Planta 138, 295-297 (1978)

PI~JH-~ 9 by Springer-Verlag 1978

Short Communication Xanthium Pith and Hypoeotyl Tissue in Culture H a n n a h Bonsey Suthers Department of Biology, Princeton University, Princeton, NJ 08540, USA

Abstract. A r a p i d m e t h o d is d e s c r i b e d o f o b t a i n i n g callus tissue cultures f r o m h y p o c o t y l s o f vegetative a n d flowering Xanthiurn strumarium L. seedlings. T h e tissue is g r o w n on M u r a s h i g e a n d S k o o g m e d i u m m o d i f i e d with 1 g/1 casein h y d r o l y s a t e a n d 5 mg/1 e a c h o f kinetin a n d ~ - n a p t h a l e n e a c e t i c acid.

Key words: Tissue culture -

Xanthium.

X a n t h i u m strumarium L. is one o f the p r i n c i p a l m a t e rials in the study o f the p h y s i o l o g y o f flowering (see review by Salisbury, 1969). S h o o t tips can be g r o w n in axenic culture a n d be i n d u c e d to flower f o r m a t i o n (Jacobs a n d Suthers, 1971). F o r studies o n certain aspects o f the r e g u l a t i o n o f flowering it w o u l d be very d e s i r a b l e to culture X a n t h i u m callus tissue in vitro a n d to o b t a i n r e g e n e r a t i o n o f shoots. F o r e x a m ple, such a system c o u l d be used to a n s w e r the question o f w h e t h e r cells f r o m an i n d u c e d p l a n t " r e m e m b e r " the flowering c o n d i t i o n . F o x a n d M i l l e r ' s (1959) a t t e m p t s to g r o w Xanthium c a m b i a l tissue on v a r i o u s synthetic m e d i a were unsuccessful, b u t with the use o f c o r n steep w a t e r s o m e g r o w t h was attained. F r a t t a r e l l i et al. (1972) got stem callus to g r o w on a M u r a s h i g e a n d S k o o g (1962) m e d i u m m o d i f i e d by a d d i n g g l u t a m i c acid, a s p a r a g i n e , kinetin a n d ~.-napthaleneacetic acid. H e r e I describe a r a p i d m e t h o d o f establishing tissue lines f r o m h y p o c o t y l tissue o f vegetative a n d flowering X a n t h i u m seedlings on a M u r a s h i g e a n d S k o o g m e d i u m with c e r t a i n new m o d i f i c a t i o n s .

To obtain the tissues, burs from the Princeton "X. pensylvanicure '" strain were germinated aseptically (Jacobs and Suthers, 1974) directly on nutrient agar in dark, then put in growth chambers at 26~ Tissue was excised from vegetative seedlings after 1-4 long days of 16 h light (31 W m- 2 from 22 cool-white VHO fluores-

cent lamps and 6 75-W incandescent lamps; Sylvania, Danvers, Mass., USA) and 8 h dark, and from induced seedlings when the terminal staminate inflorescences had open florets after ca. 30 short days of 8 h light (31 Wm -2) and 16 h dark. Pith callus tissue was cultured from 2-month-old plants grown in soil in a greenhouse under continuous light (daylight supplemented by light from 500-W incandescent lamps). Young internodes were excised from vegetative plants and fiom plants induced by short days as described above until terminal staminate florets opened, ca. 14 d. To surface sterilize the internode segments they were first scrubbed with soap, Then they were alternately dipped 3 times in a commercial sodium hypochlorite solution (5.25%) diluted 3:1 with water plus 14g/l detergent powder ("Coleo"; Colgate-Pahnolive, New York, N.Y., USA) and in 100% ethanol. Finally, the segments were twice dipped in ethanol and flamed. The epidermal layer was peeled off aseptically, and the internode segments were then sliced into 3-ram segments and oriented horizontally on the medium. Tissue was grown on a Murashige and Skoog (1962) based medium supplemented with I g/1 casein hydrolysate ("Edamin"; Sheffield Chemical, Union, N.J., USA) and different concentrations of kinetin and c~-napthaleneacetic acid (NAA). Aliquots of 10 ml were dispensed in 25-ram x 95-ram shell vials and autoclaved 15 min at 121~ The tissue was incubated at 25~ and ca. 440-600 gW m 2 of light from fluorescent lamps or in later experiments in the growth chamber described above at 16h light and 8 h dark daily. Callus tissues were difficult to establish f r o m pith o f 2 - m o n t h - o l d vegetative a n d flowering plants. G r e a t e r callus p r o d u c t i o n was o b t a i n e d f r o m the coty l e d o n s a n d h y p o c o t y l s o f vegetative a n d i n d u c e d seedlings, a n d the callus tissue was r e a d y for transfer at 3 weeks. D e c a d e dilutions o f N A A a n d kinetin in v a r i o u s c o m b i n a t i o n s f r o m 10 -'~ to 1 rag/l, a n d units f r o m 1 to i0 rag/1 were tested a n d the o p t i m a l c o n c e n t r a t i o n for growth, 5 rag/1 each o f k i n e t i n a n d N A A , was a r r i v e d at. Callus tissue f o r m e d m o r e r e a d i l y on m e d i u m with b o t h N A A a n d kinetin t h a n on m e d i u m with either alone, a n d m o r e readily at 25 ~ t h a n 35~ T h e tissues r e g e n e r a t e d r o o t s d u r i n g the first few transfers. N A A p r o m o t e d r o o t f o r m a -

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296

H.B. Suthers:

Xanthium Tissue

in Culture

Table 1. Growth of Xanthium tissue lines on basic Murashige and Skoog medium_+ supplements. Growth is expressed as fresh weight (W) after 5 weeks growth minus initial fresh weight (W0) , divided by the initial fresh weight: (W- Wo)/Wo. Data for tissue in the 9th and 1 lth transfer Tissue line a

47 51 61 71 a b

Medium b

Veg FI Veg Ft

pith pith hyp hyp

Basic

Basic +K

Basic +NAA

Basic +K+NAA

Cas

Cas +K

Cas +NAA

Cas +K+NAA

0.05 1.69 3.44 0.60

3.02 3.22 8.79 2.46

2.41 4.45 0.92 12.6I

31.47 15.35 10.56 8.17

0.I1 0.18 2.18 0.35

3.53 3.40 2.47 0.96

9.16 6.86 7.14 17.73

35.37 20.52 i3.53 29.66

V e g = f r o m vegetative plant, F l = f r o m flower-induced plant, h y p = f r o m hypocotyl K = Kinetin (6-furfurylaminopurine), 5 mg/1; NAA = c~-naphthaleneacetic acid, 5 rag/l; Cas = casein hydrolysate (Edamin)

.3.5

I

1

5.0 /

25

!

/

/~///// 2.0 I

//

ii.///

o

15/

i,0-

///

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/ 9 61 Veg hypocotyl 9 47Veg pith o 71 FI hypocotyl 51 FI pith

.5-

I

2 Xanthium callus

I

4

Fig. 1. Growth of tissue lines. The log of fresh weight at time t divided by initial fresh weight at time 0 is plotted against weeks of growth: In (W/W0). Tissue from the 8th to 1 lth transfer

tion, but if the concentration of kinetin was twice that of NAA, root formation did not occur. However, high kinetin in proportion to NAA, 10 : 1, tended to stabilize tissues in their initial forms so that callus regeneration was retarded. All four established tissue lines continued to require both kinetin and NAA (Table 1). Casein hydrolysate alone did not appear to affect the growth rate

but improved growth when kinetin and NAA were also present. The tissues were even more responsive when benzyladenine at 5 mg/1 was substituted for kinetin, with 5 rag/1 NAA. Growth rates were determined by culturing simultaneously 30 pieces of each of the four tissue lines. Six pieces of each line were weighed initially, then 6 more pieces each at weekly intervals, for 4 weeks. The vegetative tissue from either source, i.e. pith or hypocotyl, grew faster than its induced companion tissue (Fig. 1). Growth of held-over cultures continued through the 6th week, then leveled off. Several treatments were tried to regenerate vegetative shoots or flower buds, but so far with no success: 1) increased light intensity to 31 W m 2 and 16 h light and 8 h dark, daily; 2) L-glutamine (3 mM) and L-tryptophan (2.45 • 10- 5 M) added to the stock medium, to compensate for their absence in casein hydrolysate; 3) adenine ( 3 x l 0 - * M ) and thymine (10 -7 M) added to the stock medium and the NAA and kinetin ratio lowered to 0.5 mg/l: 5 mg/1; 4) substituting 5 mg/1 benzyladenine for kinetin and/or 5 mg/1 2,4-D for NAA; 5) zeatin riboside (5 rag/l) as the cytokinin and different concentrations of NAA (0.01, 0.1, and 5 mg/l) ; 6) 6(Ty-dimethylallylamino)purine at 15rag/1 as the cytokinin and NAA at 0.01 and 5 mg/1. At the time of writing these four tissue lines have been carried for 2 years. They are transferred every 3 weeks, and the growth rate has remained the same. The color of the tissue is a clear tan on the stock shelf at 440-600 I~Wm -2, turns greenish in the 31 W m -2 of the growth chamber, and darkens to brown with age. The generosity of Dr. William P. Jacobs in providing burs and the use of growth chambers, the encouragement of Dr. Frederick Meins, Jr. (presently, University of Illinois, Urbana, IlL, USA) and his help with the manuscript are gratefully acknowledged.

H.B. Suthers: Xanthium Tissue in Culture

References Fox, J.E., Miller, C. : Factors in corn steep water promoting growth of plant tissues. Plant Physiol. 34, 577-579 (1959) Frattarelli, F.H., Rier, J.P., Reid, H.B. : Growth of Xanthium stem callus on a defined culture medium. Physiol. Plantarum 27, 439~4G (1972) Jacobs, W.P., Suthers, H.B.: The culture of apical buds of Xanthium and their use as a bioassay for flowering activity of ecdysterone. Amer. J. Bot. 58, 836-843 (I97l) Jacobs, W.P., Suthers, H.B. : Effects of leaf excision on flowering

297 of J(anthium apical buds in culture under inductive and noninductive photoperiods. Amer. J. Bot. 61, 1016-1020 (1974) Murashige, T., Skoog, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plantarum 15, 473 497 (I962) Salisbury, F.B.: Xanthium strumarium L. In: The induction of flowering - so'me case histories, pp. l 4-61. Evans, L.T. ed. Ithaca: Cornell Univ. Press 1969

Received l l July; accepted 20 October 1977