regeneration via somatic embryogenesis is idealfor Harada, D. 1992. How to grow Limonium. FloraCulture International, Nov- mass propagation of genetically identical and disease-free Dec 1992, pp. 22-25. . It is superior to regeneration through organogenesis Harazy, A., B. Leshem, A. Cohen and H. D. Rabinowitch. 1985. In vitro prop agation of statice {Limonium sinuatum) as an aid to breeding. HortScience since it will avoid chimeric regenerants. Synthetic seed pro 20:361-362. duction is advantageous especially with Limonium species Janson, K. and M. Reid (1999) ATUT Cut Project: Opportunities for where seed viability is low or lost rapidly. Also, the method cut flower exports from Egypt - Economic assessment. Report on the web. could be adapted to mutant selection and gene transfer tech Kunitake, H. and M. Mii. 1990. Plant regeneration from cell culture-derived protoplasts of statice (Limonium, perezii Hubbzrd). Plant Sci. 70:115-120. niques (Gray et al., 1995). Experiments are in progress to op Linsmaier, E. M. and F. Skoog (1965) Organic growth factor requirements of timize the maturation and plant regeneration phases of tobacco tissue cultures. Physiol. Plant. 18:100-127. somatic embryogenesis in L. latifolium. Lledo, M. D., Crespo, M. B. andj. B. Amo-Marco. 1996. Micropropagation of Limonium thiniensis Erben (Plumbaginaceae) using herbarium material. Botanic Gardens Micropropagation News 2(2):l-5. Literature Cited Martin, C. and C. Perez. 1995. Micropropagation of five endemic species Limoniumfrom the Iberian peninsula. Jour. Hort. Sci. 70:97-103. Martin, C. and C. Perez. 1992. Multiplication in vitro of Limonium esteveiFdez. Aly M. A. M. and B. Rathinasabapathi. 2000. Somatic embryogenesis in Limo Casas. Ann. Bot. 70:165-167. nium bellidifolium (Plumbaginaceae). World Congress 2000 on In Vitro Bi Matsumoto, T., Y. Nako, C. Takahashi and A. Asakai 1997. Induction of in vit ology, June 10-15, San Diego, CA. (Abstr.). ro cultured masses of shoot primordia of hybrid statice and its cryopreser- Amo-Marco,J. B. and M. R. Ibanez. 1998. Micropropagation of Limonium ca- vation by vitrification. HortScience 32:309-311. vanillesii Erben, a threatened statice, from stems. Plant Murashige, T. and F. Skoog 1962. A revised medium for rapid growth and Growth Regul. 24:49-54. bioassay with tobacco tissue cultures. Physiol. Plant. 15:473-497. Devi, P. U., F. E. Solomon and A. C. Sharada. 1999. Plumbagin, aplant naphtho Rathinasabapathi, B., C. Sigua, J. Ho and D.A. Gage. 2000. Osmoprotectant quinone with antitumor and radiomodifying properties. Pharmaceutical B-alanine betaine synthesis in the Plumbaginaceae: 5-adenosyl-L-methion- Biol. 37:231-236. ine dependent N-methylation of B-alanine to its betaine is via iV-methyl Gray, D. J., M. E. Compton, R. C. Harrell and D. J. Cantliffe. 1995. Somatic and A/,Akiimethyl B-alanines. Physiol. Plant. 109:225-231. embryogenesis and the technology of synthetic seed. pp. 126-151. /wBajaj SeelyeJ., D.J. Maddocks, G. K. Burge and E.R. Morgan. 1994. Shoot regen YPS (ed.), Biotechnology in Agriculture and Forestry, Vol 30, Springer- eration from discs of Limonium perigrinum using thidiazuron. New Verlag. Zealand Jour. Crop and Hort Sci. 22:23-29.

Proc. Fla. State Hort. Soc. 113:172-174. 2000.

STRATIFICATION ENHANCES GERMINATION OF PURPLE CONEFLOWER ( ANGUSTIFOUA) AND ST. JOHN'S WORT () SEEDS

Gladis M. Zinati*, Herbert H. Bryan and Yuncong Li mination was less than 50% under dark conditions. Highest University of Florida germination of Hypericum perforatum seeds was attained with Tropical Research and Education Center stratification at 10 C for 30 days. Stratificationat 5 C for 20 18905 SW 280 Street days enhanced the rates of germination of both Echinacea and Hypericum seeds. Homestead, FL 33031

Additional index words. Light, medicinal plants, seed dor The use of purple coneflower and St. John's wort for me mancy, seed germination. dicinal purposes (Wijesekera, 1991), ornamental and land scape plantings (Cox and Klett, 1984) has increased significantly in the past few years. These herbaceous perenni Abstract. Usage of purple coneflower () and St. John's wort (Hypericum perforation) for medicinal, or als are native to North America and constitute the top selling namental and landscape purposes has increased significantly dietary supplements in the U.S. The raw material is used for in the United States. Seed germination and uniformity of emer numerous phytomedicines in Europe and many other coun gence for such herbs were found unsatisfactory without pre- tries (McKeown, 1999). treatments of seeds. Three 50-seed replicates of both The extracts produced from the purple coneflower Echinacea angustifolia and Hypericum perforatum were strat (Echinacea angustifolia var. angustifolia) stimulate the human ified in the dark at 5 and 10 C for 5,10, 20 and 30 days. Germi immune system and are commonly used for treating and nation counts on stratified and non-stratified seeds of both preventing the common colds and . On the other species were recorded on 3,7 and 10 days in darkness and in hand, the oil and extract producedfrom St. John's wort direct daylight at 24C. Echinacea angustifolia seeds had the {Hypericumperforatum) are used as an anti-depressive tonic, an highest percent germination (83%) when stratified at 1tfC for 30 days and germinated under indirect daylight, whereas, ger- anti-inflammatory and an analegesic to heal damaged tissues and nerves. In general, the economic production of perennial crops

Florida Agricultural Experiment Station Journal Series No. N-01918. requires high quality seeds with rapid synchronous germina ^Corresponding author, e-mail: [email protected]. tion. Propagation from seeds is considered the most efficient

172 Proc:Fla. State Hort. Soc. 113: 2000. method of producing plants for commercial production al Tables 1 and 2, respectively. Stratification of Echinacea seeds though they can be propagated asexually (Omidbaigi and at both 5°C or 10°C enhanced early (3rd day) germination Bernath, 1993; Zhang and Cheng, 1989; Zheljazkov et al., and total germination percentages (10th day) under indirect 1996). However, seeds of many native plants including Echina daylight for all stratification periods except 10 days (Tables 1 cea angustifolia and Hypericum perforatum have relatively com and 2). Highest rates of germination (50%) were attained by plex dormancy systems. The poor germination of these has the 3rd day when seeds were stratified for 20 days at 5 °C and been attributed to unfavorable environmental conditions then placed in indirect daylight. The highest germination during seed development, low seed vigor or to seed dormancy percent (83%) was attained when seeds were stratified for 30 (Wartidiningsih et al., 1994). days at 10°C and germinated in indirect daylight. The data The poorly understood physiological dormancy and from this study (Table 1) agree with the findings of Bratcher propagation requirements associated with many wildflowers et al., (1993) on E. purpurea in regard to the stratification pe serve to discourage growers and breeding nurseries. Dorman riod but disagree on stratification temperature. Prechilling cy ensures that seeds do not germinate until conditions are and presence of source of light during germination together optimal for seedling survival (Bewley and Black, 1982). Seeds enhanced seed germination over that of non-stratified seeds. of many species require presowing treatments to overcome On the other hand, although germination of Echinacea seeds dormancy. Seed dormancy for many plant species can be bro was enhanced by all treatments, it was less than 50% in dark ken by alternating prolonged warm stratification followed by conditions. 3 months of cold stratification (Chien et al., 1998), exposure to light during the prechill period of 12 weeks at 5°C, me chanical scarification and chemical treatments (Baskin et al., 1992). Chilling stratification is one of many presowing seed Table 1. Percent germination of E. angustifolia seeds, in indirect daylight, stratified for 5, 10, 20 and 30 days at 5 and 10°C. treatments that can be used to promote germination in spe cies with dormant seeds (Hartmann et al., 1990). Very few Indirect day light studies conducted on E. angustifolia but the results showed that soaking seeds in water or stratifying them for 8 weeks E. angustifolia Stratification percent gemination caused only 50% germination (Smith-Jochum and Albrecht, Temperature treatment 1987). Very few studies have been conducted to improve ger (°C) (day) 3rd day 7th day 10th day mination of E. angustifolia and H. perforatum seeds in a simple 5 5 32 49 59 efficient ways suitable for use by growers. 10 8 22 33 The objectives of this study were to determine the effects 20 50 61 71 of an initial treatment of stratification temperatures and 30 36 72 . 76 duration followed by retention either in darkness or exposed to indirect daylight for 10 days at 24°C on percent germina 10 5 21 33 45 tion and rate of emergence of E. angustifolia and H. perforatum 10 8 16 25 20 41 61 63 seeds. 30 41 80 83

24 5 1 11 16 Materials and Methods 10 0 16 21 20 0 16 24 Petri dish germination tests were conducted on E. angus 30 3 11 13 tifolia and H. perforatum seeds provided by Plantation Medici- nals, Inc., Felda, FL. The seeds were stored at 20 ± 4°C until beginning the stratification treatments.

Three 50-seed replications of both Echinacea and Hyperi Table 2. Percent germination of E. angustifolia seeds, in the dark, stratified cum were placed in zip-loc plastic bags and stratified at 5°C for 5, 10, 20 and 30 days at 5 and 10°C. and 10°C in darkness for 5, 10, 20 and 30 days. After the strat ification period, 25 seeds per treatment of each species were Dark

placed in 9-cm diameter plastic petri-dishes lined with blue E . anguslifolici blotting paper soaked with 10 ml of deionized water. Petri- Stratification percent gemination dishes containing prechilled seeds were then transferred to a Temperature treatment (°C) (day) 3rd day 7th day 10th day growth chambers at 24°C with indirect natural light of 12 hour daylength and without light. In addition, non-stratified 5 5 1 7 8 seeds were included for each treatment of each species. 10 34 48 48 The number of germinated seeds (radicle > 1 mm long) 20 12 23 23 of each treatment was recorded on the 3rd, 7th and 10th day. 30 17 32 33

The experiment was a completely randomized design. 10 5 3 8 9 10 5 9 12 20 12 17 17 Results and Discussion 30 28 36 37

Percent germination data of Echinacea seeds under indi 24 5 0 4 7 rect daylight and dark conditions and stratified at 5°C and 10 0 4 8 10°C for 5, 10, 20 and 30 days as well as corresponding data 20 0 1 4 7 9 from non-stratified seeds (control, 24°C) are presented in 30 0

Proc. Ra. State Hort. Soc. 113: 2000. 173 Table 3. Percent germination of H. perforation seeds, in indirect daylight, Table 4. Percent germination of H. perforatum seeds, in the dark, stratified stratified for 5, 10, 20 and 30 days at 5 and 10°C. for 5, 10, 20 and 30 days at 5 and 10°C.

Indirect day light Dark

H. perforatum H. perforatum Stratification percent gemination Stratification percent gemination Temperature trpntrnpnt — Temperature (°C) (day) 3rd day 7th day 10th day (°C) (day) 3rd day 7th day 10th day

5 5 0 75 76 5 5 0 43 57 10 1 73 73 10 0 39 45 20 31 64 67 20 3 27 27 30 4 69 73 30 3 13 21

10 5 16 71 77 10 5 0 19 21 10 15 67 67 10 4 59 59 20 19 61 64 20 1 27 28 30 5 77 85 30 1 1 1

24 5 1 25 48 Control 5 1 19 45 10 0 50 54 10 1 27 36 20 0 60 68 20 0 32 47 30 3 47 67 30 0 3 8

Similarly , the data in Table 3 show that stratification of H. Literature Cited perforatum seeds at 5°C and 10°C enhanced total germination percentage (10th day) under indirect daylight when seeds were Baskin C, J. M. Baskin and G. R. Hoffman. 1992. Seed dormancy in the prai stratified for 5, 10 and 30 days (Table 3). Hypericum perforatum rie forb Echinacea angustifolia van angustifolia (): After ripen ing pattern during cold stratification. Int. J. Plant Sci. 153:239-243. seeds stratified for 20 days at either 5 °C or 10°C did not differ Bewley, J. D. and M. Black. 1982. Physiology and biochemistry of seeds in re in percent germination from non-stratified seed in indirect lation to germination. II. Viability, dormancy and environmental control. daylight, but as the stratification period was extended to 30 Springer-Verlag, Berlin. days the percent germination of the stratified seeds was signifi Bratcher, C. B., J. M. Dole andj. C. Col. 1993. Stratification improves germi nation of five native wildflower species. HortScience 28:899-901. cantly higher than that of non-stratified seeds by the 10th day Chien, C. T., L. L. Kuo Huang and T. P. Lin. 1998. Changes in ultrastructure (85%). Possibly, the seeds developed a secondary dormancy on and abscisic acid level, and response to applied gebberelins in Taxus mairei initial exposure to low temperature, but continued stratifica seeds treated with warm and cold stratification. Annals of Botany 81:41-47. tion overcame this dormancy (Bewley and Black, 1982). Under Cox, R. A. andj. E. Klett. 1984. Seed germination requirements of native Col the dark conditions, the maximum germination percent was orado plants for use in the landscape. Plant Prop. 30:359-361. Hartmann, H. T., D. E. Kester and F. T. Davies, Jr. 1990. Plant propagation 59% when seeds were stratified for 10 days at 10°C (Table 4). principles and practices. 5th ed. Prentice Hall, Englewood Cliffs, N. J. The results of this study shows that stratification of H. per McKeown, K. A. 1999. Echinacea gives the United States an opportunity to foratum seeds for 5 days at either 5°C or 10°C will provide 76% put conservation policies into practice. Diversity 15:17-19. total germination, whereas stratification for 30 days at 10°C Omidbaigi, R. and J. Bernath. 1993. Correlation between cutting size and growth of licorice (Glycyrrhiza glabraL.). Acta Hort. 331:265-268. will provide 85% total germination under indirect daylight Smith-Jochum, C. C. and M. L. Albrecht. 1987. Field establishment of three conditions. In dark conditions, the percent germination was Echinacea species for commercial production. Acta Hort. 208:115-119. highest (59%) when seeds were stratified for 10 days at 10°C. Wartidiningsih, N., R. L. Geneve and S. T. Kester. 1994. Osmotic priming and Similar to E. angustifolia, exposure to light and prechilling chilling stratification improves seed germination of purple coneflower. were found to be important in breaking dormancy and im HortScience 29:1445-1448. Wijesekera, R. O. B. 1991. Plant-derived medicines and their role in global proving germination rate and total percent germination of health, pp. 1-18. InR. O. B.Jijesekera (ed.) The medicinal plant industry. H. perforatum seeds. CRC. Press, Boca Raton, Fla. In conclusion, the data demonstrate that dormancy can Zhang, S. Y. and K. C. Cheng. 1989. Angelica sinensis (Oliv.) Diels: In vitro be overcome and the highest total percent germination can culture, regeneration and the production of medicinal compounds. Bio- technol. Agr. For. 7:1-22. be attained when seeds of Is. angustifolia and H. perforatum are Zheljazkov, V., B. Yankov and Topalov. 1996. Comparison of three methods prechilled at 10°C in the dark for 30 days followed by germi of mint propagation and their effect on the yield of fresh material and es nation under indirect daylight. sential oil. J. Essent. Oil. Res. 8:35-45.

174 Proc. Ha. State Hort. Soc. 113: 2000.