Thebaine variations between germplasm sources within one collection of Papaver bracteatum Lindl

Sections

Abstract
Introduction
Materials and methods
Results and discussion
References and notes

Details

Author: C. B. COFFMAN, C. E. BARE, W. A. GENTNER
Pages: 41 to 46
Creation Date: 1975/01/01

Thebaine variations between germplasm sources within one collection of Papaver bracteatum Lindl

Ph.D. C. B. COFFMAN
M.S. C. E. BARE
Ph.D. W. A. GENTNER
United States Department of Agriculture, Agricultural Research Service, Beltsville, Maryland 20705, U.S.A.

Abstract

Plants of Papaver bracteatum Lindl. derived from different germplasm sources within one Iranian collection were grown for 18 and 37 weeks in the greenhouse and controlled environment chambers. Gas-chromatographic analyses showed significant differences between thebaine concentrations of root tissue for 18-weekold plants, but not for 37-week-old plants. The two age groups did not differ in thebaine yield per unit of dry weight of root tissue. However, due to more profuse root growth, the total yield of thebaine from roots of the older plants was about four times that of the younger plants.

Shoot thebaine concentrations differed significantly for 18- and 37-week-old plants. The over-all mean thebaine concentration for shoots of older plants was nearly twice that of younger plants.

The variation in thebaine concentrations in these plants suggested genetic differences between the germplasm sources. Thus, domestication of P. bracteatum will require selection of germplasm from within and between collections, for plants of the greatest economic potential.

Introduction

Papaver bracteatum Lindl. was first recognized as a species by Lindley in 1821 [ 1] . This species is being studied for its potential as an economic source of thebaine, a phenanthrene alkaloid of opium that can be converted to codeine. Papaver somniferum L. is the main source of raw material for codeine production, but the abuse of its primary alkaloid, morphine, has led to searches for alternate sources.

Domestication of P. bracteatum has many problems, such as ( a) what germplasm source should be used, ( b) what the optimum growing environment is, ( c) what pests affect the species, ( d) how pests should be controlled, ( e) how long the plants should grow before harvest, ( f) which parts of the plants should be harvested, and ( g) what harvesting procedures should be used.

Our objectives were ( a) to study the variation in thebaine content of plants derived from different germplasm sources within a collection, ( b) to evaluate thebaine production in root and shoot tissues, and ( c) to evaluate thebaine production in plants of two different chronological ages.

Materials and methods

Seed were harvested in the spring of 1973 from 1-year-old plants of P. bracteatum [ 2] field grown at Beltsville, MD. One capsule each from six plants was randomly selected for seed (germplasm). The capsules and subsequent propagules were numbered as germplasm sources 1 to 6. Seed were planted in peat pots in the greenhouse in August 1973. The silt loam growth medium (pH 6.9) contained (in kg/ha) Mg, 250; P205, 784; K20, 470; Ca, 2620; and Mn, 240. 1

Seed germinated about 2 weeks after planting. Seedlings were transferred to 800-cc soil-filled plastic pots and arranged on greenhouse benches in a randomized complete-block design with 20 replications. Plants were surface-irrigated as needed and subjected to ambient photoperiod, beginning with about 13 hours in September and decreasing to nearly 11 hours in December [ 3] .

Six of the replications were transferred from the greenhouse to a controlledenvironment chamber in December. Plants were maintained in a randomized complete block design and subjected to an 8-hour photoperiod of about 10,000 lux for 25 weeks, with photoperiod temperature 21 °C and dark-period temperature 16 °C. Dead leaf tissue was removed regularly. All plants Were harvested 37 weeks after germination.

In April 1974, other seed from the same six capsules were planted in silt-loam soil in 800-cc plastic pots and placed in a controlled-environment chamber as a randomized complete-block design with four replicates. The photoperiod was 8 hours at 26 °C with light intensity of about 10,000 lux. Dark period temperature was 18 °C. Seeds germinated 1 week after planting. Plants were harvested 18 weeks after germination.

Plants were removed from the soil, washed, and freeze-dried. Dry weights of root and shoot tissue were recorded, and tissue was ground to pass a 40-mesh sieve. Tissues were stored in amber, screw-top jars at 3 °C.

Thebaine was extracted from root and shoot tissue by use of ammoniated methanol, by the procedure described by Vincent and Gentner [ 4] , with several modifications. Extract solution in contact with plant tissue was not agitated. Plant pigments of shoot tissue were not separated from alkaloids in' extract solution. Thebaine was analysed by use of a Searle (Nuclear-Chicago) [ 5] dual-column gas chromatograph, equipped with a hydrogen-flame ionization detector. Glass columns were packed with 100-120 mesh Chromosorb Q with 3% OV-17 liquid phase. Operating temperatures were: 280 °C for injector, 270 °C for column and 300 °C for detector. Data were reduced with an Autolab System IV Digital Computing Integrator.

Thin-layer chromatography (TLC) was used to verify the qualitative aspects of the gas-chromatographic analysis of thebaine. Plates were composed of silica gel G. The solvent was acetone-toluene-ethanol-ammonium hydroxide (40:40:6:2) as per Stahl et al. [ 6] . Migration distance of the solvent front, 12.5 cm, required 40 minutes. Alkaloids were detected by a Drangendorff solution spray reagent [ 7] .

The presence of thebaine as determined by gas liquid chromatography (GLC) was confirmed by TLC.

1 Extractable elements. Soil Testing Methods. University of Maryland Soil Testing Laboratory, Agronomy Mimeo No. 37.

Results and discussion

Root and shoot measurements. Root weights or lengths did not significantly differ among germplasm sources within each age group (table 1). Roots of the older plants weighed about four times more than roots of the younger plants.

TABLE 1

Mean morphological characteristics of 18- and 37-week-old Papaver bracteatum plants

 

Morphological characteristics

Germplasm source

Weight (g)

Length (cm)

Shoot

 
18-week-old plants a
   
1 0.4ns 15.8ns 1.2ns
2 0.5 16.2 1.6
3 0.4 21.1 1.5
4 0.7 18.6 1.9
5 0.6 17.2 1.7
6 0.4 13.4 0.9
x
0.5 17.0 1.5
 
37-week-old plants b
   
1 1.7ns 9.5ns 0.6ns
2 1.8 16.5 0.7
3 1.8 14.8 0.5
4 2.5 14.7 0.7
5 2.0 14.8 0.5
6 1.4 14.3 0.6
x
1.9 14.1 0.6

aMeans of four replicate

bMeans of six replicate

Shoot dry weights did not differ significantly between treatments within either age group. Shoot dry weights of the younger plants exceeded that of the older plants. The weight differences reflected removal of nonviable leaves from the older plants. Shoot lengths are not presented, since plants were in the rosette form.

Thebaine concentrations. Among older plants root thebaine concentrations did not differ significantly; they ranged from 3,294 to 5,705 ppm, with a mean of 4,703 ppm (table 2). Among younger plants, root thebaine concentrations differed significantly (P 0.05); they ranged from 2,379 to 6,938 ppm, with a mean of 4,813 ppm. Thus, age varied root thebaine concentrations among plants derived from different sources of germplasm. The mean ratio of root thebaine concentrations of older plants to those of younger plants was about 1 (table 3). Thus, age of plants in this collection did not affect root thebaine concentration when averaged over the experimental population. However, older roots had about twice the thebaine content of younger roots for plants of germplasm source 6.

TABLE 2

Mean thebaine concentrations of root and shoot tissue of Papaver bracteatum

 

Thebaine concentration (ppm)

 

Germplasm source

Root

Shoot

Root/Shoot ratio

 
18-week-old plants a
   
1 5 401a
689ab b
7.84ab
2 6 938a 808a 8.59b
3 4 036ab 418bc 9.66b
4 5 390a 524abc 10.29b
5 4 737ab 233c 20.33a
6 2 379b 436bc 5.46b
x
4 813 518 9.29
 
37-week-old plants c
   
1 5 021ns 1 528a 3.28b
2 5 009 1 606a 3.11b
3 4 767 855ab 5.58ab
4 5 705 809ab 7.05ab
5 3 294 437b 7.54a
6 4 426 1 039ab 4.26ab
x
4 703 1 047 4.49

a Means of four replicate

bWithin column and age, values not followed by the same letter differ significantly at the 5% leve

c Means of six replicates

TABLE 3

Ratios of thebaine concentrations of 37- and 18-week-old Papaver bracteatum plants

 

Concentration ratios

Germplasm source

Roots

Shoots

1 0.93bb 2.22ab
2 0.72b 1.99ab
3 1.18b 2.04ab
4 1.06b 1.54c
5 0.70b 1.88bc
6 1.86a 2.38a
x
0.98 2.02

a 37-week-old plants/18-week-old plan

b Within a column, values not followed by the same letter differ significantly at the 5% leve

Among older plants, shoot thebaine concentrations differed significantly (P 0.05) and ranged from 437 to 1,606 ppm with a mean of 1,047 ppm (table 2). Among younger plants, shoot thebaine concentrations also differed significantly (P 0.05) and ranged from 233 to 808 ppm, with a mean of 518 ppm. The mean thebaine concentration in the shoot tissue of the older plants was twice that of the younger plants (tables 2, 3). Among the younger plants root and shoot concentrations of thebaine were highest from source 2. Source 2 shoots were also highest in thebaine among the older plants.

Thebaine stabilized in the roots and increased in the shoots. Thus, thebaine was probably synthesized in the roots and accumulated to stable concentrations. Also, additional synthesis, after thebaine stabilization in the roots, may result /n thebaine translocation to the shoots. Furthermore, as plants aged, continued thebaine synthesis resulted in increased shoot thebaine concentrations. Alternatively, thebaine may be synthesized in the shoots and translocated down until the roots are "saturated", at which time accumulation begins in the shoots. However, the actual site of thebaine synthesis in P. bracteatum and the mechanisms involved remain to be determined.

Except for sources 2 and 6, root/shoot tissue ratios of thebaine concentrations rank similarly descending from source 5 to source 1, within both age groups (table 2). This similarity indicates possible genetic variation between germplasm sources within this collection.

No significant relationships were found between thebaine concentrations and morphological measurements within either age group.

TABLE 4

Mean total yields of thebaine from roots of 37- and 18-week-old Papaver bracteatum

 

Mean yields (mg)

Germplasm source

37-week-old

18-week-old

37- wk/18-wk

1 8.0 1.8 4.44
2 9.0 3.4 2.65
3 9.0 1.9 4.74
4 15.0 3.3 4.54
5 7.0 2.9 2.41
6 7.0 0.9 7.78
x
9.2 2.4 3.83

Thebaine yield. The older plants yielded more thebaine than the younger plants (table 4. Plants from source 6 showed the largest difference in yield between age groups, with the older plants yielding nearly eight times more than the younger plants. The average thebaine yield from older plants was 3.8 times that of younger plants. Thebaine yields between age groups differed because root masses of the older plants were larger than those of the younger plants, since thebaine concentrations were essentially the same for both (tables23, ). The relationship between root age and thebaine concentration over a growing season or over several years has not been ascertained. Bohm (8) suggested that research efforts be directed toward improving the thebaine concentration of roots of P. bracteatum, since roots were the most important harvest products in Europe. However, root improvement may not be the best approach to thebaine production. If the genetics of germplasm sources varies, a reliable source of thebaine may be eliminated by harvest of roots.

References and notes

001

Kuhn, L., D. Thomas, and S. Pfeifer. Wissenschaftliche Zeitschrift der Humboldt Universitat zu Berlin, Mathmatisch-Naturwissenschaftliche Reihe 19: 1, 81-119, 1970.

002

P. I. Number 368264, ARYA I seed.

003

U.S. Naval Observatory. American Nautical Almanac, 1943.

004

Vincent, P. G., and W. A. Gentner. "Thebaine: Comparative extraction and stability." ST/SOA/SER.J/9. United Nations, 1974.

005

Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.

006

Stahl, V. E., H. Jork, E. Dumont, H. Bohrmann, and H. Vollmann. ArzneinittelForsch 19: 2, 194-199, 1969.

007

Randerath, K. Thin Layer Chromatography. Second ed. Academic Press, New York, 1968.

008

Bohm, H. Pharmazie. 21: 1, 70-71, 1974.