Planta (Berl.) 82, 189--192 (1968)

Short Communication

Tissue Culture of the Monoeot Lilium WILHA~ F. SItE~IDAN Biology Department u University New Haven, Connecticut Received May 13, 1968 The monocot Lilium, possessing very large nuclei and chromosomes, is a favorable material for studying problems of development, particularly with regard to the proteins of the nucleus (SHERIDANand STEn~, 1967). Since it would broaden the possible approaches to such study if successful, an attempt was made to establish Lilium in tissue culture. This communication reports (1) the successful establishment in both agar and liquid shake cultures of callus of Lilium longi/lorum Thunb; (2) the repeated subculturing of the callus on growth factor free media; and (3) the production of large numbers of plantlets from callus under certain growth conditions in liquid suspension cultures. Callus was established by placing tissue explants from stem apices onto the basal medium supplemented with 2 mg/1 of indoleaeetic acid (IAA). The terminal i0 cm of stems which were approximately 25 cm in height were removed and all but the smallest leaves were stripped off. The apices were washed for 5 min in a solution of the detergent tteikol, 10 min in 10 % Clorox, and 5 rain in sterile distilled water. Each apex was then placed in a sterile petri dish and the outer portion ineluding all the leaves and primordia was removed with a scapel leaving a cylinder which was free of any surface tissue. The terminal 2 cm were removed and placed on the medium. The basal medium was that of Lr~SMAIER and SKOOG (1965) containing major and minor salts, organic supplements of thiamin and inositol, and 40 g of sucrose per liter. Basal agar medium refers to the above medium solidified with 0.8 % agar. Fresh weight was used for determining growth response. Within three weeks of explantation, callus appeared on the edge of the tissue block in contact with the medium. The presence of IAA in the culture medium stimulated the production of callus by the stem tissue but was not essential for it. When explants were placed on medium lacking IAA, buds appeared on the surface of the tissue blocks and shoots and roots were formed producing new plants; on occasion callus was also formed, but usually only after the formation of roots and shoots.

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W . F . SHERIDAN:

Callus grown for 74 d a y s on b a s a l a g a r m e d i u m s u p p l e m e n t e d w i t h 2 mg/1 I A A was e x p l a n t e d (150--200 rag/flask) into flasks of b a s a l a g a r m e d i u m c o n t a i n i n g 0, 0.03, 0.1, 0.3, 1.0, 3.0, a n d 10 rag/1 I A A . S e v e n t y - n i n e d a y s later, a n a v e r a g e fresh-weight increase of 12.3-fold was f o u n d for callus in u n s u p p l e m e n t e d cultures. G r o w t h of I A A - s u p p l e m e n t e d cultures was n o t significantly different. S i m i l a r l y (Fig. 1),

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=Basa[ liquid medium with IAA . . . . Basal agar medium with ]AA ~ ' ~ B a s a l agar medium

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kl_

Io

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55

66

77

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99 days I10

Fig. l. Growth rate of Lilium callus showing fold increase in fresh weight. Each point on the graph represents the average fold increase in fresh weight for four cultures in the case of the liquid suspension cultures and five cultures in the cases of the agar grown cultures. The explants for the liquid suspension cultures were obtained from callus grown in basal liquid media supplemented with 1 rag/1 IAA. The source of explants for the agar cultures was callus which had been grown for two subcultures on basal "agar medium. All of the cultures were started with explants weighing 400 to 410 mg t h e callus grew a t a p p r o x i m a t e l y t h e same r a t e on t h e b a s a l a g a r m e d i u m lacking I A A as on t h e b a s a l a g a r m e d i u m s u p p l e m e n t e d w i t h I A A . H o w e v e r , callus grew m o r e r a p i d l y in liquid t h a n on agar. To s t u d y t h e effect of k i n e t i n on growth, callus grown in b a s a l liquid m e d i u m s u p p l e m e n t e d w i t h 0.225, 0.75, or 2.75 mg/1 I A A was e x p l a n t e d ( 1 - - 2 g/flask) into b a s a l liquid m e d i u m s u p p l e m e n t e d w i t h 2 rag/1 of I A A , a n d k i n e t i n in t h e c o n c e n t r a t i o n s 0, 0.03, 0.3, 1.0, 3.0, a n d 10.0 rag/1. A f t e r 30 d a y s , a b o u t a five-fold a v e r a g e increase in fresh weight was f o u n d for cultures in which k i n e t i n was o m i t t e d or was p r e s e n t in a conc e n t r a t i o n less t h a n 1.0 mg/1. A t k i n e t i n c o n c e n t r a t i o n s of 1.0 mg/1 or g r e a t e r t h e tissues t u r n e d b r o w n a n d grew o n l y slightly. U p o n observing t h a t t h e lily callus could grow s a t i s f a c t o r i l y on m e d i a to which n e i t h e r I A A n o r k i n e t i n was a d d e d , s u b c u l t u r e s were

Tissue Culture of the Monocot Lilium

191

established with the purpose of determining if it was possible to omit other complex organic molecules in the original medium ( L I N s M A I E R and SKOOO, 1965) and still obtain satisfactory growth. A detailed report of these studies and of the growth response of Lilium callus during repeated subeulturing in basal media is to be reported elsewhere. However it m a y be reported at this time t h a t callus is growing in its seventh subculture in basal liquid medium (no I A A or kinetin) without any diminution in rate of growth during the subculturing. This callus has undergone a theoretical increase in fresh weight of over 300000-fold during the first six subcultures which averaged 65 days in length. I n addition, callus is growing in its sixth subculture in a liquid medium containing only sucrose and the mineral salts of the basal medium without any decrease in the rate of growth during the subculturing. This callus has undergone a theoretical increase in fresh weight of over 200000-fold during the first five subcultures which averaged 79 days in length. Agar-grown callus occasionally produces plantlets with roots, leaves and buds reg~rdiess of the presence or absence of IAA or the organic supplements in the medium. Liquid shake cultures do not often produce plantlets during the usual culture period. However, after 3 or 4 months, depending on the growth rate, those liquid cultures which have become rather dense with an accumulation of callus produce large numbers of plantlets. Small dense areas appear on the surface of the callus masses and from these points roots and buds with leaves appear. Preliminary observations indicate t h a t the time and frequency of plantlet production do not differ among the various liquid media tested. Callus growing in sucrose and the mineral salts of the basal medium, as well as t h a t growing in basal liquid medium seemed to have the same propensity for producing plantlets as callus growing in basal liquid medium supplemented with IAA. I t is not known what causes the appearance of the plantlets late in the growth period of the liquid shake cultures, but it seems possible t h a t either the depletion or the accumulation of some substance in the cultures m a y be involved. The successful production of lily callus on the comparatively simple medium of LINSMAI~a and SKOOG (1965) is somewhat surprising in view of the general failure of establishing monocots in tissue culture. I t is noteworthy in this regard t h a t the recent successful tissue culture of rice (CAUTIOn, YAMADA and TAKAHASHI, 1967) and oats (u TX~AKA and Tx~:~I~SHI, 1967) were accomplished on the very similar medium of MU~ASHIG]~ and SJ~ooe (1962) or the medium of L I ~ S ~ A I ~ and SJ~oo~ (1965). These media differ from the others in general use in t h a t they are much higher in their mineral salt content, particularly nitrogenous and potassium salts. 13 Planta (Berl.), Bd. 82

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W. F. SHERIDAl~: Tissue Culture of the Monocot Lilium

I t seems likely t h a t t h e lily callus w i t h its a u t o n o m y f r o m exogenous g r o w t h factors, its large n u c l e a r size, its s u i t a b i l i t y for b i o c h e m i c a l a n d cytological studies a n d its p r o p e n s i t y for p r o d u c i n g p l a n t l e t s in suspension culture, will be of v a l u e in f u t u r e studies on p r o b l e m s of development. The author wishes to thank Professor IA~ SUSSEX of the Yale Biology Department for his facilities and encouragement. He and Drs. MARY CLUTTER and RvsSELL J. BARR~ETT were helpful in their critical examinations of the manuscript.

References CARTER, 0., NX~.YAMADA,and E. TAK~Svrr: Tissue culture of oats. Nature (Lond.) 214, 1029--1030 (1967). LI~SMAIER, E. lVi., and F. SKOOG: Organic growth factor requirements of fx)bacco tissue cultures. Physiol. Plant. 18, 100--127 (1965). S~E~IDA~, W. F., and H. STERN: Histones of meiosis. Exp. Cell Res. 45, 323--335 (1967). Y~nWADA,Y., K. T A ~ A , and E. T ~ A ~ S ~ : Callus induction in rice, Oryza sativa L. Proc. Jap. Acad. 48, 156--159 (1967). Dr. WILLIAMF. S~ERIDAN Department of Botany University of Missouri Columbia, Missouri 65202

Tissue culture of the monocotLilium.

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