemes [3,7-16]. Seed mutagenesis was used to produce sulfonylurea herbicide-resistant Arabidopsis [17] and soybean [18]. While it is obvious from these examples that resistant mutants can be obtained by various methods, it is important to consider the advantages and specific requirements of each selection strategy before chosing one. Dix [2] provides a useful discussion of selection systems and strategies as well as their inherent attributes. Radin and Carlson [19] have obtained tobacco plants showing recessively inherited tolerance to the herbicides phenmedipham and bentazon by inducing shoot formation in vitro from explanted green sectors which they had identified in situ on bleached leaves following radiation mutagenesis

0168-9452/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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and treatment of the plants with these herbicides. Using the same approach Fluhr et al. [20] have obtained whole plant mutants which exhibited chloroplast-encoded resistance to streptomycin, spectinomycin, lincomycin and chloramphenicol. McCabe et al. [21] describe the selection of cytoplasmic mutants in various Solanaceae species by culturing leaf explants for shoot formation in the presence of streptomycin. These authors have speculated that mutant selection using organogenic explants may not be restricted to chloroplast-encoded traits such as streptomycin resistance. However, selection for nuclear-encoded mutant traits via organogenesis or embryogenesis directly from cultured explants has not been reported to date. Mutagenesis of somatic tissues, followed by in vitro adventitious shoot formation, has been used with ornamental and vegetatively propagated plants in which seed mutagenesis is not an option [22] but the mutant phenotypes were evaluated after plant regeneration rather than selected in vitro. In this paper we describe the selection of plant mutants of tobacco through adventitious shoot formation directly from cultured leaf explants in the presence of primisulfuron, a new sulfonylurea herbicide. Since it obviates the need to establish regenerable cell cultures first, this method of mutant selection directly from primary tissue explants cultured in the presence of a selective agent will be widely applicable in plant species which produce an organogenic or embryogenic response from primary explants such as leaf, hypocotyl, root or stem sections, or anthers.

Chemicals Primisulfuron ((2-[3-(4,6-bis(difluoro-methoxy)pyrimidin-2-yl)-ureidosulfonyl]-benzoic acid methylester) is a new sulfonylurea herbicide for weed control in corn [25]. Cinosulfuron [26] and triasulfuron [27] are new sulfonylurea herbicides for weed control in rice and wheat, respectively. Chlorsulfuron and sulfometuron methyl are sulfonylurea herbicides used in small grain cereals and for industrial weed control, respectively [9]. Imazaquin is an imidazolinone herbicide used in soybean [28]. The compounds were kindly provided by Dr. W. F6ry, C I B A - G E I G Y A G (Basel, Switzerland). The formulae of the sulfonylureas used in this study are shown in Fig. 1. Herbicide stock solutions were made up in a small volume of acetone, filter-sterilized and added to autoclaved culture medium after cooling ( < 50°C). Selection Leaf discs (5-7 mm) were punched from leaves of tobacco shoot cultures and placed upside down on shoot induction medium consisting of MS [24] salts supplemented with 1 mg 1-1 6-benzyladenine, 0.1 mg 1-I a-naphthaleneacetic acid, 100 mg 1 i myo-inositol, 1 mg 1-l nicotinic acid, 1 mg 1-I pyridoxine, 10 mg 1-] thiamine HC1, 3% (w/v) sucrose and 0.8% (w/v) Difco Bacto agar (pH adjusted to 5.8 prior to autoclaving). Ten to 16 discs

R

Y SO 2 - N H - C O - N H - - ~ _ _

X

Materials and Methods

Plant material Shoot cultures of tobacco cv. Coker 176 and Xanthi were used as explant sources. Haploid plants were produced from cultured anthers of cv. Coker 176 as described [23]. Rooted shoot cultures were kept aseptically on basal Murashige and Skoog (MS) medium [24] without growth regulators in a lighted growth room (16 h per day photoperiod, 30-80 ~E m -2 s-I, from Growlux and cool white fluorescent lamps) at 25°C.

Z

primisulfuron triasulfuron cinosulfuron

R

X

Y

COOCH 3

CH

OCHF 2

OCH2CH2C/ OCHzCH2OCH 3

Z OCHF 2

N

OCH 3

CH 3

N

OCH 3

OCH 3

OCH 3

CH 3

CH 3

CH 3

chlorsulfuron

CI

N

sulfometuron methyl

COOCH 3

CH

Fig. I. Structural formulae of sulfonylurea herbicides used in this study.

79 were plated per 10-cm petri dish. For selection, the medium was further supplemented with 100 #g 1primisulfuron. In some experiments, leaf discs were exposed for various periods to irradiation from a 254 nm UV-lamp (Mineralight, Model R-52G, UVP Inc., San Gabriel, CA), delivering 1.25 m W cm -2 at a distance of 15 cm. Resistant shoots emerging from plated leaf discs on selection medium were excised and transferred to MS salts medium supplemented with 2% sucrose, 0.8% agar and 100 /xg 1-l primisulfuron. Resistant shoots were then kept as rooted shoot cultures on this medium or transplanted to the greenhouse for genetic analysis. For tolerance assays using leaf discs of mutant plants, the shoot induction medium was supplemented with various amounts of sulfonylurea or imidazolinone herbicides. Leaf discs were scored for green color and shoot or callus formation on herbicide containing medium after 3 - 4 weeks in comparison to leaf explants from wild type plants.

Inheritance tests Well-rooted resistant shoots from selection were transplanted into potting mix, acclimated and grown under greenhouse conditions. Flowering plants were selfed and (reciprocally) crossed with wild type plants of the same cultivar. Progeny seed were sterilized in 20°/,, (v/v) commercial Chlorox bleach (0.5% NaOC1) for 10 min followed by three rinses with sterile water. One hundred to two hundred seeds were plated, 50 per 10-cm petri dish, onto MS medium without sucrose or growth regulators but supplemented with various concentrations of sulfonylurea or imidazolinone herbicides. After 2 - 3 weeks, seedlings were scored resistant if they were rooted, green, and had developed primary leaves. Sensitive seedlings bleached completely, lacked roots and never formed primary leaves.

Enzyme assays Acetohydroxyacid synthase (AHAS) activity was extracted from leaves of shoot cultures or greenhouse-grown plants, purified and assayed essentially as described [29].

Results

Sensitivity of leaf explants to primisulfuron Prior to selection, the organogenic response of tobacco leaf discs on medium with increasing primisulfuron concentrations was determined. At 100/~g 1-1, all discs bleached. At 30/zg l -I, no callus or shoots were formed, though discs kept a light greenish appearance and became somewhat expanded. Occasional minimal callus or shoot bud formation was observed at 10 /~g 1-I primisulfuron. In the absence of primisulfuron all discs formed adventitious shoots in abundance. Based on these observations, 100 /zg 1-I primisulfuron was chosen for selection.

Selection of mutant shoots from cultured leaf discs From 1305 haploid leaf discs of cv. Coker 176, 23 (1.8%) produced green shoots on selection medium with 100/xg 1-1 primisulfuron (Fig. 2). These arose from explants that had been irradiated with UV for 2 - 3 min immediately after they were placed on the medium. In later experiments, four out of 1060 haploid leaf discs (0.38%) produced shoots without UV irradiation. One of 90 leaf discs (1.1%) of diploid tobacco (cv. Xanthi) produced resistant shoots. In all cases, the shoots emerged as dark-green buds on the edge of bleached leaf explants and grew without inhibition by the herbicide. Shoots abolat 2 cm in length were separated from the explant and transferred to basal medium containing 100/~g 1-1 primisulfuron to establish rooted shoot cultures. All green shoots obtained from selection developed normal root systems in the presence of primisulfuron. These features strongly indicated that these plantlets were herbicide resistant.

Genetic analysis In order to obtain genetic evidence for the mutant nature of the selected adventitious shoots, four plantlets were transplanted to potting soil and grown to maturity in the greenhouse. All of them had originated from haploid explants. Two of the plants (designated SU-I and SU-II) had a normal phenotype and set seed upon selfing and cross pollination with wild type plants, indicating that

8(I

Fig. 2. F o r m a t i o n o f green resistant shoots directly from leaf explants in the presence of 100 g g I t primisulfuron. Note bleaching of leaf explants.

they had spontaneously diploidized. The two other plants (SU-III and SU-IV) were small, sterile and presumed haploid. By counting the chloroplast number per guard cell (5-8 for haploid vs. 10-15 for diploid plants; 25 cells counted per plant) the haploid status of these plants has been confirmed. Leaf mid-rib sections of the haploid plants were then cultured on organogenic medium (no herbicide or colchicine added) in an attempt to produce diploid shoots. Diploid plants of SU-III were thus established and grown to maturity for genetic analysis. All seedlings from self-pollinated plants of SU-I, SU-II and SU-III were resistant to 100 #g 1-I primisulfuron (Table I), while control seedlings (wild type) bleached and lacked both roots and primary leaves. The uniform resistant phenotype of the mutants after self pollination is indicative of homozygosity for the resistance trait, i.e. a consequence of selection at the haploid level followed by dipioidization. Fl seedlings from crosses of the regenerated SU-I, SU-II and SU-III plants with wild type plants were uniformly resistant at 100 ~g i -j primisulfuron, indicating dominance of the trait and confirming the homozygous constitution of the SU-R regenerants. Seedlings representing

each of these Fis were grown to maturity. Following self pollination, seedlings of the F2 generation were found, as expected, to segregate resistant and sensitive individuals in a 3:1 ratio when plated on 100 t~g 1-1 primisulfuron-containing medium (Table I). Progeny seed from crossing heterozygous SU-R plants with wild type plants

Table 1. Segregation analysis of seed progenies derived from self and back crosses of m u t a n t s SU-I, SU-II and S U - I I | . Line

N u m b e r of seedlings ~l Resistant

Sensitive

wt b wt x SU-i R R

0 198

153 0

wt x SU-I R r wt x SU-II R R wt x S U - l l R r SU-III R R SU*III R R × wt wt x S U - I I | R R wt x S U - l l l R r SU-III R r selfed

98 194 107 199 189 199 101 136

96 0 88 0 0 0 97 51

X2

0.02 (1:1) 1.84 (1:1)

0.08 (1:1) 0.21 (3:1)

aSeeds plated on m e d i u m c o n t a i n i n g 100 # g 1 I primisulfuron. hWild-type.

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segregated 1:1, as expected. The results of analyses (Table I) clearly indicate primisulfuron-resistance in each of the mutants was inherited as a single nuclear which was dominantly expressed.

SU-I and SU-III showed the same level of tolerance as their homozygous counterparts. However, heterozygous seedlings of SU-II, while clearly tolerant, showed retarded development even at 100 t~g 1-I primisulfuron, indicating incomplete expressivity of the resistance trait in this mutant (Fig. 3). The level of tolerance to various herbicidal AHAS inhibitors of both the sulfonylurea (Fig. 1) and imidazolinone type (imazaquin) were also tested in a leaf disc assay. Leaf discs from in vitro grown shoot cultures or from greenhouse-grown plants were plated on shoot regeneration medium containing a range of herbicide concentrations. All mutant lines (shoot cultures of SU-I to SU-IV) showed prolific adventitious shoot formation from leaf discs on all sulfonylureas and imidazolinones

these that three gene

Level o f resistance and cross resistance

Populations of both homozygous and heterozygous seeds of mutants SU-I, SU-II and SU-III were plated on medium containing a range of primisulfuron concentrations to determine their level of tolerance. Homozygous seedlings of all three mutants, grown at 100, 1000 and 10 000 #g 1-1, were indistinguishable from seedlings grown in the absence of the herbicide and showed a slight retardation of development at 30 000 #g 1-I primisulfuron. Heterozygous seedlings of mutants [ n g • m1-1 ] 0

100

1000

Fig. 3. Herbicide tolerance of seedling progenies: homozygous (upper row) and heterozygous seedlings (middle row) of mutant SU-II vs. primisulfuron. The bottom row shows control seedlings (cv. Coker 176); note that the germination of control seeds was not inhibited but seedlings do not grow in the presence of primisulfuron.

~2 (a)

chlorsulfuron

0

imazaquin

100

1000

sulfometuron methyl

100

[ n g • m1-1 ]

(b)

cinosulfuron

0

triasulfuron

100

100

primisulfuron

100

[ng • m1-1 ] Fig. 4. Adventitious shoot formation from leaf explants as an assay to evaluate the hcrbicide tolerance (cross tolerance) of selected mutants. (a) Mutant SU-II (upper row) and control (bottom) vs. chlorsulfuron, imazaquin and sulfometuron methyl. (b) Mutant SUIV (upper row) and control (bottom) vs. cinosulfuron, triasulfuron and primisulfuron.

~3 Table I1.

Inhibition of acetohydroxyacid synthase (AHAS) activity from wild type and mutant plants by various herbicidal AHAS inhibitors. Figures represent nanomolar concentrations resulting in 50% enzyme inhibition. Line

wt b SU-I SU-II SU-III

Specific activity a

15 15 7 19

nM concentrations giving 50% AHAS inhibition (IC50) Chlorsulfuron

Sulfomet. methyl

Triasulfuron

Primisulfuron

Cinosulfuron

lmazaquin

6 40 27 60

7 22 85 130

15 120 90 35

9 15 30 25

35 90 80 110

155 140 250 470

aSpecific activity: pKat (pg acetoin h -I mg -I protein). bwild-type.

tested at 100 and 1000 ~tg I -I while control discs bleached and formed neither callus nor shoots (Fig. 4 a,b). These results clearly demonstrate the high-level herbicide tolerance of the selected mutants.

Inhibition of A HA S enzyme activity by sulfonylurea and imidazolinone herbicides AHAS enzyme inhibition profiles were determined in leaf extracts from the mutant lines in an attempt to characterize the resistance trait at a biochemical level. Table II shows the concentration of various herbicidal AHAS inhibitors which reduced the AHAS activity of our mutants by 50% (ICs0). These data show that all of the mutants tested have AHAS enzyme activity which is less susceptible to inhibition by herbicidal AHAS inhibitors than the wild type. This strongly suggests that the mutation leading to the herbicide resistant phenotype occurred in one of the AHAS genes. However, the level of resistance varied among mutants and was different for the different inhibitors (Table II). The fact that each of the mutants has a characteristic inhibition profile suggests that they may represent mutations at different sites in the AHAS gene. Two mutants have a specific activity equal to the wild type while one (SU-II) appears to have a reduced specific activity. Discussion Our results indicate that mutant plants can be selected by placing tissue explants directly onto a

selective medium which stimulates shoot formation from resistant cells. Since multicellular explants were used it is difficult to calculate the frequency of mutant selection per cell as is possible using other types of selection, such as plated suspension cultures [12]. For practical purposes, the frequency of mutant plants is of secondary importance as long as selection yields mutants with a reasonable experimental effort. In this regard, our method, due to its inherent simplicity, compares well with other more elaborate in vitro selection procedures. The origin of adventitious shoots forming on leaf explants may be unicellular [30] or multicellular [31]. It is therefore possible, at least theoretically, that chimeric shoots are formed with our method. In reality, though, we did not observe chimeras among the plants we obtained. It appears unlikely that herbicide-sensitive cells survive in the explant, the small amount of callus formed or the regenerating shoots, considering the stringency of the selection applied. We have obtained resistant shoots with our method from both haploid and diploid explants. Given the dominant nature of the mutations recovered, both kinds of explants can indeed be expected to yield resistant mutants. Under other selective conditions, there may be a definite advantage of using haploid explants in order to allow the recovery of recessive mutants. It appears that the natural frequency of mutant cells within the explants used was sufficient to allow selection of herbicide-resistant shoots in our system. The UV

84

treatment applied in some experiments did not appear to be essential to generate mutant cells for selection. This does not imply, however, that chemical or radiation-induced mutagenesis, as practiced for instance by Roest and Bokelmann [22], could not be used with our system to obtain mutants at a higher frequency. It is worthwhile to consider that mutagen treatments can lead to additional undesirable changes in the selected mutant plants which may reduce their usefulness or agronomic performance. Our method, by stimulating shoot formation directly from the explant, i.e., without an intervening callus phase, should also minimize the occurrence of tissue culture-induced variability (somaclonal variation) in addition to the resistance trait selected for. Mutant cells and plants resistant to sulfonylurea and/or imidazolinone herbicides have been produced by a variety of methods in a range of different plant species [3,7-18,35]. These two herbicide classes share a common target, acetohydroxyacid synthase (AHAS, ALS; E.C. 4.1.3.18) and plant species naturally insensitive to sulfonylureas or imidazolinones generally have a capacity for specific detoxification reactions [29,32-34]. By contrast, the mutants selected for resistance have been shown to possess an altered form of AHAS which is less inhibited by the herbicides. Recently, Armour et al. [35] have reported a case of A H A S gene amplification in sulfonylurea-resistant tobacco cells as another resistance mechanism. The mutants described here show altered A H A S inhibition profiles, suggesting mutation within the A H A S locus. Although genetic tests for allelism have not been completed, it is likely that our mutants represent different allelic forms of the AHAS gene. The different A H A S profiles exhibited by our mutants also suggest different mutational events. It is known [36] that the AHAS coding sequence can be altered by mutation in at least 10 different sites without loss of function but resulting in different enzyme inhibition profiles towards different herbicidal A H A S inhibitors. It remains to be seen from sequencing the SuRA and SuRB AHAS genes exactly which sites are mutated in our mutants. In principle, our method is applicable to a wide range of plant species and different kinds of explants and is not limited to a particular kind of

selective agent such as a herbicide. Unexpectedly, we were able to adapt the direct selection method for maize whereby mutant plants were produced in the presence of a selective agent via embryogenesis directly from the scutellum of explanted immature embryos (Harms et al., in preparation). Selection of mutants from cultured leaf base explants of Dactylis is also feasible, thus capitalizing on the capacity for direct somatic embryogenesis in this system. Another compelling feature of our method is that it obviates the need to establish tissue or cell cultures of the kind usually used for selection, i.e. callus or suspension cultures from which plants can be regenerated. By keeping the duration of in vitro culture and undifferentiated cell proliferation to a minimum, our method should reduce the occurrence of secondary mutations in other than the selected trait and allow the regeneration of true-to-type mutant progeny. Thus our procedure not only minimizes a source of undesirable variability ('somaclonal variation') but it also exposes cells to selection which are differentiated and whose physiology much more closely resembles that of 'normal' (as opposed to 'in vitro cultured') plant cells.

Acknowledgement We are grateful to George Lankfort and Joe Sloane for assistance with the crossing and caring of plants in the greenhouse and to Dr. E. Wernsman (Dept. Genetics, North Carolina State University, Raleigh) for suggesting mid-rib culture for diploidization.

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