AU715536B2 - Method of regeneration of medicago sativa and expressing foreign DNA in same - Google Patents

Description

S F Ref: 226521D3

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Pioneer Hi-Bred International, Inc.

700 Capital Square 400 Locust Street Des Moines Iowa 50309 UNITED STATES OF AMERICA Charisse M Buising, Dwight Tomes and Janice F Schmidt Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Method of Regeneration of Medicago sativa and Expressing Foreign DNA in Same The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845 Method of Regeneration of Medicago sativa and Expressing Foreign DNA in Same BACKGROUND-OF THE INVENTION Genetic transformati'on of plants has been one of the major advances achieved in biotechnology and its contributions to producing improved plants, improved crops, and consequently improved availability of food worldwide has been widely recognized. In certain plants, however, transformation has been especially difficult to achieve, and transformation of the valuable forage crop alfalfa, Medicago sativa has been inhibited by the peculiarities of the plant.

Transformation of alfalfa has been hampered primarily by two major limitations: constraints imposed by the method of transformation, and the poor regeneration from tissue and cell cultures of many alfalfa varieties.

The first limitation occurs because alfalfa is presently 15 primarily transformed through the use of Agrobacterium tumifaciens. Arobacterium exhibits host strain specificity and only certain Agrobacterium strains will infect a few alfalfa genctypes. The ability to transform alfalfa is considerably limited as a result. The second major inhibition of transformation of alfalfa is its own poor regeneration frequency. Only a few varieties exhibit even modest regeneration, and those elite varieties providing superior performance in the field are notoriously poor regenerators. The combination of these two problems has created a considerable bottle- 25 neck in achieving transformation of the plant.

Alfalfa exhibits other traits setting it apart from many crop plants. It is an autotetraploid and is frequently self incompatible in breeding. When selfed, the pollen may not germinate or, when it does, later stops germinating. Thus producing a true breeding parent for hybrids is not possible, which complicates breeding substantially.

It has been determined that there are nine major germplasm sources of alfalfa: M. falcata, Ladak, M. varia, Turkistan, Flemish, Chilean, Peruvian, Indian, and African.

Culture of explant source tissue, such as mature cotyledons and hypocotyls, demonstrates the regeneration frequency of 2genotypes in most cultivars is only about 10 percent. Seitz- Kris, M.H. and E.T. Bingham, In vitro Cellular and Developmental Biology 24 (10):1047-1052 (1988). Efforts have been underway to improve regeneration, and have included attempts at asexual propagation to maintain individual genotypes which possess the regeneration trait. Further, propagation by asexual methods is not practical if many genotypes are involved. Bingham and others have attempted to avoid this problem by recurrent selection. In the first cycle, regenerating genotypes were selected, crossed and recycled until regeneration was improved to 60 percent or better. The result of this was the development of Regen-S, in which two-thirds of the plants were capable of regeneration from callus tissue.

E.T. Bingham, et. al., Crop Science 15:719-721 (1975).

15 Additionally, researchers believe that somatic embryogenesis in alfalfa is inheritable, and is controlled by relatively few genes. Efforts at improving regeneration have thus been directed towards isolation of the genetic control of "embryogenesis, and breeding programs which would incorporate such information. See, e.g. M.M. Hernandez-Fernandez, and B.R. Christie, Genome 32:318-321 (1989); I.M. Ray and E.T.

Bingham, Crop Science 29:1545-1548 (1989). This is complicated by the characteristics of alfalfa noted above.

This invention relates to improvements in transformation 25 and regeneration of alfalfa by departing from these previous approaches. Direct introduction of DNA is accomplished by the *use of microprojectile bombardment. As a result of the use of bombardment, the limitations of Agrobacterium are overcome.

Furthermore, limitations in regeneration of alfalfa are overcome by selecting immature cotyledons for transformation and regeneration. It has been found that when immature cotyledons of alfalfa are used, regeneration is considerably improved, and there are no limitations on what type of alfalfa can be regenerated as a result of this method. Thus even elite varieties may be regenerated, and transformed.

Thus, it is an object of this invention to improve 3 transformation rates of Medicago sativa.

It is another object of this invention to improve regeneration of Medicago sativa.

A still further object of this invention is to allow transformation and regeneration of any variety of Medicago sativa.

Still further objects of the invention will become apparent through the following description.

Summary of the Invention The invention relates to the use of immature cotyledons of Medicago sativa for transformation and regeneration of any variety of Medicago sativa.

According to a first embodiment of the invention, there is provided a process for regeneration of alfalfa comprising initiating somatic embryogenesis of cells of immature cotyledon of alfalfa.

According to a second embodiment of the invention, there is provided a process for improving regeneration ability of an alfalfa plant comprising initiating somatic embryogenesis of cells of immature cotyledons of the plant and culturing the somatic embryo into a mature alfalfa plant.

According to a third embodiment of the invention, there is provided a process of transforming i1 foreign DNA into alfalfa plants comprising introducing the DNA into cells of immature cotyledons of alfalfa and cultivating the cells with foreign DNA into alfalfa plants.

Description of the Drawings .Fig. 1 isa map of plasmid pPHI251.

Fig. 2 is a map of plasmid pPHI256.

20 Fig. 3 is a graph showing time course harvest results plotting age of the cotyledon on the x axis S and regeneration response on the y axis.

Fig. 4 is a graph of regeneration using immature cotyledons (clear bar) and mature cotyledons (solid bar) of varieties listed.

Fig. 5 is a map of plasmid pPHI413.

2" Description of the Invention 9 Microprojectile Bombardment :.Microprojectile bombardment in order to transform plant cells is known to those skilled in the art. The general process has been described by T. M. Klein, et al., Proc. Natl. Acad. Sci. USA 85:4305-4309 (1988). This reference, as well as those cited throughout, represent knowledge of those skilled in the art and are each incorporated herein by [I:\DAYLIB\LIBFF]08539A.IC:gcc reference. The basic process includes coating DNA onto small high density particles, microprojectiles, which are then placed into the particle gun or helium gun apparatus and accelerated to a high velocity in order to penetrate plant cell walls and membranes and carry the DNA or other substance into the interior of the bombarded cell.

Previous work involved delivery of foreign genes through this method into intact plant of tobacco tissue, but its application to the economically important species alfalfa has not been successfully accomplished. Tomes, et al. Plant Molecular Biology 14:261-268 (1990). Microprojectile bombardment of alfalfa to achieve transformation has not been previously reported.

Introduction of DNA into a plant is demonstrated at first 15 by transient expression. Short term expression is noted by 6 confirming the presence of the DNA within the plant cells 24 to 48 hours after bombardment, When expressed up to 72 hours after bombardment it is demonstrated that the DNA has been t delivered via the particle gun or other method and that the DNA vector functions. When continuing to be expressed two to eight weeks after bombardment, it may be concluded the DNA is persistent and likely integrated into the plant genome. Its ability to persist at this point shows it has survived attack from nucleases which typically would attack unprotected 25 foreign DNA. When the R plants are recovered, continuing expression is further indication that stable transformation into the plant cells has occurred. Southern analysis allows confirmation of this. When crossed and the R, generation analyzed, expression and inheritibility of the DNA is further confirmed.

A variety of plant cell sources can be used for transformation by microprojectile bombardment. Hypocotyls, cotyledons of mature seed and petioles are plant tissue which can be subjected to bombardment. The applicant has discovered that when cotyledons are used, satisfactory transformation results.

While not wishing to be bound by any theory, it is proposed 5 that cotyledons may be a better source of tissue for bombardment because the cells to be bombarded are those which are capable of giving rise to plants. Mature cotyledons are also convenient sources of tissue and easy to excise from the seed.

Cotyledons from mature seed can be used in transformation, that is, seed which has reached dormancy. This seed is then placed in water, typically for one to several days, the root breaks through the seed coat, and the cotyledon is dissected. The use of immature cotyledons is discussed more fully below.

It has been found that the optimum stage for best transformation results of mature cotyledons occurs when bombarded after 24 to 120 hours of imbibing water. It has been discovered that at this point regeneration, transient transformation, and resulting transformation is at its optimum. Prior to 24 hours it is as a practical matter more V9009 difficult to remov-e the seed coat without damaging the cotyledon. After 120 hours, it is more difficult to regenerate the plants.

The tissue should be bombarded one or two times, and bombardments in excess of this would likely kill the cells.

Tissue culture was also optimized for the maximum regeneration possibilities. In the experiments described below, Regen-S, was used. As noted supra, Regen-S is known 25 for its improved regeneration potential. Set forth below are tissue cultures which were employed. The most important factor in tissue culture optimized for regeneration is high concentration of 2 4 -dichlorophenoxyacetic acid as compared to a low concentration of kinetin. Tissue/organ culture is described generally by Atanassov and Brown in Plant Cell Tissue Or__an Culture 4:111-122 (1985).

CULTURE

MEDIA

The following describes media used in regeneration of transformed and non-transformed alfalfa. It is to be understood that those skilled in the art could use media which 6varies considerably from these media and fall within the scope of the invention. The description is given by way of example.

Gamborg's Based Medium Gamborg's B-5 medium is a widely used medium for culture of plant species. It is well known to those skilled in the art and is described in detail at 0.L. Gamborg, R.A. Miller, K. Ojima, Exp Cell. Res. 50:151-158 (1968). It forms a component of media listed below.

Modified B5 Medium This medium is described at Atanassov, A. and Brown, D.C.W. Plant Cell Tissue and Organ Culture 3:149-162 (1984).

A typical mixture is that formulated by GIBCO Laboratories and 15 include: 1 mg/1 2,4-D, 0.2 mg/l kinetin, 30 g/1 sucrose, 3000 m g/1 KNO 895 mg/l CaC2 800 mg/1 1-glutamine, 500 mg/1 MgSO 7H O, 100 mg/1 serine, 10 mg/1 L-glutathione, 1 mg/1 adenine, with the modification that was used instead of gelrite reported in Atanassov, 9 g/l bacto agar. It forms a component of media listed below.

MS Medium This medium is well known to those skilled in the art and is described in detail at T. Murashige and F. Skoog, 25 Physiologia Plantarum 15:473-497 (1962). A typical mixture is that formulated by Gibco Lab and includes: S.Component mg/L NH NO 1650.0 KNO 1900.0 CaCl 2H 0 a 440.0 MgSO 7H Ob 370.0 KH2 P 6 170.0 Na *EDTA 37.3 FeO 4 7H 2 0 27.8

H

3 B0 3 6.2 MnSO 4

H

2 0 16.9 ZnSO 7H 0 8.6 K1 0.83 Na MoO .2H2 0.25 CuO 4

H

2 0 0.025 CoCl2 6H 2 0 0.025 7 Blaydes Medium and Modifications This well known medium to those skilled in the art is described in detail at D.F. Blaydes, Physiol. Plant. 19:748- 753 (1966).

BO (basal Blaydes medium) contains per liter: 300 mg KH PO 100 mg KNO 1 g Nl NO 3 347 mg Ca(NO 3 )2 4 H0, 35 mg MgSO 7 H 2 65 mg KCl, 0.8 mg KI, 1.5 mg ZnSO 4 7 H20. 1 .6 mg H BO 4.4 mg MnS0O 120, 2 mg glycine, 0.1 mg thiamine hydrochloride, 30 g sucrose, 10 g (5.57 g FeSO ;7H 2 0 in 500 ml hot distilled water with 7.45 g Na2EDTA in 500 ml hot distilled water with pH to 5.9-6.0.

BII medium is the same as BO, but contains 2 mg/l each 15 NAA, Kinetin, and 2,4-D.

BOi2Y is the same as BO, but contains 100 mg/l inositiol and 2 g/l bacto yeast extract. After embryo induction, explants must be removed from exposure to 2,4-D. 2,4-D appears to inhibit embryo development.

Schenk and Hildebrandt (SH) medium This medium is well known to those skilled in the art and Sis described in detail at B.V. Schenk and A.C. Hildebrant, Can. J. Bot. 50:199-204 (1975). SHII contains 9.05 uM 2,4- 25 dichlorophenoxy acetic acid and 9.30 uM kinetin Modified SH medium This medium is well known to those skilled in the art and is described in detail at D.H. Mitten, S.J. Sato, and T.A.

Skokut, Crop Sci. 24:943-945 (1984). Modified SH medium contained: 25 uM a-naphthaleneacetic acid (NAA) and 10 uM kinetin, callus was transferred to SH medium containing 50 uM 2,4-D and 5 uM kinetin, transferred 3 days later to regeneration medium containing BOi2Y.

The following is presented merely as examples and are not intended to limit the scope of the invention.

In each of the experiments set forth below, Regen-S, as 8 described above, was employed. This variety is known for its high regeneration potential. Genes encoding the Alfalfa Mosaic Virus coat protein (AMVcp), Phosphinotricin Acetyl Transferase (referred to here as BAR), Neomycin Phosphotransferase (NPTII) and 8-glucuronidase (GUS), were transformed into this genotype using a DuPont PDS 1000~ particle gun. The alfalfa mosaic virus coat protein may protect plants from AMV pathogens, BAR inactivates the nonselective herbicide phosphinotricin, present in Basta® medium and NPTII inactivates kanamycin. Plasmid pPHI251 encoding for NPTII, and AMVcp was used. A map of this plasmid is shown in Figure 1. Plasmid pPHI256 was separately used as indicated below in coding for BAR, AMVcp, and GUS. A map of this plasmid is found at Figure 2.

*c e S EXPERIMENT 1 Alfalfa Mature Cotyledon Particle Gun Transformation on Basta® Selection Explant: Mature Cotyledons of RegenS Plasmid: pPHI256 (GUS, AMVcp, BAR) Bombardment: 8 cotyledons per plate (8 plates) bombarded twice with 1.8 pm tungsten particles S.

S

S.

Culture: Results: Seed germinated 2 days and embryonic axis removed from cotyledon Cotyledon plated to filters soaked with 0.25 M sorbitol and adaxial surface bombarded twice Cultured on modified B5 medium 2 days 3 day post-bombardment cotyledons cultured on a modified B5 medium containing 2.5 mg/l Basta® for 9 wks 4 wks callusing/embryogenesis (B5 base, lmg/l 2,4-D and 0.2 mg/l kinetin) 2 wks embryogeny/embryo development (B5 base, 0.1 mg/1 NAA) 3 wks embryo maturation (Boi2Y base, no hormones) Rooted on 5 mg/l Basta® Shoot tips cultures initiated 60 embryos recovered 11 browned and died during selection abnormal sacrificed for GUS histochemical 9 staining (all negative) 31 abnormal recultured for callus (also

GUS

negative) 8 normal--5 survived higher selection In this experiment, five plants were recovered from culture of bombarded mature cotyledons on modified B5 media containing 2.5 mg/l Basta®. Each plant was identified to contain the AMVcp and BAR genes by the method of polymerase chain reaction amplification, as shown in Table 1. glucuronidase enzyme activity was also identified in the five plants by a GUS assay described by Rao, G. and Flynn, p., BioTechniques, Vol. 8, No. 1, pp. 38-40 (1990).

TABLE 1 Alfalfa Plants Recovered on Basta Selection

PCR

Plant AMVcpb c GUS" Slant AVcp BARC Shoot Root Assay Assay Assay Assay 25 1 2 1 2 El 4 El 3 2 2 E2 +3 2 2 E2 2 E3 2 N/A E4 2

-N/A

*E5

N/A

S3 luorometric GUS assay expressed as pgn/ g total protein. 35 Oligonucleotides target internal to AMVcp coding region.

.Oligonucleotides target CaMV promoter and 5' region of BAR coding region.

Below, PCR analysis of the parent and progeny is set forth showing 50% were positive for BAR. The first three plants are progeny followed by a maternal plant showing

BAR

expression, a paternal negative control, maternal plant positive for BAR and controls.

10 STABLE 2 PCR Analysis of Parent and Progeny Plants Sample Source BAR

AMV

BOO

1 E2 x YAE92 Progeny BOO1E2 x YAE92 Progeny BOO1E3 x YAE92 Progeny Maternal BOO1E2 Maternal YAE92 Paternal Paternal Maternal BOO1E3 Maternal RA3 11-5 controla NPTII+ AMV+ RA3 C308 control a A description of this positive control is found at Hill, et al., Bio/Technolo 9:373-377 (1991).

Southern analysis was performed on the parent plants 25 which were found to be clones and were positive by PCR for BAR and AMVcp genes. Thus, it can be seen heritable transformation of plants was achieved.

In summary, it can be seen that transformation of mature cotyledons from alfalfa can be accomplished through he use of microprojectile bombardment. However, as noted, regeneration is typically poor. Regeneration is dramatically improved by the use of immature cotyledons in transformation and regeneration.

a.

35 Immature Cotyledons Somatic embryogenesis can be direct, where embryos are formed directly from the cells, or indirect where a callus is formed which goes through dedifferentiation.

Where in the past research has centered on using a particular germplasm source, selecting for genotypes with improved regeneration, recurrent selection to create varieties having improved regeneration, or selection for genes in plant breeding techniques in developing improved regeneration lines, this invention uses an entirely different approach. See, 11 e. Mitten, et al., Crop Science, 24:943 (1984); Seitz, Kris Bingham, In Vitro, 24:1047 (1988); Brown and Atanassov, Plant Cell Tissue Organ Culture, 4:111-122 (1985). Thus, the invention relates to the use of immature cotyledons to improve regeneration, and thereby transformation of alfalfa.

The use of immature cotyledons has been found to be an important factor in regeneration. As a seed develops, from about 0-5 days past pollination the seed embryo is globular in shape and generally without form, translucent in color. At about 5 days it demonstrates a heart shaped appearance. The embryo then undergoes rotation, and at about 10 days has a visible cotyledon. The color is translucent to light green, and a scalpel placed behind the cotyledon can almost be visualized. At about 15 days the differentiation of the seed 15 parts has become more distinct, and by 20 days it has a dark green appearance. Beyond 25 days, the dark green color gives away to a yellowing. At 30 days it is creamy white in color.

It is at this point that the dormancy process is underway.

It has been found by the applicant that immature cotyle- 20 dons providing improved regeneration include those which are formed up to 25 days past pollination. At 5-7 days postpollination the heart stage is apparent, however, as a practical matter it is difficult to excise the cotyledon portion at this stage and to differentiate it from the other parts of the embryo. The cotyledon can be harvested more easily beginning at about 10 days when it has a translucent to very light green color. The time period between 10-15 days is preferred and provides for considerably improved regeneration results. The most preferable time to excise the cotyledon is at about 10 days past pollination and/or the cotyledon has a translucent to light green color. The light green color can be compared to that found at Panton Color Chart Number PMS372.

As a result of using immature cotyledons as provided herein, it is possible to regenerate varieties which have never been capable of transformation and regeneration before.

12 Thus, while, highly regenerable plants in the past have not always carried the preferted phenotypes, now one may regenerate even elite lines of alfalfa. These elite lines typically have desirable production qualities but notoriously poor regeneration.

As a further result, when immature cotyledons are used, one can obtain transformation of such elite lines which could not be regenerated previously after introduction of DNA. The transformation may occur by bombardment or the previously known use of agrobacteria, with regeneration now possible.

EXPERIMENT 2 .a 15 The typical protocol includes placing the immature o cotyledon explant on a modified B. medium. After 21-28 days somatic embryos are transferred to MS medium and allowed to mature. Obviously there are a number of variations on this protocol known to those skilled in the art and this is given 20 by way of example. The following shows improved regenerance which correlates to explant age.

Plants from two varieties were divided into three groups.

Six plants from YAE92 were placed into a first group, five plants from YAE92 were placed into a second group, and five plants from YAM93 were placed into a third group. Table 3 'below shows the background of each variety. Each group was crossed exclusively within itself. From the resulting plants, each raceme is individually identified and its integrity maintained. Harvesting occurs at timed intervals from 0-30 days past pollination, with an early harvest from a numbered raceme and later harvest from the same raceme. By maintaining the integrity of the group and harvesting from a numbered raceme over the time course of the experiment, it can be demonstrated that variation of genotype even within a particular variety does not affect regeneration as long as regeneration is from immature cotyledon. Each of the cotyledons excised at the 13 time course harvest was regenerated. A graph at Figure 3 of the results plots the age of the cotyledon post-pollination on the x-axis and the regeneration response on the y-axis. The results show that even from the same raceme there is increasing regeneration beginning at just after pollination, up to about 15 days past pollination, with declining regeneration up to maturity.

The scoring and evaluation of the time course harvest is shown in Table 4. Thus, it is clear that age of the cotyledon excised is the critical factor effecting regeneration.

TABLE 3 Percent Contribution of Germplasm varia ladak turk falc chil peru indian african flemish unk YAE92 27 8 4 6 8 7 YAM93 23 8 10 8 7 2 42 p.

14 TABLE 4 Regeneration Shown as Percent Response, From Immature Cotyledons of Different Ages From Controlled Matings Within Three Groups of Alfalfa Plants AGE Number (Days Post- Cotyledons Percent Pollination) Evaluated Response 6 44 48.

7 38 53.

8 42 52.

9 39 64 20 10 52 11 44 61.

12 51 57.

13 53 62.

14 38 25 15 42 43 16 38 34 17 42 27.

18 49 22.

19 59 17.

S 30 20 56 14 21 30 7 22 45 9 23 41 7.

24 19 25 68 1 26 73 1 27 18 0.

28 9 0 S29 17 0 30 17 0.

31 15 0.

32 11 0.

33 15 0.

34 9 0 35 10 0.

36 17 0 37 18 0 38 14 0 39 11 0.

40 12 0.

15 Thus, it can be seen that when immature cotyledons are used in regeneration of alfalfa, dramatically improved results occur.

EXPERIMENT 3 This experiment confirms that it is the immature cotyledon use which provides for the improved regeneration and may be applied to any germplasm source. A number of varieties, including those that have poor or little regeneration were regenerated using immature cotyledons. A minimum of twelve plants of each of the varieties listed in Table 5 were planted and pollinated, with the exception that 15 plants of Grimm (Pi 452472), 30 plants of Mesa Sirsa and 1 plant of RA3 clone were 15 planted and pollinated. Each raceme identified was harvested at about 10-15 days past pollination and at maturity (about days). Immature and mature cotyledons were regenerated as described in Experiment 2.

The data in Table 5 below demonstrates that use of 20 immature cotyledons substantially improves regeneration even in those varieties which traditionally have poor or no regeneration. Figure 4 graphically displays the differences in regeneration occurring in varieties that are extremely difficult to regenerate. Selected varieties and, in particular, those with the worst regeneration, are shown in terms of percent regeneration of mature cotyledons in the solid bar; and percent regeneration of immature cotyledons, represented by the hashed bar. Use of immature cotyledons resulted in improved regeneration in each instance, including those varieties with no regeneration using mature cotyledons.

16 TABLE Comparison of Percent of Regeneration of 30 Days Past Pollination Mature Cotyledons With Percent Regeneration of 10-15 Day Post-Pollination Immature Cotyledons MATURE MATURE IMMATURE

IMMATURI

ALFALFA COTYLEDONS COTYLEDONS COTYLEDONS

COTYLEDON

DESIGNATION SAMPLED REGENERATING SAMPLED

REGENERAT

Grimm (Pi 452472) 206 0 223 S 15 Norseman 152 28 198 37 Lahontan 167 2 184 Turkistan (Pi 86696) 176 8 186 18 Teton 145 3 175 16 Pi251689 140 0 129 21 Caliverde 65 138 0 167 27 SBuffalo 127 1 158 Cody 161 0 183 31 Hairy Peruvian 147 4 166 18 S. 25 Hairy Peruvian (BIG-PLH) 150 0 173 22 Mesa Sirsa 243 0 262 17 Sonora 110 11 127 DuPuits 138 6 145 24 Iroquois 143 0 158 26 Vernal 152 22 161 34 Culver 170 0 173 23 Agate 135 0 155 19 Ramsey 121 0 181 24 El Unico 149 0 190 28 RegenS/ RA3 43 54 63 72 YAM93 164 0 196 34 YAE92 179 0 187 27 17 EXPERIMENT 4 Three separate tests were conducted to determine if immature embryos could be transformed.

In the first test, cotyledons were bombarded with pPHI413 (see Figure as above, and levels of GUS expression assayed. Forty-two samples were bombarded. Optimum expression occurred 48 to 72 hours post bombardment where 26 of the 42. samples expressed GUS with a mean of 1.

7 pg/pg total protein. Five days post bombardment 6 of 30 samples showed an average of 2 pg/pg total protein, while at 17 days post bombardment 3 of 30 samples showed an average of 2 pg/pg total protein.

In the second, the effect of bombardment on alfalfa 15 regeneration under selection was studied. Immature cotyledons of Regen S were harvested 11 days post-pollination. Cotyledons were excised from the embryo, bombarded three times with Sthe plasmid pPHI251 (Figure adsorbed to tungsten particles, and cultured on modified B5 media containing 25 mg/l kanamycin sulfate. Somatic embryos were harvested approximately two months after treatment, allowed to desiccate on MS media for two months, and germinated on MS media containing 1 00 mg/l kanamycin sulfate. Leaf tissue was harvested and assayed for neomycin phosphotransferase (NPTII) activity. The 25 results are shown in Table 6.

a 18 Table 6 Plant NPTII activity pg/pg total protein CBX106 3 CBXl07 2 CBY107 CBY108 4 CBZ108- 1 CBX112 1 CBY112 3 CBZ112 1 CBA112 1 CBX115 2 CBX116 2 CBY116 3 20 CBX117 3 1 Regen S 3-11 13 2 Regen S 3-11 3 Regen S 3-11 9 Regen S Negative Control a 0 25 Rambler Positive Control b 4 AMVcp (elisa) ap a The negative control was bombarded with TE buffertreated tungsten particles and regenerated on media not containing kanamycin.

Rambler Positive Control was a previously identified transgenic alfalfa plant shown to contain and express the neomycin phosphotransferase gene (Hill et al., Bio/Technology, 9:373-377 (1991)).

In the third test, yet another embodiment of the invention is demonstrated and the affect of bombardment on he regeneration of transformed elite alfalfa varieties was examined. Immature cotyledons were excised from 11 day postpollination embryos. Somatic embryos were regenerated.

Somatic embryos were bombarded five times with tungsten particles adsorbed with the plasmid pPHI251 (Figure 1) and cultured on modified B5 media containing 25 mg/l kanamycin sulfate. Embryos were subcultured at 20 days post-bombardment to fresh modified B5 media containing 25 mg/l kanamycin sulfate. Green somatic embryos were harvested 50 days post bombardment and matured on MS medium containing 100 mg/l kanamycin sulfate. Leaf samples were taken at 80 days post- 19 bombardment and assayed for neomycin phosphotransferase activity. The results are shown in Table 7.

Table 7 Yam93 Regenerant NPTII Activity (pg/pg Total Protein) CB93.1 11 CB93.2 13 CB93.3 3 1. 5 CB93.4 8 CB93.5 4 CB93.6 9 Yam93 negative controla 0 Rambler 1 0-1- 1 b 2 a The negative control plant was regenerated from bombarded immature cotyledons bombarded with S* TE-buffer treated tungsten particles.

b Rambler 10-1-1 was a previously identified transgenic plant shown to contain and express the neomycin phosphotransferase gene.

[Hill, et al., Bio/technology, 9:373-377 (1991)].

The latter test demonstrates that when immature cotyledons are used to form somatic embryos, and then those embryos are bombarded, even more plants are recovered.

Furthermore, the resulting plant has been found to retain this ability to regenerate. Elite varieties can not only be regenerated, but also retain this property.

It can further be seen that bombardment of the immature embryos or somatic embryos does not adversely affect 20 regeneration and that DNA is expressed in these now regenerable cells and plants.

The foregoing demonstrates transformation of M1edicago sativa, transformation~ with particle acceleration, and that substantially improvea regeneration of Medicag0 sativa is possible by he use of immature cotyledons. Regeneration of varieties not previousiy regenerated or with very poor regeneration is acineved. Thus, transformation of these sawr varieties is now possi!b Thus, in can Ze seen Ehe invention accomplishes its objectives.

21

Claims (21)

1. A process for regeneration of alfalfa comprising initiating somatic embryogenesis of cells of immature cotyledons of alfalfa.

2. The process of claim 1, wherein the immature cotyledon cells are up to 25 days past S pollination.

3. The process of claim 1 or claim 2, wherein the immature cotyledon cells are 10 to days past pollination.

4. The process of any one of claims 1 to 3, wherein the immature cotyledon cells are days past pollination. to

5. The process of any one of claims 1 to 4, wherein the cotyledon is translucent to light green.

6. The process of any one of claims 1 to 5, wherein an elite variety of alfalfa is regenerated.

7. The process of any one of claims 1 to 5, wherein the variety is one of the varieties listed in Table 13

8. The process of any one of claims 1 to 7, wherein the immature cotyledon is excised from seed embryos of alfalfa, the cotyledon placed in contact with an auxin to induce cell division and growth, causing somatic embryogenesis from the immature cotyledon.

9. A process for regeneration of alfalfa, substantially as hereinbefore described with reference to any one of the examples. 20

10. A process for improving regeneration ability of an alfalfa plant comprising initiating *oo. somatic embryogenesis of cells of immature cotyledons of the plant and culturing the somatic embryo into a mature alfalfa plant.

11. The process of claim 10, wherein somatic embryogenesis is initiated of cells of immature cotyledons of varieties of alfalfa not regenerable, and the embryo cultured into a regenerable plant.

12. A process for improving regeneration ability of an alfalfa plant, substantially as hereinbefore described with reference to any one of the examples. S.

13. A process of transforming foreign DNA into alfalfa plants comprising introducing the DNA into cells of immature cotyledons of alfalfa and cultivating the cells with foreign DNA into alfalfa plants.

14. The process of claim 13, wherein the immature cotyledons are 10 to 15 days past ;0 pollination.

The process of claim 13 or claim 14, wherein the immature cotyledons are days past pollination.

16. The process of any one of claims 13 to 15, wherein the immature cotyledons are translucent to light green in colour. [1 :\DAYI-IB\LIBFF]08539A.DOC:gcc 23

17. The process of any one of claims 13 to 16, wherein the foreign DNA is attached to a carrier particle, the particle is accelerated at an immature cotyledon such that at least some of the DNA is introduced in the interior of the immature cotyledon cells, the tissue is cultured on a growth promoting medium, and the resulting growth cultivated into a whole mature alfalfa plant containing the introduced DNA.

18. The process of any one of claims 13 to 16, wherein the foreign gene is prepared as a plasmid, hosted in Agrobacteria tumefaciens and the Agrobacteria tumefaciens combined with the immature cotyledon alfalfa cells such that the foreign DNA is transformed into the alfalfa cells.

19. The process of any one of claims 13 to 18, wherein the DNA is transformed into elite in varieties of alfalfa.

An alfalfa plant regenerated by the process of any one of claims 1 to 9.

21. An alfalfa plant transformed in accordance with the process of any one of claims 13 to 19. Dated 25 November, 1999 Pioneer Hi-Bred International, Inc. Patent Attorneys for the Applicant/Nominated Person 20 SPRUSON FERGUSON 0* 0 LF. *0 [I:\DAYLIB\LIBFF]08539A.DOC:gcc