Induction of bulbing of tulip shoots in vitro

Scientia Horticulturae, 20 (1983) 377--390 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

377

INDUCTION OF BULBING OF TULIP SHOOTS IN VITRO

R.D. RICE, P.G. ALDERSON and N.A. WRIGHT University of No ttingham School of Agriculture, Sutton Bonington, Lough borough, Leics. LE12 5RD (Gt. Britain) (Accepted for publication 14 December 1982)

ABSTRACT

Rice, R.D., Alderson, P.G. and Wright, N.A., 1983. Induction of bulbing of tulip shoots in vitro. Scientia Hortic., 20: 377--390. Bulbing has been induced in adventitious shoots from floral stem explants of Tulipa 'Merry Widow'. Incubation of l-ram thick explants at 20°C on a m e d i u m containing 1 m g I-~ N A A and B A P for 14--18 weeks, followed by 4°C for 8 weeks, produced a consistent bulbing response in shoots after transfer back to 20°C. Bulbing was further enhanced by transfer to 25°C and by raising the concentration of sucrose in the medium. Microscopy indicated the presence of developing meristematic centres in shoots 12 weeks of age. Shoots over 16 weeks of age containing meristematic centres gave a variable bulbing response with applied gibbereUins and cold incubation, depending on whether gibberellins were supplied continuously in the m e d i u m or via a brief soak in sterile gibberellin solutions before sub-culturing. A G A 3 "soak" of 1.0 m g I-I was o p t i m u m for bulb production. The possible mechanisms of cold and gibberellin effects are discussed. Keywords: bulbing in vitro;growth regulators; temperature; tulip. ABBREVIATIONS

BAP = benzylamino-purine; GA = gibberellin; NAA = ~-naphthalene acetic acid; SED = standard error of difference between treatment means.

INTRODUCTION T h e in v i t r o c u l t u r e o f tulip tissues t o p r o d u c e viable p r o p a g u l e s f o r t r a n s f e r t o soil, w i t h a v i e w t o c o m m e r c i a l p r o d u c t i o n , has, until r e c e n t l y , p r e s e n t e d an area in w h i c h little p r o g r e s s has b e e n m a d e . W o r k e r s have r e p o r t e d t h e i n d u c t i o n o f b u d s , m o s t c o m m o n l y o n scale e x p l a n t s (Nishiuchi, 1 9 7 9 ; Rivi~re a n d Muller, 1 9 7 9 ) , a n d b u l b f o r m a t i o n has b e e n o b t a i n e d f r o m axillary b u d s w i t h r o o t g r o w t h a n d b u l b d e v e l o p m e n t e n h a n c e d b y l o w t e m p e r a t u r e s (Rivi~re a n d Muller, 1 9 7 6 ) . Axillary b u d c u l t u r e , h o w e v e r , r e p r e s e n t s o n l y a slight increase a b o v e t h e n a t u r a l m u l t i p l i c a t i o n r a t e o f tulips b y d a u g h t e r b u l b f o r m a t i o n . R e c e n t l y , a d v e n t i t i o u s b u d s o f scale

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378 explants of T u l i p a 'Apeldoorn' have been induced to bulb by low-temperature incubation (Nishiuchi, 1980). Large numbers of adventitious shoots have been produced on floral stem explants on a modified Murashige and Skoog medium (Wright and Alderson, 1980), and the suitability of these shoots for further development to rooting propagules is indicated by the observation of a limited degree of basal or bulbous development. A period of 4°C has been found to induce both the initiation of bulb primordia and the breaking of seed dormancy in tulip embryos (Niimi, 1978, 1980). It is also well established that prolonged periods of cold are required for satisfactory development of spring-flowering bulbs such as tulip (Rees, 1969, 1972; De Hertogh, 1974). This cold requirement has been correlated with an increase in endogenous gibberellins (Alpi and De Hertogh, 1975; Rakhimbaev et al., 1978; Hanks and Rees, 1980), and applied gibberellins have been found to substitute in part for the required cold period (Van Bragt and Zijlstra, 1971; Van Bragt and Van Ast, 1976; Van Bragt and Van Gelder, 1979). The present paper reports the application of low-temperature treatments and treatments with gibberellins for the induction of bulbing in tulip shoots in vitro. MATERIALS AND METHODS - - Tulip bulbs cultivar 'Merry Widow' were obtained from Kirton Experimental Horticulture Station and Lingarden Ltd., Lincs., England, in August 1980 and 1981, respectively. The bulbs were stored dry at 17°C. Sterilization and culture procedures were similar to those described earlier (Wright and Alderson, 1980). Floral stems were excised when between 12 and 14 mm in length, surface-sterilized in 10% (v/v) "Domestos" (a commercial bleach containing 2% available chlorine, Lever Bros., England) for 10 min, and washed 4 times in sterile distilled water.

P l a n t ma t er i a l .

o f c u l t u r e s . - - The medium for shoot production was as described previously {Wright and Alderson, 1980), containing Murashige and Skoog {1962) macro-elements, micro-elements, vitamins and casein hydrolysate (500 mg l-l). NAA (1 mg 1-1) and BAP (1 mg 1-l) were added to the medium, and the pH was adjusted to 6.1 with 0.1 N KOH. Agar (6.75 g 1-1) was dissolved by autoclaving at 121°C for 5 min, and final sterilization was achieved by autoclaving at 121°C for a further 15 min. Explants 1 mm in thickness were dissected from the bases and nodes of floral stems, and grown at 20°C with a photoperiod of 16 h and a light intensity of 30 + 10 ~E m -2 s-~ at culture level. Cold treatment of productive cultures was given after 6, 8, 10, 12, 14, 16 and 18 weeks at 20°C using a temperature of 4°C for periods of 4 or 8 weeks under the same light regime. After the cold treatment, the cultures were returned to 20°C for 16 weeks.

Growth

379 To investigate further the factors affecting bulb production, the following treatments were carried o u t on 16-week-old cultures: (1) cold incubation (8 weeks) in the light followed by transfer to 25°C in the light; (2} cold incubation (8 weeks) in darkness followed by 20°C in the light; (3) cold incubation (8 weeks) in the light followed by 20°C in the light and sub-culture to the same basal medium b u t containing 30, 40, 50, 60, 70 or 80 g 1-1 sucrose. Shoots produced on explants incubated for 16--20 weeks at 20°C were sub-cultured individually on to the basal medium with and without 1.0 or 10.0 mg 1-1 GA4÷7 or GA3, and given cold treatment at 4°C for 8 or 10 weeks. Shoot-producing explants were soaked overnight (15 h) in sterile solutions of GA3 or GA4÷~ (1.0 and 10.0 mg 1-1) in phosphate buffer pH 6.1. Individual shoots were then removed and sub-cultured on to the medium lacking gibberellins. The effect of different durations of soaks was investigated with shoots soaked in 1.0 mg 1-1 GA3 for 10, 15, 20 or 25 h. A minimum of 12 and a maximum of 20 explants were used for each treatment, and experiments were run at least twice. Histological preparations. -- Some of the cultures were fixed in formalin--

acetic acid--alcohol, dehydrated through an ethanol series, and e m b e d d e d in polyester wax. Sections 10--15 pm thick were cut using a Cambridge Rotary Microtome and stained in Safranin/Fast Green (Berlyn and Miksche, 1976) prior to examination under a Vickers M17 Photomicroscope. RESULTS The basal and first nodal segments of the floral stem were considerably more reactive than the upper nodes, producing on average between 20 and 25 shoots per explant, and therefore these cultures were used for subsequent treatments. T h e e f f e c t o f cold o n c u l t u r e s o f d i f f e r e n t ages. -- Cultures exposed for 4 or

8 weeks at 4°C exhibited a gradation of response related to their age and development (Fig. 1). Cultures allowed 6 weeks' development and then exposed to 8 weeks at 4°C exhibited retarded scale leaf and bulb growth (Fig. 2a); exposure to 4 weeks at 4°C produced pale stunted shoots (Fig. 2b). No appreciable bulb development occurred at this stage. Significant bulb production occurred in cultures allowed 10 weeks' development before cold treatment, but bulbing was greatest in terms of number and speed of develo p m e n t when cultures were grown for 16 or 18 weeks (Fig. 2c) before receiving cold treatment, with over 16% of the shoots producing bulbs. In general, 4 weeks at 4°C was less favourable for bulb production (see Fig. 1). Bulbing was reduced by cold incubation in darkness, but tended to increase slightly with transferral to 25°C after the cold period (Table I). Bulb produc-

380

8 WEEKS 4°C

3'2 2"8

WEEKS 4°E

2.Z~ F-Z

X

o-

2.0 1"6 1"2

..J e~

08

o d Z

O.Z~ 0 8

10

12

WEEKS

1~ AT

16

1B

20°C

(before cold freafmenf) Fig. 1. Mean bulb production by explants allowed 8--18 weeks of growth at 20°C before 4 or 8 weeks cold t r e a t m e n t at 4°C. Means based on variable replicate numbers. Vertical bars give S E D for m i n i m u m replication.

381

Fig. 2. Explant response to cold treatment. (a) 6 weeks growth before 8 weeks at 4°C showing retarded bulbing. (b) 6 weeks growth before 4 weeks at 4°C producing pale stunted shoots. (c) 18 weeks growth before 8 weeks at 4°C giving well-developed bulbing.

382 TABLE I Effect of darkness, 25°C incubation, and sucrose concentration on mean bulbing response of 16-week-old explants exposed to 4°C for 8 weeks

Cold treatment

4°C light (control) 4°C dark 4°C light 4°C light 4°C light 4°C light 4°C light 4°C light SED

Temperature after transferral

Sucrose conc.

Mean fresh wt. per bulb

(g 1-z)

Mean bulb production per explant

from cold (°C) 20 20 25 20 20 20 20 20

30 30 30 40 50 60 70 80

1.78 1.02 1.91 1.89 2.04 2.14 1.83 1.53

115.2 46.1 127.1 112.6 130.6 148.5 117.8 81.2

0.23

12.4

(rag)

tion was increased by raising the sucrose concentration, with the optimum concentration being 60 g 1-1 (Table I).

The effect o f GA3, GA4÷~ and cold on individual shoots. --Treatment of individual shoots with gibberellins and incubation at 4°C for 8 or 10 weeks gave a variable bulbing response (Table II), and this was partly attributable to shoot quality. However, a GA3 soak (1.0 mg 1-1) and growth at 20°C appeared most favourable, with bulbing visible within 4 weeks. GA4÷7 soaks were less effective than GA3 and required a concentration of 10 mg 1-1 to give appreciable bulbing. Cold incubation appeared to inhibit bulbing both in untreated individual shoots sub-cultured to basal medium and in subcultured shoots previously soaked in gibberellins. Media provided with GA3 or GA4÷7 produced some bulbs, and production was increased by 10 weeks of 4°C. However, the response was variable and the interaction between cold and media-provided gibbereUins was unclear. In general, gibberellins in the media gave rise to large quantities of callus (Fig. 3); GA3 was more effective than GA4÷7 in this respect. No significant difference in bulbing was noted with different durations (10--25 h) of GA3 soaks. The s u b c u l t u r e of whole explants after 1.0 mg 1-1 GA3 soak gave bulbing in an average of 12% of the shoots after 3 months of growth at 20°C. Bulbs produced in the presence of exogenous gibberellins appeared well developed (Fig. 4), and hand sectioning revealed the presence of scales, daughter bulbs, basal plates, r o o t primordia and developing meristematic centres.

383 TABLE II Percentages of shoots producing bulbs after exposure to gibberellins and low temperature (4°C). Values in parentheses are numbers of shoots giving bulbs versus total number of treated shoots. Binomial regression of transformed data indicates that temperature × method of gibberellin application, and type of gibberellin x method of application, interact significantly at P < 0.05 Incubation temperature and time

Control

20°C

4°C, 8 weeks

4°C, 10 weeks

6.3 (3/48)

2.1 (1/48)

0 (0/48)

Soak G A 3 (rag 1-~)

1.0 10.0 Soak GA~+7 (mg I-I) 1.0

10.0

65.4

12.2

2.1

(34/52)

(6/49)

(1/48)

25.0 (12/48)

0 (0/48)

0 (0/48)

14.3 (7/49) 28.3 (13/46)

0 (0/48) 5.1 (2/39)

0 (0/48) 0 (0/48)

14.3 (6/42) 0 (0/49)

6.4 (3/47) 2.1 (1/48)

28.0 (14/50) 17.4 (8/46)

19.1 (9/47) 15.2 (7/46)

10.2 (5/49) 25.00 (12/48)

10.2 (5/49) 20.8 (10/48)

Media GA~ (rag 1-1)

1.0 10.0 Media GA4+7 (mg l-l)

1.0

10.0

D e v e l o p m e n t o f areas o f m e r i s t e m a t i c activity. -- F r o m a histological study o f the s h o o t basal region, areas of meristematic activity were evident in y o u n g u n tr eated shoots at 12 weeks of age. These areas of activity were distinguished by regions of cell division and enlargement (Fig. 5a), and often by the d e v e l o p m e n t of a cavity (Fig. 5b). Some shoots allowed to develop at 20°C for 5--6 m ont hs w i t h o u t any cold t r e a t m e n t exhibited limited basal expansion. Sectioning revealed prominent s h o o t meristems or buds within a large cavity associated with areas of p r o c a m b i u m (Fig. 6). Exogenous gibberellins appeared to activate this region to give an enclosed apex (Fig. 7) possessing a darkly stained region

384

Fig. 3. Callus produced on shoots sub-cultured onto m e d i u m containing 1 m g l-' of

NAA, BAP and GA3.

suggesting meristematic activity, or procambial strands associated with a basal plate. DISCUSSION

The production of tulip bulblets in vitro from floral stem tissues can be regarded as a 2-step process, with the initial production of adventitious shoots from the epidermal layers of explants, followed by the initiation of a vegetative centre or bulb primordium within each shoot. The induction of bulbing at the proximal end of adventitious shoots in response to chilling resembles that obtained by Nishiuchi (1980) on bulb scale segments of 'Apeldoorn', as does the enhancement of bulbing by a sucrose concentration of 6% (Table I). The presence of a meristematic centre (Fig. 5a) and an e m p t y primordial cavity (Fig. 5b) in non-cold-treated cultures closely resembles the response

385

Fig. 4. Bulb produced from a sub-cultured shoot soaked for 15 h in GA~ (1.0 mg l-'s

386 of tulip embryo cultures reported by Niimi (1978), as does the breaking of dormancy in response to cold to produce a bulb. However, it is unlikely that low temperature is responsible for the complete initiation of a bulb primordium, which occurs in tulip e m b r y o cultures (Niimi, 1978; 1980), as cold treatment of cultures at an early stage of shoot development, i.e. 6--8 weeks, inhibited the bulbing response, indicating that prior development of a vegetative region at 20°C was necessary. The initiation of a bulbing response in shoots by exogenous gibberellins correlates with the comparatively high levels of extractable gibberellin activity in developing bulblets in vivo (Einert et al., 1972}, and with the promotive effect of GA3 on bulblet number (Van Bragt and Van Gelder, 1979). Physiologically, gibberellins may strengthen the "sink" relationships of the vegetative bud within the cultured shoot in the manner described by

387

Fig. 5. Sections o f shoots at 12 weeks of age. (a) Area of meristematic activity in an untreated shoot (× 80). (b) Cavity associated with an active region in an untreated shoot (×8o).

De Munk (1979), and thereby activate it. The superior effectiveness of GA3 soaks in this respect may be due to the supra-optimal level of gibberellins when provided in the medium, resulting in an interaction with auxin to produce callus (Fig. 3). However, the percentage of shoots induced to bulb by GA3 soaks was slightly lower than that produced by optimal cold treatment of cultures (12% as compared with 16%). The inhibition of bulbing by cold in individually sub-cultured untreated shoots indicates that the parent tissue is necessary for cold-induced bulbing. GA4÷7, a more effective regulator in the partial replacement of the cold requirement in vivo (Van Bragt and Zijlstra, 1971), appeared to be less effective in vitro (Table II).

388

Fig. 6. S e c t i o n o f s h o o t base at 20 w e e k s of age showing a dormant shoot meristem present within a prominent cavity (x 32).

The different treatments of cold and gibberellins possess several qualities that contribute to their comparative suitabilities for an in vitro-based system of vegetative propagation of tulip. Gibberellin-induced bulbing was more intermittent when individual shoots were sub-cultured, and was slightly less productive than cold-treated cultures when whole explants were used. This

389

...... ~

: i~¸

Fig. 7. Section of shoot meristem activated by GA~ soak. The bud is connected by procambial strands to a basal plate (x 32).

m a y have been due t o the r e q u i r e m e n t of a sufficiently pre-formed vegetative apex. By comparison, cold t r e a t m e n t induced consistent and welldeveloped bulb f or m a t i on, b u t this required a cold period of 8 weeks duration. Th e shorter time-span of bulb induction by gibberellin soaks may t h e r e f o r e provide the m o r e practical m e t h o d , but this can only be confi rm ed by establishment of the propagules in the soil.

390 ACKNOWLEDGEMENT The authors wish to thank the Ministry of Agriculture, Fisheries and Food for providing a Postgraduate Studentship. REFERENCES Alpi, A. and De Hertogh, A.A., 1975. Extractability of gibberellin-like substances from tulips grown at two temperatures in either the light or dark. Acta Hortic., 47: 297-305. Berlyn, G.P. and Miksche, J.P., 1976. Botanical Microtechnique and Cytochemistry. Iowa State University Press, pp. 97--98. De Hertogh, A.A., 1974. Principles for forcing tulips, hyacinths, daffodils, Easter lilies and Dutch irises. Scientia H o r t i c . , 2: 313--355. De Munk, W.J., 1979. Influence of plant growth regulators on the development of bulbous plants with special reference to organ relationships. Acta Hortic., 91: 207-219. Einert, A.E., Staby, G.L. and De Hertogh, A.A., 1972. Gibberellin-like activity from organs of Tulipa gesneriana L. Can. J. Bot., 50: 909--914. Hanks, G.R. and Rees, A.R., 1980. Partly cooled tulips: response to GA3 and other gibberellins. Acta Hortic., 109 : 169--176. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473--497. Niimi, Y., 1978. Influence of low and high temperatures on the initiation and the development of a bulb primordium in isolated tulip embryos. Scientia Hortic., 9: 61--69. Niimi, Y., 1980. Histological observations on the initiation of the vegetative apex in tulip seeds cultured under low temperatures. Scientia Hortic., 1 3 : 161--172. Nishiuchi, Y., 1979. Studies on the vegetative propagation of tulip. II. Formation and development of adventitious buds on excised scales cultured in vitro. J. Jpn. Soc. Hortic. Sci., 48: 99--105. Nishiuchi, Y., 1980. Studies on the vegetative propagation of tulip. IV. Regeneration of bulblets in bulb scale segments cultured in vitro. J. Jpn. Soc. Hortic. Sci., 49: 235-240. Rakhimbaev, I.R., Syrtanova, G.A. and Solomina, V.F., 1978. Effect of ~old treatment on the level of biological activity of endogenous growth regulators in tulip bulbs. Fiziol. Rast., 25: 249--253. Rees, A.R., 1969. Effects of duration of cold treatment on the subsequent flowering of tulips. J. Hortic. Sci., 44: 27--36. Rees, A.R., 1972. Preparing tulip bulbs for controlled forcing. Commerc. Grow., 3974: 349--350. Rivi~re, S. and Muller, J.F., 1976. Vegetative multiplication of Tulipa gesneriana L. variety Paul Richter through the culture in vitro of axillary buds and scales. C.R. Acad. Sci., Ser. D, 282: 533--536. Rivi~re, S. and Muller, J.F., 1979. A study on bud formation in vitro in the bulb scale of tulip. Can. J. Bot., 57: 1986--1993. Van Bragt, J. and Van Ast, K.J., 1976. Substitution of the cold requirement of tulip cv. ' A p e l d o o r n ' by GA3. Scientia Hortic., 4: 117--122. Van Bragt, J. and Van Gelder, H., 1979. Effects of gibberellic acid, 6-benzylaminopurine, s-naphthalene acetic acid and ethephon o n growth and flowering of tulip bulbs cv. ' A p e l d o o r n ' and their bulblets. Acta Hortic., 91 : 161--165. Van Bragt, J. and Zijlstra, F.A., 1971. Effects of gibberellins on flowering of tulip cv. 'Apeldoorn'. Z. Pflanzenphysiol., 64: 139--144. Wright, N.A. and Alderson, P.G., 1980. The growth of tulip tissues in vitro. Acta Hortic., 109: 263--270.