In vitro vegetative multiplication of Fuchsia hybrida

Scientia Horticulturae, 21 (1983) 67--71

67

Elsevier Science Publishers B.V., Amsterdam - - P r i n t e d in The Netherlands

IN VITRO VEGETATIVE MULTIPLICATION OF FUCHSIA H Y B R I D A

Cl. KEVERS, M.F. COUMANS-GILLES, M. COUMANS and Th. GASPAR

Hormonologie Fondamentale et Appliqu~e, Institut de Botanique (B 22), Universitd de LiJge, Sart Tilman, B-4000 LiJge (Belgium) (Accepted for publication 27 December 1982)

ABSTRACT Kevers, Cl., Coumans-Gilles, M.F., Coumans, M. and Gaspar, Th., 1983. In vitro vegetative multiplication of Fuchsia hy brida. Scientia Hortic., 2 1 : 6 7 - - 7 1 . Rapid development of axillary buds from shoot-tips and nodes of 18 cultivars of

Fuchsia hybrida was obtained on solid Murashige and Skoog medium with BAP (1 mg 1"I and an auxin ( 0 2 mg 1"1 ). NAA as the auxin appeared to be more active than IAA or IBA. Vegetative shoots were subsequently isolated and developed up to 15 supplementary axillary shoots on the same solid medium. Agitated and non-agitated liquid media of the same composition were less effective. One-cm long shoots could be rooted in 20 days in the presence of IBA before being transferred to soil. Keywords: Fuchsia hy brida; tissue culture; vegetative multiplication. ABBREVATIONS BAP = benzylaminopurine; GA3 = gibberellic acid; IAA = indoleacetic acid; IBA = indolebutyric acid; MS = Murashige and Skoog basal medium; NAA = naphthaleneacetic acid.

INTRODUCTION

Tissue and organ culture are now well established as methods for the rapid vegetative propagation of many ornamental plants (Holdgate, 1977). The system facilitates build-up of initial propagation stock (Murashige, 1974; Hussey, 1978), the production of virus-free plants (Mellor and StaceSmith, 1977) and the storage of valuable germplasm (Bajaj and Reinert, 1977). In the present paper, the regenerative capacity of Fuchsia hybrida is investigated in vitro. MATERIALS AND METHODS

Petioles, leaves, stems, flowers, shoot-tips and nodes of Fuchsia hybrida

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© 1983 Elsevier Science Publishers B.V.

68 ('Mrs. Popple') were collected from flowering plants grown in a greenhouse. In addition, 17 more 'cultivars' were tested. All plant parts were surface-sterilized b y immersion in 1% " T e e p o l " (S.A. Belgium Shell) and 2% sodium hypochloride solution (30 min), before being rinsed in 3 passages through sterile water. After sterilization, shoot-tips and nodes were reduced to 3 mm in length, petioles and stems to 10 mm and leaves into 1-cm 2 sections. Single sterile explants were placed in 50-ml flasks. The basal medium (MS) of Murashige and Skoog (1~62) was supplemented by BAP, an auxin (IAA, NAA or IBA) and/or GAs, and adjusted to pH 5.8. The growth regulators were added before autoclaving (20 min, 121 ° C, 1.2 kg cm-2). For solid culture, 0.8% Difco Bacto agar was added; 10 and 100 ml were dispensed into 50-ml and 500-ml flasks, respectively. Fifty ml of liquid medium was used in 100-ml Erlenmeyer flasks for agitated liquid culture. The cultures were maintained in a growth chamber at 24°C with 16-h photoperiod and light intensity of 1000 lux from Sylvania Gro-Lux fluorescent lamps. R o o t e d plantlets were transferred from in vitro conditions to c o m p o s t and grown in a shade-house for 15 days at 17°C with 16-h photoperiod and light intensity of 10 000 lux, before being delivered to horticulturists for commercial use. Standard deviations in the tables were calculated from 3 series of 10 explants. RESULTS O r g a n o g e n e s i s f r o m e x p l a n t c u l t u r e . - - Excised flowers, shoot-tips, nodes and segments of petioles, leaves and stems were cultured on MS solid medium supplemented with BAP at 1 mg 1-1 in combination with NAA, IBA or IAA at 0.1 or 1 mg 1"1 . On all media, petioles, leaves, stems and flowers callused or became necrotic. Shoot-tips and axillary buds grew readily. The mean number of shoots formed by these explants is given in Table I. Young nodes (taken from the terminal 10 cm of branches) showed the same capacity for shoot formation as shoot-tips proliferating new shoots. Old nodes were less active. Among the 3 auxins used, NAA at 0.1 mg 1"1 tended to give the best proliferation rates. When the BAP level was increased, the n u m b e r of shoots decreased (not in the table). S h o o t m u l t i p l i c a t i o n . - - After 5 weeks of culture, the new axillary-produced shoots (0.5--1.0 cm long) were dissected and planted separately onto solid initial explant medium with or without GA3 (Table II). Up to 15 such axillary shoots were obtained from a single shoot. GA3 at 1 mg 1"1 did not significantly modify the auxin effect. Increase in GA3 concentration (10 and 100 mg 1-1 ) reduced shoot proliferation rates (not in the table). In addition, the plantlets were weak and spindly.

69 TABLE I Mean n u m b e r of shoots f o r m e d o n Fuchsia 'Mrs. P o p p l e ' explants after 30 days as a f u n c t i o n of the auxin used and o f its concentration. All media were s u p p l e m e n t e d with 1 mg l "~ BAP Explant

Auxin IAA

NAA

0.1 mg

1 mg

Oldnodes

1.3±0.8

0

Youngnodes

9.3±0.9

Shoot~ips

8.9±1.3

0.1 mg

IBA 1 mg

0.1 mg

1 mg

2.4±0.8

0.3±0.9

1.1±0.7

0.5±0.4

3.5±0.7

10.5±1.4

6.4±1.0

8.5±1.3

4.1±0.9

4.4±1.0

10.4±1.1

5.7±0.9

8.8±1.0

3.9±0.6

T A B L E II S h o o t multiplication (mean n u m b e r after 5 weeks) by an in vitro previously-formed shoot-plantlet as a f u n c t i o n of GA~ and N A A or I A A concentration. All media were s u p p l e m e n t e d with 1 mg 1"~ BAP GA 3 (mg 1"1 )

N A A (rag 1"~ )

I A A (mg 1-~)

0.1

1

0.1

1 4 . 7 + 1.8 1 4 . 4 ± 2.1

1 0 . 5 + 1.7 1 1 . 1 + 1.3

1 3 . 8 + 2.3 8 . 3 + 1.4 1 3 . 8 ± 1.9 6 . 8 ± 2.1

1

S h o o t multiplication in 18 cultivars on solid and liquid media. - - S h o o t -

tips and nodes from the 18 cultivars listed in Table III were assayed on the MS initial explant medium. They formed shoots which could be dissected and planted separately on the same solid or liquid medium for further multiplication. The 18 cultivars multiplied b y 0 to a maximum Of 10.8 (Table III). As a general rule, the solid medium appeared to be best suited for proliferation. Six cultivars did not multiply at all on liquid medium, although they did on the solid one. Shoots formed after 1 month in such conditions were large enough (ca. 1 cm on solid and agitated liquid media, a b o u t 3 times longer on non-agitated liquid medium) to be successfully transferred on to a rooting-medium. R o o tin g . -- O n e ~ m long axillary shoots rooted (3--4 fascicular roots per

plantlet) in a b o u t 20 days on the MS medium supplemented with 1 mg 1-1 IBA. R o o t e d plantlets could be transferred to soil where 95% of them successfully developed into adult plants.

70 TABLE III Mean number of axillary shoots formed by 18 cultivars, after 30 days, as a function of medium used (solid, agitated and non-agitated). All media were supplemented with 1 mg 1-~ BAP and 0.1 mg 1-1 NAA Cultivar

Tingaling The Doctor Wasser Nymphe Charming Leonora Fulgens Corallina Boliviana Marinka Rufus Beacon Pin-up Winston Churchill Liebreitz Orange Star Trail Blazer Pink Pearl La Campanella

Solid medium

3.1 6.2 4.6 7.2 2.1 8.4 10.4 4.1 10.8 9.9 5.7 5.2 2.8 5.5 3.5 5.6 6.3 8.7

± 0.6 ± 1.4 ± 2.0 ± 1.7 ± 0.3 ± 1.6 ± 2.1 ± 1.1 ± 1.8 ± 1.6 ± 1.2 ± 0.8 ± 0.5 ± 0.9 ± 0.5 ± 0.8 ± 1.5 ± 1.3

Liquid media Agitated

Non-agitated

0 2.1 0 2.8 0 2.7 5.4 1.7 1.3 2.8 1.7 1.3 0 0 0 0 1.5 2.4

0 3.2 0 5.9 0 4.9 10.5 2.9 3.2 3.9 4.1 3.0 0 0 0 2.7 2.3 3.4

± 0.6 ± 1.2 ± 1.2 ± 1.3 ± 0.5 ± 0.3 ± 0.7 ± 0.3 ± 0.3

± 0.4 ± 0.2

± 0.5 ± 1.3 ± 0.9 ± 1.9 ± 0.8 ± 0.7 ± 1.3 ± 1.2 ± 0.6

± 0.4 ± 0.5 ± 0.7

DISCUSSION T h e i n d u c t i o n o f m u l t i p l e s h o o t s in vitro is possible in a wide range o f species (Murashige, 1 9 7 4 ) . S u c h s h o o t s are p r o d u c e d either b y stimulating a d v e n t i t i o u s b u d s t o f o r m o n t h e initial explant, o n callus i n d u c e d f r o m it, or b y e n c o u r a g i n g t h e rapid d e v e l o p m e n t o f axillary buds. S h o o t s f r o m axillary b u d s are preferable f o r vegetative p r o p a g a t i o n because o f t h e r e d u c e d risk o f genetic instability (Hussey, 1 9 7 8 ) . The rapid vegetative m u l t i p l i c a t i o n o f Fuchsia hybrida in t h e p r e s e n t w o r k was achieved t h r o u g h axillary s h o o t d e v e l o p m e n t . O u r t e c h n i q u e is d i f f e r e n t f r o m t h a t o f S t e v e n s o n a n d Harris ( 1 9 8 0 ) in t h a t it uses a solid m e d i u m instead o f liquid ones, b u t it m u s t be m e n t i o n e d t h a t o u r e x p l a n t s c a m e f r o m flowering plants, while t h o s e o f S t e v e n s o n a n d Harris were t a k e n prior t o flower initiation. This o n c e m o r e e m p h a s i z e s t h e i m p o r t a n c e o f the p h y s i o logical state o f the m o t h e r - p l a n t (Gaspar, 1 9 8 1 ) w h e n used as a s o u r c e o f explants. T h e d i f f e r e n c e s registered in t h e o r g a n o g e n e t i c capacities a m o n g t h e 18 F u c h s i a cultivars t e s t e d either p o i n t e d to genetic influences, or m i g h t be related t o their d i f f e r e n t a d v a n c e d f l o w e r i n g state at t h e mom e n t o f e x p l a n t a t i o n . Similarly, t h e relatively h i g h e r r e g e n e r a t i o n c a p a c i t y

71 o f y o u n g e r n o d e s m i g h t be discussed in r e l a t i o n to b i o c h e m i c a l g r a d i e n t s existing a l o n g v e g e t a t i v e a n d f l o w e r i n g axes ( T h o r p e et al., 1 9 7 8 ) . T h e ability to r e g e n e r a t e F u c h s i a b y tissue c u l t u r e t h e r e f o r e d e p e n d s o n t h e n a t u r e o f t h e p l a n t - p a r t used, its p o s i t i o n a n d t h e stage o f d e v e l o p m e n t of t h e s o u r c e p l a n t . ACKNOWLEDGEMENT This w o r k was s u p p o r t e d b y Belgian g r a n t E t a t - U . L g . N ° 8 0 / 1 0 . 4 7 3 .

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