A scheme for mass propagation of Lilium in vitro

Scientia Horticulturae, 18 ( 1 9 8 2 / 8 3 ) 3 5 3 - - 3 6 2

353

Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m -- P r i n t e d in T h e N e t h e r l a n d s

A SCHEME FOR MASS PROPAGATION OF LILIUM IN VITRO

S H I N S A K U T A K A Y A M A a n d M A S A N A R U MISAWA.

Tokyo Research Laboratory, Kyowa Hakko Kogyo Co. Ltd., 3-6-6 Asahimachi, Machidashi, Tokyo, 194 (Japan) ( A c c e p t e d for p u b l i c a t i o n 11 M a r c h 1 9 8 2 )

ABSTRACT T a k a y a m a , S. a n d Misawa, M., 1983. A s c h e m e for mass p r o p a g a t i o n o f Lilium in vitro. Scientia Hortic., 18: 3 5 3 - - 3 6 2 . A mass p r o p a g a t i o n s c h e m e for Lilium p l a n t s using a shake c u l t u r e t e c h n i q u e has b e e n d e v e l o p e d . A c c o r d i n g t o t h e scheme, a b o u t 1.2 X 10 ~° o f L. speciosum or 3.2 x 10 ~2 o f L. auratum b u l b s c a n t h e o r e t i c a l l y b e o b t a i n e d in o n e year f r o m o n e m e d i u m sized b u l b . T h e s c h e m e involves 4 steps: (1) e s t a b l i s h m e n t o f aseptic p r o d u c t i o n o f b u l b l e t s ; (2) t h e s t i m u l a t i o n of bulb-scale p r o d u c t i o n b y high K a n d t h e i r rapid g r o w t h in s h a k e c u l t u r e s ; (3) b u l b l e t p r o d u c t i o n ; (4) t h e e s t a b l i s h m e n t o f b u l b l e t s in soil.

ABBREVIATIONS BA = b e n z y l a d e n i n e ; DW = d r y weight, FW = fresh weight; K = k i n e t i n ; M S - m e d i u m = Murashige a n d S k o o g ' s m e d i u m ( 1 9 6 2 ) ; N A A = a - n a p h t h a l e n e a c e t i c acid. INTRODUCTION

Two methods of propagating Lilium using tissue culture techniques have been reported by many workers. In the first method, bulblets have been produced on agar media in vitro from bulb scales (Robb, 1957; Hackett, 1969; Allen, 1974; Anderson, 1977; Stimart and Ascher, 1978; Takayama and Misawa, 1979, 1980, 1982b; Novfik and P e t ~ , 1981; Van Aartrijk and BlomBarnhoorn, 1981) and in the second method, bulblets have been produced through callus (Sheridan, 1968; Sharp and Raskin, 1971; Simmonds and Cumming, 1976; Kato and Yasutake, 1977; Stimart et al., 1980). Although the theoretical propagation rate is higher using callus [6 X 10 ~2 plants per year from 1 g of callus has been reported by Simmonds and Cumming (1976)] than in the bulb-scale method [100 000 from one medium-sized bulb in 6 months by Anderson, 1977; 8000 or more bulbs from an average bulb with 100 scales in 6 weeks by Stimart and Ascher, 1978], the bulb-scale method seems to be advantageous for mass propagation of Lilium. The reasons are (1)days required for bulblet regeneration from bulb scales are fewer than from callus, and at the same time, bulb scales have stronger regenerating-ability than callus; 0304-4238/83/0000--0000/$03.00

© 1983 Elsevier Scientific P u b l i s h i n g C o m p a n y

354

(2) significant chromosomal changes occur principally in de-differentiated callus or suspension cultures (Bayliss, 1980); (3) calluses lose their regeneration-ability during long-term sub-culture (Syono, 1965), while bulb scales have a stable regeneration-ability during serial sub-culture. We have established a mass propagation scheme for Lilium through serial sub-culture of bulb scales in vitro which resembles the method for Begonia propagation (Takayama and Misawa, 1982a). The 4 main steps have been mentioned in the "Abstract". In previous experiments, we investigated the conditions for bulblet formation (Takayama and Misawa, 1979, 1980, 1982b). The present study is concerned with the establishment of the mass propagation scheme for Lilium by tissue culture. MATERIALS AND METHODS

The species used were Lilium auratum Lindl., L. speciosum Thunb. cul tivar 'Uchida' and L. longiflorum Thunb. 'Hinomoto', as previously described (Takayama and Misawa, 1979). Bulb scales for experimental purposes were obtained from a bulblet sub-cultured in vitro every 2 months. They were grown on MS agar medium supplemented with 90 g 1-1 sucrose, 0.1 mg 1-1 NAA and 8 g 1-1 agar at 25°C under cool white fluorescent light (Toshiba FL40SW-100V/40W lamps) and a photoperiod of 24 h. The bulb scales were transferred to MS agar medium containing 0.1 mg 1-1 NAA, 30 g 1-1 sucrose, 8 g 1-1 agar and 10 g 1-1 K. After 60 days of incubation at 25°C, numerous leafy adventitious bulb scales were regenerated which developed further by shake culture. The bulb scales thus obtained were transferred to MS liquid medium, then incubated at 25°C under 2.5 W m-: of fluorescent light. A 300-ml conical beaker containing 100 ml of MS liquid medium was used as a culture vessel, unless otherwise indicated. A rotary shaker at 180 r/min was used for shake culture. RESULTS

Establishment o f aseptic culture and maintenance o f stock plants in vitro. A stock-plant bulb grown in soil (Fig. l-A) was sterilized using sodium hypochlorite and ethanol solutions (Fig. l-B), then sectioned aseptically into -

-

Fig.1. S c h e m a t i c d i a g r a m o f t h e steps used in a rapid p r o p a g a t i o n process for L i l i u m b y tissue culture. A = b u l b of s t o c k p l a n t ; B a n d C = sterilization o f b u l b scales; D a n d E = b u l b l e t f o r m a t i o n f r o m a bulb-scale s e g m e n t ; F = r e s u l t i n g b u l b l e t ; G = b u l b scale f r o m b u l b l e t ; H a n d I = f o r m a t i o n o f n u m e r o u s a d v e n t i t i o u s b u l b scales in a high c o n c e n t r a t i o n o f c y t o k i n i n ; J a n d K = r a p i d g r o w t h o f m u l t i p l e a d v e n t i t i o u s b u l b scales b y s h a k e cult u r e at 180 r / r a i n ; L = m u l t i p l e b u l b scales; M, N, O a n d P = b u l b l e t f o r m a t i o n for t r a n s f e r i n t o soil o n agar plate c u l t u r e (M a n d N) or b y s h a k e c u l t u r e (O a n d P); Q = r e s u l t i n g b u l b l e t ; R = t r a n s f e r i n t o soil; S = leaf e m e r g e n c e a n d s u b s e q u e n t g r o w t h ; T = f l o w e r i n g (anthesis).

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0.5--1-cm pieces (Fig. I C). These pieces were transferred to 25 × 1 2 5 - m m test tubes containing fresh M S agar medium supplemented with 30--90 g l-I sucrose, 0.1 m g l-' N A A , 8 g l-I agar and cultured at 25°C under 2.5 W m - : of continuous irradiance (Fig. l-D) until bulblet formation (Fig. I-E). Bulblet formation was influenced by degree of dormancy of a stock plant bulb of L. auratum. O n 11 September, just after harvest from the field (bulbs in dormant state), efficiency of bulblet formation was low (I0.2%), but after 18 or 100 days of 5°C treatment (29 September and 20 December), efficiency of bulblet formation was increased to 45.2 or 94.0%, respectively. Bulblet formation was fairly good at the proximal and inner parts of bulb scales. However, external bulb scales provided insufficient material for bulblet formation because of the low efficiency of bulblet formation (especially in the dormant state) and because of the high frequency of bacterial and fungal contamination. The internal bulb scales presented no such problems. Shoot-tip culture is another method for the establishment of aseptic culture. Shoot tips about 0.2 m m long grew better on M S liquid medium (floated) than on M S agar medium. The 30 g l-' sucrose in the medium stimulated bulblet growth, whereas 90 g l-I sucrose inhibited growth and stimulated callus formation (Fig. 2). Maintenance of species or cultivars in vitro was achieved by recycling D, E, F and G in Fig. 1.

i m

Fig. 2. Effect of sucrose concentration on bulblet regeneration from shoot-tips (0.2 m m long) in Lilium longiflorum. The media used were M S containing 0.1 m g I-I N A A and 30 (left) or 90 (right) g l-I sucrose.

357

Increase in n u m b e r o f bulb scales. -- To propagate L i l i u m bulblets efficiently,

we tried to use bulb scales from bulblets produced in vitro. Our results revealed that numerous primordium-like adventitious bulb scales were regenerat ed (Fig. 1-I) when bulb scales (Fig. l-G) dissected from a bulblet grown in vitro were further transferred to MS agar medium containing high concentrations of cytokinins (3--10 mg 1-~ K or BA) (Fig. l-H). These bulb scales grew rapidly in shake culture (Fig. l-J, K) using liquid medium without K, and yielded numerous secondary bulb scales (Fig. l-L) which were used as the materials for bulblet formation (Fig. l-M, N, O, P; details described in the next section). Numbers of bulb scales can be increased rapidly b y recycling the procedures H, I, J and K in Fig. 1. In the bulb scale multiplication procedure (Fig. l-H, I), 90 g 1-~ sucrose in the medium inhibited the action of cytokinin in multiplying bulb scales, while bulb scale multiplication was stimulated by cytokinin in the medium containing 30 g 1-~ sucrose (Takayama and Misawa, 1982b). The o p t i m u m concentration of cytokinin required to multiply bulb Scales differed between species. B u l b l e t f o r m a t i o n f r o m bulb scales. -- Bulblets are formed from bulb scales

(Fig. l-L) on agar (Fig. l-M, N) or in liquid media (Fig. 1-O, P). The conditions for bulblet formation on agar medium have been described previously (Takayama and Misawa, 1979, 1980). This section describes the use of liquid medium for producing L i l i u m bulblets. Effect of strength of MS medium on the differentiation of bulblets or roots in a static-culture liquid medium are shown in Fig. 3. The number of E

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MS MEDIUM STRENGTH Fig. 3. E f f e c t o f the strength o f M S - m e d i u m o n the d i f f e r e n t i a t i o n and g r o w t h o f b u l b l e t s a n d r o o t s in L i l i u m auratum in static culture. The m e d i u m used w a s MS containing 30 g 1-1 sucrose a n d 0.1 mg 1-1 N A A . T h e culture vessel used was a 25 x 1 2 5 - m m t e s t t u b e c o n t a i n i n g 2 ml o f the m e d i u m . Cultures w e r e i n c u b a t e d for 100 days at 25°C u n d e r 2.5 W m -2 o f c o n t i n u o u s irradiance. S y m b o l s : 0 0, bulblets; X . . . . X, roots. The p o i n t s are m e a n s o f 10 replicates _+ SE.

358 TABLE I D i f f e r e n t i a t i o n o f b u l b s a n d r o o t s o f Lilium speciosum in various c u l t u r e vessels. Bulb scales f r o m a b u l b l e t p r o d u c e d b y tissue c u l t u r e were i n o c u l a t e d in e a c h c u l t u r e vessel a n d i n c u b a t ed for 29 days at 25°C u n d e r 0.5 W m -2 of c o n t i n u o u s irradiance. M S - m e d i u m was used w i t h 0.1 m g 1-1 N A A a n d 30 g 1-1 sucrose (A, B, C, D). In E x p e r i m e n t D, 8 g 1-1 agar a n d 5 g 1-1 c h a r c o a l p o w d e r were a d d e d . A = S h a k e c u l t u r e at 180 r / r a i n in a conical b e a k e r w i t h 100 ml o f liquid m e d i u m . B = As for A e x c e p t for static c o n d i t i o n s . C = Static c u l t u r e in a 300-ml R o u x - f l a s k c o n t a i n i n g 50 ml o f liquid m e d i u m . D --- C u l t u r e in a 90 x 20 m m Petri dish cont a i n i n g 50 ml o f agar m e d i u m . Values in p a r e n t h e s e s = % of survived e x p l a n t s Experiments

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CUMULATIVE NUMBEROF BULB SCALES CULTURED IN VITRO Fig.4. E f f e c t o f c u l t u r a l c o n d i t i o n s o n t h e d i f f e r e n t i a t i o n a n d g r o w t h of b u l b l e t s o f Lilium auratum. A = d i f f e r e n t i a t i o n a n d g r o w t h of n e o - f o r m e d b u l b l e t s in liquid s h a k e c u l t u r e ( " s h a k e " ) ; B a n d C = s u b s e q u e n t g r o w t h o f a b u l h l e t a f t e r t r a n s f e r t o liquid m e d i u m ("liquid") (B) or t o agar m e d i u m ("agar") (C). MS m e d i u m was used w i t h 0.1 m g 1-1 N A A

and 30 (a), 60 (b) or 90 (c) g 1-1 sucrose. 300-ml Erlenmeyer flasks were used containing 50 ml of the medium. In each culture vessel, 20 bulb scales were incubated for 40 days.

359

bulblets differentiated was not affected by the concentration of MS-medium between 1/8 and standard strength, while their DW increased proportionately to the strength of the MS-medium. Double strength MS-medium inhibited both the growth of bulblets and the numbers differentiated. R o o t number and growth responded somewhat differently from bulblets; lower ( 1 / 8 - - 1 / 2 ) strengths stimulated, but higher ones (1--2) inhibited. The effects of various culture vessels on differentiation and growth are shown in Table I. In 300-ml conical beakers containing 100-ml of medium, shake culture markedly stimulated the differentiation and growth of bulblets and roots (Table I-A), but static culture suppressed them (Table I-B). Rouxflasks (Table I-C) also suppressed differentiation and growth. In Petri dishes (Table I-D), bulblet differentiation and growth were almost the same as in shake culture, while root differentiation and growth were suppressed. The effect of sucrose on growth of bulblets by shake culture is shown in Fig. 4. Bulblet differentiation and growth from a bulb scale in shake culture I0

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360

incubated for 1 m o n t h (Fig. 4-A) were almost the same in 30, 60 or 90 g 1-1 sucrose media (Fig. 4A-a, b, c). These small bulblets were transferred to liquid medium for shake culture (Fig. 4-B "Liquid") and agar medium (Fig. 4-C "Agar"), and subsequent growth was compared. Best growth was observed in liquid shake culture (Fig. 4-B) with 60 or 90 g 1-1 sucrose, but in agar, growth was suppressed at a higher concentration of sucrose. Differentiation and growth of roots were similar to those of bulblets (data not shown). For bulblets on shake culture, which had been pre-cultured on agar medium, the time course of the growth was analyzed as shown in Fig. 5. Most bulblets and roots were obtained with L. auratum. The growth rate of bulblets, expressed as logarithms of dry weight, were almost the same for the 3 species tested (Fig. 6). The doubling-time of dry weight was 15 days for L. auratum and L. speciosum, and 20 days for L. longiflorum. 500

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Cultivation of in vitro-produced plants in soil. -- The bulblets produced by the in vitro technique (Fig. l-Q) had their dormancy broken by treatment at 5°C before being transplanted into soil (Fig. I-R). Bulbs produced emergent shoots (Fig. l-S) and flowers (Fig. l-T). Lilium longiflorum flowered within the first year of culture in soil, while L. speciosum and L. auratum required 2--3 years for flowering. Theoretical propagation rate. -- The experiments on Lilium plants indicated that 2000 bulbs can be produced from a medium-sized bulb in 45 days. Numbers of bulbs can be further increased 200-fold in L. auratum and 50-

361

fold in L. speciosum from bulbs thus obtained by re-cycling from steps H--L in Fig. 1 every 75 days. Bulb scales produced by this procedure were transferred to fresh medium to produce new bulblets (M,N and O,P in Fig. 1). The ultimate number of bulblets produced is estimated to be 1.25 X 101° in L. speciosum and 3.2 X 1012 in L. auratum. DISCUSSION

Tissue culture techniques facilitate mass-propagation of plants (Murashige, 1974, 1978; Holdgate, 1977; Winton, 1978), and allow a million-fold increase in plant numbers in a year (Murashige et al., 1974). Propagation of Lilium through tissue culture has also been studied by several groups (Simmonds and Cumming, 1976; Anderson, 1977; Stimart and Ascher, 1978; Nov~k and Petru, 1981) using techniques in which bulblets were propagated by successive transfer of bulb scales or calli on media. In practice, however, these techniques require a large number of vessels as well as considerable labour to produce numerous bulblets. The method presented in this study is efficient for the mass propagation of Lilium plants using shake culture with liquid media in a similar way to that used to produce begonia plants (Takayama and Misawa, 1981, 1982a). The growth rates of bulblets in shake culture were quite similar in the 3 species of Lilium tested and the doubling time of Lilium bulblets at 15--20 days was lower than that of begonia shoot cultures (at 3.6 days). The difference may relate to the surface contact area between cultures and medium; larger surface contact areas of the cultures stimulate growth. This is verified, in part, in that Lilium tissues in shake culture (aggregate of numerous adventitious bulb scales with larger surface contact area with the medium) had a higher growth rate than that obtained in cultures of the usual bulblet formation in vitro (small contact area with the medium). An established mass-propagation scheme reflects the results presented in this and in previous studies (Takayama and Misawa, 1979, 1980, 1982b). The scheme is characterized by (1) regeneration of multiple adventitious bulb scales by a high cytokinin level in the medium (Takayama and Misawa, 1982b), and these multiple bulb scales were (2) grown rapidly by shake culture. The multiplication processes of bulb scales could be recycled to produce numerous bulb scales after one year. These multiplied bulb scales will regenerate bulblets when on agar medium or by shake culture in liquid medium. Although the shake-culture method has great advantages in developing a mass-culture system and for assessing a theoretical multiplication efficiency, the adaptation of the technique for other species, or to larger culture systems such as jar fermentors, need further development, and experiments to this end are in progress in our laboratory. ACKNOWLEDGEMENT

We thank Mrs. H. Ogawa for her technical assistance.

362 REFERENCES Allen, T.C., 1974. Control of viruses in lilies. In: Lilies and Other Liliaceae 1974. R. Hortic. Soc., London, pp. 3--10. Anderson, W.C., 1977. Rapid propagation of Lilium, cv. Red Carpet. In Vitro, 13: 145. Bayliss, M.W., 1980. Chromosomal variation in plant tissues in culture. In: I.K. Vasil (Editor), Perspectives in Plant Cell and Tissue Culture. Int. Rev. Cytol., Suppl. 11A, Academic Press, New York, pp. 113--144, ISBN 0-12-364371-6. Hackett, W.P., 1969. Aseptic multiplication of lily bulblets from bulb scales. Proc. Int. Plant Propag. Soc., 19: 105--108. Holdgate, D.P., 1977. Propagation of ornamentals by tissue culture. In: J. Reinert and Y.P.S. Bajaj (Editors), Applied and Fundamental Aspects of Plant Cell, Tissue and Organ Culture. Springer Verlag, pp. 18--43, ISBN 0.540-07667-8. Kato, Y. and Yasutake, Y., 1977. Plantlet formation and differentiation of epidermal tissues in green callus cultures from excised leaves of Lilium. Phytomorphology, 27 : 390--396. Murashige, T., 1974. Plant propagation through tissue cultures. Annu. Rev. Plant Physiol., 25: 135--166. Murashige, T., 1978. The impact of plant tissue culture on industry. In: T.A. Thorpe (Editor), Frontiers of Plant Tissue Culture 1978. Book Store, University of Calgary, Canada, pp. 15--26. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473--497. Murashige, T., Serpa, M. and Jones, J.B., 1974. Clonal propagation of Gerbera through tissue culture. HortScience, 9 : 175--180. Nov~k, F.J. and Petrfl, E., 1981. Tissue culture propagation of Lilium hybrids. Scientia Hortic., 14: 191--199. Robb, S.M., 1957. The culture of excised tissue from bulb scales of Liliurn speciosum Thunb. J. Exp. Bot., 8: 348--352. Sharp, W.R. and Raskin, R.S., 1971. Haploidy in Lilium. Phytomorphology, 21: 334--337. Sheridan, W.F., 1968. Tissue culture of the Monocot. Lilium. Planta, 82: 189--192. Simmonds, J.A. and Cumming, B.G., 1976. Propagation of Lilium hybrids. II. Production of plantlets from bulb-scale callus cultures for increased propagation rates. Scientia Hortic., 5: 161--170. Stimart, D.P. and Ascher, P.D., 1978. Tissue culture of bulb-scale sections for asexual propagation of Lilium longiflorum Thunb. J. Am. Soc. Hortic. Sci., 103: 182--184. Stimart, D.P., Ascher, P.D. and Zagorski, J.S., 1980. Plants from callus of the interspecific hybrid Lilium "Black Beauty". HortScience, 15: 313--315. Syono, K., 1965. Changes in organ forming capacity of carrot root calluses during subcultures. Plant Cell Physiol., 6: 403--419. Takayama, S. and Misawa, M., 1979. Differentiation in Lilium bulbscales grown in vitro. Effect of various cultural conditions. Physiol. Plant., 46 : 184--190. Takayama, S. and Misawa, M., 1980. Differentiation of Liliurn bulbscales grown in vitro. Effect of activated charcoal, physiological age of bulbs and sucrose concentration on differentiation and scale leaf formation in vitro. Physiol. Plant., 4 8 : 1 2 1 - - 1 2 5 . Takayama, S. and Misawa, M., 1981. Mass propagation of Begonia × hiemalis plantlets by shake culture. Plant Cell Physiol., 22: 461--467. Takayama, S. and Misawa, M., 1982a. Factors affecting differentiation and growth in vitro, and a mass-propagation scheme for Begonia × hiemalis. Scientia Hortic., 1 6 : 6 5 - - 7 5 . Takayama, S. and Misawa, M., 1982b. Regulation of organ formation by cytokinin and auxin in Lilium bulbscales grown in vitro. Plant Cell Physiol., 23: 67--74. Van Aartrijk, J. and Blom-Barnhoorn, G.J., 1981. Growth regulator requirements for adventitious regeneration from Lilium bulb-scale tissue in vitro, in relation to duration of bulb storage and cultivar. Scientia Hortic., 14: 261--268. Winton, L.L., 1978. Morphogenesis in clonal propagation of woody plants. In: T.A. Thorpe (Editor), Frontiers of Plant Tissue Culture 1978. Book Store, University of Calgary, Canada, pp. 419--426.