Rapid vegetative propagation of Hyacinthus orientalis L. in vitro

Scientia Horticulturae, 3 (1975) 293--297 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

RAPID V E G E T A T I V E P R O P A G A T I O N O F H Y A C I N T H U S O R I E N T A L I S L. IN V I T R O

R.L.M. PIERIK and A.J.M. POST Department of Horticulture, Agricultural University, Wageningen (The Netherlands) Publication 407 (Received November 29th, 1974)

ABSTRACT

Pierik, R.L.M. and A.J.M. Post, 1975. Rapid vegetative propagation of Hyacinthus orientalis L. in vitro. Scientia Hort., 3: 293--297. A simple in vitro system was developed to increase the vegetative propagation rate of hyacinth, Hyacinthus orientalis L. Excised bulb scale segments were induced to regenerate bulblets on the basal sides of the inverted explants. Within 12 weeks, 240--300 bulblets were obtained from a single 17--18 cin bulb when cut into basal scale segments, 3--4 cm long and 0.5 cm wide.

INTRODUCTION With the classical propagation m e t h o d s (scooping and scoring) t he commercial i n t r o d u c t i o n of a new h y a c i n t h cultivar takes m any years. F o r t h a t reason an in vitro m e t h o d was developed t o accelerate the multiplication rate and to m a rk ed ly increase the potential n u m b e r o f bulblets (Pierik and Woets, 1971; Pierik and Ruibing, 1973; Pierik and Steegmans, 1975). This paper describes the the m o s t economical m e t h o d o f cutting explants from the scales and h o w 2 4 0 - - 3 0 0 bulblets can be obtained from one 17--18 cm bulb. MATERIAL AND METHODS All experiments were done with bulb scale segments o f the hyaci nt h (Hyacinthus orientalis L.) cv. 'Pink Pearl'. Details on plant material, preparation and sterilization of scales, inoculation, basic culture m edi um and growth conditions have been previously described (Pierik and Ruibing, 1973). All experiments were c o n d u c t e d in 1973 and replicated twice. Data given represent the averages o f two experiments which had c o m pl et el y similar results. Experimental m e t h o d s for each e x p e r i m e n t are outlined in Figs 1--3.

294

cm 4

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b

c

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c

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distat

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--basat

Figs 1--3. Various methods of cutting explants. Fig.1. Comparison of basal (a), distal (b) and basal and distal (c) explants at constant surface area. Fig.2. Effect of the length of the explants at constant width. Fig.3. Effect of the width of the explants at constant length.

RESULTS Position o f the explant in the bulb scale at constant surface area (Fig.1 a, b, and c). -- Three m e t h o d s of cutting explants from a hyacinth bulb scale were compared. A characteristic difference in regeneration and bulblet weight between basal (a) and distal (b) explants is shown in Table I (exp. 1). Although the numbers of bulblets per explant are identical in a and c, a p r o n o u n c e d difference in bulb growth occurred. A great difference was apparent in the total n u m b e r of bulblets produced per scale area of 4 × 2 cm in 2 ( l a + l b ) and 4 ( l c ) , 4.4 and 7.6 respectively, and in the total bulblet weight produced in 2 ( l a + l b ) and 4 ( l c ) , 76 and 268 mg, respectively. It is evident that m e t h o d c was the most effective way of cutting explants. Effect o f the length o f the explant at constant width (Fig.2 a, b, c and d). -The n u m b e r o f bulblets per explant increased nearly linearly by increasing the length of the explants (Table I, exp. 2). Bulblet weight was also greatly increased. This experiment showed that the bulb regeneration factors were distributed at random over the various parts of the bulb scale, at least when each explant contained a part of the basal portion of the scale. The distribution of the bulblet growth factors over the various parts of the scale was n o t as regular since the increase of bulblet growth was maximum when the length of the explant was increased from 2 to 3 cm and was lower when the explant length was increased from 3 to 4 cm. Effect o f the width o f the explant at constant length (Fig.3 a, b and c). -Table I (exp. 3) shows that the greatest number of bulblets per cm 2 were obtained by m e t h o d a (width 0.5 cm). The highest bulblet weight per cm 2 explant was obtained by applying m e t h o d b or c.

295 C o r r e l a t i o n b e t w e e n s u r f a c e area and regeneration. - - T h e c o r r e l a t i o n b e t w e e n

s u r f a c e a r e a a n d r e g e n e r a t i o n ( b u l b l e t s p e r e x p l a n t a n d t o t a l fresh w e i g h t o f t h e b u l b l e t s p e r e x p l a n t ) is given in Fig.4 in w h i c h values o f exp. 2 a n d 3 f r o m T a b l e I are used. Fig. 4 (left) clearly s h o w s t h a t t h e n u m b e r o f b u l b l e t s p e r e x p l a n t increased n e a r l y linearly b y increasing t h e surface area o f t h e e x p l a n t s . W h e n t h e s u r f a c e area w a s p l o t t e d against t o t a l fresh w e i g h t p e r e x p l a n t (Fig.4, right) an e x p o n e n t i a l r e l a t i o n s h i p existed b e t w e e n 0 a n d 2 c m 2 a n d a linear o n e b e t w e e n 2 - - 6 c m 2 s u r f a c e area. T o t a l b u l b l e t p r o d u c t i o n p e r bulb. - - F r o m p r e v i o u s e x p e r i m e n t s a n d t h e d e t e r m i n a t i o n o f t h e t o t a l s u r f a c e a r e a o f scales p e r b u l b o f 1 7 - - 1 8 c m , t h e p o t e n t i a l n u m b e r o f b u l b l e t s p e r b u l b was c a l c u l a t e d t o b e a p p r o x . 2 0 0 - - 2 5 0 . Since 1 0 - - 2 0 % i n f e c t i o n c a n b e e x p e c t e d , 1 6 0 - - 2 0 0 b u l b l e t s p e r b u l b c a n be h a r v e s t e d a f t e r 12 w e e k s . I f in this research t h e a u x i n I A A h a d b e e n a p p l i e d t o t h e m e d i u m at 10 mg/1 (Pierik a n d S t e e g m a n s , 1 9 7 5 ) , t h e n u m b e r o f b u l b lets c o u l d h a v e b e e n i n c r e a s e d b y a p p r o x . 50%, bringing t h e b u l b l e t p r o d u c tion per bulb ultimately to 240--300.

DISCUSSION T h e e f f i c i e n c y o f this p r o p a g a t i o n m e t h o d a p p e a r s to b e s t r o n g l y d e p e n d e n t o n t h e w a y in w h i c h e x p l a n t s are c u t . Essential p r e r e q u i s i t e s f o r g o o d regenerat i o n a n d b u l b l e t g r o w t h are: t h e p r e s e n c e o f t h e basal p a r t o f a scale a n d a TABLEI Regeneration and bulblet growth on excised bulb scale segments of hyacinth after 12 weeks. For explanation of the treatments see Figs 1--3. Exp. 1. The effect of the origin of the explants (0 x means less than 0.5 rag). Exp. 2. The effect of the length of the explants at constant width. Exp. 3. The effect of the width of the explants at constant length. Experiment

1

2

3

Treatment

Fig.la Fig.lb Fig.lc Fig.2a Fig.2b Fig.2c Fig.2d Fig.3a Fig.3b Fig.3c

Bulblet regeneration (%)

Number of bulblets per explant total

per cm ~

91 24 88 72 89 89 90 83 91 96

1.8 0.4 1.9 1.2 2.0 2.9 3.8 2.5 3.6 5.2

0.9 0.2 0.9 1.2 1.0 1.0 0.9 1.2 0.9 0.9

Total weight per explant (mg) fresh dry total per total per cm; cm 2 37 1 67 20 60 131 173 54 175 289

18 0x 33 20 30 44 43 27 44 48

10 0x 16 5 15 31 39 13 40 65

5 0x 8 5 8 10 10 7 10 11

296

totat fresh weight per exptant (rag) 300 --

number of burblers per exptant 6--

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Fig.4. Correlation between surface area and number of bulblets per explant (left) and between surface area and total fresh weight per explant. relatively small width and large length of the explants. The observation in horticultural practice that hyacinths must n o t be scooped t o o deeply, agrees with our observation that the basal part of a scale appears to be essential to obtain the o p t i m u m number of bulblets. In addition to the 'regeneration factors' contained in the basal part of a scale, however, the distal parts also contribute to the increase in number of bulblets. Bulb growth appears to be strongly due to explant size and thereby the total food reserve or some other u n k n o w n c o m p o n e n t in the explant. Both basal and distal parts o f a scale contribute to bulblet growth, although the distribution of bulblet growth factors over the various parts o f a scale is n o t at random (Table I, exp. 2). However, since 'regeneration factors' are less c o m m o n in the distal parts, bulblet growth can hardly manifest itself in distal explants. In practical bulb propagation, growers can regulate the number of bulblets and bulblet weight by choosing b e t w e e n scooping or scoring. The in vitro system which we have developed permits an analogous choice: the largest n u m b e r of small bulblets can be obtained b y taking explants of 0.5 cm width or a smaller n u m b e r of large bulblets can be harvested b y cutting explants of 1.0 or 1.5 cm width. ACKNOWLEDGEMENTS The authors are greatly indebted to the Royal Netherlands Bulb Nurseries, Van Zanten Brothers, Hillegom, The Netherlands, for the supply o f the hyacinth bulbs. Thanks are also due to Miss H.H.M. Steegrnans for her valuable

297 t e c h n i c a l assistance a n d t o P r o f e s s o r D r W.J. K e n d e r f o r critically c o m m e n t i n g upon the manuscript.

REFERENCES Pierik, R.L.M. and Ruibing, M.A., 1973. Regeneration of bulblets on bulb scale segments of hyacinth in vitro. Neth. J. Agric. Sci., 21: 129--138. Pierik, R.L.M. and Steegmans, H.H.M., 1975. Effect of auxins, cytokinins, gibberellins, abscisic acid and ethephon on regeneration and growth of bulblets on excised bulb scale segments of hyacinth. Physiol. Plant., in press. Pierik, R.L.M. and Woets, J., 1971. Regeneration of isolated bulb scale segments of hyacinth. Acta Hortic., 23 (2): 423--428.