Scientia Horticulturae, 19 (1983) 387--394 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
387
THE STIMULATORY EFFECT OF LIQUID INDUCTION MEDIUM ON SHOOT PROLIFERATION OF R USCUS HYPOPHYLLUM L.
MEIRA ZIV
The Hebrew University of Jerusalem, Faculty of Agriculture, Department of Agricultural Botany, Rehovot (Israel)
(Accepted for publication 21 June 1982)
ABSTRACT
Ziv, M., 1983. The st imulatory effect of liquid induction medium on shoot proliferation of Ruscus hypophyllum L. Scientia Hortic., 19: 387--394.
Pre-culture of shoot tip and inflorescence explants of Ruscus hypophyllum (popular names are "butcher ' s b room'~or "mouse thorn") in shake liquid culture increased both axillary and adventit ious shoot proliferation 6--10 fold. Explants pre-cultured in agitated Linsmaier and Skoog liquid medium containing 1 mg 1-1 each of 2,4-dichloroacetic acid and 6-benzylamino purine had to be sub-cultured after 15 days to a solid medium with a higher (5:1) cytokinin: auxin ratio for shoot development. Explants remaining in liquid medium increased in size and produced only few shoots. The initial agitation in liquid medium provided the explants wi~b opt imum contact with the medium and the subsequent release from apical dominance after transfer to stat ionary culture. In addition, the agitation in liquid medium induced adventitious shoot proliferation from the epidermal tissue of the phylloclades.
INTRODUCTION
Ruscus hypophyllum L. (Liliaceae) is a perennial shrub, native to the Mediterranean region. Its commercial cultivation for the product ion of orna- mental evergreen branches is dependent mainly on vegetative propagation by division of its subterranean rhizomes. The developmental morphology of I~uscus in relation to environmental effects has recently been described (Kigel et al., 1981). The growth cycle of plants grown in Israel is limited to two sea- sons; a short one in early autumn and one from late winter to early spring. Although 7--8 vegetative buds appear on each rhizome, only one or two have regenerative potential. The slow plant development and the limited growth period, determined by environmental conditions, combine to make the com- mon practice of propagation inadequate to keep pace with the demand for new selected clones.
The culture in vitro of excised tissue of several horticultural crops and their ability to redifferentiate has rendered this technique a useful tool for asexual propagation (Murashige, 1974; Holdgate, 1977). Reports on successful regener ation of ornamental members of the Liliaceae through adventitious bud
proliferation or bulblet production were reviewed recently by Hughes (1981). Hussey (1976) promoted axillary bud proliferation in Liliaceae through release from apical dominance by high cytokinin. In vitro propagation techniques deve- loped so far have been limited mostly to herbaceous species, not to woody types like Ruscus.
This paper describes the inductive effects of pre-culture in an agitated liquid medium on the production of both axillary and adventitious buds from Ruscus hypophyllum shoot tip and inflorescence explants.
MATERIALS AND METHODS
Ruscus plants were grown in the phytotron of the Faculty of Agriculture in Rehovot. A 16-h photoperiod was achieved with extension of natural day- length by incandescent light of 1 X 10 -3 W cm -2 at 17/12°C day and night temperature.
Explants were taken from the terminal or lateral buds of exposed rhizomes or from the phyUoclades of newly formed shoots. The rhizomatous buds were exposed from their outer scales, rinsed for 24 h under running water and dipped in 70% ethanol. Surface sterilization with 9% CaHC104 for 15 rain was followed by rinsing 3 times in sterile water. Shoot tip explants, 0.5--0.8 cm long, were dissected on a sterile filter paper soaked with 1% ascorbic acid. Explants 0.3 X 0.5 cm, including the inflorescence bud, were removed from young, partly expanded phylloclades (not larger than 0.5 × 1.0 cm). Follow- ing sterilization (4.5% CaHC104) and prior to culturing the explants, the outer protective scales of the inflorescence bud were removed.
The basal medium (LS) of Linsmaler and Skoog (1965) was supplemented with different concentrations of 6-benzylaminopurine (BA) or kinetin (K) in combination with either naphthyl-acetic acid (NAA), indole-acetic acid (IAA) or 2,4-dichloroacetic acid (2,4-D). Explants were cultured in liquid or on solid medium. Twenty ml of liqt~id medium were dispensed into 50-ml Erlenmeyer flasks into which 5 explants were introduced. The cultures were agitated on a horizontal shaker at 120 strokes per minute. Ten ml of 1% agar solidified medium were dispensed into 2.0 × 10.0 cm culture tubes in which one explant was planted. The cultures were placed in an environment-control- led chamber, under a 14-h photoperiod of 1.5 X 10 -3 W cm -2 at a constant temperature of 20 + I°C.
For microscopic examination, inflorescence samples were placed on scan electron microscope (SEM) stubs, frozen in liquid nitrogen, freeze-dried at -40°C and coated with gold. The samples were viewed in a JSM35C Jeol Model at 25 KV.
RESULTS
Ruscus shoot tips placed on LS medium, containing different combina- tions of auxins and cytokinins, grew slowly and produced only a few buds after 12 weeks in culture. Six buds developed from explants cultured on LS
389
m e d i u m c o n t a i n i n g 5 m g 1-1 K a n d 1 m g 1-1 2 ,4-D, wh i l e all o t h e r a u x i n /
c y t o k i n i n c o m b i n a t i o n s p r o d u c e d st i l l f ewe r b u d s ( T a b l e I). T h e s a m e t rea t -
m e n t s d id n o t i n d u c e a d v e n t i t i o u s b u d s f r o m e i t h e r t h e p h y l l o c l a d e s o r t he
TABLEI
The effect of different combinations of growth regulators on bud proliferation from Ruscus shoot tip explants, expressed as percent cultures proliferating and mean number of buds per explant ± SE
Fig. 1. Shoot tip cultured continuously for 58 days in agitated LS liquid culture con- taining 1.0 mg 1 "! each of BA and 2,4-D. Growth and callus formation with some bud regeneration can be observed (x 2.5).
390
inflorescence, except for a small amount of callus which was induced on the cut ends of phyUoclade explants. However, callus redifferentiated into buds, following excision and subcul ture to a high (5 mg l "1 ) cytokinin medium, after 20 weeks in culture.
The possibility that limited adventitious bud regeneration was due to slow growth-substance penetration into the explants suggested the use of an agi- tated liquid medium. Proliferation was not observed to increase in explants which were continuously cultured in agitated liquid medium for several weeks, nor in those which were further sub-cultured to hormone-free static media. Continuous shake liquid culture induced bud growth with slight pro- liferation and callus production (Fig. 1 ).
Further transfer from liquid agitated culture to solid medium containing 5 mg 1 "I BA and 1 mg 1 "1 2,4-D increased bud regeneration (Fig. 2). Of the different growth-regulator combinations tried, BA and 2,4-D (1 mg 1 "1 each) were found opt imum for pre-culture. As depicted in Fig. 4A, pre-culture on solid induction medium had little effect on bud regeneration of both shoot and inflorescence explants. Pre-culture of shoot tip explants in agitated li- quid medium for 15 days, followed by sub-culture to solid medium, in- creased bud regeneration by about six-fold, inducing both axillary and ad- ventitous bud formation. Adventitious buds also developed on newly-form- ed thick phylloclades (Fig. 2).
Fig. 2. Effect of liquid pre-culture (see F ig . l ) for 15 days followed by transfer to solid medium containing 5.0 mg 1 "1 BA and 1.0 mg 1 "12,4-D, on terminal shoot tip prolifera- tion. Both axillary (a) and adventitious (b) buds developed after 42 days in stationary culture (x 3.0).
391
Fig. 3. Floral (f) and vegetative (v) bud development from inflorescence explants pre- cultured for 15 days in liquid LS medium followed by transfer to solid LS medium with 5.0 mg I'ZBA and 1.0 mg 1 "1 2,4-D for an additional 56 days (X 2.5).
i-/-,0 z , - - . I Q. I..iJ
_~20
O
0
I I 1 I I
_ ~ I I I I 5 10 15 20 25
I I I I I
-® T SD
~ • A A
0 5 10 15 20 25 DAYS IN INDUCTION MEDIUM
Fig. 4. The effects of pre-culture for 15 days in shake liquid medium (open symti~ls) or agar medium (closed symbols) with 1.0 mg 1" each of BA and 2,4-D, on bud regenera- t ion from shoot tip (A) and inflorescence (B) explants. Vegetative (--o-- ; --e--) and floral (--~--, --A--) buds produced upon transfer to agar solidified medium with 5 mg 1 "1 BA and 1.0 mg 1 "1 2,4-D.
392
Similarly, inflorescence bud proliferation was found to increase signifi- cantly by the use of agitated liquid pre-culture (see Fig. 4B). Inflorescence explants pre-cultured for 15 days regenerated 34 vegetative and 24 floral buds; a total of 58 buds per explants (Figs. 3 and 4B).
Fig. 5 AB. SEM of inflorescence bud explants showing vegetative (A) and flower (B) bud development after pre-culture for 15 days in agitated liquid medium (see Fig. 1) (A x 32; B x 24).
393
Pre-culture periods longer than 15 days decreased the number of prolife- rating buds from both shoot tip and inflorescence explants (Fig. 4A,B). Vegetative bud formation from inflorescence explants was induced by the agitation in liquid medium. These buds appeared in the same scale axils where flower buds normally develop (Fig. 5A,B).
DISCUSSION
The physical state of the medium had a pronounced effect on Ruscus shoot proliferation. Proliferation of Ruscus explants was observed to in- crease about 6- and 10-fold in shoot tip and inflorescence explants, respec- tively, by pre-culture in agitated liquid medium. Pre-culture in liquid me- dium had to be followed by sub-culture to BA- and 2,4-D-containing solid medium to induce shoot development. Explant culture in static culture induced proliferation of very few buds.
It is possible that the failure, or slow organ-forming potential in Ruscus and other species in stationary cultures, could be due to lack of, or slow, pene- tration from the medium of growth substances or other constituents. This may have been overcome in Ruscus by agitation in liquid medium for 15 days, which also provided better aeration of the explants. The decreased prolifera- tion observed in prolonged shake liquid culture could be due to the accumu- lation of toxic substances. In addition, agitation could serve to break apical dominance and induce axillary bud growth, as observed in some monocoty- ledonous plants by high cytokinin treatment (Hussey, 1976). Species belonging to the Liliaceae required high concentrations of BA to promote axillary branch- ing (Hussey, 1977). In Ruscus, both liquid pre-culture and a high cytokinin were required for axillary and adventitious bud development. The cauline nature of the phylloclades, which are heavily cutinized, could have contri- buted to these requirements.
Callus originating from the cut ends of the phylloclade explants organized when placed on a high cytokinin medium. Ruscus explants required both BA and 2,4-D after sub-culture from the inductive medium. Adventitious shoot proliferation from new phylloclades in culture was observed to develop from the epidermal layer of liquid agitated shoo t tip explants. The agitation could have prevented inter-tissue correlative inhibition effects (Tran Thanh Van Kiem et al., 1974; Tran Thanh Van Kiem, 1981) existing in static cultures, promoting bud regeneration from epidermal cells without the need for tissue excision and separation from the explant.
Unlike flower formation observed in vivo, as many as 24 flower buds developed from a single inflorescence in vitro, which usually produces only 3--4 flowers. Observation under SEM indicated that vegetative buds did not exist in the inflorescence prior to sub-culthre from liquid to solid medium, and could have been induced by the agitation in the inductive medium.
The observation that better shoot proliferation took place in explants which were sub-cultured from agitated to stationary cultures may indicate
394
a two-step response. Initial agitation released the explants from apical domi- nance and inter-tissue correlative inhibition (Tran Thanh Van Kiem, 1981), while sub-culture to static culture allowed bud development and growth, as long as growth substances were supplied to the medium.
ACKNOWLEDGEMENT
The author wishes to thank Miss Geula Gadasi for her skillful technical help.
REFERENCES
Holdgate, D.P., 1977. Propagation of ornamentals by tissue culture. In: Applied and Fundamental Aspects of Plant, Tissue and Organ Culture. J. Reinert and Y.S.P Bajaj (Editors), Springer Verlag, Berlin, pp. 18--42.
Hughes, K.W., 1981. Ornamental species. In: B.V. Conger (Editor) , Cloning Agricultural Plants via in vitro Techniques, CRC Press, Boca Raton, FL, pp. 5--50.
Hussey, G., 1976. In vitro release of axillary shoots from apical dominance in mono- cotyledonous plantlets. Ann. Bot., 40: 1323--1325.
Hussey, G., 1977. In vitro propagation of some members of the Liliaceae, Iridaceae and Amaryllidaceae. Acta Hortic., 78 : 303--309.
Kigel, J., Seeman, V. and Mayak, S., 1981. Environmental effects on the developmental morphology of Ruscus hypophyllum L. Israel J. Bot., 30: 65--74.
Linsmaier, E.D. and Skoog, F., 1965. Organic growth factor requirements of tobacco tissue culture. Physiol. Plant., 18: 100--127.
Tran Thanh Van Kiem, M., 1981. Control of morphogenesis in vitro cultures. Annu. Rev. Plant Physiol., 32: 291--311.
Tran Thanh Van Kiem, M., Chlyah, H. and Chlyah, A., 1974. Regulation of organogenesis in thin layers of epidermal and sub-epidermal cells. In: H.E. Street (Editor), Tissue Culture and Plant Science. Academic Press, London, pp. 101--139.
