AU599887B2 - Nitrate-tolerant soybean - Google Patents

Description

"0 p~* t L-__~Ci~iCi

A

I 599887 FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-1962 COMPLETE SPECIFICATION (Original) FOR OFFICE USE: Class Int. Class Application Number: 54.338 1 5 Lodged: Complete Specification Lodged: Accepted: Published: Priority: This document contains the amendments made under Section 49 and is correct for printing.

Related Art: Name of Applicant: Address of Applicant: Actual Inventor(s): THE AUSTRALIAN NATIONAL UNIVERSITY Acton

ACT

tK t jt

,Z:

i t PETER M GRESSHOFF, BERNARD J CARROLL and DAVID L McNEILL Ct C c Address for Service: DAVIES COLLISON, Patent Attorneys, AMP Building, Hobart Place, Canberra, ACT 2601 Complete Specification for the invention entitled: "NITRATE-TOLERANT SOYBEAN" The following statement is a full description of this including the best method of performing it known to us invention, 1

I.

Introduction The invention relates in general to leguminous plants, in particular soybean varieties, having the phenotypes of nitrate-tolerant nodulation, and of supernodulation. Novel, genetically modified nitrate-tolerant and supernodulating soybean plants are disclosed, distinguished from wild-type and domesticated cultivars in having the phenotype of nitrate-tolerant nodulation or of supernodulation. A method of plant breeding, for introducing the nitrate-tolerant nodulation and supernodulation traits into domestic cultivars is disclosed.

SFurther, a process for genetic manipulation to produce and select for nitrate-tolerant plants or for supernodulating plants is disclosed. Plants having nitrate-tolerant nodulation are agronomically useful in that they are able to form effective root nodules at an earlier growth stage than conventional cultivars, in soils having endogenous nitrate. Early nodulation enhances the effectiveness of any added 7 soil inoculant, and it prevents a growth lag commonly associated with the depletion of endogenous nitrate during plant growth and prior to the establishment of effective root nodules. Supernodulation similarly 'I 25 enhances the effectivenss of soil inoculants and contributes to decreased dependence on exogenous nitrogen sources.

t 4,.

Background Nitrate-tolerant nodulation is here defined as the J 30ability of the plant to form effective root nodules when grown in a support medium having a nitrate level of about 5-6 mM, more particularly about 5.5 mM, in Sthe presence of an inoculating 2- Ci 5 lc I amount of symbiotic bacteria. Effective root nodules are those capable of carrying out nitrogen fixation.

Nitrogen fixation, the process of reducing dinitrogen to biologically usable forms of nitrogen, such as ammonia, is conveniently measured by reduction of acetylene, since the enzyme catalyzing dinitrogen reduction, nitrogenase, is also capable of catalyzing acetylene reduction. Therefore, the ability to reduce acetylene is deemed equivalent to the ability to fix nitrogen and is diagnostic of the presence of active nitrogenase enzyme activity. Support medium is the term used herein to denote any material used to support the normal erect growth of a plant, including without limitations soil, sand, vermiculite and the like. Symbiotic microorganism means any Ct E organisms, most typically bateria, of the genus 0. Rhizobium, capable of forming an effective root nodule under appropriate conditions. A nodulating amount of a symbiotic microorganism is simply a sufficient number of such organisms distributed in the support medium to allow a wild-type or conventional cultivar to form sufficient numbers of nodules to support normal growth in an essentially nitrogen-free support medium.

25 Nitrate level is defined as the amount of nitrate per unit of soil volume, obtained by supplying nitrate in solution at a specified concentration, supplied to the plant under defined conditions. The nitrate level is defined in terms of the concentration of solution added because the measured soil concentration may vary due to soil drying, nitrate assimilation and other such factors. A nitrate level of 5mM, for example, is that obtained by the equivalent of watering the plants daily in 12" deep, 3 t--

C

diameter pots with 1/4 1 of 5mM KNO 3 solution, as described herein. Such a procedure provides a relatively constant nitrate level, given the difficulties of obtaining constant concentrations of soluble material in soil.

Supernodulation is defined as the ability of the plant to form effective nodules in greater number and greater mass of nodules per plant than wild-type, by a factor of at least two-fold, when grown in a support medium in the presence of an inoculating amount of symbiotic bacteria. Supernodulating plants may be, but are not necessarily, nitrate-tolerant.

The phenotype of supernodulation is observable in the absence of added nitrate.

Normal wild-type legumes and domestic cultivars of agronomically important leguminous crops, such as t i; soybean, do not form effective root nodules in the presence of low levels of nitrate in the soil. Both nodule size and the ability to fix nitrogen are substantially reduced in soil containing nitrate, when compared with controls grown in essentially nitrate-free soil in the absence of added •nitrate. Residual nitrate is commonly found in soils where crop rotation is practiced and a fertilized 4 a 25 crop, such as corn, was planted the previous season.

Soybeans are commonly grown in rotation with fertilized crops. High soil nitrate levels may occur simply by the action of nitrifying bacteria or organic matter in soil. Some soils, especially those of volcanic origin, ,ze naturally high in nitrate.

3 Under such circumstances, effective nodulation is prevented or delayed until t-e residual soil nitrate is substantially depleted, at which time a lag in -4- 4 f j irplant growth may be observed. The lag, which is sometimes accompanied by transient yellowing, is due to the delay in establishing nodulation. Time to maturity, susceptibility to stress, both during the lag phase and subsequently, and final crop yield are affected adversely. The value of soil inoculation during planting is decreased if the delay in establishing nodulation results in a diminished or ineffectual population of symbiotic microorganisms in the soil. The nitrate-tolerant nodulation phenotype is agronomically advantageous in preventing or reducing such adverse effects.

Nitrate leached from soils is rapidly becoming a S" serious pollutant of rivers and aquifers. The 15 pollution is generated by continual applications of 4" fertilizer to soil and leaching from soil by rain and irrigation water. Since soybeans obtain only about 70% of their total N by nitrogen fixation, reducing the dependence on added fertilizer, this helps to alleviate the pollution problem. Nitrate tolerant and supernodulating mutants reduce the need for added fertilizer.

The nitrate-tolerantran supernodulation phenotypes are disclosed herein are obtained by genetic modification. The term genetic modification as used herein includes any means of altering the genotype of .a plant other than conventional cross-breeding. Such genetic modification means include, but are not I limited to, mutagenesis, followed by selection for the desired phenotype; in vitro construction of recombinant DNA followed by transformation and selection for a desired phenotype or such other means for deleting, inactivating or altering the function 5 5

_I

~~I

it ii of existing plant genes, or for the introduction of new genes into the plant, as may occur to those of ordinary skill in the art, following the teachings of the invention as disclosed herein. Nitrate-tolerant nodulation has not been observed in populations of wild or domestic cultivars of soybean. Prior to the present invention, it was not known or understood that genetic manipulation could achieve a plant phenotype of nitrate-tolerant nodulation, since both plant and bacterial genes are involved in establishing effective root nodules and genetic studies involving genetic manipulation of the symbiosis has been confined to Rhizobium. An unusual feature of the present invention is the discovery of mutations resulting in an enhancement of plant function, the.making of greater than normal numbers of effective nodules. In general, a mutuation providing enhancement of plant function is an "up" mutation wherein the mutant plant produces more of a product, structure or process than wild-type plants. Since enhancements of plant function are rarely achieved by mutagenesis, the disclosed method of mutagenesis and selection will be useful for obtaining genetically modified plants 25 having an enhanced plant function.

Summary of the Invention The present invention outlines a mutagenesis and selection procedure used to isolate 15 independent nitrate-tolerant soybean mutant lines, demonstrates the stability of the nitrate-tolerance phenotype for one generation to the next and describes the Pyw-rxmypiwyeQic d, C6.nc\~ Qharacteristics of soybean lines having the nitrate-tolerance phenotype. Unexpectedly, some of t 4 i I I t k X t t t II

I(

6 the mutants isolated were also supernodulators. A method for mutagenesis of seeds and selection of nitrate-tolerant plants and supernodulating plants is disclosed. The mutagenesis and selection methods described herein are applicable to a wide variety of plants, in particular for mutants having an enhanced plant function, and where the desired phenotypic trait is resistance to a stress. The nitrate-tolerant nodulation phenotype is usually a recessive trait which segregates according to normal Mendelian genetic principles. Supernodulation has been found in both recessive and dominant (or semi-dominant) mutations that behave as single Mendelian traits. Therefore, conventional plant breeding techniques may be used to introduce the phenotypes of nitrate-tolerant nodulation and supernodulation into commercial soybean cultivars.

The invention is exemplified by its application to Ssoybean (Gylcine max) cv. Bragg; however, its it 20 operating principles may be applied to other cultivars of soybean, for example "Williams", and is i not limited to any particular soybean cultivar, but may be applied generally to any plant varieties of I the genus Glycine, whether wild, domestic or hybrids I 25 of the two. The term soybean is used herein to denote the species Glycine max and all domestic I cultivars thereof.

Detailed Description of the Invention The genetic manipulations used herein to generate a 30 phenotype of nitrate-tolerant nodulation were mutagenesis followed by selection. It will be understood that other genetic manipulations, such as the application of recombinant DNA techniques or the 7 use of transposable elements, may be applied as alternative means of genetic manipulation. Seeds were mutagenised in trial experiments with ethyl methane sufonate (EMS), sodium azide and gamma irradiation. EMS was judged preferable because it was the most efficient in generating chlorophyll-deficient mutants which are readily detected by observation of plants with yellow or white leaves. Since mutagenesis of the embryo was most likely to yield a chimeric embryo, due to the existence of more than one germ line cell in the embryo, and it appeared likely that the majority of mutations would be recessive, the screening was carried out in M2 generation. (The seeds subjected to the mutagen are termed the Ml generation, plants grown from those seeds are Ml plants, the seeds produced by the Ml plants are termed M2 seeds the plants grown from M2 seeds are termed M2 plants, and so forth. Families of seeds are those harvested from a single plant. Therefore, a M2 family of seeds includes all the descendants of a single mutagenised seed.) 1 Having chosen EMS mutagenesis as the preferred means f of genetic manipulation, a large scale mutagenesis and selection was done. Ml seeds were mutagenised with EMS in 2 batches, one at 0.44% for 4 hours exposure, or at 0.5% for 6 hours exposure, and then planted. Selection for nitrate-tolerant I r nodulation was not carried out with the Ml plants; 30 however, survival rates and frequency of appearance Sof chl mutations was noted, in order to get a general estimate of mutation frequency and lethality -8

T

1 of the mutagenesis. At maturity, M2 seed families were collected and kept separately, harvesting the M2 seeds in bulk.

Grouping the individual M2 families has several advantages in the selection process. First, it is possible to recognize which mutants arose from the same mutation event. Second, if a given selected variant were to be lost before it produced seed, it was possible to go back to the designated family and reisolate the remaining mutant individuals (siblings) from the family.

For selection of nitrate-tolerant mutants, 10-12 seeds from each family were planted at 2cm depth in pots using washed river sand as the support medium.

The plants were cultured in the presence of nitrate and a commercial soil inoculant (USDA 136) for 5-7 weeks, then carefully removed from the sand and visually screened for extent of nodulation. Pots were initially watered 3 times per week, but this was increased to daily waterings as the demand increased I with growth. Out of 2500 M2 families, by screening more than 25,000 individual plants in the manner described, 15 nitrate-tolerant mutants were obtained.

The concentration of nitrate use8for selection was determined by preliminary experiments with the parent cultivar, Bragg. Figure 1 shows the results obtained, measuring nodule fresh weight as a function Sof KNO 3 level during growth. At 5-6 mM KNO 3 nodule i size was drastically reduced and a small increase in tolerance by an individual plant would be manifest in a substantially increased nodule fresh weight. The selected variants showed significantly increased 9 nodulation in the presence of 5 mM KNO 3 compared to wild-type siblings and the parent cultivar. Since the selection process was time consuming and laborious, only those individuals displaying obviously increased nodulation upon visual inspection were selected. Many more variants having marginally increased nodulation were also observed. It is possible that further analysis, for example by measurement of acetylene reduction, would reveal the existence of additional nitrate-tolerant variants, since an increase in nitrogen-fixing capacity or efficiency of individual nodules would produce a phenotype of nitrate tolerance.- Since these mutants have not been characterized to date, the invention is exemplified by characterization of the mutants that displayed signficantly increased nodulation. Nodule numbers per plant for selected mutants and their wild-type siblings are shown in Table 1. The wild-type siblings of nitrate-tolerant mutants had a nodule number not significantly different from that of Bragg. Nodule number for the mutants ranged from 16 nodules per plant for nts65 up to 370 nodules per I plant for nts2062. In the case of nts2062, the I nodule number as a function of plant mass was times greater than in the wild-type (Bragg) grown under identical conditions, 5mM KNO 3 i Comparison of the mutant grown on nitrate to the wild-type grown in the absence of nitrate was equally I!I striking: nts2062 grown in the presence of 5mM KNO 3 30 which causes a 56% reduction in nodules per plant mass in the wild-type has 4.5 times as many nodules as does the wild-type grown in the absence of nitrate, conditions under which the wild-type is 7 presumably forming its maximum number of nodules.

35 Similarly, measurements of nodule mass showed that t nts2062 plants grown on nitrate have 12 times the nodule mass as wild-type grown on nitrate, and 3. times the nodule mass of wild-type plants grown in the absence of nitrate.

Figure 2 shows nodule fresh weight as a function of plant size (fresh weight) for nts2062 grown in 5mM4 KNO 3and Bragg grown in 0, 4 and 6mM KNO 3*The data demonstrate that nodule fresh weight per plant mass is greater in nts2062 cultured in 5mM KNO 3 than wild-type Bragg grown in the absence of nitrate.

Figure 4 also shows data points corresponding to 3 plants of nts2282 cultured on 5mM KNO In contrast to nts2062, these 3 points approximate the values for Bragg grown in the absence of nitrate. Furthermore, wild-type siblings of nts2062 and nts2282 cultured on KN0 3 approximate the values obtained for the parent cultivar, Bragg, cultured on 4-6mM KNO 3 High nodule numbers were also observed for nts382.

Figure 3 shows a comparison of nts3)82 with Bragg 01 under a variety of growth conditions. As the nitrate concentration increased, the number of nodules per plant actually increased with nts382, while for Bragg the number decreased. The effect of other nitrogen-containing compounds in the support me~dium is also shown. The presence of ammonium at reduced the number of nodules per plant; however, the mutant continued to form significantly more nodules 4 .than the wild-type. The ef fect of ammonium may be :.wholly or partially accounted for by pH changes in roots that have taken up large amounts of ammonium ion. when. the number of nodules per gram of plant fresh weight was measured, the variety nts382 remai.ned constant up to 3-1'VS 3 whereas the ,t wild-type decreased. The presence of urea and ammonium decreased the nodule number of both the mutant and the wild-type; however, the mutant was able to form nodules under conditions where the wild-type was not.

Acetylene reduction assay was used to estimate nitrogen fixation. Conversion of acetylene to ethylene was measured by gas chromatography. Intact plants were placed in 1040ml airtight jars in a 6% acetylene atmosphere, incubated over a minute period. Subsequently, the seedlings were replanted and grown to maturity. In some experiments, detached nodules from mutants or wild-type were assayed instead of whole plants.

Figure 4-shows a comparison of acetylene reduction rates between wild-type and nts2062 at 6 weeks after planting. In wild-type soybeans cultured in the absence of nitrate, nitrogenase activity per plant and per plant mass was about 16 times higher than 20 wild-type grown on 5mM KNO 3 In comparison, nts2062 plants cultured on 5mM KNO had approximately times the activity of wild-type grown under identical conditions. At harvest,6 weeks after planting, the nitrogenase activity of nts2062 plants grown on nitrate was about 55% of that of wild-type grown in the absence of nitrate. Plants of mutant line nts2264 had 4 times the nitrogenase activity of wild-type siblings. Expressing the data for nts2264 per plant fresh weight the activity of the variants was about 6 times that of the wild-type, which, in r 5,l.'t *part, reflected the smaller size of the mutants.

Similar values were obtained from mutants nts382 and ntslll6. Data comparing nts?82 and Bragg are shown f,t:i' in Figure 5. For the wild-type, nitrogenase activity 12 was sharply reduced at a KNO 3 level of 2.75mM and reduced even more at 5.5mM KNO3. In contrast, nitrogenase activity of nts382 remained constant or increased with increasing KNO 3 levels up to Also noteworthy is the observation that, although urea at 5.5mM (total N 11mM) the nitrogenase activity of both wild-type and mutant were reduced. However, nts382 retained many times greater nitrogenase activity than did the wild-type.

Figure 6 shows the nitrogenase activity per gram of nodule fresh weight (specific nitrogenase activity) in nts2062. Activity was the same as for the wild-type cultured under identical conditions.

Specific nitrogenase activity of both nts2062 and 15 wild-type cultured on 5mM KNO. was about 20% of that -j of the wild-type grown under nitrogen-free conditions. In separate experiments, cultures of two other high-nodulating lines, nts382 and ntsl007, showed a similar trend. Specific nitrogenase activity of Bragg, nts382 and ntsl007 grown on nitrate were not significantly different from one another.

I Plants displaying the nitrate-tolerant phenotype were generally smaller than wild-type siblings and the parent cultivar, Bragg. For example, mutants nts246 and ntsl007 were significantly shorter and had I significantly smaller leaf area than the respective it wild-type siblings. Data is shown in Table 2.

SGrowth of nts mutants depends on the nitrogen 30 availability in first few weeks of growth as N-free grown nts mutants are smaller than nitrate grown nts materials. Composite data for several nts #t lines compared with their wild-type siblings, chosen 13 as a better control for this purpose than Bragg wild-type which had not undergone mutagenesis, shows that at approximately 6 weeks after planting, the mutants had grown to about 84% the height and had about 80% the leaf area of the wild-type siblings.

The same trend was reflected in measurements of plant fresh weight, the mutants averaging somewhat less in weight than the wild-type siblings, although for some strains, nts2282, the difference was not significant.

The rate of nodulation during growth was studied, comparing nts382 and wild-type Bragg, both inoculated with Rhizobium USDA 110, grown in sand gravel with V daily waterings of nutrient media plus a supplement, either 5.5mM KNO cr 5.5mM KC1. At intervals, a 3 number of test plants were removed from pots to determine the number of nodules per plant. Results are shown in Table 3. Nodulation rates of nts382 were consistently higher throughout the growth of the 20 plants than were the wild-type rates, both in the S presence and absence of nitrate. The results also Sdemonstrate that the observed phenotypes are not related to strain specificity of the inoculating StRhizobium, since both USDA 110 and USDA 136 nodulated nts382 with substantially similar results.

A comparison of nts382 with wild-type Bragg was carried out at 0, 2.75 and 5.5mM KNO 3 measuring j t nodule number per plant, nodule fresh weight as a Spercent of root fresh weight, and nitrogen fixation I 30 ability as rate of acetylene reduction per gram of plant fresh weight. The results are shown in Table 4. In addition to the strikingly higher nodule a numbers per plant and greater nodule weight per 14 :i

*L

plant, characteristic of the supernodulation phenotype, the ability of nts382 to fix nitrogen was greater than the wild-type at all nitrate levels studied. All of the nts mutants studied had a higher nitrogenase activity per plant and per plant fresh weight than the wild-type siblings when cultured on nitrate. In contrast, in the absence of nitrate, nitrogenase activity per plant at time of harvest was greater in wild-type than for nts2062 grown on nitrate. The specific nitrogenase activity of wild-type and nts2062 cultured on nitrate was identical, and in both cases it was 20% of the activity of wild-type grown under nitrogen-free conditions. A similar trend was shown in measurements with detached nodules of nts382 and ntsl007 cultured on 5mM KNO 3 compared with wild-type grown in the presence and absence of nitrate. A more detailed analysis of nts382 has shown that it behaves similarly to nts2062. The results also suggest that S 20 these nts mutants are capable of using both endogenously fixed nitrogen and exogenous nitrate, a ,,matter of considerable agronomic significance.

The genetics of the nitrate-tolerant mutants suggests that most but not all of them behave as Mendelian recessives, The frequency of appearance of mutant in the M2 generation, where recessive mutants must be homozygous to be detectable, is a function not only of the normal Mendelian frequency of appearance of homozygous recessives but also of the S i 30 genetically effective cell number (GECN). The GECN "is a measure of the number of germline cells contained within an embryo, any one of which may give rise to the mature plant, assuming each of them has an equal probability of doing so. For example, if 15 t I GECN 1, the M2 segregation ratio will be 3:1, if it is 2, the ratio with be 7:1, and if GECN 3, the segregation ratio will be 11:1, and so forth.

Judging by the segregation ratios observed for the recessive nitrate-tolerant mutants, the GECN for soybean is estimated at between 3 and 5. It follows that M2 segregation ratios significantly lower than 11:1 may indicate that the mutant is not recessive.

Further evidence of recessivenss is provided by measuring segregation ratios in M3 plants. If a mutant is a true recessive, its appearance in the M2 generation will be manifest only in the homozygous state and its M3 progeny should not segregate. On the other hand, if there is segregation in the M3 generation, it may be presumed that the mutant is dominant or semi-dominant. For example, in the case of nts246, there were two nitrate-tolerant plants U observed in the M2 family of 10 members.

jFurthermore, in the M3 generation, one of the two isolates yielder wild-type segregants, indicating it was heterozygous in the M2. Therefore, nts246, uniquely among the 15 nitrate-tolerant strains isolated to date, represents a dominant, or semi-dominant, mutation.

On the basis of their genetic behaviour and on the characteristics of nodulation in the presence of nitrate, the nitrate-tolerant soybean varieties f t isolated to date can be classified into four S. complementation groups. Group 1, represented by t 30 nts246, is characterized as dominant or i semi-dominant, and behaves as a supernodulator.

(Supernodulation is defined as an increase of at least two-fold in nodule number and nodule mass over i -wild-type, when comparing mutant and wild-type grown 16 li---L in the absence of added nitrate.) Class 2 is represented by nts strains 382, 2062, 1116 and 1007.

Mutants in this group are recessive, have supernodulation, the rate of nitrogen fixation per plant is equal to or greater than wild-type in the presence or absence of nitrate and the specific activity of the nitrogenase is essentially equal to wild-type. Class 3 is represented by nts65. Class 3 mutants are recessive, display normal nodule number in the presence of 5mM nitrate, but increase nodule size, nodule mass per plant is greater than wild-type. Class 4 is represented by .strains nts97 and 225. Mutants are recessive. Nodule number and mass in plants grown on 5mM nitrate ranges from equal to greater than wild-type grown in the absence of nitrate and is thus characterized as intermediate.

However, in addition the nodules are found more frequently on the peripheral roots and less frequently on the tap roots, suggesting that in these varieties nodulation may be occurring late in plant growth as an adaptive response to high nitrate.

Sn The finding that some mutants had the phenotype of S* supernodulation was unexpected. Supernodulation is t.t distinguished from nitrate erance in that the former is manifested in the absence of added nitrate.

Therefore, some but not all supernodulators are nitrate tolerant. Had selection been carried out for Z t c, supernodulation, by screening M2 plants in the t* absence of nitrate, supernodulating strains that lacked nitrate tolerance could have been isolated.

The phenotype of nitrate tolerant nodulation is S+ distinguishable from an inability to utilize nitrate in that the latter are simply unresponsive or less -17 responsive to the effects of nitrate than wild-type, depending on the leakiness of the mutant. If one were to compare nodule fresh weight as a function of plant fresh weight in such a mutant on a plot of the type shown in Figure 2, one would expect nitrate utilization deficient mutants to fall somewhere within the range of behaviours for wild-type, between 0mM and 6mM KNO 3 That is, individual plants of such a mutant line would plot on Figure 2 between the fitted lines for Bragg (or other wild-type progenitor) cultured on the various nitrate levels.

The exact location between these fitted liles would depend on whether or not the nitrate assimilation mutant was leaky. This was not the case for nts2062 and other supernodulating mutants, which produced greater nodule mass than wild-type when grown in the absence of nitrate. The strain nts382 has also been shown to behave as a typical supernodulator in forming greater nodule fresh weight per gram of plant than wild-type at all nitrate levels. Furthermore, direct experiments have shown that nts382 can assimilate nitrate. Therefore, the phenotypes of i nitrate tolerant nodulation and supernodulation are j 2 distinguishable from deficiencies in nitrate "1 25 utilization of nitrate assimilation. The degree of symbiotic advantage is indicated by the results detailed in Table Specific aspects and features of the present invention are further illustrated by the following examples.

18 -18 'f

.I

Example 1: EMS mutagenesis of plant seeds Since EMS is highly mutagenic even in the volatile (gaseous) form, all procedures were carried out using a fume hood, neoprene gloves and vapor traps. EMS mutagenesis requires an actively respiring embryo.

This is best achieved by imbibing seeds in water bubbled with air or under running tap water prior to EMS exposure. The following is a soybean technique and may be modified for other plant seeds.

Only seeds having high viability as determined by pre-experiments were used. Seeds (5,000 as a minimum) were counted and packaged in plastic wire-netting envelopes (1,000 seeds per bag, fly-screen plastic netting works very well).

The seed bags were placed in a container and flushed with tap water (280C) through the seeds for 12 hours.

Seeds were then transferred to 0.1M KH2PO 4 pH 6.0 (1 liter/1,000 seeds) and ethyl methane sulfonate (EMS) (Sigma) was added to give a final concentration of 0.5% The solution was bubbled with air (to effect agitation and aeration) and the fumes were removed through a vapor trap. The vapor trap solution was 2% potassium hydroxide plus sodium thiosulphate. All the steps involving handling of EMS werecarried out in a fumehood.

I Afte six hours exposure to EMS, the aeration waw stopped and the mutagen solution was decanted through a hose connecting a tap on the bottom of the 1 mutagenesis vessel to a similar vessel for inactivation. The inactivation vessel already had a 19

I

freshly prepared concentrated KOH sodium thiosulphate solution in it. See "EMS inactivation solution", infra. The tap was closed and the mutagenesis chamber was filled with water. ;Air was bubbled through the chamber to effect agitation for a few minutes, then the rinse was decanted into the inactivation vessel. The seeds (still in rinsed mutagenesis vessel) were washed for 3 hours, then the seed bags were removed, using fishing line attached to assist removal, and washed for a further 1 hour under the tap. The inactivation solution was agitated with bubbling air for several hours, then decanted down the fumehood sink with copious amounts of water.

Seeds were removed from seed envelopes and planted immediately. Those handling the seeds should do so only where it is well ventilated and should wear protective gloves. Two planting procedures were used: field planting: directly into the furrow followed by quick hand watering; pot plants: 10 inch diameter pots 1 foot deep (filled with sand plus slow release fertilizer) were planted with 10-14 soybean S° f seeds per pot. Seeds were spread onto the surface of Ithe pre-filled pots, then covered by about 2cm of additional sand. Attempts at drying back the seeds with a hair dryer to permit transport between the lab and field site resulted in increased seed loss.

ii~I The M1 generation was grown and compared for vigor t *I with unmutagenised control plants. Survival numbers *29 30 were scored to get a general estimate of viability and mutagenicity was estimated by noting the i frequency of sectoring of chlorophyll deficiencies.

Prospective chimeric plants were labeled for future analysis.

Seeds were allowed to mature completely. Seeds of the M2 generation were collected from each plant and bagged separately. Separate collection of M2 families allowed the determination of GECN (genetically effective cell number) and more rapid screening. For example, it is certain that any mutant isolated in the M2 screen is separate from any other. Also on those occasions when one loses a selected plant, it then is possible to go back to the family and select a sibling.

EMS inactivation solution: Dissolve 8g KOH in 400ml H2 0 (this stabilizes thiosulphate). Add 40g sodium thiosulphate and dissolve. Submerge all articles for 24 hours, then Sdiscard in running water inside a fume hood (an odd P, tsmell will still persist). Use the inactivating 20 solution also in the vapor trap. Although the EMS s was presumed to be substantially inactivated by the l, ~foregoing solution, no measurements of residual EMS concentration were made.

C Example 2: Screening for nitrate-tolerant mutants 25 M2 families were planted into sand pots (1I family per S. pot), inoculated with commercial peat inoculum, and t r grown for 4-6 weeks. Plants were watered with a standard nutrient solution containing KNO 3 5mM. To S ensure a constant nitrate concentration level, the 30 pots (10 inches diameter, 1 foot depth) were flushed 21 with about 1.4 1 of solution at each watering. Pots were initially watered 3 times per week, and more frequently as demand increased with growth. All plants were carefully removed from the -sand and visually screened for extent of nodulation and for segregation (depends on GECN.); if GECN 1, 3:1 mutant, if GECN 2, 7:1, if GECN 3, 11:1 if GECN 4, 15:1 (assuming recessive mutations). The M2 families were also screened for chl (chlorophyll deficiency). This parameter helps to evaluate the GECN and the mutagenic efficiency.

Selection for the supernodulation phenotype is conducted in the same fashion, except that KNO 3 is not included in the watering solution.

r- I P St ,Ltt Lu f 22 :i1~ FLOW CHART OF MUTANT SELECTION PROGRAM BAGG Nitagenised ~ith 0.44% EMS 4h I PLANTED ii Ii ii Mutagenised. with EMS 6h HARVESTED Planted

HARVESTED

M2 FAMILIES M2 FAMILIES 2500

FAMILIES

Selected POT or/ TRAY screened 2.

3.

4.

6.

7.

nts nts nts nts nts nts 382 2062 2282 246 65 1116 2264 nts nts nts nts nts nts nts nts 1007 97 183 225 501 733 739 761

C

t 4 14 ~4 4 4114 44 gis 4 4,44 1 23 t Example 3: Soybean breeding method Nitrate-tolerant nodulation soybean varieties and ,supernodulation soybean Warieties can -be agenerated using commercial wild-type cultivars as the wild-type starting material. Such wild-type varieties as Bragg and Williams have been described by way of example; however, it will be apparent to those of ordinary skill in the art that other commercial cultivars may be employed as progenitor strains. Once a variety with desired pheontypic traits has been obtained by the above-described mutagenesis and selection, it may be preferred to transfer the trait to other commercial cultivars by conventional plant breeding methods to achieve a new variety combining the desired phenotype (nitrate tolerance, supernodulation) with other valuable agronomic traits.

The desired commercial cultivar is crossed by conventional plant hybridization with a mutant variety having the phenotype of nitrate-tolerant nodulation or of supernodulation. Whether the mutant parent is preferably used as the male parent or the female parent is a matter which may be readily *determined by making test crosses, or by comparing the results of mass crossings made both ways, Where the mutant is recessive, the Fl progeny plants will display wild-type nodulations characteristics. The Fl hybrids are selfed to produce F2 hybrids in which S the recessive phenotype reappears in a portion of the S 30 F2 plants. F2 hybrid parents are then selected, to retain those having the desired phenotype, either i i. nitrate-tolerant nodulation or supernodulation, using mn selection means appropriate and specific for the 24 r desired phenotype, as described in Example 2. The F2 plants are then backcrossed with the parental commercial cultivar in a recurrent manner, repeating .thesteps just described in selecting a strain which combines the desired *agronomic traits of the commercial cultivar parent with the nitrate-tolerance or supernodulation phenotype, as desired. Selections in all cases are carried out as previously described for the mutagenesis procedure.

Further research undertaken bythepresent inventors has now established the following: nts382, ntsl007 and nts501 are in one genetic linkage group which is separate from ntslll6 and nts246; nts246 and nts733 are codominant alleles; nts382 and ntsl007 sigregate in 3:1 (wt vs.

mutant) in M3 populations derived from M2 wildtype heterozygote plants; in nts382 and ntslll6 the supernodulation is shoot-controlled; nts382 roots grown on nitrate for 5 days show an alteration of 34-36 kilo daltons peptide as S.determined by in vitro translation of total RNA 7 t and 2-D electrophoresis of 35 S-labelled peptides; I c the mutations nts382, ntslll6 and ntsl007 are recessive; the supernodulation ability of mutants is illustrated by the results detailed in S" t 30 Tables 6 and 7. The nitragenase activity of 4 nts mutants is detailed in Table 8; i -25 '1 0 -LC 1I *r supernodulation is not controlled by bacterial strains nor the general mode of culture (soil vs.

vermiculite vs. hypoponics); supernbdulation mutant :nts382 is -characterised -by altered nodulation interval and percentage of the root nodulated as illustrated in the results detailed in Table 9; and different roots (tapus lateral) have different nodule numbers as illustrated by the results detailed in Table It will be understood that the number of possible biochemical and physiological traits associated with nitrate tolerance or supernodulation is not exhausted by the present data disclosed herein. Therefore, it may be possible to employ other selection means, less time-consuming or more specifically associated with a given class of nitrate tolerant or supernodulatiig mutants, and that such selection or detection means may be employed as equivalents to the techniques disclosed herein. Similarly further studies may result in the elucidation of additional complementation groups. All such additional information, deemed cumulative and supplementary to the teachings herein, is deemed to fall within the 25 scope of the claims.

Seeds of Glycine max cv. Bragg nts382 have been placed on deposit at Agrigenetics Advanced Research Laboratory, 5649 East Buckeye Road, Madison, Wisconsin 53716. Access to the material on deposit 30 will be available during the pending period of the patent application to one determined by the Commissioner to be entitled thereto under 37 C.f.r.

1.14 and 35 USC 122, and all restrictions on the i i

I

f i.

r i: i!: 8. i A St C 5 5i *O t C St .551 S 15 St rt 1* a 41*4 26 r availability to the public of the materials so deposited will be irrevocably removed upon the granting of the patent.

The -materials on -eposit or viable replications thereof shall be maintained on deposit at the above given address for the life of the patent.

Strain nts382 was chosen for the deposit from among the many strains isolated to date because it exemplifies both the nitrate tolerant nodulation phenotype and the supernodulation phenotype. This deposit is made to further exemplify the invention.

It is not intended as in any way limiting the scope of the invention.

I

tII !:ii i *e tS 4 It

SI

I' 4 5*41

S.

27 2 ~~1

I

Table 1: Nodule number for Bragg, nts mutants/ variants and wildtype siblings cultured on KNO for 5-7 weeks. Unless notedithat data\is nodule number plant 1

S.E.)

selected family mutants/variants wildtype siblings Bragg 19 11 382 146 24 26 12 1007 179 12 13 12 1116 b 79 246 b 115 15 8 17 733 213 26 18 14 183 269 21 19 17 c 16 9 17 9 7 d 120 32 21 225 d 75 17 12 501 d 251 19 17 2062 370 26 38 12 aparent cultivar bdata from M3 plants Ctwo nts plants, both had 16 nodules ddata for nts mutants/variants from 1 plant only 4

I

.'c 28

-C

2 Tab le 2 selected phenotype height (cm) leaf area family S.E. S.E.

246 mutants 22.6 2.4 162 siblings 35.5 T 2.5 227 T ratio 0.64 0.71 1007 mutants 36.4 2.0 158 12 siblings 36.9 T 1.6 206 T 13 ratio 0.78 -0.77- 382 mutants 43.5 6.0 166 16 siblings 52.7 1.8 281 T 3 ratio 0.83*- 0.59f~ r t ~t I I

I

not significantly different

I

II I III 4 II I I 4 *4 S t ''Ii *4

S

S

29 i Table 3 Nodulationof nts382 versus Bragg wild-type in the presence of N0 3 Nodule per plant after S.D.) Day 9 Day 15 Day 22 Day 29 Bragg KCl) 6 4 22 +10 37 +14 39 6 nts 382 KCl) 54 17 103 35 294 70 320 48 Bragg 5.5mM 5 2 9 4 19 8 22 11 KNO 3 nts382 5.5mM 63 19 380 43 483 114 693 142 KNO 3 inoculated with USDA 110, grown in sand gravel with daily waterings of nutrient media plus supplement KN0 3 or KCl)

A

11 t t 30 Table 4 SUMMARY OF nts-382 MUTANT PERFORMANCE (APRIL 19840, Linp* N-Source Nodule Nodule fresh weight Nitrogen fixation number of root fresh ability (nmol C 2

,H

4 per plant weight) -i~ 1 mng plant fresh weight) Bra 0 MM (KCl1control) 3 7 ntV 2 QmiM (KCl1control) 33 9 B~l g 2.75 mM KNO 3 27 nts 382: 2.75 mM KNO 3 474 Br 5.5 mM KNO 3 25 6.12 9.50 0.60 13.54 0.33 9.73 nt 4 5.5 MM KNO I AM 13 783 ?wn in sand, inoculated with CB 1809 (=USDA 136); flushed daily and :vested at 4 weeks.

11 4 .4 .4 .4 .4.4.4.4 .4 .4 .4.

14.4 .4 U .4 .4 4..tQ .4W 4 Lu SYMBIOTIC PARAMETERS OF nts382 EXPRESSED 'AS A PROPORTION OF ERAGG Each datum in the table was computed by dividing the value of the symbiotic parameter for nts382 by the value for Bragg cultured under identical conditions nts382 Bragg). Nitrogen sources are listed in order of increasing severity on nodulation in Bragg. Plants were harvested 4 weeks after planting. The inoculant strain was R. japonicum USDA 110.

Symbiotic Parametera Nitrogen Source Nodules Nodules/g Nitrogenase /plant plant fresh activity wt 0 mM (5.5 mM KC1) 9 21 1.6 2.75 mM KNO 3 18 51 22.6 mM KNO 3 31 73 29.5 mM urea 65 132 36.0 mM NH 4 Cl 1 b 0 mM NH 4

NO

3 b co o Ct t I i i

I

ants382 Bragg.bNH4Cl and NH4NO3 totally prevented nodule formation in Bragg.

II

It 1 lit I 1* I I 11 t,

I

14~ Ii S I III S 32 ~17~ Table 6 ~o~ue~w er± it an'~i -type, plants (rcu1tured 'on "S inM KNOI for 'B Veeks ii 11 4 ii ii

[I

ii j Nodules per plant (mean SD) Selected f amily nts mutants Wild type 382 1 1007l1 1116 2 246 2 733 1 183 1 97 4 501 4,1 14 6 71 179 39 79 A= 60 115 *74 213 *177 269 70 120 251 233 45 409 *148 26 *11 13 4 19 7 3 8* 2 18 19 ±8 32 7 19 8 34 19 12 6 3 2062 2264 2 Unless noted, the data are for M2 plants and wild type refers to wild-type siblings of respective nts mutants. The inoculant strain for these data was R japonicum, CB1809 USDA 136); however, R japonicum USDA 110 also elicited the nts phenotype in the six mutant lines so far tested.

1 The nts phenotype also was elicited when R japonicum USDA110 was used as the inoculant strain.

2 Data for M3 plants.

3 Parent cultivar Bragg.

4 Dat for nts mutants from one plant.

C Ct Vt tc~ e

C

C

u~ CC C Vt C C

C,

C

ICC C 33 *1 I

<WV'

I

Nodule fresh weight for nts mutants and wild-type plants cultured on 5 mM KNO 3 for 6-9 weeks Nodule fresh weight per plant mg (mean 4 SD) Selected family nts mutants Wild type 1007 2461 733 183 501 20622 2264 1196 t 377 1537 647 1086 349 1715 411 445 m 252 1929 391 941 307 62 251 198 134 86 53 187 911 64 176 1181 86 53 3

I

U

11 Unless noted, the data are for M3 plants and wild type refers to parent cultivar Bragg. The inoculant strain was R japonicum CB1809 (=USDA 136).

1 Non-nts siblings of respective mutants were used as wild-type contiols.

2 Data from M4 and M2 plants, respectively.

tt C; t f 34 t 1 Table 8 Nitrogenase activity of mutant and wild type plants ii ii ii *t 41 I Nitrogenase activity nmol C 2

H

4 per plant per min nts KNO 3 LSD 1 mutants mM Mutant Wild type (p(<0.05) 3822 0 17.0 19.2 NS 3 2.75 34.2(3.5) 3.4 -(0.8)4 1116 4 5 143.7 21.4 39.0 22644 5 171.9 20.5 71.6 10074 5 90.1 10.95 21.3 Mutant (M3 or M4 generation) and wild type plants were compared after culture in the presence of nitrate, for nts382, a comparison also was made in the absence of nitrate.

Nitrogenase (acetylene reduction) activity is expressed per plant. Unless otherwise noted, wild type refers to parent cultivar Bragg. The incculant strain was R. japonicum CB1809 USDA 136). Each entry for nitrogenase activity in the table is the mean for 4-9 plants.

1 Least significant difference test.

2 Harvested after 4-wk culture in sand pots watered daily with nutrient solution.

3 Mutant and wild-type plants were not significantly different.

Raw data required loge transformation to satisfy assumption for an analysis of variance, means and LSD of transformed data are shown in parentheses.

after culture for 9-wk (for 1116 experiment), 8-wk (for 2264 experiment), or 7-wk (for 1007 experiment) in sand pots watered three times a week with 5 mM KNO 3 6 3 Non-nts 1007 siblings (wild-type phenotype) were used for comparison.

35 i TAP ROOT NODULATION PATTERN OF N DEPENDENT nts382 AND BRAGG PLANTS Plants were inoculated with R. japonicum USDA 110 and harvested 9 weeks after planting. The pots were watered daily with N-free nutrient solution throughout the experiment.

Tap Root a b Parameter nts382 Bragg LSD0.

0 Root length (cm) 16.6 28.1 3.1 Nodulation interval (cm) 14.4 7.6 2.1 Nodulation interval of root length) 86.5 72.1 6.7 Nodule density on root length (nodules cm- 1 4.32 (2.0) c 0,47(0.67) -(0.15) Nodule density on nodulation interval (nodules cm- 1 5.07 2.10 0.99 aEach entry in the table for nts382 represents the mean of seven plants.

bEach entry in the table for Bragg represents the mean of 28 plants.

CRaw data required square-root transformation to satisfy assumptions for an analysis of variance; means and LSD of transformed data are shown in parentheses.

Ct C 4 C. t Si 4 4 4144

I:":U

4 44 4 4 4-44 4 4: 4; *i 4 4: 44 44 4 4 44,4 i 44 46 36 i i zt~w e:AII ~i rlj Nodulation and Nitrogenase (Acetylene Reduction) Activity in KNO Grown nts and Wild-Type Segregants from a nts382 M3 Family This family was derived from a wild-type M2 plant. The segregation ratio was 14 nts:40 wild type which approximates 1:3 (chi-square 0.00 and was not significant).

Plants were inoculated with R. japonicum USDA 110 and harvested after 7 weeks growth on 5 mM KNO 3 Data are expressed per g plant fresh weight.

Symbiotic Parameter Wild-typeb

LSD

0 Nodule number g plant fresh wt-l on tap root on lateral roots mg nodule fresh wt g plant fresh wt-l nmol C.H. g plant fresh wt-l'.mln-1 9.6(2.22)c 0.7(-0.45) 19.2(9.4) d 1.5(1.1) (0.20) (0.4) (0.76) 145.3(12.

0 8.0( 2.05) c 6.2(2.5) 1.1(0.00) i i' i ;:i 7 abEach entry in the table is the mean of 14 and 18 plants, for nts and wild-type, respectively, except that acetylene reduction data are the means of seven nts and nine wild-type plants.

C'dRaw data required either loge or square-root transformation to satisfy assumptions for an analysis of variance; means and LSD of tranformed data are shown in parentheses.

iti 1*f rt

IC

37 ":1

Claims (16)

1. A method of producing a genetically modified soybean plant having a desired phenotypic trait of nitrate-tolerant nodulation and/or supernodulation comprising the steps of treating actively respiring soybean seeds with a mutagenic agent, growing Ml plants to maturity and harvesting M2 seeds, planting M2 seeds in family groups derived from single Ml plants, testing individual M2 plants for the desired phenotypic trait, and growing tested M2 plants to maturity and harvesting M3 seeds of plants having the desired phenotypic trait, whereby a genetically modified soybean plant having the desired phenotypic trait is obtained.

2. The method of claim 1 wherein the treatment with a mutagenic agent is conducted under conditions where mutation frequency is maximised without substantially decreasing viability.

3. The method of claim 1 wherein ethyl methane sulfonate is the mutagenic agent.

4. The method of claim 1 wherein testing for the S desired phenotypic trait of nitrate-tolerant nodulation at 'is carried out by observing changes in plant response when grown in a support medium having a nitrate level of The method of claim 1 wherein the desired phenotypic trait is nitrate-tolerant supernodulation. i iu~l-~ i~ n Ii* ti th t t

6. A method of soybean breeding to produce a soybean variety having a phenotype of nitrate-tolerant nodulation, comprising the steps of: crossing a first parental variety with a genetically modified second parental variety having the phenotype of nitrate-tolerant nodulation, recovering Fl hybrid progeny plants, selfing the Fl hybrid plants, selecting for F2 hybrid plants having the phenotype of nitrate-tolerant nodulation to identify nitrate-tolerant hybrids, and back-crossing the nitrate-tolerant hybrids recurrently with the first parental variety and repeating b, c and d, thereby producing a soybean variety having the nitrate-tolerant nodulation phenotype.

7. The method of claim 6 wherein the step of selecting for nitrate-tolerant nodulation includes growing F2 plants in the presence of a nitrate level of

8. The method of claim 6 wherein the genetically modified second parental variety is selected from the group nts382, nts2062, nts2282, ntslll6, nts2264, ntsl007, nts97, ntsl83, nts501 or nts733 (as hereinbefore described).

9. A method of soybean breeding to produce a soybean variety having a phenotype of nitrate-tolerant nodulation, comprising the steps of: crossing a first parental variety with a genetically modified second parental variety having a phenotype of nitrate-tolerant nodulation, r ri J F 8; 4* 11 JI f r, r r I! i sele nitr tole back recu repe vari pher i icting Fl hybrid plants having the phenotype of ate-tolerant nodulation to identify nitrate- rant hybrids, and -crossing the nitrate-tolerant hybrids irrently with the first parental variety and ating step thereby producing a soybean ety having the nitrate-tolerant nodulation lotype. The method of claim 9 wherein the step of selecting for nitrate-tolerant supernodulation includes growing Fl plants in the presence of a nitrate level of

11. The method of claim 9 wherein the genetically modified second parental variety is nts246 (as hereinbefore described).

12. A method of soybean breeding to produce a soybean variety having a supernodulation phenotype comprising the steps of: crossing a first parental variety with a genetically modified second parental variety having the phenotype of supernodulation, recovering Fl hybrid progeny plants, selfing the Fl hybrid plants, selecting for F2 hybrid plants having the phenotype of supernodulation to identify supernodulating hybrids, and back-crossing the supernodulating hybrids recurrently with the first parental variety and repeating b, c, and d, thereby producing a soybean variety having the phenotype of supernodulation.

13. The method of claim 12 wherein the step of selecting for supernodulation includes examining the roots for F2 plants and comparing nodule number per plant tl F P~. It N1 M r *r ar*n~ i I rcr .clar a i~ I 41 and nodule mass per plant with the first parental variety, grown under essentially similar culture conditions.

14. The method of claim 12 wherein the genetically modified second parental variety is selected from the group nts382, nts2062, ntslll6, or ntsl007 (as hereinbefore described). A method of soybean breeding to pruce a soybean variety having a supernodulation phenotypep .:prising the steps of: crossing a first parental variety with a genetically modified second parental variety having the phenotype of supernodulation, selecting Fl hybrid plants having the phenotype of supernodulation to identify supernodulating hybrids, and back-crossing the supernodulating hybrids, recurrently with the first parental variety and repeating step thereby producing a soybean variety having the supernodulating phenotype.

16. The method of claim 15 wherein the step of selecting for supernodulation includes growing Fl plants in the presence of a nitrate level of

17. The method of claim 15 wherein the genetically modified second parental variety is nts246 (as hereinbefore described).

18. A method according to claim 1, claim 6 or claim 9, or claim 12 or claim 15, substantially as hereinbefore described with reference to the Examples. I (C- C ;l A" i J Ii~a r 44 -Ii C C I LI i u 1 42

19. A genetically modified soybean plant, produced by the method of any one of claims 1 to 18. Dated this 23rd day of May, 1990, THE AUSTRALIAN NATIONAL UNIVERSITY. By its Patent Attorneys, DAVIES COLLISON c t g c Z r6 1 1 iitA" r