Ipomoea, Rivea and Argyreia Tissue Cultures: Influence of Various Chemical Factors on Indole Alkaloid Production and Growth
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Ipomoea, Rivea and Argyreia Tissue Cultures: Influence of Various Chemical Factors on Indole Alkaloid Production and Growth R. H. DOBBERSTEIN1 AND E. JOHN STABA2 (UnIversity OF NebrasKa. Lincoln. NEBrasKA 68508) Seeds and plants of certain members of the Convolvulaceae contain ergoline alkaloids. The principal ergot-alkaloid containing plants are IpOMOEA vIOLAEEA L. (morning-glory, Badoh Negro), RIOEA eorymbosa (L.)Hall. f. (Ololiuqui, wild morning-glory) and ArGyreia NErvosa Boj. (baby Hawaiian wood rose). Extracts of these plants may (6, 11, 17) or may not (9, 12) elicit a lysergic acid diethylamide- like response. Stab a and Laursen (19) reported the presence of indole alkaloids in static tissue cultures of IPOMOEA vIOLAEEA var. pearly gates and flying saucer and RIOEA eorymbosa. The principal objective of this study was to examine suspension cultures of IPOMOEA vioLAEEA var, pearly gates, Rivea EOrymbosA and Argyreia. NErvosa and static cultures of RivEA COrymbOsa in media containing various chemical factors that might stimulate indole alkaloid production. MATERIAL A D METHODS Plant tissue origin and media.-Seeds of L. viOLACEA var. pearly gates and R. Corymbosti were sterilized and grown in static culture as previously described (19). A. NEruOsa seeds! were scraped with a razor blade to remove the hair. notched with a scalpel and soaked in tap water for 24 hr before sterilization. They were sterilized in a .5.25%sodium hypochlorite solution! diluted 1:3 with sterile distilled water, for 15 min IN vAcuO. These seeds were placed in Petri plates con- taining 20 ml sterile tap water.After germination, root, stem and leaf callus cultures were ob- tained by placing sterile roots, stems and leaves in one-oz dry squares containing 18 ml revised tobacco medium (RT)(13) with 0.1 ppm 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.1% agar. The Argyreia callus tissue used for this study was of leaf origin; the Ipomoea and Rivea callus tissues were of seed origin. All callus tissues had been maintained on solid medium for at least one year before they were grown as suspension cultures. The basal medium used for Ipomoea was RT medium with 0.1 ppm 2,4-D and 10 ml/liter of the following amino acid stock solution in g/liter: L-glutamine, 20.0; L-glutamic acid, 5.0; L-argenine HCI, 2.5; L-asparagine, 20.0; glycine, .5.0;L-methionine, 0..5; L-cystine, 0..5; and D-alanine, 5.0. The basal medium used for Rivea was RT medium with 0.1 ppm 2,4-D; that used for Argyreia was RT medium with 0.1 ppm 2,4-D and 0.2 ppm kinetin. All media were adjusted to pH 6.5 with 10% KOH or 10% NaOH and autoclaved. Basal media modifications.- The following modifications were made to each of the three basal media: (i) LOw potassiuMMEDiuM (K-1).-The RT medium contains 783.8 mg/liter of potassium. The inorganic stock solution was made as previously reported (13), but the 38.0 g/2 liters KNO, was replaced with 32.0 g/2 liters NaN03 and the pH was adjusted with 10% NaOH instead of 10% KOH.Thus prepared, the low potassium medium contained 49.0 mg Zliter of potassium. This medium was used because it has been shown that in intact higher plants, a low potassium level (1,564 mg/lOOg dry weight of soil) favors free tryptophan accumulation or biosynthesis, a low indoleacetic acid (IAA) content and an increased amount of total alkaloids in DAtura tatuZa and RauwOLFIA serpentINE (30). (ii) HIGH phosphatEMEDiuM (P-2).-The RT medium contained 118.6 mg/liter of phosphate and the high phosphate medium prepared, contained 593.2 mg /liter of phosphate.To obtain this additional phosphate without altering the potassium concentration, 1.3 g a2HPO. were added to each liter of medium. It is known that in some species of CLtuNCEps pur-puree (Fr.) Tul. (ergot), a high phosphate concentration (0.1-3.0 g/liter) causes increased alkaloid formation (18, 22). 'Based on portions of a thesis submitted by R. H. Dobberstein to the Gradua te College, university of Nebraska in partial fulfillment of the requirements for the M.S. degree. 'Present address: Department of Pharmacognosy, University of Minnesota, Minneapolis, Minn. 55455. 30btained from Dr. Ara Der Marderosian, Philadelphia College of Pharmacy and Science, Philadelphia, Penn. +Purcx Corp., Ltd., South Gate,Calif. 141 142 LLOYDIA [VOL. 32, xo 2 (iii) SPECIALCArbohyDratE MEDIum (C-3).-In this medium, the concentration of sucrose was reduced from 3% to 0.5%.In addition, 1.0% sorbitol and 0.5% fumaric acid were added. In order to dissolve all of the fumaric acid,NaOH was added, one pellet at a time, until the furmaric acid dissolved. This medium was used because a reduced carbon source should be depleted early, forcing the tissue into nitrogen shunt metabolism (21). At the same time, car- bohydrate alcohols (1, 5, 21, 23, 24, 27) and Krebs-cycle acids (3) have been reported to in- crease alkaloid production in ergot submerged cultures. (Iv) Precursor MEDIuM 0II-4).-In this experiment, 100 mg /liter i.-tryptophan and 1.0 mg /liter mevalonic acid lactone were added to the medium. Mevalonic lactone (3.5 mg) was added to 10 ml double distilled water and the PH was adjusted to approximately 11. The solu- tion was allowed to stand overnight at room temperature, and then the PH was adjusted to approximately 6.5.This treatment hydrolyzes mevalonic lactone to mevalonic acid.The L-tryptophan (350 mg) was added to the solution, and diluted to 46.6 ml. After cold steriliza- tion, the solution was aseptically transferred to a specially constructed flask connected to a multiple dose inoculation syringe (14), and 0..5 ml of this solution was aseptically added to each flask. Mevalonic acid and L-tryptophan are ergoline alkaloid precursors (8, 16, 25, 26, 2S). (v) Tween-CHICK PEAMEAL MEDium (T-5).-This medium consisted of 0.1% Tween-SO' and 1.0% chick pea meal added to basal RT medium. The untreated chick peas! were ground to a ::lO-mesh powder in a Wiley mill.The chick pea meal did not completely dissolve, so the medium was well shaken before being dispersed into each flask or dry square. Since Tween-80 is a sur- face active agent, it might favorably change the cell membrane permeability. Chick pea meal has been shown to increase alkaloids in certain submerged ergot strains (4). TABLE 1. GrOwth INDEx AND AGE OFA rgyrEIA, IPOMOEAAND RIvEA susPensION tissues AND RIOEA CALLus tissuE. Argyreia Ipomoea Rivea Rivea Callus Media- , G.I.b Age G.I.h Age c.r.» Age G.I.b Age (wk) (wk) (wk) (wk) ---- Control I . .. 2.9 3 1.7 4 1.1 3 3.6 10 Control II. ... 5.8 5 2.1 8 8.0 5 - - C-3 . ..... 1.1 5 0.9 8 1.5 5 0.8 10 M-4 .... 3.7 5 2.0 8 2.3 5 2.5 10 T-5. 2.1 5 1.8 S 2.0 5 3.7 10 Control III .. ... .. 2.1 6 1.6 7 1.8 S 2.5 15 K-l. ..... ... - . l.5 6 0.9 7 l.7 8 2.3 15 P-2 .. .. ... .. 1.1 6 0.7 7 2.1 S 2.7 15 1.1 1.4 :\-6. ... ' . ..... 0.4 6 0.6 7 S 15 "Tissues grown in media Control III, K-l, P-2 and N-6 were grown for two generations. Argyreia was grown for three weeks, the old media decanted and fresh media added. Ipomoea was grown for four weeks, the old media decanted and fresh media added. Rivea was grown for three weeks, the old media decanted and fresh media added. Rivea callus was grown for ten weeks, transferred to fresh media. "Grow th Index (G.I.)=final wet weight of tissue/wet weight of inoculum. (vi) AuxIN MEDIuM (N-6).-To the basal RT medium, 2.0 mg /liter naphthalene acetic acid (NAA) and 0.2 mg /liter kinetin were added in place of 2,4-D, or kinetin and 2,4-D. It has been shown that lAA, NAA, 2,4-D, indolepropionic acid and indolebutyric acid can cause increased alkaloid production in certain submerged ergot strains (29). Growth Conditions.-For each test medium modification and its control, five loopfuls of tissue were transferred with a sterile, sieved, stainless steel transfer cup of one-ml capacity, to twenty 250 ml Erlenmeyer flasks containing 75 ml of the test or control media. After inoculation, the flasks were placed on a reciprocating shaker (SO strokes/min; stroke length 5 ern) at room temperature (25-30°). The light conditions were not controlled, thus the cell suspensions received partial light exposure during the day and none at night.The Growth Index (G.!.) and the age of the tissue are given in table 1. The Rivea static cultures were grown at room temperature (25-30°) in one-oz dry squares containing 18 ml of media.For each test media modification and its control, 75 dry squares were inouc1ated with tissue by using a sterile scalpel. 5Atlas Power Co., Wilmington,Delaware. 6Garbanzas, EI Molino Mills, Alhambra, Calif. JUNE 1969] DOBBERSTEIN AND STABA: INDOLEALKALOIDS 143 Analytical Methods.-(i) GrOwth.-After the specified growth periods, the contents of each flask were filtered and the wet weight of the tissue from each flask determined. The Rivea callus tissues from each dry square were combined and the total wet weight determined. All tissues C and media were frozen (-30 ) until extracted.