Frequency and timing of leaf removal affect growth and podophyllotoxin content of Podophyllum peltatum in full sun.

Podophyllotoxin is a pharmaceutical compound found in leaves and rhizomes of American mayapple (P. peltatum L.), a species being investigated as an alternative to that of the Indian mayapple (P. emodi). Leaves alone can serve as a renewable source of podophyllotoxin (and other lignans) leaving rhizomes undisturbed to produce leaf biomass in subsequent years. It is not known, however, how frequently or severely plants can be defoliated without adversely affecting future plant growth, lignan content, or podophyllotoxin yield (g.m(-2)). This study compared harvest strategies that were mild to severe in frequency and timing of leaf removal. A wild population in full sun was subjected to leaf removal treatments of varying frequency (every year, every 2nd or 3rd year) and timing (early or late). Control plots not previously harvested were included every year. Plots were 1.0 m2 and established during spring of 2001. Duration of the study was four years. P. peltatum plants did not tolerate the most severe harvest treatment: annual harvest frequency in combination with early harvest time. Early annual harvests reduced total leaf dry mass and total leaf area in a consistent and linear manner. In contrast, plants tolerated annual harvests when conducted late in the growing season and tolerated early harvests when conducted every 2nd or 3rd year. The number of sexual shoots was reduced to zero by early annual harvests. Podophyllotoxin content was 2.7 to 6.5 times greater in leaves harvested early compared to those harvested late, though content was significantly greater in only two out of four years. In conclusion, we can recommend leaf removal every year from well-established P. peltatum populations grown in full sun if harvests are conducted late in the growing season. This harvest strategy ensures maximum podophyllotoxin yield without jeopardizing future leaf biomass yield. Leaves harvested early appear to have greater podophyllotoxin content, but we discourage early harvest every year. Instead, our results indicate that leaves can be harvested early every other year without reducing long-term performance of P. peltatum populations.

Arbuscular mycorrhizal inoculation enhances survival rates and growth of Micropropagated plantlets of Echinacea pallida.

In an attempt to induce positive effects on the acclimatization of in vitro propagated Echinacea pallida, four arbuscular mycorrhizal (AM) fungi, Glomus mosseae, Gigaspora ramisporophora, Scutellospora fulgida and Entrophospora colombiana were selected to aid the soil adaptation process. Fungal inocula affected the survival of E. pallida plantlets ranging from 83 % to 92 %, depending on the AM species and also contrasting with 58 % survival of the non-inoculated plantlets. Growth and development were faster in mycorrhizal treated plantlets than in non-treated ones, especially among those treated with Glomus mossae and Scutellospora fulgida. The presence of well-formed arbuscules and vesicles in Echinacea infested roots was confirmed by microscopic examinations in addition to 90 % success in the survival rate of vigorous plants indicated that mycorrhization is a valuable tool to overcome Echinacea acclimatization shock.

Variation of podophyllotoxin in leaves of Eastern Red Cedar (Juniperus virginiana).

Leaves of Eastern red cedar (Juniperus virginiana L. Cupressaceae) have been reported to contain podophyllotoxin, a pharmaceutical compound used to manufacture drugs for treatment of cancer, rheumatoid arthritis, genital warts, psoriasis, and multiple sclerosis. Podophyllotoxin content of leaves of immature, mature male, and mature female plants (approximately 1.45 mg x g -1) was significantly higher than that of leaves of juvenile plants (0.60 mg x g -1). Sampling date also affected podophyllotoxin content. Leaves harvested in January and April exhibited higher podophyllotoxin contents (1.56 and 1.45 mg x g -1, respectively) than leaves harvested in February and June (1.06 and 1.08 mg x g -1, respectively). There was no obvious pattern or trend in the data due to sampling date. There was no significant interaction between plant type and sampling date. These results indicate that foliage of mature Eastern red cedar, a waste product of the lumber industry, could be a low-yielding, but relatively stable, source of podophyllotoxin.

Micropropagation of Hydrastis canadensis: Goldenseal a North American endangered species.

An in vitro propagation protocol for rapidly producing Hydrastis canadensis L., Goldenseal, plantlets from disk tissue of young leaves was developed. Leaf explants were inoculated on MS medium supplemented with various concentrations of NAA and TDZ for production of callus. Two-month-old calli were sub-cultured on MS media containing cytokinins (BA, kinetin, TDZ) in different concentrations for shoot initiation. The optimum level of callus induction and maintenance was in 5.3 microM NAA in combination with 2.2 microM of TDZ. Shoot multiplication was achieved on MS medium with 2.2 microM TDZ in combination with 0.5 microM NAA. The alkaloid profile of micropropagated plantlets was similar to the profile of the mother plants. These results suggest that our in vitro propagation protocol will produce a positive impact in the conservation of H. canadensis.

In vitro plant regeneration from leaf-derived callus of Cimicifuga racemosa.

Cimicifuga racemosa (L.) Nutt., also known as Black Cohosh, is among the top 10 selling medicinal herbs in the United States. The rhizomes have been used to relieve menopausal discomfort. This plant is wild crafted and conservationists have expressed concerns with the sustainability of C. racemosa. Excised tissues from young leaves of C. racemosa were cultured on Murashige and Skoog's medium (MS) supplemented with various concentrations of NAA and TDZ for production of callus. The optimum callus growth and maintenance was in 1.0 microM NAA plus 0.5 microM TDZ. Two-month-old calli were sub-cultured on different concentrations of cytokinins (BA, kinetin, 2ip, TDZ) or in combination with GA(3) for shoot induction. The rate of shoot induction and proliferation was higher in MS media supplemented with 2.0 or 4.0 microM of TDZ. Concentrations of TDZ greater than 4.0 microM suppressed shoot growth. Adding 3.5 microM of GA(3) into media containing BA increased shoot growth. The presence of GA(3) with kinetin or TDZ did not affect shoot production. For rooting, shoots were transferred to MS medium with activated charcoal supplemented with various auxins (IAA, IBA and NAA), roots were noticed 20 days after transference. Activated charcoal was an essential component for vigorous rooting formation. Our results suggest that conservation of C. racemosa is possible through in vitro multiplication of leaf-derived callus.

Evaluation of Podophyllum peltatum accessions for podophyllotoxin production.

In an effort to develop a sustainable source of podophyllotoxin for the production of anticancer drugs such as etoposide, teniposide and etopophos, Podophyllum peltatum accessions with podophyllotoxin-rich leaf biomass were identified and transplanted to different growing conditions by vegetative cuttings. Results indicate that the lignan profile in leaves does not change over time or due to environment conditions. Podophyllotoxin and alpha-peltatin content in the blades seems to be stable with an inverse relationship of concentration between these compounds. A podophyllotoxin-rich leaf accession showed low biosynthetic capability to synthesize alpha- and beta-peltatin and the converse was also true, indicating that selection and cultivation of high-yielding podophyllotoxin leaf biomass may reduce production costs.