# Patent

## Title
Process for production of optically active oxazolidinone derivative

## Abstract

Beneficial growth of roots in dicotyledonous plants is achieved by genetic transformation with root-inducing A. rhizogenes, preferably the novel strain A. rhizogenes ATCC 39207. The A. rhizogenes not used to transform the plant is reduced to noninfective levels with A. radiobacter, preferably A. radiobacter K-84.

## Background
Agrobacterium rhizogenes is the causal organism of what has been termed "the hairy root disease" in a number of species of higher plants. A. rhizogenes can be isolated from the soil, and is a species of agrobacterium which has the following characteristics: aerobic, rod-shaped (0.8.times.1.50-30 micron), one to four peritrichous flagella, Gram negative, and nonspore forming. The common species are A. tumefaciens, which is tumor-causing; A. rhizogenes, which causes hairy-root disease; and A. radiobacter, which is nonpathogenic.

The potential of using A. rhizogenes to the benefit of man was mentioned in an article entitled, "Studies on Infectious Hairy Root of Nursery Apple Trees," Journal of Agricultural Research, Vol. 41, No. 7, pp. 507-540, A. J. Riker et al. The authors state at 537, "In preliminary experiments cuttings or layers of certain plants treated with the hairy-root organism have rooted sooner and more vigorously than those untreated. These results suggest the possibility of using this organism to stimulate root production in the propagation of certain plants. More work is necessary before conclusions can be drawn." Despite this invitation, A. rhizogeneshas not been actively pursued to the benefit of man, and remains regarded as an undesired pathogen.

It has now been found that one can effectually cause the controlled beneficial growth of roots in dicotyledonous plants by genetic transformation with root-inducing A. rhizogenes, preferably a novel strain designated as Agrobacterium rhizogenes ATCC 39207. The control exists in that functional A. rhizogenes has or exhibits an undesired potential to contaminate soil, either can be effectively controlled by the use of Agrobacterium radiobacter, preferably A. radiobacter K-84, used in conjunction with or following genetic transformation of the plant with A. rhizogenes. Independent of which A. rhizogenes is used, a plant is transformed by contact of the exposed pericycle of the plant with A. rhizogenes in an aqueous suspension or other medium, such as peat, preferably at an effective concentration of at least about 10.sup.8 cells per ml, for a period of time to achieve transformation. Uniform results are obtained by an exposure of at least about 20 hours. The presently preferred exposure for the stated concentration is at least about 24 hours. Irreversible transformation occurs. Any A. rhizogenes then remaining on the surface of the plant is superfluous to root development. It can therefore be reduced to noninfective levels by exposure to A. radiobacter. A simple dip is all that is required in an aqueous suspension in which the A. radiobacter concentration is from about 10.sup.7 to about 10.sup.9 cells/ml. Conveniently, it has been found that transformation may be accomplished by combining A. rhizogenes with A. radiobacter in the same treating solutions, in a cell ratio of at least about 4 to 1, preferably from about 4 to 1 to about 8 to 1 A. rhizogenes to A. radiobacter. Presently preferred solutions contain about 10.sup.9 cells of A. rhizogenes and about 2.5.times.10.sup.8 cells of A. radiobacter per ml. Contact, again, is for at least about 20 hours. With such treatment, essentially no viable A. rhizogenes remains on the exposed surface of the transformed plant to create a problem of soil contamination.

In either event, both A. rhizogenes and A. radiobacter, alone or in combination, will withstand pelletizing by centrifugation and lyophilization to yield dry, viable cells. In addition, they may be, and preferably are, supported by other media such as neutral to alkaline peat to be used directly by application to the receptive zone of the plant for the prescriptive period of time or from which the organism(s) are extracted for use in aqueous solution.

A. rhizogenes ATCC 39207 has been established to be more effective than its source for root development exhibiting the potential of doubling root growth in the same amount of time.

FIG. 1 is a photo of a control North Star cherry tree taken 45 days after planting, illustrating the degree of leaf development.

FIG. 2 is a photo of a transformed North Star cherry tree taken 45 days after planting, illustrating the increased leaf development.

FIG. 3 illustrates carrot disks cut from the same carrot. With reference thereto, disk 10 is a control as was not transformed. Disks 12 and 14 were transformed with A. rhizogenes TR 105. Disk 16 was transformed with A. rhizogenes ATCC 39207.

According to the present invention, there is provided a novel strain of A. rhizogenes, namely, A. rhizogenes ATCC 39207, which was isolated from A. rhizogenes TR 105, which may be used alone or in combination with other root-inducing A. rhizogenes for genetic transformation of dicotyledonous plants to enhance root development, with control of the A. rhizogenes being available through the use of A. radiobacter, preferably A. radiobacter K-84.

A plant genetically transformed with A. rhizogenes may be subsequently treated with A. radiobacter or the two combined, to achieve transformation without leaving an infective residue of A. rhizogenes on the surface of the plant. If the two are combined, they may be combined in a cell ratio of about 4 to 1 or more A. rhizogenes to A. radiobacter, preferably from about 4 to 1 to about 8 to 1, with A. radiobacter serving to substantially prevent the A. rhizogenes contaminating the soil without interfering with plant transformation. Transformation of a plant with A. rhizogenes in all cases requires exposure to the pericycle, since the organism is noninvasionary. Exposure is for a time sufficient for genetic transformation to occur. It is presently preferred that effective exposure to transforming A. rhizogenes be for at least about 20 hours, preferably at least about 24 hours. Transformation generally requires exposure of A. rhizogenes to a wound of the plant, either created by means of a cut (whether or not new) or simply through severed roots. A. rhizogenes concentration in a treating medium is normally at least about 10.sup.8 cells/ml, preferably at least about 10.sup.9 cells/ml. Lower concentrations are also useful but may require longer exposure for transformation to occur. It may be provided as an aqueous solution or through a carrier, such as peat, which is brought into contact with the receptive, exposed surface of the plant, and from which transformation occurs by cell diffusion. In the alternative, the contained organism(s) may be extracted from the peat into an aqueous solution used for transformation of the plant.

Benefits of the use of A. rhizogenes in accordance with the instant invention are many. It may be employed, first of all, to promote early rooting of bare-root stock, leading to more leaf development, both in size and number; less die-back of limbs and stems; an increase in opportunity for fruit development during first-season planting; reduced pruning of the tree prior to planting; and, significantly, increasing the potential of the plant to survive drought. The same benefits may be induced to plant cuttings, where cut stems are placed in mist benches until sufficient roots develop. In this instance, more root development per unit of time can be expected by practice of the instant invention. The invention may also be employed in transplanting of larger plants, such as trees, which are well known to undergo shock during the process of transplanting. Transformation with A. rhizogenes through cut roots can be used to promote rapid development of new roots at locale of root cleavage, allowing adequate moisture take-up by the tree to prevent leaf-drop and tree death.

Finally, plants suffering from root damage or disease may also be benefited by treatment with A. rhizogenes to promote the rapid development of new root growth to counteract those undergoing degeneration.

Despite the potential benefits of the invention, A. rhizogenes may remain regarded as a soil pathogen. In this instance, its effect can be eliminated or diminished to noninfective levels through the use of A. radiobacter. A. radiobacter K-84 is presently preferred. A. radiobacter K-84 is cultured and sold by AgBioChem, Inc., 3 Fleetwood Court, Orinda, Calif. 94563. One can simply dip the transformed plant into an aqueous solution of A. radiobacter, preferably present in a concentration in excess of about 10.sup.7 cells/ml, for but seconds to effectively terminate the active A. rhizogenes.

More conveniently, the two may be combined in a common transforming medium. When present in a cell ratio of A. rhizogenes to A. radiobacter of at least about 4 to 1, preferably from about 4 to 1 to about 8 to 1, transformation can occur without leaving a residue of active A. rhizogenes on the plant. In this instance, as in the instance of the use of A. rhizogenes alone, transformation requires exposure of a receptive plant area to the transforming organism. However, the A. radiobacter does not appear to interfere with the transformation process.

Peat has been found to be an excellent host for the organisms. Organisms have been found to thrive in essentially neutral to alkaline (pH) peat, which serves as a convenient carrier and an effective treating medium. Lyophylization can also be employed. If peat is employed, the A. rhizogenes is kept separate from the A. radiobacter and the peat units carrying them are combined at the time of use.

Without limitation, the following illustrate the invention. In connection with plantings in ground, plantings, unless otherwise indicated, were made during the normal planting season for the 45th parallel for the State of Montana, United States of America.

Bare-root plum trees averaging from 4 to 5 feet in height, were root-wounded, then treated with A. rhizogenes TR 105, provided by Dr. Nester, Department of Microbiology and Immunology, University of Washington, Seattle, Wash. 98195, by submersing the wounded area of the plant into a solution containing A. rhizogenes at a cell-suspension concentration of about 9.times.10.sup.9 cells per millimeter for 24 hours. The control roots were submerged in water alone. Treated and control stocks were stored at 40.degree. F. until time for planting, and were then planted in a soil/vermiculite 1:1 mix in the field. The trees were watered every three days for nine days, then natural rainfall was relied upon for root development. After a period of about 30 days, the plants were gently removed from the soil and rinsed with water. The new roots were picked, with the aid of tweezers, from the old roots, then dried and weighed. The results are shown in Table I. ______________________________________ NEW ROOT WEIGHT Transformed Example  Plant (g)       Control  (g) ______________________________________ 1        .025            A        .004 2        .018            B        .009 3        .019            C        .014 4        .012            D        .006 -x       .019            x        .008 ______________________________________ Significant difference is at the level

Significant difference is at the 0.1 level

Other plum trees from the same source as those used for Examples 1 to 4 and Controls A to D were treated in the same manner, but measured for leaf development. The measurements were respectively taken at the end of midsummer and about two weeks later. The results are shown in Table II. ______________________________________ LEAF DEVELOPMENT Transformed Plant Leaf Size  No. of  Con- Leaf Size No. of Example (cm)       Leaves  trol (cm)     Leaves ______________________________________ 5      3.919 length 155     E    2.028 length 53 2.725 width             1.740 width 6      4.183 length 170     F    1.920 length 104 2.253 width             1.680 width 7      5.124 length 113     G    2.529 length 115 4.001 width             2.109 width 8      4.501 length 158     H    2.348 length 147 4.173 width             2.440 width Total             596                   419 ______________________________________

All differences between treatments and controls are significant at the 0.01 level.

North Star cherry trees averaging about 4 feet tall taken from bare-root stock were root-wounded for both the treatment and controls. Following the procedure of Examples 1 to 4, injured roots were transformed with a solution containing about 1.6.times.10.sup.10 cells of A. rhizogenes TR 105 per milliliter, soaked overnight, and left in dark, cold storage for 18 days. Planting occurred early in the planting season. The transformed plants blossomed five days after budding. It took the controls an additional 10 days to blossom. Approximately 45 to 50 days after planting, cherries were observed on the transformed plants, and a leaf, stem and cherry count was made in midsummer. With significant difference at the 0.05 level, the comparison of transformed to control, the number of leaves, number of cherries, number of branches, die-back and leaf length were observed, as reported in Table III. FIG. 1 is a photo of control plant about 45 days after planting. FIG. 2 is a photo of a transformed plant which has been exposed to the same growth conditions as those of the plant shown in FIG. 1, except for the use of A. rhizogenes to promote root growth. In addition to the apparent differences, there was also more root spur formation, which is indicative of higher flower and root development in the following years of growth. ______________________________________ No. of    No. of No. of Ripened   Branches  Leaf Leaves Cherries  Died Back Length (cm) ______________________________________ Example #9 590     3         6/13    6.5 Example #10 707     9         5/14    6 Example #11 593     0         1/07    6 Control I 0      0         15/15   -- Control J 351     0         10/16   4.2 Control K 374     0         6/10    2.5 ______________________________________

A. rhizogenes ATCC 39207 was derived from Agrobacterium rhizogenes TR 105 by screening isolated single colonies of TR 105 on carrot root disks until a change was observed in the degree of root development. A treating solution was made of Agrobacterium rhizogenes TR 105 to a to a cell concentration of 2.1.times.10.sup.9 cells per milliliter, and a comparison solution of A. rhizogenes ATCC 39207 at a cell concentration of 4.times.10.sup.9 cells per milliliter. Carrot disks were treated at a concentration of 0.1 milliliter per carrot disk and incubated for three weeks at room temperature. The results of the degree of root formation are shown in Table IV and illustrated in FIG. 3, explained at page 4 herein. ______________________________________ Carrot #1   Carrot #2  Carrot #3  Carrot #4 ______________________________________ A. rhizogenes TR 105 .0025      .0054      .0000    .0006 .0010      .0015      .0060    .0003 .0011      .0009      .0037    .0016 .0030      .0027      .0020    .0016 .0043      .0015      .0003    .0009 .0076      .0027      .0017    .0009 .0030      .0001      .0034    .0001 .0053      .0044      .0042    .0012 .0030      .0046      .0013    .0016 .0096      .0021      .0047    .0005 -x   .0040      .0026      .0027    .0009 A. rhizogenes ATCC 39207 .0024      .0000      .0001    .0000 .0101      .0003      .0015    .0013 .0036      .0010      .0022    .0003 .0113      .0012      .0007    .0006 .0118      .0063      .0012    .0005 .0093      .0018      .0035    .0027 .0078      .0004      .0058    .0002 .0123      .0034      .0019    .0031 .0127      .0035      .0011    .0010 .0006      .0059      .0051    .0002 -x   .0082      .0024      .0023    .0010 ______________________________________

Data x for Carrot #1 are different TR 105 from ATCC 39207 at 0.05 level.

In the above work, cell density was about 130 cells per square inch of carrot-disk surface.

If a plant has the potential of being an excellent rooter, A. rhizogenes ATCC 39207 will at least double the dry weight of roots; otherwise, no difference is shown. A. rhizogenes ATCC 39207 is on viable deposit at the America Type Culture Collection, 12301 Parklawn Dr., Rockville, Md. 20852, and was initially given the identification Agrobacterium rhizogenes MT 232 by me.

Avocado-stem cuttings 10-12 cm in length were used for the control (80 test plants) and for transformation (100 test plants). To transform the plants, the cuttings were dipped in a suspension containing A. rhizogenes TR 105 at a concentration of 10.sup.9 cells/ml for 15 minutes. This insured inoculation with sufficient cells to achieve transformation. The cuttings were planted in a rooting mixture of 40% by weight perlite and 60% by weight peat. The cuttings were placed on a cloud 9 fogging system mist bench, and every 10 minutes the mist was applied during daylight hours only (16-hour days). Two months after planting, it was determined that 5 of the 80 control plants were alive and had produced leaves and roots, while 61 of the 100 transformed plants were alive and had produced leaves and roots.

The following is to establish that A. rhizogenes is effective in promoting root development in plants that are vegetatively propagated. Cuttings of stems with leaves of violets were dipped into a suspension of A. rhizogenes at a concentration of about 10.sup.9 cells per milliliter, and placed on the mist bench until rooting occurred. In this instance, the control plants gave an average of 0.002 grams dry weight of root development per plant, whereas transformed cuttings gave an average of 0.005 gram of roots per plant on a dry-weight basis. This is a significant difference at the 0.1 level. The observations were made two weeks after placement on the mist bench and shown in Table V. ______________________________________ DRY WEIGHT OF ROOTS PRODUCED ON VIOLET-STEM CUTTINGS Control M                 Example 14 ______________________________________ .0015                 .0043 .0042                 .0038 .0011                 .0040 .0054                 .0063 .0002                 .0030 .0020                 .0028 .0010                 .0036 .0022                 .0034 .0014                 .0103 .0012                 .0026 .0012                 .0054 .0004                 .0064 .0025                 .0056 .0023                 .0086 .0028                 .0050 -x =    .0020          -x =   .0050 ______________________________________

Mango cuttings of approximately 10 cm. in length were transformed by dipping the lower 7 cm. into a bacterial suspension containing 7.times.10.sup.5 cells of A. rhizogenes per milliliter. Forty mango cuttings were used for each test. The transformed cuttings and controls were placed on the mist bench and maintained at 70.degree.-80.degree. F. In the case of the controls, 41.0.+-.7.5% of the cutting developed plants, whereas the A. rhizogenes-treated cuttings had 79%.+-.7% develop into plants.

To establish that A. rhizogenes ATCC 39207 does not contaminate the rhizoplasm or nearby soil, Agrobacterium radiobacter K-84 was used as a co-inoculant to treat the plant following treatment with A. rhizogenes separately or in conjunction therewith. Treatments following the contact with A. rhizogenes was only by a dip in the suspension of A. radiobacter. When used in combination, the cell ratio of A. rhizogenes to A. radiobacter was approximately 4 to 1, A. rhizogenes being present in a concentration of about 10.sup.9 cells per milliliter, and the A. radiobacter in a concentration of 2.5.times.10.sup.8 cells per milliliter. In all instances, transformation with A. rhizogenes occurred during 24 hours. After rooting three to four weeks, carrot disks were rinsed with 0.1 milliliter of water, the rinse did not yield any infectious colonies of A. rhizogenes when placed onto another carrot disk.

## Claims

1. Biologically pure Agrobacterium rhizogenes ATCC 39207.

2. A composition for the genetic transformation of dicotyledonous plants for the beneficial growth of roots which comprises: provide an aqueous suspension of cells in a concentration sufficient to genetically transform the dicotyledonous plant to enhance root growth; and substantially noninfective levels.

3. A composition as claimed in claim 2 in which the A. rhizogenes and the A. radiobacter are contained as separate units, with at least one being present in peat.

4. A composition for the genetic transformation of dicotyledonous plants for the beneficial growth of roots which comprises: aqueous suspension of cells thereof in a concentration of at least about 1.07 cells/ml; and cells per milliliter such that cell ratio of A. rhizogenes to A. radiobacter is at least about 4 to 1.

5. A composition as claimed in claim 4 in which the A. rhizogenes and the A. radiobacter are contained as separate units, with at least one being present in peat.

6. A composition as claimed in claim 4 in which the cell ratio of A. rhizogenes to A. radiobacter is from about 4 to 1 to about 8 to 1.

7. A composition as claimed in claim 5 in which the cell ratio of A. rhizogenes to A. radiobacter is from about 4 to 1 to about 8 to 1.

8. A composition as claimed in claim 4 in which the A. rhizogenes comprises A. rhizogenes TR 105 and in which the A. radiobacter comprises A. radiobacter K 84.

9. A composition as claimed in claim 4 in which the A. rhizogenes comprises A. rhizogenes ATCC 39207.

10. A composition as claimed in claim 4 in which the A. radiobacter comprises A. radiobacter K 84.

11. A composition as claimed in claim 9 in which the A. radiobacter comprises A. radiobacter K 84.

12. A process for genetically transforming dicotyledonous plants which comprises contacting roots of the dicotyledonous plant having exposed pericycle to the transforming action of root-inducing A rhizogenes for a time sufficient to transform the dicotyledonous plant for enhanced root growth, and contacting the plant with A. radiobacter for a time sufficient to diminish the A. rhizogenes to substantially noninfective levels.

13. A process as claimed in claim 12 in which the A. rhizogenes comprises A. rhizogenes TR 105.

14. A process as claimed in claim 12 in which the A. rhizogenes comprises A. rhizogenes ATCC 39207.

15. A process as claimed in claim 11 in which the A. radiobacter comprises A. radiobacter K-84.

16. A process for genetically transforming dicotyledonous plants which comprises contacting roots of the dicotyledonous plant having exposed pericycle to the action of A. rhizogenes in a medium in which root-inducing A. rhizogenes is present in a concentration equivalent of an aqueous suspension of at least 10.sup.8 cells/ml for at least about 20 hours and sufficient to genetically transform the dicotyledonous plant and enhance root growth, then contacting the plant with A. radiobacter present in a medium in which the concentration of A. radiobacter is present in a concentration equivalent a suspension of about 10.sup.7 cells/ml for a time sufficient to diminish A. rhizogenes to substantially noninfective levels.

17. A process as claimed in claim 16 in which contact with A. rhizogenes is for at least about 24 hours.

18. A process as claimed in claim 17 in which the A. rhizogenes comprises A. rhizogenes TR 105.

19. A process as claimed in claim 17 in which the A. rhizogenes comprises A. rhizogenes ATCC 39207.

20. A process as claimed in claim 17 in which the A. radiobacter comprises A. radiobacter K-84.

21. A process as claimed in claim 18 in which the A. rhizogenes comprises A. rhizogenes TR 105.

22. A process as claimed in claim 18 in which the A. rhizogenes comprises A. rhizogenes ATCC 39207.

23. A process for genetically transforming dicotyledonous plants which comprises exposing roots of a dicotyledonous plant having a pericycle receptive to A. rhizogenes to a mixture of root-inducing A. rhizogenes and A. radiobacter contained in a medium wherefrom the A. rhizogenes can transform the dicotyledonous plant upon exposure of at least about 20 hours, the A. rhizogenes being present in the medium in a quantity equivalent of a suspension concentration of at least 10.sup.8 cells/ml of water and in which the cell ratio of A. rhizogenes to A. radiobacter is at least 4 to 1.

24. A process as claimed in claim 23 in which transformation occurs in at least about 24 hours.

25. A process as claimed in claim 23 in which the medium is aqueous and the A. rhizogenes and the A. radiobacter are contained in an aqueous suspension and in which the cell ratio of A. rhizogenes to A. radiobacter is from about 4 to 1 to about 8 to 1.

26. A process as claimed in claim 23 in which the A. rhizogenes and the A. radiobacter are provided from peat.

27. A process as claimed in claim 25 in which the A. rhizogenes and the A. radiobacter are provided from peat.

28. A process as claimed in claim 23 in which the A. rhizogenes comprises A. rhizogenes ATCC 39207.

29. A process as claimed in claim 23 in which the A. rhizogenes comprises A. rhizogenes ATCC 39207.

30. A process as claimed in claim 25 in which the A. rhizogenes comprises A. rhizogenes ATCC 39207.

31. A process as claimed in claim 12 in which the A. radiobacter comprises A. radiobacter K-84.

32. A process as claimed in claim 24 in which the A. radiobacter comprises A. radiobacter K-84.

33. A process as claimed in claim 25 in which the A. radiobacter comprises A. radiobacter K-84.

34. A process as claimed in claim 26 in which the A. radiobacter comprises A. radiobacter K-84.

35. A process as claimed in claim 27 in which the A. radiobacter comprises A. radiobacter K-84.

36. A process for genetically transforming dicotyledonous plants which comprises exposing roots of a dicotyledonous plant having a pericycle receptive to A. rhizogenes to A. rhizogenes ATCC 39207 in a medium wherefrom the A. rhizogenes ATCC 39207 can transform the dicotyledonous plant upon exposure of at least about 20 hours.

37. A process as claimed in claim 36 in which the transformation occurs in at least about 24 hours.

38. A composition for the genetic transformation of dicotyledonous plants for the beneficial growth of roots which comprises: rhizogene TR 105 and ATCC 39207 in a concentration sufficient to provide an aqueous suspension of cells thereof in a concentration of at least about 10.sup.7 cells/ml; and in cells per milliliter such that cell ratio of A. rhizogenes to A. radiobacter is at least about 4 to 1 to about 8 to 1.

39. A process for genetically transforming dicotyledonous plants which comprises exposing roots of a dicotyledonous plant having a pericycle receptive to A. rhizogenes to a mixture of root-inducing A. rhizogenes selected from A. rhizogenes TR 105 and A. rhizogenes ATCC 39207 and A. radiobacter K-84 contained in a medium wherefrom the A. rhizogenes can transform the dicotyledonous plant upon exposure of at least about 20 hours, the A. radiobater K-84 being present in the medium in a quantity equivalent of a suspension concentration of at least 10.sup.8 cells/ml of water and in which the cell ratio of A. rhizogenes to A. radiobacter is at least 4 to 1 to about 8 to 1.