Plant Cell, Tissue and Organ Culture 9:253-261 (1987) © Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands

Short communication

The influence of cytokinin pulse treatments on adventitious bud formation on vegetative buds of Picea abies

SARA VON ARNOLD & ELISABETH TILLBERG Department of Plant Physiology, University of Uppsala, Box 540, S-751 21 Uppsala, Sweden

Received 26 November 1986; accepted in revised form 4 March 1987

Abstract. Resting vegetative buds of Picea abies collected from phytotron-grown rooted cuttings of 24-year-old trees or a 12-year-old hedge were tested for their capacity to form adventitious buds after various cytokinin treatments. The most effective method for obtaining a high yield of adventitious buds within 8 weeks was to pulse treat the buds in 250/~M BA for 3 h and then culture them on medium containing 5 #M each of BA and kinetin for 1 week. The developmental pattern for adventitious bud production, with the formation of 10 to 20 adventitious buds per bud, was similar for all tested genotypes, although the number of buds giving rise to adventitious buds varied significantly. The capability of some clones to form adventitious buds was correlated to endogenous cytokinin content. The clone which contained most endogenous cytokinin in its resting bud had the highest potential for adventitious bud formation.

Introduction

Important progress has been made in vegetative propagation of coniferous species by in vitro techniques. For thorough reviews see John [12] and Sommer et al. [13]. With a few exceptions, such as in the case of needle fascicles of Pinus pinaster [10], lateral branches of Thuja plieata [8] and basal juvenile sprouts of Sequoia sempervirens [7], in vitro produced plantlets have been regenerated from juvenile plants or embryos. As trees go through the maturation process it becomes more difficult to induce adventitious buds and to regenerate plantlets. To some extent, the bud producing capability of Picea abies increased after pregrowing cuttings from 24-year-old trees in a phytotron but marked differences in the ability to form adventitious buds were observed among various clones [2]. Similarly the rooting potential of the tested clones increased after such pretreatment although the cuttings still exhibited mature growth characteristics (Dormling, personal communica- collected from: (I) a 12-year-old hedge and (II) different clones of rooted cuttings growing in a phytotron. The cuttings from the clones were taken in 1978 from 24-year-old trees growing at Bogesund, close to Stockholm, Sweden and rooted. Since then they have been grown in a phytotron as described by Dormling and Kellerstam [9] and run for two to three growing

254

Picea abies develop faster and with less variation between different experi- ments when the embryos are pulse-treated with benzyladenine (BA) than when they are cultured on medium containing cytokinin [4]. Dormant buds of Picea abies also formed adventitious buds after a BA pulse, although there were great clonal differences [11].

The aim of this work has been to find out if it is possible to adapt the technique for rapid and reproducible formation of adventitious buds on juvenile plant material of Picea abies to vegetative buds from trees of Picea abies. We have chosen to develop the technique with buds from a 12-year- old hedge and to test the technique on buds from cloned cuttings from 24-year-old trees, which exhibit mature growth characteristics.

Materials and methods

Plant materials

In the present study, resting vegetative buds of Picea abies (L.) Karst. were collected from: (I) a 12-year-old hedge and (II) different clones of rooted cuttings growing in a phytotron. The cuttings from the clones were taken in 1978 from 24-year-old trees growing at Bogesund, close to Stockholm, Sweden and rooted. Since then they have been grown in a phytotron as described by Dormling and Kellerstam [9] and run for two to three growing seasons per year. The presented results are based on buds collected 1984, 1985 and 1986. Resting, vegetative buds were collected from the hedge during winter (December to March) and from plants growing in the phyto- tron in the middle of the cold treatment period.

The buds were surface sterilized by shaking in 7.5% (w/v) calcium hypochlorite containing a few drops Tween 20 for 20min, and in 70% ethanol for 2min. They were finally washed in three changes of sterile, distilled water. Buds were excised and bud scales removed. All buds were excised just above the crown.

Culture conditions

LP medium [3] was used as basal medium, with macro elements at half strength and micro elements and vitamins at full strength. It was solidified with 0.7% agar and supplemented with 3% sucrose.

Five to eight buds were cultured in each 5 cm Petri dish containing about 10 ml medium. The plates were sealed with Parafilm ® and incubated in

255

growth chambers at 20 °C under a photoperiod of 16 h. Irradiance was 2-3 Wm -2 (400--700 nm) supplied by Philips TL 25 W 33 white fluorescent tubes.

Induction of adventitious buds

Newly isolated buds were either cultured directly on medium containing 5/~M each of kinetin and BA for 8 weeks or first pulse-treated with BA. The pulse treatments were carried out by shaking the buds at 20 °C for various periods of time, ( lmin to 4h) in a filter-sterilized BA solution (250 or 500/~M) with pH adjusted to 5.5. After pulse treatment, the buds were briefly blotted on sterile filter paper before being placed on the surface of the medium. The pulse-treated buds were cultured either directly on medium lacking cytokinin or first on medium containing 5/~M each of kinetin and BA for 1 to 8 weeks and then on medium lacking cytokinin. In one experi- ment the buds were pulse-treated in 250pM BA-solutions supplemented with 0.025 to 250/~M IBA for 3 h and then cultured on medium lacking plant growth regulators.

After 8 weeks in vitro, all buds were transferred to 9 cm Petri dishes containing about 25 ml medium without cytokinin. The buds were subcul- tured every fourth week. If not stated otherwise the experiments were repeated 2 to 5 times with 30 to 50 buds per treatment. Bud formation was followed microscopically. Depending on the experiments the buds were examined every week or every month.

Determinations of endogenous cytokinins

Descaled buds were immediately put into liquid nitrogen. Each sample consisted of about 300 buds with a total fresh weight of about 0.3 g. After about one week's storage at - 8 0 °C the buds were homogenized in saline phosphate buffer (pH 7.4), boiled for 15 min and centrifuged (2000 x g). The supernatants were filtrated through Whatman GF/C glass fibre discs and used for comparative determination of cytokinin levels between dif- ferent clones. Quantification of the cytokinins was made with a competitive enzyme-linked immunosorbent assay (ELISA) according to the procedure described by Barthe and Stewart [6]. Polyclonal antibodies against trans- zeatin riboside (ZR) and isopentenyladenosine (IPA) were obtained from rabbits by established methods [6, 15].

256

Results

Buds from the hedge

Initial studies for developing optimal conditions for bud induction were carried out with buds from the hedge. Adventitious buds could be induced either by culturing the buds on medium containing 5 #M each of BA and kinetin for 8 weeks (the technique described previously for vegetative buds [2]), or by pulse treating the buds in 250/~M BA and then culturing them on medium lacking cytokinin (the technique described previously for embryos [4]). The percentage of buds yielding adventitious buds could not be in- creased by combining the two techniques, i.e. culturing BA-pulsed buds on medium containing cytokinin (Table 1).

The effect of the pulse treatment time was tested by treating the buds in 250 or 500~M BA for 1 min to 4h and then culturing them on medium lacking cytokinin (Fig. 1). Only about 5% of the buds formed adventitious buds after being pulse-treated in 250 or 500/~M BA for 1 min. The number of buds increased to about 35% when the buds were treated for 30min. Prolonged treatment, up to 4 h, had no significant effect on bud formation. Addition of IBA (0.025 to 250k~M) to the BA solution had no significant effect on the bud formation frequency. A treatment for about 3 h with 250/~M BA was the most practical and was therefore chosen as routine.

By comparing buds cultured on medium supplied with cytokinin for 8 weeks and buds pulse-treated with BA (250 #M) for 3 h it was found that the

Table 1. Percentage of buds from Picea abies which formed adventitious buds after various BA treatments. Newly isolated buds were either cultured directly on medium containing 5/IM each of BA and kinetin for 8 weeks or first pulse-treated with 250/~M BA for 3 h and then

cultured on medium containing 5 #M each of BA and kinetin for 0 to 8 weeks. Results are given as the percentage of buds forming adventitious buds after 4 months in culture. Data are based on 100 to 500 buds per treatment. Different superscripts indicate significant differences (p < 0.01) within each clone (xZ-test).

BA Time on medium Source of buds pulse containing BA

and kinetin Hedge

(weeks)

Clone

4:1 40:5 44:4 1:5

% of buds forming adventitious buds

No 8 25 a Yes 0 27 a Yes 1 27 a Yes 2 39 a Yes 4 25 a Yes 8 31 a

13 b 22 b 30 b 8

7" 3 a 6 a 0 43 ~ 61 a 59 c 0 37 c 47 ca 38 b 0

16 b 43 c 40 b 0 18 b 21 b 42 b 6

257

5 0

4O

.o 30

2 O

E o

m

i i i i i i i i

30 60 120 180 240

Time (min)

Fig. 1. Effect of treatment time in BA on adventitious bud formation. Newly isolated buds from the Picea abies hedge were pulse-treated in 250 ( - - e - - ) or 500 ( - - I I - - ) / t M BA for various periods of time and then cultured on medium lacking cytokinin. Results are given as the percentage of buds forming adventitious buds after two months in culture. Data are based on 30 to 40 buds per treatment.

pulse treatment was as effective after 2 months as the conventional treatment after 4 months (Fig. 2). The variation among different experiments was also lower after the pulse treatment. The number of adventitious buds per bud could not be counted exactly, it varied from 2 to 50, but usually each bud formed 10 to 20 well developed adventitious buds (Fig. 3A). This number was not correlated to the cytokinin treatment. Two to five adventitious buds on each bud usually started to elongate during the third month (Fig. 3B). However, it has not yet been possible to regenerate vigorously growing adventitious shoots.

Buds from different clones

The ability to form adventitious buds on medium containing cytokinin varied among different clones (Table 1). Only a few buds from clone 4:1, 40:5 and 44:4 and no buds from clone 1:5 formed adventitious buds after being pulse-treated with BA and then cultured on medium lacking cyto- kinin. This yield could not be improved by transferring the buds a second time, after 1 to 8 weeks, to fresh culture medium lacking cytokinin. How- ever, the number of buds forming adventitious buds increased significantly for clone 4: 1, 40:5 and 44:4 if the buds were cultured on medium containing cytokinin for one week after the pulse treatment. With this treatment 43 to

258

o

2c

E

4 8 12 16 20

Time (weeks)

Fig. 2. Time-course for the development of adventitious buds after two different cytokinin treatments. Buds from the Picea abies hedge were either cultured on medium supplied with 5/~M each of BA and kinetin ( - - I I - - ) or pulse-treated with BA for 3 h and then cultured on medium lacking cytokinin ( - -o - - ) . Data are based on at least 100 buds per week and treatment.

Fig. 3. Picea abies bud with adventitious buds. Fig. A after two months and Fig B after three months in culture. Scale bars indicate 1 mm.

6 1 % o f the b u d s f o r m e d in a v e r a g e 10 to 20 wel l d e v e l o p e d a d v e n t i t i o u s

buds . A f t e r p r o l o n g e d cu l t u r e on m e d i u m c o n t a i n i n g c y t o k i n i n the y ie ld o f

a d v e n t i t i o u s b u d s dec reased . T h e r e q u i r e m e n t fo r a d d i t i o n a l c y t o k i n i n in

the cu l t u r e m e d i u m c o u l d n o t be r e d u c e d b y i nc r ea s ing the pu l se B A

c o n c e n t r a t i o n to 500 # M o r b y p r o l o n g i n g the t r e a t m e n t t ime up to 4 h.

259

By comparing buds cultured for 8 weeks on medium containing cytokinin with buds pulse-treated with BA for 3 h and then cultured on medium containing cytokinin for one week, it was found that the pulse-treated buds had reached a maximum of adventitious buds after 3 months and the buds only cultured on cytokinin containing medium after 4 months. When the pulse treated buds were cultured on medium containing cytokinin for more than two weeks, the development of adventitious buds was delayed.

Cytokinin content in resting buds in clone 44:4, which easily formed adventitious buds after pulse treatement with BA, was compared with cytokinin content in clone 1 : 5, which did not form any buds after the same treatment. Big differences between the two clones were obtained. Clone 44:4 gave the highest yield, 224 ZR eqv. and 27 IPA eqv. (g fresh weight) -1. Clone 1:5 contained only 38 ZR eqv. and 14 IPA eqv. (g fresh weight) -1. Although the determinations with the ELISA test were found to be repro- ducible, they must only be regarded as relative ones. As the samples were not purified, there is a possibility that the determination cytokinin levels are too high.

Discussion

By comparing buds cultured on medium supplied with BA and kinetin for 8 weeks and buds pulse-treated with BA for 3 h and cultured on medium containing BA and kinetin for zero or one week, it was found that the variation between different experiments was smaller and the development of adventitious buds faster after a pulse-treatment. The maximum number of pulse-treated buds yielding adventitious buds was reached after two months and for buds cultured on cytokinin containing medium first after four months. Similar time-courses for development of adventitious buds were obtained with embryos of Picea abies that were either pulse-treated with BA or cultured on medium containing cytokinin [4]. This shows that the formation of adventitious buds can take the same time, whether they are formed on vegetative buds from trees or on embryos.

The yield of adventitious buds on buds from the hedge was not affected by the type of cytokinin treatment. In contrast, there was a significant increase in adventitious bud formation in clone 4: 1, 40:5 and 44:4 if the buds were pulse-treated with BA and then cultured on medium containing cytokinin for one week, compared to buds cultured on medium lacking cytokinin. It is reasonable to assume that most of the cytokinin, taken up during the pulse-treatment, is released to the culture medium, as is the case with pulse-treated embryos of Picea abies where only 10% of the cytokinin initially taken up remained in the embryos after 24h [14]. Culture of the embryos on medium containing released cytokinin for one week was enough

260

to give rise to the optimal yield of adventitious buds [5]. It is not clear why pulse-treated buds from the tested clones did not produce many adventitious buds when cultured on medium containing only released cytokinin for one week but had to be cultured on medium containing additional cytokinins. It might be that these buds require a higher cytokinin concentration than the buds from the hedge and the embryos during the first week.

Although the cytokinin analysis was performed only on a few samples, it clearly showed that the buds of various clones contained different levels of endogenous cytokinin. Based on observations in the field, the Picea abies clones used in this study could be ranked in the following growth order: 1:5, 4:1, 40:5, 44:4 (1:5 had the highest growth rating). The same order was obtained when the field-grown trees were ranked with regard to rooting potential of cuttings [9]. Clone 1:5, which contained less endogenous cyto- kinin in its resting buds, had the highest ratings for growth and rooting potential and the lowest rating for adventitious bud formation capacity. In contrast, clone 44:4, which contained more endogenous cytokinin, had the lowest ratings for growth and rooting potential and the highest rating for adventitious bud formation. Similarly, plants of Fitis vinifera with poor growth had a higher content of cytokinin in the shoots compared to plants with more intense growth [1]. Difference in the endogenous cytokinin con- tent might at least partly explain the differences in the ability to produce adventitious buds.

Now that a high yield of adventitious buds (10 to 20 adventitious buds on 30~60% of the initial buds) can be obtained from vegetative buds from trees of Picea abies within two months, more emphasis can be placed on regeneration of shoots and plantlets.

Acknowledgements

We thank Professor T. Eriksson for his support in this research and K. Bjelke for technical assistance. Grant was provided from the Swedish Coun- cil for Forestry and Agricultural Research.

References

1. Andonova TA, Lilov DT (1977) Changes of the free cytokinins in the shoots and leaves in plants differing in their growth. CR Acad Bulg Sci 30:905-908

2. von Arnold S (1984) Importance of genotype on the potential for in vitro adventitious bud production of Picea abies. Forest Sci 30(2): 314-318

3. von Arnold S, Eriksson T (1981) In vitro studies of adventitious shoot formation in Pinus

contorta (Englm.). Can J Bot 59:870-874

261

4. von Arnold S, Eriksson T (1985) Initial stages in the course of adventitious bud formation on embryos of Picea abies. Physiol Plant 64:41-47

5. von Arnold S, Gr6nroos R (1986) Meristematic zone formation and peroxidase activity during early stages of adventitious bud formation on embryos of Picea abies. Bot Gaz 147: 425-431

6. Barthe GA, Stewart J (1985) Enzyme immunoassay (EIA) of endogenous cytokinins in Citrus. J Agric Food Chem 33:293-297

7. Boulay M (1974) Multiplication et clonage rapide du Sequoia sempervirens par culture in vitro. AFOCF4L, l~tudes et Recherches 12:49-55

8. Coleman W, Thorpe T (1977) In vitro culture of western red cedar (Thuja plicata Don). I Plantlet formation. Bot Gaz 138(3) 298-304

9. Dormling I, Kellerstam H (1981) Rooting and rejuvenation in propagating old Norway spruce cuttings. Symposium on clonal forestry, Swedish University of Agricultural Scien- ces. Research notes 32, p 65. ISBN 91-576-0959-4

10. Franclet A, David A, David H, Boulay M (1980) Premi6re mise en 6vidence morpho- logique d'un rajeunissement de m6ristemes primaries caulinaires de Pin maritime ag6 (Pinus pinaster Sol.). CR Acad Sci Paris 290, S6rie D: 927 930

11. Jansson E, Bornman CH (1983) Morphogenesis in dormant embryonic shoots of Picea abies: Influence of the crown and cold treatment. Physiol Plant 59:1-8

12. John A (1983) Tissue culture of conferous trees. In: Dodds JH (ed.) Tissue Cultures of Trees. Croom Helm Ltd, pp 6-24

13. Sommer HE, Wetzstein HY, Merkle SA (1986) Application of tissue culture to forest tree improvement. Symposium paper. 8th Long Ashton Symposium

14. van Staden J, Forsyth C, Bergman L, von Arnold S (1986) Metabolism of benzyladenine by Picea abies embryos. Physiol Plant 66:427-434

15. Weiler E (1980) Radioimmunoassays for trans-zeatin and related cytokinins. Planta 149: 155-162