Plant Cell Reports

Plant Cell Reports (1986) 5:471-474

© Springer-Verlag 1986

Plant regeneration and thebaine content of plants derived from callus culture of Papaver bracteatum K. B. Day, J. Draper, and H. Smith Department of Botany, University of Leicester, Leicester, LE1 7RH, UK Received October 22, 1986 - Communicated by M. H.Zenk

ABSTRACT Plants have been regenerated from embryogenic callus cultures of two v a r i e t i e s of Papaver bracteatum and successfully transplanted to s o i l . Regeneration occurred in good y i e l d , around 40 plants to soil w i t h i n 5 months per O.4g piece of o r i g i n a l c a l l u s . Thebaine concentrations comparable to those in seed-grown plants were obtained in callus-derived plants, i t is suggested that there is potential f o r mass micropropagation of P. bracteatum, which may be useful in developing a g r i c u l t u r a l l y - i m p r o v e d l i n e s . ABBREVIATIONS MS, Murashige and Skoog (1962) basal medium. INTRODUCTION Over the l a s t decade Papaver bracteatum has been under consideration as an a l t e r n a t i v e source of the medicall~valuab~ analgesic and cough-suppressing drug, codeine. The t r a d i t i o n a l commercial source is P. somniferum (opium poppy) which accumulates mainly morphine. About 90-95% of the l e g a l l y - e x t r a c t e d morphine is converted to codeine f o r medical uses, but morphine or diacetyl morphine (heroin) is also a widely-abused i l l i c i t drug. P. bracteatum, on the other hand, accumulates the morphinan a l k a l o i d , thebaine, but lacks s i g n i f i c a n t a c t i v i t y of the enzymes f o r i t s demethylation to codeine and morphine. Thebaine can be chemically converted e a s i l y into codeine but is barely open to abuse since i t s conversion to morphine and heroin is d i f f i c u l t . (See Seddigh et a l , 1982). P. bracteatum, native to Western Asia, has been under t r i a l as a crop in the United States. Further domestication would be b e n e f i c i a l , p a r t i c u l a r l y selection f o r such characters as high thebaine y i e l d in the aerial parts, u n i f o r m i t y of plants for ease of harvest, and disease resistance (Vincent et a l , 1977; Seddigh et a l , 1982). I t is suggested here that tissue culture techniques may be useful in the development of improved P. bracteatum s t r a i n s . For instance, regenerated plants may be screened for mutations in the thebaine biosynthetic pathway, or f o r other t r a i t s induced or altered during tissue culture. Micropropagation of selected, conventionally-grown plants may also prove useful. Both of these techniques require the e f f i c i e n t mass regeneration of plants from callus or explants.

Offprint requests to: K. B. Day

The regeneration of P. somniferum from callus to a stage suitable f o r t r a n s f e r to soil (Nessler 1982) and to maturity and seed set (Yoshikawa and Furuya 1983) has been described. This level of regeneration has not been reported in P. bracteatum. Ikuta et al (1974) reported, f o r both P. bracteatum and P. somniferum cultures, the f a i l u r e of shoots to develop beyond 1.5cm in height. Kutchan et al (1983) have investigated the e a r l y stages of cytod i f f e r e n t i a t i o n in P. bracteatum c a l l u s , leading~to p l a n t l e t formation. Separate root and shoot cultures have been established by organogenesis in P. bracteatum callus in l i q u i d media (Zito and Staba 1982; Staba et al 1982). Two research groups reported trace amounts of thebaine in unspecialised callus cultures of P. b~acteatum (Kamimura and Nishikawa 1976; Shaffiee and Lalezari 1976) w h i l s t others reported i t to be absent (Ikuta et al 1974; Lockwood 1981; Kutchan et al 1983). Only with c y t o d i f f e r e n t i a t i o n and organogenesis does i t s accumulation increase (Kamimura et al 1976; Staba et al 1982; Zito and Staba 1982; Kutchan et al 1983). Thebaine levels in the separate roots or shoots or in young, r e d i f f e r e n t i a t i n g p l a n t l e t s were s t i l l low in comparison with mature, seed-grown plants. The major aim of our work was to achieve e f f i c i e n t , m a s s regeneration of plants from P. bracteatum c a l l u s , s u f f i c i e n t l y developed to transplant to s o i l . The thebaine concentration in regenerated plants was measured to look f o r any somatic variation induced or selected by tissue culture. The potential f o r micropropagation of t h i s species is also discussed. MATERIALS AND METHODS Plant material: Seeds of P. bracteatum var. Arya I I (1978 season) were a g i f t from Dr G.B.Lockwood, Dept. of Pharmacy, U n i v e r s i t y of Manchester. P. bracteatum " I s . " ( l i n e 59) seeds were a g i f t f ~ m Prof. D.Lavie, Dept. of Organic Chemistry, Weizmann I n s t i t u t e of Science, Rehovot, I s r a e l . (From experimental plots in I s r a e l , 1982). Cell cultures: Seeds were surface s t e r i l i s e d (5s in 70% alcohol, 3 min in 10% household bleach), rinsed in d i s t i l l e d water and germinated on 0.8% agar in the dark at 25°C. Seedlings, 5-20mm long were transferred to Murashige and Skoog (1962) basal medium with 3% sucrose (MS) containing k i n e t i n (0.47

472 ~mol 1-1 ) and 2,4-dichlorophenoxyacetic acid (4.52 or 0.45 ~mol I - i ) and incubated at 25°C in continuous l i g h t (15-20 Nmol m-2 s- l ) or darkness f o r P, bracteeatum "Is" and Arya I I respectively. Callus formed was subcultured monthly on the same medium. Plant reqeneration: P. bracteatum "Is" callus was maintained f o r 18 months and Arya I I f o r 2 years. 4-0.45g batches of callus (meristemoids) derived from a single seedling of each v a r i e t y were transferred, one month from t h e i r l a s t subculture, to s o l i d i f i e d , hormone-free MS medium in 175mi screw cap jars kept loosely-closed and sealed with Nescof i l m (Nippon Shoji Kaisha Ltd, Japan). These cultures were incubated at 20°C with a 16h photoperiod (60-65 ~mol m-2 s - i ) . Further callus (0.8O.9g per f l a s k ) was transferred to 50ml l i q u i d Ms medium containing 2% sucrose in 250mi Erlenmeyer flasks shaken at 120 rpm, 20°C in continuous l i g h t (90-95 Nmol m-2 s - l ) . When of s u f f i c i e n t size (about 40mm), regenerated plants were removed from tissue c u l t u r e , planted in Fisons Levington compost/ sand (3:1) and grown in a greenhouse at 25°C with 16h natural l i g h t supplemented with mercury vapour l i g h t s . Plants were kept under an i n t e r m i t t e n t - m i s t spray u n i t for the f i r s t 8 weeks while they hardened off. Analysis of thebaine: Fresh tissue (O.l-2g) was homogenised in 20ml 5% acetic acid with a polytron (Kinematica, Luzern-Sweiz, FRG) and centrifuged at 3000 rpm to remove debris. Supernatant was adjusted to pH 9.0 with conc. NH40H and extracted twice with 20 ml chloroform/propan-2-ol (3:1). The pooled organic extracts were dried over anhydrous Na2SO4, f i l t e r e d (Whatman INm PTFE polypropylene-backed f i l t e r s ) and evaporated to dryness under vacuum. Samples were taken up in O.5-1.Oml methanol f o r analysis by HPLC. HPLC technique was a modification of Fairbairn and Steele (1981), with mobile phase hexane + (methanol/ chloroform/triethylamine 153:65:0.5), 83 + 17, flow rate 2 ml min-i in a 250 x 5mm i . d . column contain-

ing P a r t i s i l 5Nm s i l i c a . RESULTS AND DISCUSSION The o r i g i n a l callus derived from both v a r i e t i e s consisted of l i g h t brown, loosely-packed c e l l s and clusters of compact, white meristemoids. The l a t t e r regions (Fig. IA) were transferred to regeneration media. Such meristemoid structures were described by Torrey (1966) as meristematic masses of c e l l s capable of forming organised shoot or root meristems or b i polar embryos. Similar structures have been recognised in P. somniferum callus by Nessler and Mahlberg (1979). Embryogenesis and plant regeneration from P. somniferum meristemoids is enhanced on transfer to hormone-free medium (e.g. Nessler 1982) and at lower temperatures, 16-18°C, rather than 25°C. (Yoshikawa and Furuya 1983). Similar conditions were used f o r P. bracteatum in our experiments. Green shoots, up to 8mm long, were produced from some of the white, meristemoid regions w i t h i n 3-4 weeks of t r a n s f e r to s o l i d i f i e d MS (20°C, l i g h t ) . At t h i s stage the cotyledons were emerging at the apex of "germinating" somatic embryos, as described in P. somniferum (Nessler 1982). A f t e r 8 weeks of growth on MS, cultures of P. bracteatum "Is" and Arya I I were at various developmental stages from callus and meristemoids, through e a r l y , emerging p l a n t l e t s (approximately 4mm long), to p l a n t l e t s with d e f i n i t e leaves and roots (up to 15mm excluding roots). Each piece of o r i g i n a l callus (approx. 0.4g) had produced 16 to 30 p l a n t l e t s greater than 6mm in height by t h i s time. In addition P. bracteatum Arya I I produced some abnormal shoots with narrow, c u r l y leaves up to 5mm in length. At 12 and 16 weeks P. bracteatum "Is" had grown up to 50mm and 100mm respectively, while Arya I I reached 20mm and 60mm. A small proportion of shoots

Figure 1 A P. bracteatum "Is" meristemoids. Graduations = 1 mm. B and C P. bracteatum "Is" p l a n t l e t s 12 and 14 weeks a f t e r t r a n s f e r to MS. _D P. bracteatum"Is"plantsregeneratedonMSfollowedby7monthsand3.5monthsin s o i l .

473 formed without roots but appeared otherwise normal. The small, curly shoots showed l i t t l e f u r t h e r development. Somemeristemoids p r o l i f e r a t e d without f u r t h e r d i f f e r e n t i a t i o n and then germinated into p l a n t l e t s a f t e r f u r t h e r subcultures on the same medium. This may be a r e s u l t of the formation of new embryogenic centres or simply germination of e x i s t i n g ones. Thus in each culture vessel several stages of development were always represented at any one time (Fig. 1B). I t is unclear whether an external f a c t o r t r i g g e r s germination or whether internal competence to d i f f e r e n t i a t e f u r t h e r must be attained, f o r example by growth or maturation of a proembryogenic c e l l cluster (Ammirato 1983). Rooted plants 40-50mm and above (Fig. 1C) were suitable f o r transplanting to soil where they continued to develop in a fashion s i m i l a r to normal seedlings in the greenhouse, remaining in the rosette habit during the f i r s t year (Fig. ID). Plants are presently being grown to flowering (second year and beyond) to check f o r seed set and v i a b i l i t y . Kinetin has sometimes proved helpful f o r regeneration of P. somniferum although the requirement and s e n s i t i v i t y d i f f e r s between v a r i e t i e s (Nessler 1982, Yoshikawa and Furuya 1983; own observations). A range of kinetin concentrations in the n u t r i e n t medium was tested to see i f regeneration could be improved in Table 1. Thebaine concentration and size ofP. bracteatum ~ " p l a n t l e t s derived from callus maintained f o r d i f f e r e n t times on MS. a

Time on MS (months)

Leaf length (cm)

Root length (cm)

6.5 9 12 12 12 12

1-3 7 4.5-5 5 1.5-2.5 0.5-1.2

0.6-1.1 5.5 4.5-6.0 0.1 0.i-1.2 None

F.wt. of plantlet (g) 0.017 0.211 0.200 0.135 0.083 0.034

b

Thebaine (l~g/g f.wt.) 276.0 30.6 86.5 203.1 17.5 93.3

(3) (I) (5) (1) (4) (7)

a Straight length between upper and lower l i m i t s of root mass b

Data are averaged f o r one p l a n t l e t (number of plantlets in brackets)

P. bracteatum cultures. Plantlets formed on medium w-~thO~.~ ~ I i -~ kinetin but less e f f i c i e n t l y than on hormone-free medium. Shoots were very poorly organised and f a i l e d to continueldevelopment with kinetin at 2.33 and 4.65 Nmol i ' - . On hormone-free MS the two P. bracteatum lines used here regenerated consistently whereas our own previous observations with three P. somniferum lines showed that there was some d i f f i c u l t y in root formation and unsatisfactory growth was exhibited. Embryogenesis and p l a n t l e t formation in l i q u i d media has been described for P. o r i e n t a l e and P. somniferum callus. This technique allowed greater p r o l i f e r a t i o n of embryoids and hence the potential f o r higher y i e l d s of p l a n t l e t s (Schuchmann and Wellmann 1983). P. bracteatum Arya I I callus (approx. O.9g per flask) was transferred to l i q u i d , hormone-free MS with 2% sucrose in the l i g h t and produced healthy shoots, 1-3mm long, in i0 days. About 15 p l a n t l e t s were produced per flask in 20 days. Green excrescences formed, but not normal shoots, from clusters of embryoids, i f kinetin (4.65 Nmol 1" I ) was included in the medium. After an i n i t i a l burst of "germination" in MS, remaining meristemoids f a i l e d to show f u r t h e r d i f f e r e n t i a tion and would only r a r e l y produc@ shoots by t r a n s f e r to a kinetin medium (4.65 Nmol i - i ) . Shoots transferred to solid MS a f t e r 2 or 3 weeks in l i q u i d MS developed into plants as described above. Those grown in l i q u i d f o r 7 weeks became vitreous in appearance, showing some c a l l u s - l i k e growth on the l e a f and stem surfaces, and died on solid media. Thebaine was absent from o r i g i n a l callus and meristemoids of both lines but accumulated during d i f f e r e n t i a t i o n of p l a n t l e t s . Thebaine concentrations in P. bracteatum "Is" (Tables 1 and 2) showed no obvious c o r r e l a t i o n with the morphological data. Shoots lacking roots also contained thebaine. Thebaine was undetectable in the 12mm p l a n t l e t s of P. bracteatum Arya I I grown in l i q u i d f o r 7 weeks which were showing signs of callus formation. But the abnormal, curly shoots, a f t e r 15 weeks on s o l i d i f i e d MS, contained low l e v e l s , 9.6 ~g/g f.wt. S i g n i f i c a n t l y more thebaine was found in plants transferred to soil and grown in the greenhouse. A maximum of 517 ~g thebaine/g f.wt. in the leaves was measured in one P. bracteatum "Is" plant tested a f t e r 5 months on MS plus 5 months in the greenhouse. Table 2 presents f u r t h e r values f o r a random sample of plants transplanted at one time. These levels are

Table 2. Root and shoot thebaine concentrations and size of 8 P. bracteatu m "Is" plants derived from callus a f t e r 13.5 months on MS and 9 ~eeks in s o i l . Shoot:

Root:

Whole plants:

Sample

Av. l e a f length

No. of leaves

F.wt. (g)

Thebainea content

Length b (cm)

F.wt. (g)

Thebainea content

Thebainea content

1 2 3 4 5 6 7 8

10.0 11.5 9.0 6.8 6.8 6.1 4.5 3.1

5 3 6 3 3 3 3 4

0.718 0.634 0.690 0.278 0.273 0.247 0.230 0.202

75.4 148.9 21.5 14.9 241.4 58.7 24.0 48.8

7.5 7.0 5.5 5.0 4.5 4.5 3.0 8.0

0.565 0.404 0.323 0.178 0,110 0.113 0.080 0.107

1174.3 1202.4 910.0 1206.9 765.5 857.8 397.1 937.5

559.4 559.0 309,8 390.9 381,9 310.3 120,3 356.6

3.75

0.409

79.2

5.6

0.229

931.4

372.9

0.227

78.4

0.180

274.5

142.7

Mean S.D.

7.2

aThebaine concentrations expressed as ~g per gram of fresh tissue bstraight length between upper and lower l i m i t s of root mass

474 are comparable to normal, seed-grown plants, as Vincent et al (1977) reported 11 months old P. bracteatum Arya I I plants containing an average of 472 Ng/g shoots (S.D. 221.1 Ng/g) and 1553 ~g/g roots (S.D. 471.9 Ng/g) when data are expressed on a fresh weight basis.

capable of accumulating thebaine to levels found in plants c u l t i v a t e d normally. I t is l i k e l y that selection f o r desirable characters in both parent material and variants a r i s i n g during tissue culture w i l l y i e l d plants with improved and h e r i t a b l e characteristics.

I t is not possible to d i s t i n g u i s h from t h i s experiment the contributions of genetic and environmental factors to the v a r i a t i o n in thebaine content among plants regenerated from the same callus l i n e in e s s e n t i a l l y the same conditions. The times of i n i t i a l embryo]d germination were not synchronous. Subsequently p l a n t l e t s grown together in one vessel may have been subject to unequal competition f o r n u t r i e n t s and l i g h t . Both factors have been found to a f f e c t morphinan a l k a l o i d content in P. somniferum plants (Nowacki et al 1976; Bern~th and T~t~nyi 1979). Limitation of gas exchange might also prove a s i g n i f i c a n t variable. I f genetic i n s t a b i l i t y caused by tissue culture were a major cause of the v a r i a b i l i t y t h i s would be problematic to propagation of p a r t i c ular l i n e s via mass embryogenesis. On the other hand somaclonal v a r i a t i o n might provide high thebainey i e l d i n g l i n e s , useful morphological variants or even some disease resistance. In sugar cane f o r example, resistance to a fungal t o x i n has been expressed in somaclones and was g e n e t i c a l l y i n h e r i t a b l e . (Scowc r o f t and Larkin 1982).

ACKNOWLEDGEMENTS

In practical terms plant regeneration via embryo genesis rather than organogenesis is advantageous because embryoids possess both root and shoot apices and so the need f o r sequential induction by changes of medium is avoided, Large numbers can be cultured and they separate e a s i l y into single p l a n t l e t s . In our case about 15 p l a n t l e t s per O.4g of c a l l u s could be transferred to soil a f t e r 3 months on MS and at least two or three times t h i s number by 5 months, representing around 75-110 plants per gram of o r i g i n a l callus. Scaling up to larger vessels allowing more space per p l a n t l e t may increase t h i s level of e f f i c iency and would f a c i l i t a t e handling of material. The spontaneous formation of meristemoids in callus shows v a r i e t a l differences (also in P. somniferum, Nessler 1982) and might be a r e q u i s i t e f o r e a ~ regeneration. This may l i m i t the general applicab i l i t y o f the procedure described in t h i s paper. However, lack or loss of embryogenic potential in callus is not necessarily permanent and in some cases has been shown to be reversible by manipulation of key variables, such as k i n e t i n levels. (Reviewed, Ammirato 1983). Thus, using the conditions described or minor modifications, i t should be possible to propagate large numbers of P. bracteatum plants. These are

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