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Effects of abscisic acid and analogues on the maturation of white spruce (Picea glauca) somatic embryos
Plant Science, 58 (1988)77-- 84
77
Elsevier Scientific Publishers Ireland Ltd.
EFFECTS OF ABSCISIC ACID AND ANALOGUES ON THE MATURATION OF WHITE SPRUCE (PICEA GLA UCA) SOMATIC EMBRYOS
DAVID I. DUNSTAN~', FAOUZI BEKKAOUP, MARC PILON,, LARRY C. FOWKEband SUZANNE R. ABRAMS,
•Plant Bioteelmology l~stitute, NationalResearch Co~ndl Canad~ 110 Gymnasium Road, Saskatoon, Saskatchewan, S7N OW9 and bDepartment of Biology, University of Saskatchewan, Saskatoon, Saskatcheu~m, STN OWO(Canad~ (Received February 10th, 1988) (Revision received May 20th, 1988) (Accepted May 25th, 1988)
Somatic embryo-eompotent cultures of white spruce, Picea glanca (Meench) Vou, were grown on ( ± )-abscisie acid (ABA) and three analogues known to be biotrausformed by intnct plants into ABA: ( ± )-methyl abscisate (MeABA); ( ± )~thyl-(2E,4E) and ~,E,4Z)-5-(l'2'~poxy-2',6',6~-trimethylcyelohexyl)-3-methylpentadienoate (epoxyester, EE); and ( ± )-(2E,4E)and -(2E,4Z~5(l'~2'-epoxy-2',6',6'-trimethylcyciobexyl)-3-methylpentadienoic acid (epoxyacid, EA). ABA between 8 and 12 ~M most efficiently promoted embryo maturation and led to the maximum recovery (4%) of stage 4b embryos when used in the absence of auxin or cytokinin.MeABA in the absence of auxin and cytokinin also promoted embryo maturation, EE promoted only the early stages in maturation, and EA was unable to promote any maturation of somatic embryos. Phytohormone-f~ee medium produced very similar responses to EA, though in rare eases stage 3 embryos were found. Neither ABA nor the AnAloguespromoted embryo maturation when used in combination with 9 ~M 2,4-dichloropbenoxyacetic acid (2,4-D}and 4.4 ~M NS-benzyiadenine (BA).
Key words: Picea glauc~, somatic embryogenesis; abscisic acid; maturation; abscisic acid analogues
Introduction The plant growth regulator ABA is involved in all stages of plant growth and development [1]. With particular reference to embryos, ABA is implicated both in zygotic embryo maturation [2] and in the maintenance of seed dormancy [3]. Addition of ABA to coniferous tissue cultures has been investigated by Gupta and Durzan [4], who used 0.5 ~ ABA to promote embryo maturation from protoplasts of Ioblolly pine, and by Durzan and Gupta [5] who reported the promotion of embryo maturation by 0.5 *To whom all correspondence should be addressed. NRCC n0. 28886. Abbreviations: ABA, (±)-abscisic acid; ac÷, medium with 2,4~D and BA; ac-, medium without 2,4-D and BA; BA, N ~benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid; EA, (±)-(2E,4E) and -(2E,4Z)-5-(l',2'epoxy-2',6',6'-trimethylcyciohexyl)-3-methylpentadienoie acid; EE, (±)-ethyl-(2E,4E) and -(2E,4Z)-5-(l'2'epoxy-2',6',6'trimethylcyciohexyl)-3-me. thylpontadienoate; MeABA, ( ± )-methyl abseisate.
ABA in suspension cultures of Douglas fir. More recently, von Arnold and Hakman [6]* have shown that maturation of Norway spruce embryos occurred after treatment with 7.6/~I ABA. Because of the potential for the use of ABA, we decided to compare the effects of ABA and active analogues as chemical agents to promote the maturation of embryos in cultures of white spruce. This comparison was of interest because a very few analogues of ABA, in particular epoxy beta-ionylideneacetie acid, its esters and esters of ABA, have been found to have similar or greater activity than ABA [7,8] and have been shown to be biotransformed into ABA by intact plants [9]. We present here our findings on the effects of exogenous supplements of ABA, MeABA, *Since going to press this manuscript and that of Hakman and yon Arnold, Physiol. Plant., 72 (1988) 579-587, have been published. We have amended our embryo classification to integrate with these authors.
0168-9452/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
78 EE and EA on somatic embry~competent cultures of white spruce. Materials and methods Plant material Embryogenic cell suspension cultures were prepared as previously described [10]. Suspensions were maintained by subculturing 0.5 g fresh weight of ceils, at 7~lay intervals, to 50 ml of fresh yon Arnold and Eriksson's LP medium [11] containing 1% (w/v) sucrose, 9 i~I 2,4-D and 4.4 ~ I BA. To this was added 15 ml of filtered spent (conditioned) medium. Cultures were contained in 250 ml Delong flasks agitated at 150 rev./min on a gyratory shaker (New Brunswick, Edison, N.J., U.S.A.) and incubated at 25°C with continuous incident photosynthetically active radiation (PAR) [12] of 15--20 ~E m -2 s -1. The cultures had been maintained for 18 months prior to the start of these experiments. These cell suspension cultures (designated WS1) contained a heterogeneous population consisting of free cells, clusters of isodiametric cells, and pro-embryos in a repressed state of development (stage 1, described later). Filter disc cultures Filter disc cultures were prepared after the technique of Horsch and King [13], with the modifications described below. Cell suspensions were harvested by filtration and were washed twice with 50 ml of phytohormone-free LP medium described above, before being resuspended as a 200/0 w/v suspension in the same medium. Aliquots of 0.75 ml (0.15 g fresh wt.) were dispensed onto gridded black MiUipore filter discs (0.8 /an, diam. 47 ram, no. AABG04750, Bedford, Mass., U.S.A.) placed on the experimental media described below. The filters facilitated subsequent observations, and were useful in retaining the inoculum in a rectangular grid configuration by surface tension. To estimate the number of embryos that were plated, 3 × I ml samples of the 200/0 w/v suspension were observed in a cell counting chamber (Sedgewick Rafter 150 micro litre cell, Graticules Ltd., Tonbridge, U.K.). Filter disc
Abscisic acid (ABA)
Epoxy acid (EA)
Methyl Abscisate
(MeABA)
Epoxy ester (EE)
Fig. 1. Structural formulae of ABA and analogues used in study.
cultures were incubated on 25 ml of experimental medium, contained in sterile disposable Petri dishes (100 × 15 ram). These were left unsealed, and were placed in loosely tied plastic sleeves, 10 dishes per sleeve collected randomly from across the range of media tested. Cultures were incubated at 25°C with an incident PAR of 85 I~E m -2 s -1 during a 16-h/day photoperiod. Ezperimental media ABA, 99%, was obtained from Sigma (St. Louis, Mo., U.S.A., no. A2784). MeABA, 99%; EE, 98% (3:1 ratio of ZE,4E and 2E,4Z isomers); and EA, 980/0 (3:2 ratio of 2E,4E and 2E,4Z isomers), were prepared according to published techniques (MeABA after Milborrow [7]; EE modified after Tamura and Nagao [14]; EA after Tamura and Nagao [14]). Each compound (Fig. 1) was dissolved in the minimum quantity of ethyl alcohol prior to dilution with distilled water. Aliquots of the stock solutions so prepared were added to autoclaved medium described above, after filter-sterilisation. The stability of each compound was evaluated before and after each repeat by gas-liquid chromatography and thin layer chromatography. All compounds were found to be stable in solution during the experiment. Each compound was compared at 0 (control), 4, 8 and 12 ~u~I, either in the presence (ac÷) or the absence (ac-) of 9 ~ 2,4-D and 4.4 ~II BA. The experiment was repeated on two separate occasions, the first with an average of 1386 stage I
79 embryos per treatment, the second with an average of 2600 stage 1 embryos per treatment. In the second repeat 16 ~M ABA was also used. Control ac-, control ac*, and ABA at 4 and 8 ~M were repeated three times, receiving a total of 8000 embryos each.
Fig. 5}. (N.B. von Arnold and Hakman [6] do not differentiate early and late stage 4 embryos; we differentiate these because of the differences in results we obtained for each, as reported below). Results
Continued development Filter disc cultures were removed from the above experimental media after 35 days, and were placed on phytohormone-free LP medium. When embryo maturation proceeded sufficiently, individual embryos were removed for culture on the same medium contained in deep Petri dishes (100 × 25 mm), 25 ml per dish. A comparison was made of removal at stage 4a and at stage 4b. Media used for radicle extension are described in the text, in each ease deep Petri dishes were used. When used, charcoal was obtained from Sigma (C-4386, St. Louis, Mo., U.S.A.). Data collection Cultures were observed throughout the experiment for qualitative changes in morphology. Quantitative data were collected from day 21. This monitored the progression of embryo stages toward maturation. The stages shown are a modification of the enumeration of yon Arnold and Hakman [6] as described below. Stage 1. Repressed pro-embryo with translucent suspensor, and semi-translucent, deuselystaining embryo with slightly irregular outline (Fig. 2, large arrow) (see also Refs. 10 and 15). Stage ~ Embryo is prominent, smooth in outline and opaque, cream to pale yellow, subtended by suspensor (Fig. 2, small arrow). Stage 3. Embryo with primordial cotyledons clustered around a central meristem, cream to pale green in colour (Fig. 3}. Stage l,a {early). Embryo with distinct, partly elongated, cotyledons clustered around a central meristem, green (Fig. 4, smaller). Stage ~b flare). Embryo with elongating cotyledons and elongating hypocotyl, green, with rudimentary radical development adjacent to suspensor remnants (Fig. 4, (arrow);
MorpAological appearance and embryo maturation After 35 days cultures growing on control ac÷ medium were usually light brown in colour. Cultures grown on ac÷ in combination with ABA or MeABA were generally green. Cultures grown in the presence of EA and EE in combination with ac* were more variable in their growth pattern. In all the above ac* media only stage 1 embryos were found, indicating that no somatic embryo maturation had occurred. In comparison, 35 day~ld ac- controls were completely brown and appeared dossicated. Stage 1 embryos in poor condition were found on these plates, very rarely did more advanced stages occur (using a total of 8000 stage 1 embryos over three repeats). However, ac- used in combination with ABA or analogues produced cultures which contrasted with the ac- controls in the occurrence of embryos at stage 2, 3 and 4a on ABA (4, 8, 12 and 16 ~Vl), MeABA (4, 8 and 12 ~,M), and EE (12 ~M}. Only the use of the EA analogue did not result in slmilsl, maturation. Cultures grown on ac- with ABA or analogues had a similar morphological appearance to ac" controls, though browning was delayed in the presence of ABA. At 35 days ac* control filter disc cultures had ceased to grow (data not shown). This was taken arbitrarily as the time to remove all the filter disc cultures for placement onto phytohormone-free LP medium to encourage further development. From this time any stage 1 or 2 embryos that remained on ac- media ceased to grow, only embryos in stage 3, 4a or 4b developed further. Table I shows the maturation of embryos obtained for the above media, data from repeat 2. On ac- ABA at 12 ~.M, stage 2
80
embryos were observable prior to day 21 (data not shown), and stage 3 embryos peaked on day 28 and subsequently diminished, this indicated that there was a repression of the generation of new stage 1, 2 or 3 embryos. Stage 4a embryos
were present by the 21st day on ac- ABA at 12 /~I, and stage 4b embryos, which were first observed in 35-day~)ld cultures, reached their maximum on day 49. The maximum cumulative recovery of stage 4b embryos by the 60th day
Fig. 2. Filter disc with overlay of suspension culture solution, containing stage 1 somatic embryos (large arrow), stage 2 somatic embryos (small arrow) and associated cells, x 21. Fig. 3.
Somatic embryo in stage 3, with primordial cotyledons clustered around a central meristem, x 27.
Fig. 4. Three stage 4a somatic embryos (smaller) with elongating cotyledons and one stage 4b somatic embryo (arrow) with elongating cotyledons and elongating hypocotyl, x 6. Fig. 5.
Isolated stage 4b somatic embryos with suppressed radicle development, x 6.
Fig. 6.
Stage 4b with radicle extension and true needles, x 4.
81 T a b l e I. P r o g r e s s i o n t h r o u g h somatic e m b r y o m a t u r a t i o n stages. Data are m e a n s of n u m b e r s of e m b r y o s p e r filter disc, at various s t a g e s in maturation, ± S.E. (4--5 replicate discs p e r treatment}. N u m b e r of e m b r y o s (stage I} p e r filter disc at s t a r t w a s 650. Medium (Se-)
ABA (4 ~tM)
Embryo
Time (days}
stageb
21
4a 4b 4a 4b 3• 4a 4b 4a 4b 4a 4b 4a 4b 4a 4b 4a 4b
ABA (8 IAM) ABA (12 ~M)
ABA (16 ~LM} MeABA (4 ~M) MeABA (8 ~M) MeABA (12 ~M) Control
28
1.5 ± 1.3
1.8 -0.8 -35~ 2.0 2.5 -1.8 1.0 2.0 -
0.5 -28.3 1.3 0.5 -1.0 1.2 1.0 .
± 1.0 ± 9.4 ± 0.5 ± 0.6 ± 1.2 ± 0.8 ± 0
.
$5" ± 1.5 ± 1.0 ± 9.2 ± 0 ± 1.0 ± 0.8 ± 1.0 ± 1.0
.
49
7.0 0.3 8.8 0.7 24.8 13.8 1.0 8.8 3.0 4.0 5.4 12.5 1.2 1.0 .
± ± ± ± ± ± ± ± ± ±
2.6 0.5 6.4 0.9 7.6 4.9 1.2 5.7 0~ 1.6
60
12.5 7.0 6.7 12.7 4.8 14.3 21.8 3.8 13.5 4.2 0.8 7.0 6.2 9.8 9.0 1.0
± 1.1 ± 5.8 ± 0.9 ± 2.0
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
7.2 3.0 5.5 4.9 3.1 2.6 4.7 2.9 6.7 1.1 0.5 3.3 2.8 5.7 2.2 2.0
7.0 6.0 4.0 3.3 2.0 8.0 6.0 1.0 3.0 2.5 2.2 4.8 4.8 3.8 4.8 -
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.2 3.0 2.6 3.5 1.2 1.6 1.4 2.0 1.4 1.9 1.7 2.3 2.5 2.9 2.2
.
sAt 35 days filter discs w e r e r e m o v e d from experimental media and w e r e placed on phytohormone-free L P medium. bStages 3 and 4a w e r e left in situ, figures t h e r e f o r e r e p r e s e n t the changing population. Stage 4b's w e r e r e m o v e d at each observation ~ o m 35 days, figures are not cumulative. eData for s t a g e $'s from o t h e r media s h o w e d similar trends.
Table II. The r e c o v e r y of stage 4 e m b r y o s , data after 60 days in culture. A v e r a g e n u m b e r s of s t a g e 1 e m b r y o s p e r t r e a t m e n t w e r e 1386 (repeat 1) and 2600 (repeat 2). Data are totals p e r t r e a t m e n t . Medium
ae-ABA
Cone.
Repeat 2
Repeat I
0A 4 8 12 16
Embryos (st. 4b)
%
Embryos (st. 4b)
0 29 64 52 -
2 5 4
56 67 115 78
Embryos (st. 4a)
%
Embryos (st. 4b)
%
ae-MeABA
4 8 12
2 3 35
0.1 0.2 3
0 0 0
----
15 55 60
ae-EA
4 8 12
0 0 0
----
0 0 0
----
0 0 0
ae-EE
4 8 12
0 0 0
----
0 0
0
---
ae-Control
0
-
0
-
0
as*Control
0
-
0
-
0
---
%
2 3 4 3
0.5 2 2
82
of repeat 2, occurred on ac- ABA at 12 (Table II). Differences between the two repeats in the total numbers of stage 4b embryos recovered for each medium are shown in Table II. For example, in repeat 1 the recovery of stage 4b embryos on ac- ABA at 8 and 12 ~M was similar. In neither repeat were stage 4b embryos recovered from phytohormone-free controls, nor from any E E concentrations. When 0 . 4 / ~ I ac- ABA was evaluated, stage 2 embryos were present by day 26 but continued maturation to stages 3, 4a and 4b was not obtained. This indicated that exogenous ABA was physiologically most active when provided between 8 and 12 ~ under ac- conditions. A comparison of data from both repeats showed that the efficiency of the maturation process under the maximal conditions described (i.e. comparing ac- ABA at 12 ~M), was about 4.0°/0; that is, in repeat 1 an average of 1386 stage 1 embryos were plated while an average of only 62 stage 4b embryos were recovered, in repeat 2 an average of 2600 stage 1 embryos were plated while only 115 stage 4b embryos were recovered (Table II).
Optimum time for embryo removal To determine the best maturation stage for subsequent radicle extension and shoot growth, a comparison was made of the development of stage 4a and 4b embryos, removed from the filter disc cultures. This was accomplished by sequentially removing either stage 4a or stage 4b embryos at each observation date that they occurred, commencing on day 35. The results showed that on phytohormone-free LP medium it was possible to obtain a limited maturation of stage 4a into stage 4b embryos. This was greatest for those previously grown on ac- ABA at 8 or 12 pM (41O/o or 46%, respectively). However, in a majority of cases radicle extension was inhibited, and shoot growth was relatively slow. This material eventually ceased to grow and was not experimented with further. Conversely, when maturation was allowed to proceed to stage 4b while still on the filters, better shoot quality and greater radicle extension (e.g. 560/0 of stage 4b embryos previously
grown on ac- ABA at 12 /n~I) was observed. Stage 4b embryos collected in this way showed increases in shoot vigour with increasing ABA concentration, the most vigorous being harvested from filter discs previously grown on acABA at 16 /~M. Stage 4b embryos collected from discs previously grown on ac- at 4 /~M were poor by comparison. Shoot vigour was also related to time, with the most vigorous shoots of a medium being stage 4b embryos collected on day 49. Radicle extension of stage 4b embryos was often inhibited (Fig. 5~. Inhibition was greatest for those previously grown on ac- ABA at 16 /~I and least for those from ac- ABA at 12 ~Vl. Consequently, stage 4b embryos were placed onto one of three media designed to promote radicle extension. These were as follows: agarsolidified LP with charcoal at 0.05%, LP liquid with cheesecloth support, and agar-solidified 0.5 x LP; each medium was compared to control phytohormone-free LP. Between 10 and 25 embryos from ac- ABA concentrations 8, 12 and 16 ~M were compared. Results for stage 4b embryos previously grown on ac- ABA at 12 ~M, showed that none of these treatments significantly improved radicle extension compared to the phytohormone-free LP control (560/0). Charcoal marginally improved radicle extension on stage 4b embryos derived from other media, with the greatest improvement being obtained for ac- ABA at 16/~d (from 260/0 for control to 530/0 for charcoal). Some embryos, with expanding needles and extending radicles (Fig. 6). were transferred to test-tubes containing sterile vermiculite moistened with 0.5 x Knop's solution [16], incubated under the conditions described for filter disc cultures. After 1 month approx., they were planted in soilless medium in a growth room, where they have slowly continued their development. However, the conditions necessary for their survival and continued development have not yet been satisfactorily established. Discussion
Abscisic acid is normally added to plant tis-
88
sue cultures in the R S ( ± )- form, a synthetic racemic mixture. The natural phytohormone has the S( + )-configuration. The R( - ~enantiomer has biological activity in some circumstances e.g. growth inhibition [8], and the racemic mixture can give nearly the same activity as the natural phytohormone in some circumstances. The natural phytohormone is known to play an active role in coniferous tissues in vivo [3], particularly in embryo ripening (maturation) [2]. This was also the observed effect of exogenous supplements of racemic ABA supplied to our white spruce cultures in vitro, when used in the absence of auxin and cytokinin. ABA is associated with the maintenance of dormancy in seed embryos, e.g. of Tazus [3]; the removal of ABA in the latter example was found to be coincident with the first signs of radicle extension. A similar effect may have lead to the suppression of radicle development in our experiments. MeABA has been shown to have a higher activity than ABA in some studies [8,17], but from our data MeABA had approximately onehalf the activity of ABA at the 12 ~ concentration on day 49. The physiological effects of ABA in vivo are concentration dependent [18]. Therefore, although stage 4b embryos were recovered using MeABA, our concentration range may not have allowed us to see its maximum effect. The epoxy ester (EE) and epoxy acid (EA) in the (2E,47,) forms have high activity in some bioassays in plants [8]. By analogy with ABA, the activity ascribed to the (2E,4E) forms [8] is likely to be due to reversible photoisomerisation; because our experiments were to be performed in the light over long periods of time, the isomeric mixtures were used. EA did not however promote embryo maturation at any concentration, and only early maturation stages were found to occur on EE at 12/~M. These results may be due to the inability of the cultures to biotransform the analogues in vitro into ABA, or to inadequate final concentrations of ABA for the maturation process. Gupta and Durzan have shown that they could recover mature somatic embryos from callus on phytohormone-free medium without
addition of ABA [19]. A similar observation was recently made for white spruce after cryopreservation of stage 1 somatic embryos [20]. We were unable to recover stage 4 somatic embryos using phytohormone~free medium in the absence of ABA. These results may reflect the use of different species and/or culture conditions. Recently, yon Arnold and Hakman [6] showed for Norway spruce that they could induce the development of stage 3 embryos using 7.6 ~M ABA. These could be removed for development into green plantlets on ABA-free medium. We found that similar levels of ABA promoted embryo maturation, but removal of embryos at stage 3 or 4a was not optimal with our cultures of white spruce. Our future studies will attempt to optimise the recovery of stage 4b somatic embryos, investigating the effects of plating density, duration of ABA treatment and the effects of other medium components. Acknowledgements The authors gratefully acknowledge the technical assistance given by Mr. Terry Bethune, Ms. Angela Shaw and Mr. Bill Stirtan during the course of these experiments. References 1 F.T. Addicott and R~F.M. Van Steveniek, The significance of abseisic acid in the life of plants, in: F.T. Addicott (Ed.), Abseisie Acid, Praeger Speeial Studies, Praeger, New York, 1983, pp. 581 - 586. 2 G. Fellenberg, Developmental physiology. Prog. Bot., 44 (1982) 205-- 221. 8 M.-T. LePage-Degivry, Aeide abseisique et dormance chez los embryons de T a z ~ baccata L. C.R. Aead. Sei. Paris, 271 (1970) 482-- 484. 4 P.K. Gupta and D.J. Durzan, Somatic embryos from protoplasts of lobloIly pine proembryonal cells. Bio/ Technology, 5 (1987b) 7 1 0 - 712. 5 D.J. Durzan and P.K. Gupta, Somatic embryogenesis and polyembryogenesis in Douglas f'tr cell suspension cultures. Plant Sci., 52 (1987)229-235. 6 S. von Arnold and I. Hakman, Regulation of somatic embryo development in Picea abies, with emphasis on ABA effects. J. Plant Physiol., 132 (1988) 164-169. 7 B.V. Mflborrow, The chemistry and physiology of abscisic acid. Ann. Rev. Plant. Physiol., 25 (1974) 259-307. 8 D.C. Walton, Structure-activity relationships of abs-
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9
10
II
12
13
14
elsie acid analogs and metabolites, in: F.T. Addicott (Ed.), Absc/sic Acid, Praeger Special Studies, Praeger, New York, 1983, pp. 113-- 146. T. Oritani and K. Yamashita, Synthesis and plant growth inhibitory activities of (+)- and (-)- (2Z,4E)5-(l ',2'-epoxy-2',6',5'-trimethylcyelohexyl)~3-methyl-2,4pentadienoic acid. Phytochemistry, 22 (1983) 1909-1912. I.C. Hakman and L.C. Fowke, An embryogeulc cell suspension culture of Picea glauca (white spruce). Plant Cell Rep., 6 (1987)20--22. S. yon Arnold and T. Eriksson, In vitro studies of adventitious shoot formation in Pinus cwntorta. Can. J. Bot., 59 (1981)870-874. D.T. Krizek, Guidelines for measuring and reporting environmental conditions in controlled-environment studies. Physiol. Plant., 56 (1982)231 -235. R.B. Horsch, J. King and G.E. Jones, Measurement of cultured plant cell growth on filter paper discs. Can. J. Bot., 53 (1980)2402--2406. S. Tamura and M. Nagao, Synthesis and biological activities of new plant growth inhibitors structurally
related to abseisic acid. Agr. Biol. Chem., 34 (1970) 1393 1401. I.C. Hakman, P. Rennie and L.C. Fowke, A light and electron microscope study of Picea glauca (white spruce) somatic embryos. Protoplasma, 140 (1987) 100 109. W. Knop, Quantative untersuchungen fiber den ernahzungsprozess der pflanzen. Landw. Versuchs. Stat, 7 (1865)93-- 107. M.C. Astle and P.H. Rubery, Carrier-mediated ABA uptake by suspension cultured Phaseolus coccineus L. cells: stereospecificity and inhibition by Ionones and ABA esters. J. Exp. Bot., 38 (1987) 150-- 163. T-h.D.Ho, Biochemical mode of action of abscisic acid, in: F.T. Addicott (Ed.), Abscisic Acid, Praeger Special Studies, Praeger, New York, 1963, pp. 147-- 170. P.K. Gupta and D.J. Durzan, Biotechnology of somatic polyembryogenesis and plantlet regeneration of lobolly pine. Biof~echnology, 5 (1987a) 147-151. K~K.Kartha, L.C. Fowke, N.L. Leung, K.L. Caswell, I. Hakman, Induction of somatic embryos and plantlets from cryopreserved cell cultures of white spruce (P/~ cea g/auca). J. Plant Physiol., 132 (1988)529--539. -
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Elsevier Scientific Publishers Ireland Ltd.
EFFECTS OF ABSCISIC ACID AND ANALOGUES ON THE MATURATION OF WHITE SPRUCE (PICEA GLA UCA) SOMATIC EMBRYOS
DAVID I. DUNSTAN~', FAOUZI BEKKAOUP, MARC PILON,, LARRY C. FOWKEband SUZANNE R. ABRAMS,
•Plant Bioteelmology l~stitute, NationalResearch Co~ndl Canad~ 110 Gymnasium Road, Saskatoon, Saskatchewan, S7N OW9 and bDepartment of Biology, University of Saskatchewan, Saskatoon, Saskatcheu~m, STN OWO(Canad~ (Received February 10th, 1988) (Revision received May 20th, 1988) (Accepted May 25th, 1988)
Somatic embryo-eompotent cultures of white spruce, Picea glanca (Meench) Vou, were grown on ( ± )-abscisie acid (ABA) and three analogues known to be biotrausformed by intnct plants into ABA: ( ± )-methyl abscisate (MeABA); ( ± )~thyl-(2E,4E) and ~,E,4Z)-5-(l'2'~poxy-2',6',6~-trimethylcyelohexyl)-3-methylpentadienoate (epoxyester, EE); and ( ± )-(2E,4E)and -(2E,4Z~5(l'~2'-epoxy-2',6',6'-trimethylcyciobexyl)-3-methylpentadienoic acid (epoxyacid, EA). ABA between 8 and 12 ~M most efficiently promoted embryo maturation and led to the maximum recovery (4%) of stage 4b embryos when used in the absence of auxin or cytokinin.MeABA in the absence of auxin and cytokinin also promoted embryo maturation, EE promoted only the early stages in maturation, and EA was unable to promote any maturation of somatic embryos. Phytohormone-f~ee medium produced very similar responses to EA, though in rare eases stage 3 embryos were found. Neither ABA nor the AnAloguespromoted embryo maturation when used in combination with 9 ~M 2,4-dichloropbenoxyacetic acid (2,4-D}and 4.4 ~M NS-benzyiadenine (BA).
Key words: Picea glauc~, somatic embryogenesis; abscisic acid; maturation; abscisic acid analogues
Introduction The plant growth regulator ABA is involved in all stages of plant growth and development [1]. With particular reference to embryos, ABA is implicated both in zygotic embryo maturation [2] and in the maintenance of seed dormancy [3]. Addition of ABA to coniferous tissue cultures has been investigated by Gupta and Durzan [4], who used 0.5 ~ ABA to promote embryo maturation from protoplasts of Ioblolly pine, and by Durzan and Gupta [5] who reported the promotion of embryo maturation by 0.5 *To whom all correspondence should be addressed. NRCC n0. 28886. Abbreviations: ABA, (±)-abscisic acid; ac÷, medium with 2,4~D and BA; ac-, medium without 2,4-D and BA; BA, N ~benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid; EA, (±)-(2E,4E) and -(2E,4Z)-5-(l',2'epoxy-2',6',6'-trimethylcyciohexyl)-3-methylpentadienoie acid; EE, (±)-ethyl-(2E,4E) and -(2E,4Z)-5-(l'2'epoxy-2',6',6'trimethylcyciohexyl)-3-me. thylpontadienoate; MeABA, ( ± )-methyl abseisate.
ABA in suspension cultures of Douglas fir. More recently, von Arnold and Hakman [6]* have shown that maturation of Norway spruce embryos occurred after treatment with 7.6/~I ABA. Because of the potential for the use of ABA, we decided to compare the effects of ABA and active analogues as chemical agents to promote the maturation of embryos in cultures of white spruce. This comparison was of interest because a very few analogues of ABA, in particular epoxy beta-ionylideneacetie acid, its esters and esters of ABA, have been found to have similar or greater activity than ABA [7,8] and have been shown to be biotransformed into ABA by intact plants [9]. We present here our findings on the effects of exogenous supplements of ABA, MeABA, *Since going to press this manuscript and that of Hakman and yon Arnold, Physiol. Plant., 72 (1988) 579-587, have been published. We have amended our embryo classification to integrate with these authors.
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78 EE and EA on somatic embry~competent cultures of white spruce. Materials and methods Plant material Embryogenic cell suspension cultures were prepared as previously described [10]. Suspensions were maintained by subculturing 0.5 g fresh weight of ceils, at 7~lay intervals, to 50 ml of fresh yon Arnold and Eriksson's LP medium [11] containing 1% (w/v) sucrose, 9 i~I 2,4-D and 4.4 ~ I BA. To this was added 15 ml of filtered spent (conditioned) medium. Cultures were contained in 250 ml Delong flasks agitated at 150 rev./min on a gyratory shaker (New Brunswick, Edison, N.J., U.S.A.) and incubated at 25°C with continuous incident photosynthetically active radiation (PAR) [12] of 15--20 ~E m -2 s -1. The cultures had been maintained for 18 months prior to the start of these experiments. These cell suspension cultures (designated WS1) contained a heterogeneous population consisting of free cells, clusters of isodiametric cells, and pro-embryos in a repressed state of development (stage 1, described later). Filter disc cultures Filter disc cultures were prepared after the technique of Horsch and King [13], with the modifications described below. Cell suspensions were harvested by filtration and were washed twice with 50 ml of phytohormone-free LP medium described above, before being resuspended as a 200/0 w/v suspension in the same medium. Aliquots of 0.75 ml (0.15 g fresh wt.) were dispensed onto gridded black MiUipore filter discs (0.8 /an, diam. 47 ram, no. AABG04750, Bedford, Mass., U.S.A.) placed on the experimental media described below. The filters facilitated subsequent observations, and were useful in retaining the inoculum in a rectangular grid configuration by surface tension. To estimate the number of embryos that were plated, 3 × I ml samples of the 200/0 w/v suspension were observed in a cell counting chamber (Sedgewick Rafter 150 micro litre cell, Graticules Ltd., Tonbridge, U.K.). Filter disc
Abscisic acid (ABA)
Epoxy acid (EA)
Methyl Abscisate
(MeABA)
Epoxy ester (EE)
Fig. 1. Structural formulae of ABA and analogues used in study.
cultures were incubated on 25 ml of experimental medium, contained in sterile disposable Petri dishes (100 × 15 ram). These were left unsealed, and were placed in loosely tied plastic sleeves, 10 dishes per sleeve collected randomly from across the range of media tested. Cultures were incubated at 25°C with an incident PAR of 85 I~E m -2 s -1 during a 16-h/day photoperiod. Ezperimental media ABA, 99%, was obtained from Sigma (St. Louis, Mo., U.S.A., no. A2784). MeABA, 99%; EE, 98% (3:1 ratio of ZE,4E and 2E,4Z isomers); and EA, 980/0 (3:2 ratio of 2E,4E and 2E,4Z isomers), were prepared according to published techniques (MeABA after Milborrow [7]; EE modified after Tamura and Nagao [14]; EA after Tamura and Nagao [14]). Each compound (Fig. 1) was dissolved in the minimum quantity of ethyl alcohol prior to dilution with distilled water. Aliquots of the stock solutions so prepared were added to autoclaved medium described above, after filter-sterilisation. The stability of each compound was evaluated before and after each repeat by gas-liquid chromatography and thin layer chromatography. All compounds were found to be stable in solution during the experiment. Each compound was compared at 0 (control), 4, 8 and 12 ~u~I, either in the presence (ac÷) or the absence (ac-) of 9 ~ 2,4-D and 4.4 ~II BA. The experiment was repeated on two separate occasions, the first with an average of 1386 stage I
79 embryos per treatment, the second with an average of 2600 stage 1 embryos per treatment. In the second repeat 16 ~M ABA was also used. Control ac-, control ac*, and ABA at 4 and 8 ~M were repeated three times, receiving a total of 8000 embryos each.
Fig. 5}. (N.B. von Arnold and Hakman [6] do not differentiate early and late stage 4 embryos; we differentiate these because of the differences in results we obtained for each, as reported below). Results
Continued development Filter disc cultures were removed from the above experimental media after 35 days, and were placed on phytohormone-free LP medium. When embryo maturation proceeded sufficiently, individual embryos were removed for culture on the same medium contained in deep Petri dishes (100 × 25 mm), 25 ml per dish. A comparison was made of removal at stage 4a and at stage 4b. Media used for radicle extension are described in the text, in each ease deep Petri dishes were used. When used, charcoal was obtained from Sigma (C-4386, St. Louis, Mo., U.S.A.). Data collection Cultures were observed throughout the experiment for qualitative changes in morphology. Quantitative data were collected from day 21. This monitored the progression of embryo stages toward maturation. The stages shown are a modification of the enumeration of yon Arnold and Hakman [6] as described below. Stage 1. Repressed pro-embryo with translucent suspensor, and semi-translucent, deuselystaining embryo with slightly irregular outline (Fig. 2, large arrow) (see also Refs. 10 and 15). Stage ~ Embryo is prominent, smooth in outline and opaque, cream to pale yellow, subtended by suspensor (Fig. 2, small arrow). Stage 3. Embryo with primordial cotyledons clustered around a central meristem, cream to pale green in colour (Fig. 3}. Stage l,a {early). Embryo with distinct, partly elongated, cotyledons clustered around a central meristem, green (Fig. 4, smaller). Stage ~b flare). Embryo with elongating cotyledons and elongating hypocotyl, green, with rudimentary radical development adjacent to suspensor remnants (Fig. 4, (arrow);
MorpAological appearance and embryo maturation After 35 days cultures growing on control ac÷ medium were usually light brown in colour. Cultures grown on ac÷ in combination with ABA or MeABA were generally green. Cultures grown in the presence of EA and EE in combination with ac* were more variable in their growth pattern. In all the above ac* media only stage 1 embryos were found, indicating that no somatic embryo maturation had occurred. In comparison, 35 day~ld ac- controls were completely brown and appeared dossicated. Stage 1 embryos in poor condition were found on these plates, very rarely did more advanced stages occur (using a total of 8000 stage 1 embryos over three repeats). However, ac- used in combination with ABA or analogues produced cultures which contrasted with the ac- controls in the occurrence of embryos at stage 2, 3 and 4a on ABA (4, 8, 12 and 16 ~Vl), MeABA (4, 8 and 12 ~,M), and EE (12 ~M}. Only the use of the EA analogue did not result in slmilsl, maturation. Cultures grown on ac- with ABA or analogues had a similar morphological appearance to ac" controls, though browning was delayed in the presence of ABA. At 35 days ac* control filter disc cultures had ceased to grow (data not shown). This was taken arbitrarily as the time to remove all the filter disc cultures for placement onto phytohormone-free LP medium to encourage further development. From this time any stage 1 or 2 embryos that remained on ac- media ceased to grow, only embryos in stage 3, 4a or 4b developed further. Table I shows the maturation of embryos obtained for the above media, data from repeat 2. On ac- ABA at 12 ~.M, stage 2
80
embryos were observable prior to day 21 (data not shown), and stage 3 embryos peaked on day 28 and subsequently diminished, this indicated that there was a repression of the generation of new stage 1, 2 or 3 embryos. Stage 4a embryos
were present by the 21st day on ac- ABA at 12 /~I, and stage 4b embryos, which were first observed in 35-day~)ld cultures, reached their maximum on day 49. The maximum cumulative recovery of stage 4b embryos by the 60th day
Fig. 2. Filter disc with overlay of suspension culture solution, containing stage 1 somatic embryos (large arrow), stage 2 somatic embryos (small arrow) and associated cells, x 21. Fig. 3.
Somatic embryo in stage 3, with primordial cotyledons clustered around a central meristem, x 27.
Fig. 4. Three stage 4a somatic embryos (smaller) with elongating cotyledons and one stage 4b somatic embryo (arrow) with elongating cotyledons and elongating hypocotyl, x 6. Fig. 5.
Isolated stage 4b somatic embryos with suppressed radicle development, x 6.
Fig. 6.
Stage 4b with radicle extension and true needles, x 4.
81 T a b l e I. P r o g r e s s i o n t h r o u g h somatic e m b r y o m a t u r a t i o n stages. Data are m e a n s of n u m b e r s of e m b r y o s p e r filter disc, at various s t a g e s in maturation, ± S.E. (4--5 replicate discs p e r treatment}. N u m b e r of e m b r y o s (stage I} p e r filter disc at s t a r t w a s 650. Medium (Se-)
ABA (4 ~tM)
Embryo
Time (days}
stageb
21
4a 4b 4a 4b 3• 4a 4b 4a 4b 4a 4b 4a 4b 4a 4b 4a 4b
ABA (8 IAM) ABA (12 ~M)
ABA (16 ~LM} MeABA (4 ~M) MeABA (8 ~M) MeABA (12 ~M) Control
28
1.5 ± 1.3
1.8 -0.8 -35~ 2.0 2.5 -1.8 1.0 2.0 -
0.5 -28.3 1.3 0.5 -1.0 1.2 1.0 .
± 1.0 ± 9.4 ± 0.5 ± 0.6 ± 1.2 ± 0.8 ± 0
.
$5" ± 1.5 ± 1.0 ± 9.2 ± 0 ± 1.0 ± 0.8 ± 1.0 ± 1.0
.
49
7.0 0.3 8.8 0.7 24.8 13.8 1.0 8.8 3.0 4.0 5.4 12.5 1.2 1.0 .
± ± ± ± ± ± ± ± ± ±
2.6 0.5 6.4 0.9 7.6 4.9 1.2 5.7 0~ 1.6
60
12.5 7.0 6.7 12.7 4.8 14.3 21.8 3.8 13.5 4.2 0.8 7.0 6.2 9.8 9.0 1.0
± 1.1 ± 5.8 ± 0.9 ± 2.0
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
7.2 3.0 5.5 4.9 3.1 2.6 4.7 2.9 6.7 1.1 0.5 3.3 2.8 5.7 2.2 2.0
7.0 6.0 4.0 3.3 2.0 8.0 6.0 1.0 3.0 2.5 2.2 4.8 4.8 3.8 4.8 -
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.2 3.0 2.6 3.5 1.2 1.6 1.4 2.0 1.4 1.9 1.7 2.3 2.5 2.9 2.2
.
sAt 35 days filter discs w e r e r e m o v e d from experimental media and w e r e placed on phytohormone-free L P medium. bStages 3 and 4a w e r e left in situ, figures t h e r e f o r e r e p r e s e n t the changing population. Stage 4b's w e r e r e m o v e d at each observation ~ o m 35 days, figures are not cumulative. eData for s t a g e $'s from o t h e r media s h o w e d similar trends.
Table II. The r e c o v e r y of stage 4 e m b r y o s , data after 60 days in culture. A v e r a g e n u m b e r s of s t a g e 1 e m b r y o s p e r t r e a t m e n t w e r e 1386 (repeat 1) and 2600 (repeat 2). Data are totals p e r t r e a t m e n t . Medium
ae-ABA
Cone.
Repeat 2
Repeat I
0A 4 8 12 16
Embryos (st. 4b)
%
Embryos (st. 4b)
0 29 64 52 -
2 5 4
56 67 115 78
Embryos (st. 4a)
%
Embryos (st. 4b)
%
ae-MeABA
4 8 12
2 3 35
0.1 0.2 3
0 0 0
----
15 55 60
ae-EA
4 8 12
0 0 0
----
0 0 0
----
0 0 0
ae-EE
4 8 12
0 0 0
----
0 0
0
---
ae-Control
0
-
0
-
0
as*Control
0
-
0
-
0
---
%
2 3 4 3
0.5 2 2
82
of repeat 2, occurred on ac- ABA at 12 (Table II). Differences between the two repeats in the total numbers of stage 4b embryos recovered for each medium are shown in Table II. For example, in repeat 1 the recovery of stage 4b embryos on ac- ABA at 8 and 12 ~M was similar. In neither repeat were stage 4b embryos recovered from phytohormone-free controls, nor from any E E concentrations. When 0 . 4 / ~ I ac- ABA was evaluated, stage 2 embryos were present by day 26 but continued maturation to stages 3, 4a and 4b was not obtained. This indicated that exogenous ABA was physiologically most active when provided between 8 and 12 ~ under ac- conditions. A comparison of data from both repeats showed that the efficiency of the maturation process under the maximal conditions described (i.e. comparing ac- ABA at 12 ~M), was about 4.0°/0; that is, in repeat 1 an average of 1386 stage 1 embryos were plated while an average of only 62 stage 4b embryos were recovered, in repeat 2 an average of 2600 stage 1 embryos were plated while only 115 stage 4b embryos were recovered (Table II).
Optimum time for embryo removal To determine the best maturation stage for subsequent radicle extension and shoot growth, a comparison was made of the development of stage 4a and 4b embryos, removed from the filter disc cultures. This was accomplished by sequentially removing either stage 4a or stage 4b embryos at each observation date that they occurred, commencing on day 35. The results showed that on phytohormone-free LP medium it was possible to obtain a limited maturation of stage 4a into stage 4b embryos. This was greatest for those previously grown on ac- ABA at 8 or 12 pM (41O/o or 46%, respectively). However, in a majority of cases radicle extension was inhibited, and shoot growth was relatively slow. This material eventually ceased to grow and was not experimented with further. Conversely, when maturation was allowed to proceed to stage 4b while still on the filters, better shoot quality and greater radicle extension (e.g. 560/0 of stage 4b embryos previously
grown on ac- ABA at 12 /n~I) was observed. Stage 4b embryos collected in this way showed increases in shoot vigour with increasing ABA concentration, the most vigorous being harvested from filter discs previously grown on acABA at 16 /~M. Stage 4b embryos collected from discs previously grown on ac- at 4 /~M were poor by comparison. Shoot vigour was also related to time, with the most vigorous shoots of a medium being stage 4b embryos collected on day 49. Radicle extension of stage 4b embryos was often inhibited (Fig. 5~. Inhibition was greatest for those previously grown on ac- ABA at 16 /~I and least for those from ac- ABA at 12 ~Vl. Consequently, stage 4b embryos were placed onto one of three media designed to promote radicle extension. These were as follows: agarsolidified LP with charcoal at 0.05%, LP liquid with cheesecloth support, and agar-solidified 0.5 x LP; each medium was compared to control phytohormone-free LP. Between 10 and 25 embryos from ac- ABA concentrations 8, 12 and 16 ~M were compared. Results for stage 4b embryos previously grown on ac- ABA at 12 ~M, showed that none of these treatments significantly improved radicle extension compared to the phytohormone-free LP control (560/0). Charcoal marginally improved radicle extension on stage 4b embryos derived from other media, with the greatest improvement being obtained for ac- ABA at 16/~d (from 260/0 for control to 530/0 for charcoal). Some embryos, with expanding needles and extending radicles (Fig. 6). were transferred to test-tubes containing sterile vermiculite moistened with 0.5 x Knop's solution [16], incubated under the conditions described for filter disc cultures. After 1 month approx., they were planted in soilless medium in a growth room, where they have slowly continued their development. However, the conditions necessary for their survival and continued development have not yet been satisfactorily established. Discussion
Abscisic acid is normally added to plant tis-
88
sue cultures in the R S ( ± )- form, a synthetic racemic mixture. The natural phytohormone has the S( + )-configuration. The R( - ~enantiomer has biological activity in some circumstances e.g. growth inhibition [8], and the racemic mixture can give nearly the same activity as the natural phytohormone in some circumstances. The natural phytohormone is known to play an active role in coniferous tissues in vivo [3], particularly in embryo ripening (maturation) [2]. This was also the observed effect of exogenous supplements of racemic ABA supplied to our white spruce cultures in vitro, when used in the absence of auxin and cytokinin. ABA is associated with the maintenance of dormancy in seed embryos, e.g. of Tazus [3]; the removal of ABA in the latter example was found to be coincident with the first signs of radicle extension. A similar effect may have lead to the suppression of radicle development in our experiments. MeABA has been shown to have a higher activity than ABA in some studies [8,17], but from our data MeABA had approximately onehalf the activity of ABA at the 12 ~ concentration on day 49. The physiological effects of ABA in vivo are concentration dependent [18]. Therefore, although stage 4b embryos were recovered using MeABA, our concentration range may not have allowed us to see its maximum effect. The epoxy ester (EE) and epoxy acid (EA) in the (2E,47,) forms have high activity in some bioassays in plants [8]. By analogy with ABA, the activity ascribed to the (2E,4E) forms [8] is likely to be due to reversible photoisomerisation; because our experiments were to be performed in the light over long periods of time, the isomeric mixtures were used. EA did not however promote embryo maturation at any concentration, and only early maturation stages were found to occur on EE at 12/~M. These results may be due to the inability of the cultures to biotransform the analogues in vitro into ABA, or to inadequate final concentrations of ABA for the maturation process. Gupta and Durzan have shown that they could recover mature somatic embryos from callus on phytohormone-free medium without
addition of ABA [19]. A similar observation was recently made for white spruce after cryopreservation of stage 1 somatic embryos [20]. We were unable to recover stage 4 somatic embryos using phytohormone~free medium in the absence of ABA. These results may reflect the use of different species and/or culture conditions. Recently, yon Arnold and Hakman [6] showed for Norway spruce that they could induce the development of stage 3 embryos using 7.6 ~M ABA. These could be removed for development into green plantlets on ABA-free medium. We found that similar levels of ABA promoted embryo maturation, but removal of embryos at stage 3 or 4a was not optimal with our cultures of white spruce. Our future studies will attempt to optimise the recovery of stage 4b somatic embryos, investigating the effects of plating density, duration of ABA treatment and the effects of other medium components. Acknowledgements The authors gratefully acknowledge the technical assistance given by Mr. Terry Bethune, Ms. Angela Shaw and Mr. Bill Stirtan during the course of these experiments. References 1 F.T. Addicott and R~F.M. Van Steveniek, The significance of abseisic acid in the life of plants, in: F.T. Addicott (Ed.), Abseisie Acid, Praeger Speeial Studies, Praeger, New York, 1983, pp. 581 - 586. 2 G. Fellenberg, Developmental physiology. Prog. Bot., 44 (1982) 205-- 221. 8 M.-T. LePage-Degivry, Aeide abseisique et dormance chez los embryons de T a z ~ baccata L. C.R. Aead. Sei. Paris, 271 (1970) 482-- 484. 4 P.K. Gupta and D.J. Durzan, Somatic embryos from protoplasts of lobloIly pine proembryonal cells. Bio/ Technology, 5 (1987b) 7 1 0 - 712. 5 D.J. Durzan and P.K. Gupta, Somatic embryogenesis and polyembryogenesis in Douglas f'tr cell suspension cultures. Plant Sci., 52 (1987)229-235. 6 S. von Arnold and I. Hakman, Regulation of somatic embryo development in Picea abies, with emphasis on ABA effects. J. Plant Physiol., 132 (1988) 164-169. 7 B.V. Mflborrow, The chemistry and physiology of abscisic acid. Ann. Rev. Plant. Physiol., 25 (1974) 259-307. 8 D.C. Walton, Structure-activity relationships of abs-
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II
12
13
14
elsie acid analogs and metabolites, in: F.T. Addicott (Ed.), Absc/sic Acid, Praeger Special Studies, Praeger, New York, 1983, pp. 113-- 146. T. Oritani and K. Yamashita, Synthesis and plant growth inhibitory activities of (+)- and (-)- (2Z,4E)5-(l ',2'-epoxy-2',6',5'-trimethylcyelohexyl)~3-methyl-2,4pentadienoic acid. Phytochemistry, 22 (1983) 1909-1912. I.C. Hakman and L.C. Fowke, An embryogeulc cell suspension culture of Picea glauca (white spruce). Plant Cell Rep., 6 (1987)20--22. S. yon Arnold and T. Eriksson, In vitro studies of adventitious shoot formation in Pinus cwntorta. Can. J. Bot., 59 (1981)870-874. D.T. Krizek, Guidelines for measuring and reporting environmental conditions in controlled-environment studies. Physiol. Plant., 56 (1982)231 -235. R.B. Horsch, J. King and G.E. Jones, Measurement of cultured plant cell growth on filter paper discs. Can. J. Bot., 53 (1980)2402--2406. S. Tamura and M. Nagao, Synthesis and biological activities of new plant growth inhibitors structurally
related to abseisic acid. Agr. Biol. Chem., 34 (1970) 1393 1401. I.C. Hakman, P. Rennie and L.C. Fowke, A light and electron microscope study of Picea glauca (white spruce) somatic embryos. Protoplasma, 140 (1987) 100 109. W. Knop, Quantative untersuchungen fiber den ernahzungsprozess der pflanzen. Landw. Versuchs. Stat, 7 (1865)93-- 107. M.C. Astle and P.H. Rubery, Carrier-mediated ABA uptake by suspension cultured Phaseolus coccineus L. cells: stereospecificity and inhibition by Ionones and ABA esters. J. Exp. Bot., 38 (1987) 150-- 163. T-h.D.Ho, Biochemical mode of action of abscisic acid, in: F.T. Addicott (Ed.), Abscisic Acid, Praeger Special Studies, Praeger, New York, 1963, pp. 147-- 170. P.K. Gupta and D.J. Durzan, Biotechnology of somatic polyembryogenesis and plantlet regeneration of lobolly pine. Biof~echnology, 5 (1987a) 147-151. K~K.Kartha, L.C. Fowke, N.L. Leung, K.L. Caswell, I. Hakman, Induction of somatic embryos and plantlets from cryopreserved cell cultures of white spruce (P/~ cea g/auca). J. Plant Physiol., 132 (1988)529--539. -
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