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r by De Novo Mutagenesis
lajsb
a Institute of Microbiology, Academy of Sciences of the Czech Republic, Víde
ská l083, 142 20 Prague 4, Czech Republic
b Univ. of South Bohemia, Studentská 13, 370 05 
eské Bud
jovice, Czech Republic
* Corresponding author (novak{at}biomed.cas.cz)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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r. Ethyl methanesulfonate (EMS) treatment of large seeds was optimized in a closed system to scale-up the process and to improve safety. The 18-h exposure of non-presoaked seeds to 0.15% (w/w) EMS solution resulted in 30% field emergence in the M1 generation and in 1.04% of chlorophyll mutants and 3.64% of mutants classified as symbiotic in the M2. However, only 48.4% of the growth chamber-detected and 21.6% of the field-detected presumable symbiotic mutants were found to be stable genetic deviations in the M3 to M5. Twenty-two preliminary characterized stable mutants comprised one nonnodulating, one with reduced nodulation, one with markedly delayed nodulation, and 19 lines with affected nodule development and function. Further evaluation under controlled conditions and in field trials identified the nonfixing line Budfix2 as a suitable reference line for field estimates of symbiotic nitrogen fixation. This line was shown to be physiologically equivalent to the original cultivar when grown under asymbiotic conditions, i.e., when the symbiotic fault was compensated for by exogenous mineral nitrogen. The availability of Budfix2 allows for direct application of the difference method in the particular pea cultivar Bohatr, as well as in further varieties after trait introgression. The additional symbiotic mutants generated in the course of the search for a reference line are expected to enhance the variability available for the genetic dissection of rhizobial nodulation.
Abbreviations: DM, dry mass • EMS, ethyl methanesulfonate • FM, fresh mass • SNA, specific nitrogenase activity • TNA, total nitrogenase activity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Legume symbiotic mutants are a valid tool for the genetic dissection of the process of symbiosis development (e.g., Guinel and Geil, 2002; Stracke et al., 2002). In spite of the growing number of induced symbiotic mutants in the model legumes (e.g., Szczyglowski et al., 1998), the number of symbiotic mutants accumulated in pea represents a sufficient collection for a parallel line of work in this economically important host (Jacobsen, 1984; Jacobsen and Feenstra, 1984; Kneen et al., 1994; Duc and Messager, 1989; Engvild, 1987; Borisov et al., 1993). Extending the number of pea symbiotic mutants should produce additional information about symbiosis development, albeit depending on the degree of their further characterization.
Asymbiotic lines of legumes are also a convenient tool for the field determination of the effect of symbiotic nitrogen fixation. When an asymbiotic reference line is grown side by side with an isogenic cultivar, the difference in their yield characteristics indicates the contribution of symbiotic nitrogen fixation (Sagan et al., 1993; Biedermannová et al., 2002). In the presence of soil populations of nodule bacteria, the traditional arrangement of the difference method, based on the comparison of inoculated and uninoculated plants, is not applicable. The alternative approaches using nitrogenase activity determination or N isotope dilution are indirect and technically demanding (Danso et al., 1993; Minchin et al., 1986; Biedermannová et al., 2002). On the other hand, the asymbiotic line-based difference method automatically integrates any deviations in soil properties, weather, and agronomic measures throughout the growing season.
To date, only a few mutant pea lines that are free of pleiotropic effects of the symbiotic mutation have been shown to be suitable as reference lines (Sagan et al., 1993; Biedermannová et al., 2002). Another requirement imposed on a reference line is that it be sufficiently isogenic with the cultivar of interest so as to avoid interference from other traits (Hartwig, 1994).
Therefore, this work focused on obtaining an isogenic reference line for a modern pea variety. The widely cultivated pea cultivar Bohatr was chosen as the parental line for mutagenesis and thus the source of the genetic background to facilitate the use of the new reference line. In parallel, we pursued the aim of extending the number of symbiotic mutants of pea available for further studies.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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r, kindly supplied by the seed producer Oseva Bor
ov (Borov, Czech Republic), were mutagenized in 4 L of 0.15% (w/w) solution of ethyl methanesulfonate (EMS) in a 0.006 M phosphate buffer (K2HPO4 0.75 g/L, KH2PO4 0.25 g/L) for 18 h at 7°C. The supply of oxygen and the continuous mixing of the EMS solution were provided by bubbling air through the seed layer. The used EMS was hydrolyzed with an excessive volume of concentrated (32%) ammonia solution (Engvild, 1987). The seeds were washed with tap water at a temperature of 7°C for 20 h to increase seed viability (Konzak et al., 1970). The seeds were stored for up to 48 h and transported to the sowing location in a plastic bag kept on ice. For safety reasons (National Institutes of Health, 1982), the seeds were treated in a closed chemical apparatus with a negative differential pressure to minimize the leak of EMS vapors. The discharged air was cleaned by bubbling through ammonia solution and water. The M1 seeds were sown on a location with favorable soil conditions (Orthic Luvisol) at a lower density of 50 seeds m–2 to facilitate the emergence and growth of mutagen-damaged plants. In parallel, the laboratory germinability of the treated seeds was determined by standard procedures.
Screening M2 Plants
One pod was harvested from each M1 plant to reduce the detection of the same mutation. Seed families from individual pods were subjected to screening for symbiotically deficient phenotype.
In the growth chamber, expanded clay pellets (fraction 4–8 mm) were used as a substrate permitting easy root phenotype inspection and replanting. The extensively washed pellets were placed into polypropylene containers 36 cm long x 14 cm wide x 10 cm deep. The containers were half-filled with a nitrate-free nutrient solution according to krdleta et al. (1980), capped with aluminum foil, and autoclaved at 120°C for 30 min. After reaching room temperature, the containers were planted with M2 seed families in perpendicular rows 4 cm apart. Before sowing, the seeds were surface-sterilized with 2% (w/w) chloramine B for 20 min without rinsing. The seeds were germinated in the growth chamber Conviron S10H (Conviron, Winnipeg, Canada) at a temperature of 18/16°C under a 16/8 h day/night regime. One day after sowing, the seeds were inoculated with 40 mL of rhizobial suspension poured evenly onto the substrate surface. The inoculum was prepared by suspending a 4-d culture of a wild-type strain Rhizobium leguminosarum bv. viciae 248 (Josey et al., 1979), grown on a yeast extract-mannitol agar (Vincent, 1970) at 28°C, in 500 mL of sterile distilled water to obtain a final concentration of ca 2 x 106 cell mL–1. Five days after sowing, the aluminum foil cover was removed and the day temperature raised to 22°C, while the illumination of 500 µmol m–2 s–1 of photosynthetically active radiation was provided by equal numbers of 75-W bulbs (Tungsram, Budapest, Hungary) and 115-W cool-white fluorescent lamps (type F48T12/CW/VHO from Osram Sylvania, Danvers, MA). The nutrient solution was changed twice a week.
Two weeks after sowing, M2 plants differing in pigmentation from the wild type, presumably chlorophyll mutants, were removed. Four weeks after sowing at the 8-leaf stage (including two rudimentary leaves), the green plants of the wild type were removed, while the plants showing nitrogen deficiency symptoms (yellowing of the basal leaves and growth retardation) were inspected for the presence and appearance of symbiotic nodules. Plants of mutant symbiotic phenotype were recovered by replanting and changing the nitrate-free nutrient solution for a solution containing 10 mM NO–3 (krdleta et al., 1980). Phenotype normalization after nitrate addition was considered as evidence of the causal role of a symbiotic defect in the aberrant phenotype formation. The stability of the symbiotic defect was verified in M3 plants grown under the same conditions.
For capacity reasons, the M2 screening was performed in the field in parallel. A sandy soil location with a low nitrogen level in combination with an efficient rhizobial population as known from previous experiments (Biedermannová et al., 2002) was chosen to intensify the symptoms in asymbiotic plants. Therefore, the soil factors provided contrasting symbiotic/asymbiotic phenotypes.
Mutant Phenotype Characterization
In the M6 generation, 12 mutant lines with the most pronounced symbiotic phenotype changes were further characterized in a growth chamber under the conditions described above. Each line was grown both on the low and high nitrate level in four replicates, and the plants were evaluated 6 wk after sowing, at the stage of early flowering. The assumption about the defect in nitrogen fixation was verified by the determination of bacterial nitrogenase activity (E.C. 1.18.6.1) by the acetylene reduction technique as modified by krdleta et al. (1987). Subsequently, plant stem length, the number of leaves, shoot fresh mass, and root and nodule dry mass were recorded. The flowering index 0 through 3 was assigned to individual plants to quantify the stage of development: before buttonization, green buttons, white buttons, and blossom, respectively.
Field Test of Physiological Equivalency
New mutant lines were compared with the wild-type plants by their ability to grow and develop under field conditions with respect to their potential use in the difference method. Fifty seeds of the M6 generation of the new mutant lines and of the parental cv. Bohatr, obtained from field-grown plants, were sown on 2.0-m2 plots in an agricultural location with low soil nitrogen (evtín, Czech Republic). Half of the plots were saturated with mineral nitrogen provided weekly after sowing and every even week after blossom in the form of 10 L of 4 g L–1 NH4NO3 solution, this dose corresponding to 7 g N m–2 (70 kg ha–1). Control plots were watered with the same volume of water. Other growing measures were as described previously (Biedermannová et al., 2002). All variants were established in duplicate plots. The plants were evaluated individually for dry seed yield at the end of the growing season.
Statistical Treatment
Ninety-five percent confidence intervals for mean values as based on the least significant difference (LSD) were found by means of multiple range analysis in Statgraphics version 2.6 program. The sixth root of the primary data was used as the input for calculation to stabilize the variance (Armitage, 1971).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The closed system possesses several advantages over the traditional way of treating large-seeded legumes in open shallow trays for seed imbibition and mutagenization (Kamra and Brunner, 1970; Speckmann, 1964). In the traditional way, the large volume of the mutagen solution and the resulting EMS vapors are a health hazard (National Institutes of Health, 1982). The additional protective features in the closed system were the negative differential pressure inside the apparatus, inactivating EMS from the discharge air, and manipulating of the mutagen solution and washing the seed within the closed system.
Results of M2 Screening
A sufficient level of mutagenization was indicated by approximately 10% of chlorophyll chimeras observed in the M1 after treatment with 0.15% EMS. Among 1816 M2 plantlets subjected to screening in the growth chamber, 1.04% of chlorophyll mutants were observed, comprising 0.44% of chlorina type and of 0.60% xantha and lutea as denoted according to Lamprecht (1960) and Speckmann (1964). In addition, 2.75% of morphological mutants observed in the M2 were as follows: 2.36% dwarf, 0.22 compacta, and 0.17 of other types (dense lateral roots, delayed lateral root initiation, bushy shoot and root). Some plants (0.22%) demonstrated necrotic spots on the leaves resembling vario-maculata and vario-micro-maculata types according to Lamprecht (1960). In 4000 M2 plants screened under field conditions, the frequency of phenotypic deviations corresponded to the growth chamber data except for the absence of dwarf mutants, which probably did not survive in the open ground.
The altered symbiotic phenotype was expressed as the absence of nodules, altered nodule pattern, or as white, green, or brown coloration of the nodules which differed from the wild-type pink. In the growth chamber, 3.64% of the M2 plants appeared to be symbiotically affected, comprising 1.71% classified as Nod–, 1.65% as Fix–, 0.11% as delayed nodulation, and 0.17% as copious nodulation. Detection in the field was less sensitive, yielding 1.50% of putative symbiotic mutants.
After successful recovery with nitrate, 70 lines were derived from symbiotically affected M2 plants. The recovery rate was about 50% and equal in the growth chamber and in the field. However, the assumed presence of a symbiotic mutation was confirmed only in 22 cases of M3 offspring. The diagnosis of mutant plants in M2 was more reliable when performed in the growth chamber (48.4% of correct classifications) than in the field (21.6%). On the other hand, the lower efficiency of field screening can be compensated for by a larger scale.
Characteristics of the Obtained Mutant Lines
Twelve of the stable mutant lines were further characterized in M6 by a set of symbiotic and developmental traits under controlled conditions (Table 1). The lines were denoted as Budnod (nodule initiation or nodule pattern affected) or Budfix (nodule function and appearance affected) with a serial number attached. In most of the 11 Budfix lines, a drop in total nitrogenase activity (TNA) was observed (Table 1). In the lines Budfix6 through Budfix9, the difference from the wild type became significant when the primary data were used instead of the sixth root in the statistics. Lines Budfix10 and Budfix11, albeit indistinguishable by TNA, steadily demonstrated signs of preliminary nodule degradation. It is assumed that the nodule deterioration had not progressed enough by the time of the evaluation to affect TNA in these lines.
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Additional mutants were recovered in the screen but have not been characterized in detail. They comprise eight Fix– mutants with residual TNA, one line with reduced, and one line with delayed nodulation.
Identification of a Reference Line for the Difference Technique
Four lines from the tested set (Budfix1–Budfix4) had practically no residual nitrogenase activity but still formed a significant mass of nodules comparable to the wild-type plants when grown on low N under controlled conditions (Table 1). Therefore, these lines were tested under field conditions as candidates for a reference line applicable in the difference method. The results of the 2003 season are presented in Table 2. The mutant phenotypes proved to be stable in the open ground since nitrogenase activity was absent or remained negligible. Most importantly, yield of the mutant lines was restored by supplementing the soil with a growth-saturating amount of NH4NO3 (Table 2). No further differences in morphology or environmental stress sensitivity from the wild type were observed when compensation was made for symbiosis. Consistent results were obtained in the 2004 season (not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The choice of EMS as a mutagen was based on the comparison of chemical mutagens for symbiotic mutant induction in pea made by Engvild (1987). In that work, a 16-h treatment of dry seeds with 0.1% EMS provided the highest yield of symbiotic mutants per kilogram M1 at a favorable emergence rate of 30%. The EMS solution of 0.15% applied in our work demonstrated a comparable mutagenic efficiency, yielding 1.04% chlorophyll mutants in the M2 compared with 5.4%, obtained as a mean of published values for 0.1 and 0.2% EMS treatments (Engvild, 1987). Similarly, the obtained total frequency of chlorophyll and morphological mutants of 3.79% is comparable to the 8% value resulting from a 24-h treatment of the pea cv. Aureol with 0.13% solution of EMS (Speckmann, 1964). The observed ratio of chlorophyll to the morphological mutants (27.4%) corresponded well to the 24 and 34% published for similar treatment temperatures 6 and 12°C, respectively (Speckmann, 1964). Nevertheless, further optimization of treatment conditions is conceivable. An M2 mutation frequency was reported as high as 31.4% with an acceptable 50% M1 germinability for treatment with 0.33% EMS for 7.5 h at 23°C in the pea cv. Pauli (Heringa, 1964).
The observed improvement in M1 germination by seed cooling during the mutagen treatment corresponded to the increase in pea seed viability as reported by Speckmann (1964) for temperatures below 12°C. In spite of the drop in the yield of all mutant types in the M2 from 50% at 24°C to 8% at 6°C (Speckmann, 1964), cooling seems to be an indispensable factor in the submerged treatment.
The comparison of the achieved mutation frequency to the published data indicates that the level of mutagenization stayed at 15 to 20% of the maximum permitted by M1 lethality. The modest level of mutagenization allows one to assume a low incidence of multiple mutations in the symbiotic mutant lines. Therefore, there is hope that no backcrosses to the original cultivar need to be done before using the generated lines in the difference method.
Symbiotic Mutation Yield
Twenty-two additional pea mutants were generated in cv. Bohatr, 12 of them being characterized in detail. Surprisingly, the frequency of symbiotic mutations of 1.76% found in our work (after correction for a 50% lethality of M2 suspects) was substantially higher than the 0.15% reported by Engvild (1987). This difference might be ascribed to the more homogeneous conditions for M2 screening in the growth chamber, resulting in a higher sensitivity of detection.
The ratio between symbiotic and chlorophyll mutants provides an independent estimate of the number of symbiotic loci in pea. Assuming for simplification that the mutation rate is equal in all loci, the symbiosis/chloroplast-affecting loci ratio is 1.69. Even a stringent estimate based on the most distinct seven mutants (Budnod1, Budfix1–Budfix5) gives a ratio 0.185. We can conclude that as a minimum the number of pea symbiotic genes is of the same order as the number of genes controlling chloroplast development.
Phenotypic Relation of the New Lines to Other Symbiotic Mutants
To date, at least 40 symbiotic loci are known in pea as inferred from more than 200 symbiotic mutants (Brewin et al., 1993; Borisov et al., 1999; Kneen et al., 1994; Novák, 2003). Without completing allelism tests, the phenotype of newly obtained mutants was preliminary related to the mutant collection originating from cv. Finale (Engvild, 1987; Novák et al., 1993) by the three most informative traits: nodule color, single nodule dry mass, and specific nitrogenase activity. It was an advantage that a matching set of traits and identical growth conditions were used to characterize 27 lines of the Finale collection in the previous work (Novák et al., 1993).
While a white nodule color indicates an early developmental block preceding leghemoglobin synthesis, a greenish and brown coloration can be treated as the premature degradation of the nodules (Kneen et al., 1990; Provorov et al., 2002). Similarly, low single nodule mass indicates an early block, while low specific nitrogenase may signify an early block, a disturbance in nodule physiology, or tissue degradation.
It is obvious that the group of mutants Budfix1 through Budfix4, characterized by small white nodules and negligible nitrogenase activity, are blocked before leghemoglobin synthesis and nitrogenase activity onset. This group might correspond to the lines RisfixO and RisfixL that are affected in the locus sym32 (Novák et al., 1993; Borisov et al., 1999). Another early, low-activity mutant is Budfix5. This line shows signs of nodule degradation and might be homologous to the lines Risnod3 or RisfixB.
Budfix7 and Budfix8, which form nodules with a wild-type appearance but decreased nitrogenase activity, resemble mutants RisfixI and Risnod15, respectively. A similar type of nodules has also been reported for the pea mutant FN1 obtained in cv. Rondo (Postma et al., 1990).
Lines Budfix6, Budfix10, and Budfix11 form large nodules that degrade. From the previously characterized lines Risnod3 and RisfixB (Novák et al., 1993), this group differs in nodule size. Alternatively, these lines might correspond to RisfixM/RisfixT and RisfixQ affected in the sym26 and sym27 loci, respectively, that control nodule persistence (Borisov et al., 1999).
On the contrary, Budfix9 forms large white nodules with surprisingly high nitrogenase activity and has no counterpart among the previously published pea mutants.
It is noticeable that the mutants forming early blocked nodules (Budfix1-Budfix4, Budfix5, Budfix7) tended to form an increased number of nodules (Table 1), consistent with the behavior of early mutants from the Finale set (Novák et al., 1993). In the earliest blocked lines Budfix1-Budfix4, a nodule number exceeding 150% of the wild-type value was observed. However, no supernodulating and hypernodulating lines (Delves et al., 1987; Novák et al., 1997) were present. Similarly, the addition of nitrate did not reveal nitrate resistant (nts) nodulation as found in other pea mutant sets (Jacobsen and Feenstra, 1984; Sagan and Duc, 1996; Novák et al., 1997; Sidorova and Shumnyi, 1998).
Bohatr is, along with cvs. Sparkle (Kneen et al., 1994), Rondo (Jacobsen, 1984), Finale (Engvild, 1987), Frisson (Duc and Messager, 1989), Sprint-2 and SGE (Borisov et al., 1993), and Ramonsky-77 (Sidorova and Shumnyi, 1998), another pea cultivar in which a collection of symbiotic mutations has been generated. It is expected that the obtained mutants will bring additional information about rhizobial symbiosis after their genetic and structural characterization.
Line Budfix2 as a Reference Line for the Field Assessment of the Nitrogen Fixation Effect
An important feature of the new reference line Budfix2 is the fact that the mutation was obtained in the widely grown commercial variety Bohatr, thus enabling its immediate use with a modern cultivar. The contribution of symbiotic nitrogen fixation under particular conditions can be determined from the yield difference of cv. Bohatr and the line Budfix2. To date, pea reference lines confirmed by field tests have been reported only for cv. Frisson (Sagan et al., 1993) and for cv. Finale (Biedermannová et al., 2002). The availability of this option in cv. Bohatr should yield more data about the symbiotic performance of this cultivar in different locations and conditions, providing a necessary feedback for agronomic measures.
The Budfix2 mutation was characterized already in the background of cv. Bohatr; therefore, a changed phenotype is improbable after introgression of this mutation into modern pea varieties. Their relatedness has been documented (Ellis et al., 1998) and in breeding many of them, Bohatr was involved as a parent. In this way, a reference line for a particular pea cultivar of interest can be obtained. Similarly, a nonnodulating reference line carrying the allele rj1 instead of Rj1, and thus near-isogenic with soybean cv. Lee, has been developed by trait introgression (Hartwig, 1994).
We have shown that a mutation suitable as a reference line can be found among a comparatively limited set of new symbiotic mutants. Therefore, de novo mutagenesis is a realistic approach to obtain reference lines in pea. The rapid identification of a reference line is surprising in light of extensive pleiotropy and phenotypic instability ascribed to legume symbiotic mutants previously (Francisco and Harper, 1994; Jacobsen, 1984; Novák et al., 1994). More efficient approaches for mutation identification in pea using the methods of reverse genetics can be anticipated in the future (Triques et al., 2004). However, the phenotypic characterization of a new allele as well as field trials will remain an indispensable step in preparation of asymbiotic reference lines.
Although the mutation in Budfix2 does not directly improve the desirable trait and thus does not fit the concept of mutational breeding (Ahloowalia and Maluszynski, 2001), its indirect effect toward a better estimation and exploitation of symbiotic nitrogen fixation might be positive itself.
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ACKNOWLEDGMENTS |
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Received for publication November 19, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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krdleta, M. Kropá
ová, L. Lisá, and M. Nmcová. 1997. Interaction of two genes controlling symbiotic nodule number in pea (Pisum sativum L.). Symbiosis 23:43–62.krdleta, and L. Lisá. 1994. Plant growth, photosynthetic pigments, and nitrate assimilation in symbiotic mutants of garden pea. J. Plant Nutr. 17:1265–1273.krdleta, M. Nmcová, and L. Lisá. 1993. Symbiotic traits, growth, and classification of pea nodulation mutants. Rostl. Vyroba 39:157–170.krdleta, V., A. Gaudinová, M. Nmcová, and A. Hyndráková. 1980. Symbiotic dinitrogen fixation as affected by short-term application of nitrate to nodulated Pisum sativum L. Folia Microbiol. 25:155–161.krdleta, V., L. Lisá, and M. Nmcová. 1987. Comparison of peas nodulated with a hydrogen-uptake positive or negative strain of Rhizobium leguminosarum. I. Nodulation, acetylene-reducing, dihydrogen and carbon dioxide-evolving activities. Folia Microbiol. 32:226–233.Related articles in Crop Science:
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