Proceedings of the South Dakota Academy of Science,Vol. 82 (2003) 113

MORPHOLOGICAL EXAMINATION OF PRAIRIE TURNIP (PSORALEA ESCULENTA PURSH) ROOT

April L. Stahnke and R. Neil Reese Biology & Microbiology South Dakota State University Brookings, SD 57007

ABSTRACT

Psoralea esculenta Pursh is an herbaceous perennial legume, native to the Great Plains, that has a tuberous-thickened taproot. We have been examining its potential food value and unique anatomy as part of a larger ethnobotany program because this plant has long used been used by Native American pop- ulations as food source. The Psoralea root has a tough inedible outer cover- ing (bark) and a fleshy interior that contains starch and protein reserves. The fleshy root shows development of visible rings early in its first year of growth and contains numerous isolated vascular strands. Root tissues from mature and young roots were fixed and embedded in JB-4 plastic. Differential staining methods using Safranin and Fast Green, Toluidine Blue, IKI, and Aniline Blue were used to examine the tissue morphology. Digital pictures were made us- ing a Fuji camera attached to an Olympus bright field microscope. The root of Psoralea esculenta possesses a unique arrangement of vascular tissues embed- ded in secondary parenchyma with regions of cells containing large deposits of either protein or starch. Further analysis at different developmental stages will help to clarify the unique anatomical features observed in the root of Pso- ralea esculenta.

INTRODUCTION

Psoralea esculenta Pursh is an herbaceous perennial legume, native to the Great Plains, that has a tuberous-thickened taproot. The plant emerges in late spring to early summer in the Northern Great Plains. Flowering occurs from the first to the middle of July, with fruit maturation near the end of July. Abscis- sion of the shoot system happens with the dehiscence of the legume, as a re- sult of the formation of an abscission layer that is usually about 2-5 cm below ground. The upper, herbaceous portion of the stem is then released and the seeds are scattered as the shoot system tumbles across the prairie. Formation of shoot buds occurs on the underground crown of the woody portion of the stem, with the development of the tuber-like growth an additional 2-5 cm be- low the crown (Fig. 1). Native Americans used the root of the prairie turnip as a staple in their di- et. The tuber has been found to contain about 7.5% protein and up to 70% starch (Perera and Reese, 2002). Field observations suggest that the starch and protein are stored in parenchymous tissues that expand laterally each year. 114 Proceedings of the South Dakota Academy of Science,Vol. 82 (2003)

MATERIALS AND METHODS

Collection of plant material

Plant materials from the greenhouse and field were examined. Seeds were scar- ified in November, planted into four inch pots, and placed on a mist-bench. After three months the seedlings were removed Figure 1. Photograph of a herbarium mount of Psoralea esculenta showing the general structure, from the pots and cleaned with the underground crown and storage tissues with water, cut into pieces labeled. and fixed in AFA (50 parts ethanol, 6 parts formalin, 4 parts acetic acid and 40 parts deionized water) in preparation for plastic infiltration. Field material was collected at the end of January from the Native garden test plots at SDSU. Roots, from plants that were 3 years old, were excavated, rinsed in DI water and prepared as above for plastic infiltration.

JB-4 Hydrophilic plastic infiltration of field and seedling root material

JB-4 hydrophilic plastic (Polysciences Inc.) was used to embed the plant tissue following the protocols provided by the manufacturer. .

Sectioning tissue

Cross sections, tangential sections, and longitudinal sections were made from the seedling roots and field root material. The plant tissue was sectioned to a thickness of 4 microns using an Olympus Cut 4060E Rotary Microtome with a steel knife. The sections were removed with forceps and placed into wa- ter containing drops of ammonium hydroxide to relax the plastic. The sections were then placed onto glass slides previously coated with poly-L-lysine and stained appropriately.

Staining for protein and general morphology

After the tissue had dried the tissue sections were stained with 1% aque- ous toluidine blue, fast green & safranin (1% aqueous safranin followed by 0.5% Fast Green in 50 % ethanol), iodine (2% KI with 0.2% Iodine), or 1% Coomassie Brilliant Blue in 50% ethanol. Toluidine blue (hydrophilic) is a com- mon stain which stains proteins, cell walls, nucleic acids, and RNA. Fast green (lipophilic) and safranin (hydrophilic) were used in combination. Fast green stained the cytoplasm and membranous tissue green and safranin stained the Proceedings of the South Dakota Academy of Science,Vol. 82 (2003) 115

Figure 2. Micrographs of Psoralea esculenta root sections stained with Toluidine Blue. A) Cross section of the upper portion a 2 month old plant showing the presence of a central pith in the developing storage tissue - 10X objective. B) Cross section of the base of a mature storage root showing xylem vessel elements in the central vascular cylinder - 40X objective. cell walls and phloem red. Iodine was used to stain for starch while Coomassie Brilliant Blue was used to stain for protein.

Micrographs made with an Olympus AX70 bright field microscope

Micrographs were made with a Fuji film cam- era attached to an Olympus AX70 bright field micro- Figure 3. Near median cross section of a Psoralea scope. The objectives used esculenta storage root stained with Safranin and were the 4X, 10X, 20X, and Fast Green. Elongation of cells can bee seen to be 40X. in a radial orientation - 4X objective.

RESULTS AND DISCUSSION

The thickened root of Psoralea esculenta appears to be developmentally similar to that of Daucus and Beta in that the storage tissue seems to contain both hypocotyl tissue and the upper portion of the taproot (Esau, 1965). Cross sections through the upper portion of the storage tissue reveal that there is a central pith surrounded by the xylem, while cross sections of the base show a 116 Proceedings of the South Dakota Academy of Science,Vol. 82 (2003)

central vascular cylinder with the xylem vessel ele- ments centrally distributed forming a loose diarch to triarch pattern (Fig. 2 A and B). The developing storage tissue appears to be pre- dominantly xylem parenchyma laid down by vascular cambia with much lateral elongation. In cen- tral sections of mature stor- age tissues, many of the cells are 3 or more times as long in the lateral (radial) direction as compared to Figure 4. Cross section of a 2 month old Psoralea their vertical axis. In some esculenta storage root. The annular pattern of vas- younger tissues (1 to 2 cular tissue deposition is visible - 4X objective. months old), there appears to be more than one “an- nual” ring, which may indi- cate that the production of the xylem is periodic or that, as with sugar beets, the product of supernumer- ary cambia (Esau, 1965). The storage of starch and proteins occurs predominantly in the xylem parenchyma. Clusters of cell containing either starch or protein are found juxta- posed to each other, but do Figure 5. IKI-stained section of mature Psoralea not appear to commingle. esculenta storage-root tissue. The large starch The starch and protein can grains are darkly stained. Protein bodies, un- be seen to be in membrane stained, can be observed in adjacent cells - 40X bound vesicles. Those con- objective. taining starch are large and appear to represent amylo- plasts, while the proteins occur in smaller protein storage vesicles similar to those found in seeds (Taiz and Zeiger, 2002) Analyses of these storage tissues indicates that they contain 7.5% protein and 70% starch by weight (Perera and Reese, 2002). Psoralea has been a staple of the indigenous peoples of the northern plains (Kindscher, 1987). Morphologically their development appears to follow that of other root crop species. However, the development of both protein and starch Proceedings of the South Dakota Academy of Science,Vol. 82 (2003) 117 storage parenchyma makes them somewhat unique. Planting of this species in the South Dakota State Uni- versity Plant Science Re- search Farm in Brookings, SD indicates that develop- ment of this structure re- quires several years to reach maturity. Growth of the storage organ occurs as a result of the production of xylem tissues by the vas- cular cambium and per- Figure 6. Mature Psoralea esculenta storage-root haps additional supernu- stained with Coomassie Brilliant Blue. Small pro- merary cambia. This aspect tein-containing vesicles are darkly stained. Un- of development, however, stained starch grains can be seen in adjacent cells - 10X objective. requires further study for complete elucidation.

REFERENCES

Esau, K. (1965) The Root, Plant Anatomy (2nd Edition) John Wiley and Sons, Inc., New York. Pp. 481-530. Kinscher, K (1987) Edible wild plants of the prairie. Univ. Press Kansas, Law- erence. Pp. 183-189. Perera, G. and Reese, R.N. (2002) Nutritional value of Psoralea esculenta. SD Acad. Sci. 81:271. Polysciences Inc.2003. JB-4 Embedding Kit. Warrington, PA. Taiz, L. and Zeiger, E. (2002). Plant Physiology ( 3rd Edition) Sinauer Associ- ates, Inc. Sunderland, MA. Pp. 484-486.