- Email: [email protected]
Leaf Dimorphism in Taraxacum officinale during in vitro Culture of Shoot Tips
Leaf Dimorphism in Taraxacum officinaJe during in vitro Culture of Shoot Tips MARiA
C.
POMAlt.. EsrAN ISLAVA SI.ABNIK, OSVALDO
H.
CASO
and
HILDA
DIAZ
Centro de Ecoflsiologla Vegetal (FEClC-CONICET-FUND. M. LILLO) Serrano 665, 1414 Buenos Aires, Argentina Received May 22, 1985 . Accepted August 15, 1985
Summary Foliar morphogenesis was studied in Taraxacum officinale through the shoot apex culture of plants from different o ntogenic stages. Apices from young plants only produced juvenile leaves (entire). On the other hand, apices from adult plants differentiated juvenile and adult (runcinate) leaves. If adult plants were induced to produce juvenile leaves upon treatment with gibberellic acid (GAl)' the isolated apices produced a higher proportion of juvenile leaves. This change is associated with a prolonged apparent plastochron. alike the one observed when whole plants are ismilarly treated. These observations indicated that the apical region has enough information to express its own development. [n agreement with results of treatments to whole plants, when apices isolated from adult plants were irradiated with FR light during culture, or incubated with GAl, the production of runcinate leaves by the apex reverted to less complex fo rms. This is related either to changes produced by FR-light or GAl on the apical zone, o r directly on the earl y differentiation of primordia, regardless of t he influence of other parts of the plant. On the other hand. the plastochron was not affected. thus suggesting that this property is acquired by the apical zone through its interaction with the metabolism of the plant as a whole.
Key words: Taraxacum officinale; meristem-culture; leaf morphogenesis; gibberellic acid; far-red light.
Introduction In heteroblastic plants. the size and shape of the different leaves along the stem are a consequence of bot h thenumber and orientation of cell divisions during the formation of primordia, and of cell enl argement (Wareing and Phillips, 1975). It has also been shown that light, mediated by its intensity and quality, plays a decisive role on leaf shape differentiation in T. o./ficinale. Responses si milar to those obtained with a low light intensit y could be elicited either by a short FR irradiation at the end of the photoperiod, i.e. a phytochrome-mediated reaction, (Sanchez and Cogliatti, 1975) or by an exogenous application of GA" which delayed the appearance of the adult type of leaf shape (Arnozis et ai., 1977). When adult plants we re treated with either GA, or FR-light, primordia of more than 0.5 mm long with differentiated lobes would not be reverted to a more juvenile type. On the other hand, these treatments are quite effecAbbreviation list: FR far-red light; GA j gibberellic acid; MS Murashige-Skoog medium; IBA 4-{3-indoly l) butyric acid; SAP 6-bt:nzylaminopurine.
J.
Plant Physiol. Vol. 122. pp. 413-421 (1986)
414
MAlliA C. POMAR., EsrANISLAVA SLABNIX, OSVALDO H. CASO and HILDA DiAz
dve on smaller primordia, and the treated plants will differentiate juvenile leaves (Cogliatti and Guitman, 1984). Several hypotheses have been proposed in order to evaluate the influence of differ· ent factors on the determination of leaf inception sites and their further differentia· tion (Halperin, 1978). Moreover, different experiments support the idea of a relative metabolic autonomy of the apical region, that might be influenced by certain stimuli derived from more mature organs (Allsopp, 1964). The technique of in vitro culture might serve as a tool for the study of the responses of shoot meristems, disregarding the correlative influences on their metabolism of other plant parts (such as more mature leaves or the root) on it. Different authors have studied the effect of hormones and!or sucrose on the leaf form differentiation, either in isolated shoot apical meristems {Fukui et al., 1981; Engelke et al., 1973} or excised leaf primordia (Feldman and Cutter, 1970). In the present work, this technique has been used to obtain information about the regulation of leaf morphogenesis in T. officina/e. Hence, the responses of excised shoot meristems from plants of different ages and! or treatments were analysed. Also, the effect of changes in the medium composition and!or in the environmental condi· tions during culture were studied.
Materials and Methods Production of sterik young plants Achenes of T. officinale were washed by agitating them for a few minutes in tap water with a few drops of a commercial detergent. After rinsing, sterilization was performed for 30 min in a NaCIO solution {2.0% Ch)+0.025% Triton X·100 on a magnetic stirrer. Following several sterile water rinses, they were germinated on filter paper in Petri dishes at a temperature of 24±1°C and a 16h photoperiod with an irradiance of 1800erg · cm- 2 sec- l . Upon germination, the seedlings were transferred to 20 x 120 mm tubes containing 10ml of MS mineral salts (Murashige-Skoog, 1962) supplemented with 2% sucrose and solidified with 0.8% agar. Shoot apices were dissected from plants showing 2 co 4 juvenile leaves.
Production ofadult plants and sterilization of the apical regions Non sterile seedlings were potted on a mixture of soil; sphagnum-turbe (2: 1) (v/v») and grown in a greenhouse. Shoot apices were dissected from those plants bearing 10 to 12 expanded leaves, which showed the runcinate type of leaf shape from the 5th-6th leaf and on. Simultaneously, plants of the same age were induced to keep forming only the juvenile leaf shape by daily applications of a 10,J drop olan aqueous GA, solution (0.1 "g ·1- I GA,) to the apex, since the appearance of the third leaf. For in vitro culture, all the leaves except the smaller ones were removed, and the apical re-gioos were washed with water with a few drops of a commercial detergent. After rinsing they were desinfected by gentle agitation in a NaCIO solution (0.04% Ch) for 5 min, then in 70% ethanol for 1 min, and then in a NaClO solution {1.2% Ch)+0.025% Triton X-l00 for 15 min. Finally, the material were rinsed several times with sterile distilled water and the shoot apices were excised.
,. Plant P/rysiol. Vol. 122. pp. 413 - 421 (1986)
Leaf-shapes in meristem culture
A
415
B
Fig. 1: Leaf-types produced in the culture of shoot tips of T. officinale (x 10). A: .. entire»; B: «runcinate>J.; C: ..toothed».
In vitro culture 0/ shoot apices Shoot apices from young and adult plants, including the meristematic dome with 1 or 2 leaf primordia, not longer than 0.3 mm without any differentiated lobes, were individually cultured in 25 x 150 mm test tubes containing 10 ml medium each. The medium for culture was the MS (Murashige-Skoog, 1962) formula, supplemented with 3 % sucrose and 0.8 % agar and adjusted to pH 5.5. The hormones employed were 4-{3-indolyl)butyric acid (lBA) and 6-benzylaminopurine (BAP) both at a concentration of 0.5 mg ·1- I determined in previous work as the best ratio for the culture of shoot apices. GA3 was sterilized by filtration and added to the warm agar media. Culture conditions were a temperature of 20±2 °C and a photoperiod of 10 h light from four daylight fluorescent tubes (40W) and four HPL lamps (400W), with an irradiance of 20,000 erg' cm - 2sec - 1).
Irradiation with FR·iight Shoot apices of adult plants were irradiated daily for 20 min during the dark period. The light source was a 150W incandescent lamp with internal reflector, filtered through Schott R6-N° 9 glass filter (3 mm thickness) and a cuvette containing water to lOcm depth. The irradiance was 3,OOOerg'cm - 2sec 1.
J. P/"nt PhysiQl. Vol.
122. pp. 413-421 (/986)
MARiA C. POMAll, EsrANISLAVA SLABNIK, OSVALDO H. CAW and HIWA DiAz
416
TIME (days)
30
o
j
Adult Plants
Young Plants
Fig.2: Responses by cultured shoot meristems from young and adult plants in the differentiation of leaf-types (%): entire leaves (closed bars); toothed leaves (crossed bars) and runcinate leaves (open bars) (15 replications).
Recording of experimental results and leaf shapes The leaves formed during the experimental period were recorded every seven days, and classified as follows (Fig. 1): A) 4\entires_, with entire margins Guveniles), B) .runcinates .. with profound lobes (adults) and C) .toothed.. , intermediate between A and B. Each treatment involved 10 to 15 replications. During the experimental period all the explants showing contamination, adventitious buds and/or callus proliferation were discarded. Experiments were repeated at least twice and explants were cultured for 20 - 30 days, since longer periods always resulted in the induction of adventitious buds in some of the explants.
Results
Culture shoot apices from young and adult plants Fig. 2 compared the growth within four weeks of shoot apices excised from young
and adult plants. Apices from young plants produced in culture only entire leaves Guvenile). In contrast, when apices of adult plants are cultured, the meristems differentiated varying proportions of runcinate, entire an thoothed leaves.
Culture ofshoot apices from aduit GA,-treated and untreated piants Fig. 3 A shows formation of leaves by shoot apices from adult GAl-treated and untreated plants, cultured for a period of 4 weeks. By the end of the experiment, fewer
J.
P["nt P/rysiol. VoL 122. pp. 413-421 (1986)
Leaf-shapes in meristem culture
417
A
• ,i• ••
--
,
.--.. $As
TIM£
'"
(days)
B
27
••
~
Plants
Plants
(-GA,)
1+ GA,J
Fig. 3: Responses by cultured shoot meristems from adult control and GAl-treated plants, in the production and differentiation of leaf-types (15 replications). A: Leaves produced per apex (Mean number ± SE). B: Types of leaves differentiated (%): entire leaves (closed bars); toothed leaves (crossed bars) and runcinate leaves (open bars).
leaves were formed in the apices from GA,-treated plants, (3.53±0,47Ieaves/explant) when compared to those from controls (5.17±O.66 leaves/explant). Fig. 3 B also shows that while in control apices a higher number of the toothed and runcinate types of leaves was formed, in those apices from GA 3-treated plants about 80 % of the leaves belong to the juvenile type.
Effect ofFR -light and of GA, on the growth ofapices from adult plants Apices excised from adult plants were cultured in the same conditions as in the previous experiments and divided into two groups, one of which was daily submitted to
an FR-irradiation. Fig. 4 B shows the effect of such irradiation on the shape of leaves formed at different times during the experimental period. FR induced the reversion of the adult type of leaf to a more juvenile one, as it is shown by the very low percentage of runcinate leaves (8 %) present after 17 days in culture, when compared with more than 90 % in [he control apices. Fig. 5 B shows how [he addition of GAl to
J. Plant Physiol. Vol.
122. pp. 413-421 (1986)
418
MAlliA. C.
POMAJ., EsrANISLAVA SLABNII., OSVALDO H.
CAS<> and
HUDA
DiAz
7
• ,• l
•
•
• •,
I ,
..J
" TIME
20
30
40
( day.)
B
..•
~
Apices
Apicn
I-FR)
{+FR}
Fig. 4: Effect of FR~irradiations «in culture» of the shoot meristems from adult plants, on the production and differentiation of leaf~types (10 replications). A: Leaves produced per apex (Mean number ± SE). B: Types of leaves differentiated (%): entire leaves (closed bars); toothed leaves (crossed bars) and runcinate leaves (open bars).
the culture medium, (1 and 10 mg ·1 - '), induces the apices of adult plants to revert leaf production from the runcinated to less complex leaf shapes, as was the case with FR irradiation. When applied to isolated apices, both treatments seem not to affect
the apparent plastochron (Figs. 4 A and 5 A).
Discussion
.In vitro. grown apices of T. officinale excised from plants in different ontogenic stages, or plants treated to alter their foliar morphogenesis, differentiated leaves with a shape similar to that of leaves originating in the whole plant (Fig. 2 and Fig. 3 B). Apices from adult plants, of the different experiments (Figs. 2, 3 B, 4 B and 5 B) produced between 45 % and 90 % of runcinate leaves. These different responses could be attributed to the variations in the environmental conditions in the greenhouse during
growth of the «mother» plants. However, this variability does not invalidate the different responses with respect to the apices of the young plants with 100 % of entire leaves (Fig. 2), or the apices of adult GA,..treated plants with only 3-5% runcinate
J. Plan, l'hysiol. Vol.
122. pp. 413-421 (1986)
Leaf-shapes in meristem culture
..• . 1
:
,:
419
A
! • • ~
--lA,
...-..... u.( .... . r" o-- ·-o· ... ,to ....r
ID
20
30
40
TtM£ (da,.1
B
••
~
o
10
....rIIlA,
Fig. 5: Effect of GAl addition (0, 1 and 10 mg '1 - ') to the culture medium on the production and differentiation of leaves, by the shoot meristems from adult plants (10 replications). A: Leaves produced per apex (Mean number ± SE). B: Types of leaves differentiated (%); entire leaves (dosed bars); toothed leaves (crossed bars) and runcinate leaves (open bars).
ones (Fig. 3 B). However, apices from GA,·treated plants showed a decrease in the number of leaves produced during culture if compared with those from control plants (Fig. 3 A). A longer apparent plastochron in adult T. officinale plants, has al· ready been related with conservation of the juvenile leaf shape (Cogliatti and Sanchez, 1982). Therefore, it seems that the inception of leaf primordia and the differentiation of leaf shape are associated with metabolic and, possibly, morphological changes in the apical wne. These changes would be determined by modifications during the devel· opment of the plants, allowing self-expression by the apex. The production of more complex foliar structures in Darlingtonia cali/arnica has been related to an increase in size of the apical zone. Changes in structure and cytohistological zonation have also been described (Franck, 1976). Moreover, stable modifications of the phyllotaxis af· fected by exogenously applied growth regulators have been related to changes in the shape and size of the apex (Varnell and Vasil, 1978; Halperin, 1978).
J. PL.", P/rysioL Vol. 122. pp. 413-421 (1986)
420
MAID C. Po....., ESTANlSI.AVA SLABNI1, OsvAIDO H.
c.so and tiu.DA DiAz
Organ correlations are considered important in morphogenesis. When plants of Silene armeria growing in non-inductive conditions for flowering were treated with only one GA, application to the apical meristem, Besnard-Wibaut and col. (1983), found an increased mitotic activity. Repeated applications provoked a decrease in the cellular activity at the apex and an increase in the stem growth. This suggested competition for nutrition between both organs. This might well be the case in T. officinale, where either FR or GA, treatments induced also a greater growth of both the petiole and the main vein of the leaves, when compared with untreated plants (Cogliatti and Sanchez, 1982). However, there was a reversion (0 the juvenile leaf type (Figs. 4 B and 5 B) when apices excised from adult plants were grown in vitro, in presence of GA, or irradiated with FR light. This might suggest that the differentiation of leaf-type in the apex is independent of the correlative influences of the changes olr served in the more mature regions of the plant.
Nevertheless, when excised apices were irradiated with FR light or treated with GA" only the leaf shape was affected, but not the average number of leaves formed per explant (Figs. 4 A and 5 A). This result counteracts the feature observed in treated plants, where both processes were affected (Cogliatti and Sanchez, 1982). This behaviour corroborated that the sites of leaf-inception could be determined by the characteristics acquired by the apex. Leaf initiation has been related to the presence of provascular traces and their further differentiation. In Papulus deltoides. a procarnbial strand is initiated 13 plastochrons before the leaf emerges (Larson, 1975). Even though Lyndon (1970) observed in Pisum sativum that there was little or no increase in the rate of cell divisions associated with leaf initiation, and that its formation was a consequence of changes in the direction of apical growth, Hussey (1972) found in the same species that the initiation of a new primordium can
be traced to a local increase
in the rate of cell divisions some plastochrons before it is well formed. In T. officinal..
it is possible that at the cellular level, more sites for the inception of future new primordia were already determined, even if only one or two primordia were visible. GAl or FR light should provoke some modifications on the apical region, or more
directly, on the growth of primordia and their early differentiation (Feldman and Cutter, 1970). The effect of the FR light could be mediated by changes in the endogenous levels of gibbereUins. Different authors have established biosynthetic relationships between GA, and phytochrome, (Hilton and Smith, 1980). However, nutritional factors other than hormones may be involved in this process. For a normal in vitro growth, shoot meristem of T. officinale showed an absolute requirement for the presence of sucrose, an auxin (IBA) and a cytokinin (BAP) in the culture medium. The requirement for these substances for organogenesis in tissue cuJtUJ'e and for plant micro-
propagation through meristem culture are vety well documented. In the whole plant, these substances should be translocated to the apex from the leaves and!or from the root. At present, experiments are underway to examine the interaction of different
sugar and hormone levels in the inception of leaf primordia and their final shapes.
J. Plant P/rysiol. VoL
122. pp. 413-421 {1986}
Leaf-shapes in meristem culture
421
Acknowledgements The authors are grateful to Ing. Agr. Sergio J. Ochatt for the help and critical review of this manuscript.
References Au.sopp, A.: Shoot morphogenesis. Ann. Rev. Plant Physiol. 15. 225-254 (1964), ARNOZIS, P. A" O. H. CASO, and D. H. COGUA1n: Efeeto del GAJ Y 1a intensidad de luz sobre la morfogenesis foliar de Taraxacum offlCinale. Phyton 35 (1), 1-9 (V-1977). Bf.SNARD-WIBAUT. C, M. NOIN, and J. A. D. ZEEVAAIlT: Mitotic activities and levels of nuclear DNA in the apical meristem of Silene anneria (strain S 1.2) following application of gibberellin A J . Plant Cell PhysioL 24 (7), 1269-1279 (1983). COGLlATII, D. H. and R- SANCHEZ: lnflucncia del fitocromo sobre eI crecimiento foliar en Taraxacum offlCinale. Phyton 42 (2), 191-199 (1982). COGUATTI, D. H. and M. R. GUITMAN: Some observations on the effect of gibberellic acid on the shape of Tar=um officinale leaf primordia. j. Plant PhysioL 115, 97 -103 (1984). ENGELKE, A. L.. G. H. HAMzi, and F. SKOOG: Cytokinin-gibberellin regulation of shoot development and leaf form in tobacco plantlets. Amer. j. Bot. 60 (6),491-495 (1973). FELDMAN, L. J. and E. H. CUTIER: Regulation of leaf form in Centaurea solstitialis L. II. The developmental potentialities of excised leaf primordia in sterile culture. Bot. Gaz. 131, 39-49 (1970). FRANCK, D. H.: Comparative morphology and early leaf histogenesis of adult and juvenile leaves of Darlingtonia californica and their bearing on the concept of heterophylly. Bot. Gaz. 1.17 (1),20-34 (1976). FUt.U1, H., S.lMAKAW.... and T. TAMURA: Effects of growth regulators and sugar on the growth of apple ap;cal ,hoot. J. Japan_ Soc. Hart. Sci. 49 (4),549-556 (1981). HALPERIN, W.: Organogenesis at the shoot apex. Ann. Rev. Plant Physiol. 29, 239-272 (1978), HILTON, J. R. and H, SMITH: The presence of phytochrome in purified barley etioplast and its in vitro regulation of biologically active gibberellin levels in etioplast. Planta 148, 312-318 (1980). HUSSEY, G.: The mode of origin of a leaf primordium in the shoot apex of the pea (Pisum sativum).J. Exp. Bot. 23, 675-682 (1972). L ... RSON, P. R.: Development and organization of the primary vascular system in Populus deltoides acco,ding to phyllotaxy_ Am. J. Bot. 62, 1084-1099 (1975)_ LYNDON, R. F.: Rates of cell division in the shoot apical meristems of Pisum. Ann. Bot. 34, 1-17 (1970). MUII..ASHIGE, T. and F. SKOOG: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473 - 497 (1962). SANCHEZ, R. A. and D. H. COGUATII; The interaction between phytochrome and white light irradiance in the control of leaf shape in Taraxacum officinale. Bot. Gaz. 136 (3), 281-285 (1975). V ARNELL, R. J. and I. K. VASIL: Experimental studies of the shoot apical meristem of seed plants. I. Morphological and cytochemical effects of IAA applied to the exposed meristem of Lu· pinus albus. Amer. J. Bot. 65 (1), 40-46 (1978). W ... REING, P. f. and I. D. 1- PHILLIPS: The control of growth and differentiation in plants. Pergamon Press. 39 (1975).
]. Plant Plrysiol. Vol 122. pp. 413-421 {1986}
C.
POMAlt.. EsrAN ISLAVA SI.ABNIK, OSVALDO
H.
CASO
and
HILDA
DIAZ
Centro de Ecoflsiologla Vegetal (FEClC-CONICET-FUND. M. LILLO) Serrano 665, 1414 Buenos Aires, Argentina Received May 22, 1985 . Accepted August 15, 1985
Summary Foliar morphogenesis was studied in Taraxacum officinale through the shoot apex culture of plants from different o ntogenic stages. Apices from young plants only produced juvenile leaves (entire). On the other hand, apices from adult plants differentiated juvenile and adult (runcinate) leaves. If adult plants were induced to produce juvenile leaves upon treatment with gibberellic acid (GAl)' the isolated apices produced a higher proportion of juvenile leaves. This change is associated with a prolonged apparent plastochron. alike the one observed when whole plants are ismilarly treated. These observations indicated that the apical region has enough information to express its own development. [n agreement with results of treatments to whole plants, when apices isolated from adult plants were irradiated with FR light during culture, or incubated with GAl, the production of runcinate leaves by the apex reverted to less complex fo rms. This is related either to changes produced by FR-light or GAl on the apical zone, o r directly on the earl y differentiation of primordia, regardless of t he influence of other parts of the plant. On the other hand. the plastochron was not affected. thus suggesting that this property is acquired by the apical zone through its interaction with the metabolism of the plant as a whole.
Key words: Taraxacum officinale; meristem-culture; leaf morphogenesis; gibberellic acid; far-red light.
Introduction In heteroblastic plants. the size and shape of the different leaves along the stem are a consequence of bot h thenumber and orientation of cell divisions during the formation of primordia, and of cell enl argement (Wareing and Phillips, 1975). It has also been shown that light, mediated by its intensity and quality, plays a decisive role on leaf shape differentiation in T. o./ficinale. Responses si milar to those obtained with a low light intensit y could be elicited either by a short FR irradiation at the end of the photoperiod, i.e. a phytochrome-mediated reaction, (Sanchez and Cogliatti, 1975) or by an exogenous application of GA" which delayed the appearance of the adult type of leaf shape (Arnozis et ai., 1977). When adult plants we re treated with either GA, or FR-light, primordia of more than 0.5 mm long with differentiated lobes would not be reverted to a more juvenile type. On the other hand, these treatments are quite effecAbbreviation list: FR far-red light; GA j gibberellic acid; MS Murashige-Skoog medium; IBA 4-{3-indoly l) butyric acid; SAP 6-bt:nzylaminopurine.
J.
Plant Physiol. Vol. 122. pp. 413-421 (1986)
414
MAlliA C. POMAR., EsrANISLAVA SLABNIX, OSVALDO H. CASO and HILDA DiAz
dve on smaller primordia, and the treated plants will differentiate juvenile leaves (Cogliatti and Guitman, 1984). Several hypotheses have been proposed in order to evaluate the influence of differ· ent factors on the determination of leaf inception sites and their further differentia· tion (Halperin, 1978). Moreover, different experiments support the idea of a relative metabolic autonomy of the apical region, that might be influenced by certain stimuli derived from more mature organs (Allsopp, 1964). The technique of in vitro culture might serve as a tool for the study of the responses of shoot meristems, disregarding the correlative influences on their metabolism of other plant parts (such as more mature leaves or the root) on it. Different authors have studied the effect of hormones and!or sucrose on the leaf form differentiation, either in isolated shoot apical meristems {Fukui et al., 1981; Engelke et al., 1973} or excised leaf primordia (Feldman and Cutter, 1970). In the present work, this technique has been used to obtain information about the regulation of leaf morphogenesis in T. officina/e. Hence, the responses of excised shoot meristems from plants of different ages and! or treatments were analysed. Also, the effect of changes in the medium composition and!or in the environmental condi· tions during culture were studied.
Materials and Methods Production of sterik young plants Achenes of T. officinale were washed by agitating them for a few minutes in tap water with a few drops of a commercial detergent. After rinsing, sterilization was performed for 30 min in a NaCIO solution {2.0% Ch)+0.025% Triton X·100 on a magnetic stirrer. Following several sterile water rinses, they were germinated on filter paper in Petri dishes at a temperature of 24±1°C and a 16h photoperiod with an irradiance of 1800erg · cm- 2 sec- l . Upon germination, the seedlings were transferred to 20 x 120 mm tubes containing 10ml of MS mineral salts (Murashige-Skoog, 1962) supplemented with 2% sucrose and solidified with 0.8% agar. Shoot apices were dissected from plants showing 2 co 4 juvenile leaves.
Production ofadult plants and sterilization of the apical regions Non sterile seedlings were potted on a mixture of soil; sphagnum-turbe (2: 1) (v/v») and grown in a greenhouse. Shoot apices were dissected from those plants bearing 10 to 12 expanded leaves, which showed the runcinate type of leaf shape from the 5th-6th leaf and on. Simultaneously, plants of the same age were induced to keep forming only the juvenile leaf shape by daily applications of a 10,J drop olan aqueous GA, solution (0.1 "g ·1- I GA,) to the apex, since the appearance of the third leaf. For in vitro culture, all the leaves except the smaller ones were removed, and the apical re-gioos were washed with water with a few drops of a commercial detergent. After rinsing they were desinfected by gentle agitation in a NaCIO solution (0.04% Ch) for 5 min, then in 70% ethanol for 1 min, and then in a NaClO solution {1.2% Ch)+0.025% Triton X-l00 for 15 min. Finally, the material were rinsed several times with sterile distilled water and the shoot apices were excised.
,. Plant P/rysiol. Vol. 122. pp. 413 - 421 (1986)
Leaf-shapes in meristem culture
A
415
B
Fig. 1: Leaf-types produced in the culture of shoot tips of T. officinale (x 10). A: .. entire»; B: «runcinate>J.; C: ..toothed».
In vitro culture 0/ shoot apices Shoot apices from young and adult plants, including the meristematic dome with 1 or 2 leaf primordia, not longer than 0.3 mm without any differentiated lobes, were individually cultured in 25 x 150 mm test tubes containing 10 ml medium each. The medium for culture was the MS (Murashige-Skoog, 1962) formula, supplemented with 3 % sucrose and 0.8 % agar and adjusted to pH 5.5. The hormones employed were 4-{3-indolyl)butyric acid (lBA) and 6-benzylaminopurine (BAP) both at a concentration of 0.5 mg ·1- I determined in previous work as the best ratio for the culture of shoot apices. GA3 was sterilized by filtration and added to the warm agar media. Culture conditions were a temperature of 20±2 °C and a photoperiod of 10 h light from four daylight fluorescent tubes (40W) and four HPL lamps (400W), with an irradiance of 20,000 erg' cm - 2sec - 1).
Irradiation with FR·iight Shoot apices of adult plants were irradiated daily for 20 min during the dark period. The light source was a 150W incandescent lamp with internal reflector, filtered through Schott R6-N° 9 glass filter (3 mm thickness) and a cuvette containing water to lOcm depth. The irradiance was 3,OOOerg'cm - 2sec 1.
J. P/"nt PhysiQl. Vol.
122. pp. 413-421 (/986)
MARiA C. POMAll, EsrANISLAVA SLABNIK, OSVALDO H. CAW and HIWA DiAz
416
TIME (days)
30
o
j
Adult Plants
Young Plants
Fig.2: Responses by cultured shoot meristems from young and adult plants in the differentiation of leaf-types (%): entire leaves (closed bars); toothed leaves (crossed bars) and runcinate leaves (open bars) (15 replications).
Recording of experimental results and leaf shapes The leaves formed during the experimental period were recorded every seven days, and classified as follows (Fig. 1): A) 4\entires_, with entire margins Guveniles), B) .runcinates .. with profound lobes (adults) and C) .toothed.. , intermediate between A and B. Each treatment involved 10 to 15 replications. During the experimental period all the explants showing contamination, adventitious buds and/or callus proliferation were discarded. Experiments were repeated at least twice and explants were cultured for 20 - 30 days, since longer periods always resulted in the induction of adventitious buds in some of the explants.
Results
Culture shoot apices from young and adult plants Fig. 2 compared the growth within four weeks of shoot apices excised from young
and adult plants. Apices from young plants produced in culture only entire leaves Guvenile). In contrast, when apices of adult plants are cultured, the meristems differentiated varying proportions of runcinate, entire an thoothed leaves.
Culture ofshoot apices from aduit GA,-treated and untreated piants Fig. 3 A shows formation of leaves by shoot apices from adult GAl-treated and untreated plants, cultured for a period of 4 weeks. By the end of the experiment, fewer
J.
P["nt P/rysiol. VoL 122. pp. 413-421 (1986)
Leaf-shapes in meristem culture
417
A
• ,i• ••
--
,
.--.. $As
TIM£
'"
(days)
B
27
••
~
Plants
Plants
(-GA,)
1+ GA,J
Fig. 3: Responses by cultured shoot meristems from adult control and GAl-treated plants, in the production and differentiation of leaf-types (15 replications). A: Leaves produced per apex (Mean number ± SE). B: Types of leaves differentiated (%): entire leaves (closed bars); toothed leaves (crossed bars) and runcinate leaves (open bars).
leaves were formed in the apices from GA,-treated plants, (3.53±0,47Ieaves/explant) when compared to those from controls (5.17±O.66 leaves/explant). Fig. 3 B also shows that while in control apices a higher number of the toothed and runcinate types of leaves was formed, in those apices from GA 3-treated plants about 80 % of the leaves belong to the juvenile type.
Effect ofFR -light and of GA, on the growth ofapices from adult plants Apices excised from adult plants were cultured in the same conditions as in the previous experiments and divided into two groups, one of which was daily submitted to
an FR-irradiation. Fig. 4 B shows the effect of such irradiation on the shape of leaves formed at different times during the experimental period. FR induced the reversion of the adult type of leaf to a more juvenile one, as it is shown by the very low percentage of runcinate leaves (8 %) present after 17 days in culture, when compared with more than 90 % in [he control apices. Fig. 5 B shows how [he addition of GAl to
J. Plant Physiol. Vol.
122. pp. 413-421 (1986)
418
MAlliA. C.
POMAJ., EsrANISLAVA SLABNII., OSVALDO H.
CAS<> and
HUDA
DiAz
7
• ,• l
•
•
• •,
I ,
..J
" TIME
20
30
40
( day.)
B
..•
~
Apices
Apicn
I-FR)
{+FR}
Fig. 4: Effect of FR~irradiations «in culture» of the shoot meristems from adult plants, on the production and differentiation of leaf~types (10 replications). A: Leaves produced per apex (Mean number ± SE). B: Types of leaves differentiated (%): entire leaves (closed bars); toothed leaves (crossed bars) and runcinate leaves (open bars).
the culture medium, (1 and 10 mg ·1 - '), induces the apices of adult plants to revert leaf production from the runcinated to less complex leaf shapes, as was the case with FR irradiation. When applied to isolated apices, both treatments seem not to affect
the apparent plastochron (Figs. 4 A and 5 A).
Discussion
.In vitro. grown apices of T. officinale excised from plants in different ontogenic stages, or plants treated to alter their foliar morphogenesis, differentiated leaves with a shape similar to that of leaves originating in the whole plant (Fig. 2 and Fig. 3 B). Apices from adult plants, of the different experiments (Figs. 2, 3 B, 4 B and 5 B) produced between 45 % and 90 % of runcinate leaves. These different responses could be attributed to the variations in the environmental conditions in the greenhouse during
growth of the «mother» plants. However, this variability does not invalidate the different responses with respect to the apices of the young plants with 100 % of entire leaves (Fig. 2), or the apices of adult GA,..treated plants with only 3-5% runcinate
J. Plan, l'hysiol. Vol.
122. pp. 413-421 (1986)
Leaf-shapes in meristem culture
..• . 1
:
,:
419
A
! • • ~
--lA,
...-..... u.( .... . r" o-- ·-o· ... ,to ....r
ID
20
30
40
TtM£ (da,.1
B
••
~
o
10
....rIIlA,
Fig. 5: Effect of GAl addition (0, 1 and 10 mg '1 - ') to the culture medium on the production and differentiation of leaves, by the shoot meristems from adult plants (10 replications). A: Leaves produced per apex (Mean number ± SE). B: Types of leaves differentiated (%); entire leaves (dosed bars); toothed leaves (crossed bars) and runcinate leaves (open bars).
ones (Fig. 3 B). However, apices from GA,·treated plants showed a decrease in the number of leaves produced during culture if compared with those from control plants (Fig. 3 A). A longer apparent plastochron in adult T. officinale plants, has al· ready been related with conservation of the juvenile leaf shape (Cogliatti and Sanchez, 1982). Therefore, it seems that the inception of leaf primordia and the differentiation of leaf shape are associated with metabolic and, possibly, morphological changes in the apical wne. These changes would be determined by modifications during the devel· opment of the plants, allowing self-expression by the apex. The production of more complex foliar structures in Darlingtonia cali/arnica has been related to an increase in size of the apical zone. Changes in structure and cytohistological zonation have also been described (Franck, 1976). Moreover, stable modifications of the phyllotaxis af· fected by exogenously applied growth regulators have been related to changes in the shape and size of the apex (Varnell and Vasil, 1978; Halperin, 1978).
J. PL.", P/rysioL Vol. 122. pp. 413-421 (1986)
420
MAID C. Po....., ESTANlSI.AVA SLABNI1, OsvAIDO H.
c.so and tiu.DA DiAz
Organ correlations are considered important in morphogenesis. When plants of Silene armeria growing in non-inductive conditions for flowering were treated with only one GA, application to the apical meristem, Besnard-Wibaut and col. (1983), found an increased mitotic activity. Repeated applications provoked a decrease in the cellular activity at the apex and an increase in the stem growth. This suggested competition for nutrition between both organs. This might well be the case in T. officinale, where either FR or GA, treatments induced also a greater growth of both the petiole and the main vein of the leaves, when compared with untreated plants (Cogliatti and Sanchez, 1982). However, there was a reversion (0 the juvenile leaf type (Figs. 4 B and 5 B) when apices excised from adult plants were grown in vitro, in presence of GA, or irradiated with FR light. This might suggest that the differentiation of leaf-type in the apex is independent of the correlative influences of the changes olr served in the more mature regions of the plant.
Nevertheless, when excised apices were irradiated with FR light or treated with GA" only the leaf shape was affected, but not the average number of leaves formed per explant (Figs. 4 A and 5 A). This result counteracts the feature observed in treated plants, where both processes were affected (Cogliatti and Sanchez, 1982). This behaviour corroborated that the sites of leaf-inception could be determined by the characteristics acquired by the apex. Leaf initiation has been related to the presence of provascular traces and their further differentiation. In Papulus deltoides. a procarnbial strand is initiated 13 plastochrons before the leaf emerges (Larson, 1975). Even though Lyndon (1970) observed in Pisum sativum that there was little or no increase in the rate of cell divisions associated with leaf initiation, and that its formation was a consequence of changes in the direction of apical growth, Hussey (1972) found in the same species that the initiation of a new primordium can
be traced to a local increase
in the rate of cell divisions some plastochrons before it is well formed. In T. officinal..
it is possible that at the cellular level, more sites for the inception of future new primordia were already determined, even if only one or two primordia were visible. GAl or FR light should provoke some modifications on the apical region, or more
directly, on the growth of primordia and their early differentiation (Feldman and Cutter, 1970). The effect of the FR light could be mediated by changes in the endogenous levels of gibbereUins. Different authors have established biosynthetic relationships between GA, and phytochrome, (Hilton and Smith, 1980). However, nutritional factors other than hormones may be involved in this process. For a normal in vitro growth, shoot meristem of T. officinale showed an absolute requirement for the presence of sucrose, an auxin (IBA) and a cytokinin (BAP) in the culture medium. The requirement for these substances for organogenesis in tissue cuJtUJ'e and for plant micro-
propagation through meristem culture are vety well documented. In the whole plant, these substances should be translocated to the apex from the leaves and!or from the root. At present, experiments are underway to examine the interaction of different
sugar and hormone levels in the inception of leaf primordia and their final shapes.
J. Plant P/rysiol. VoL
122. pp. 413-421 {1986}
Leaf-shapes in meristem culture
421
Acknowledgements The authors are grateful to Ing. Agr. Sergio J. Ochatt for the help and critical review of this manuscript.
References Au.sopp, A.: Shoot morphogenesis. Ann. Rev. Plant Physiol. 15. 225-254 (1964), ARNOZIS, P. A" O. H. CASO, and D. H. COGUA1n: Efeeto del GAJ Y 1a intensidad de luz sobre la morfogenesis foliar de Taraxacum offlCinale. Phyton 35 (1), 1-9 (V-1977). Bf.SNARD-WIBAUT. C, M. NOIN, and J. A. D. ZEEVAAIlT: Mitotic activities and levels of nuclear DNA in the apical meristem of Silene anneria (strain S 1.2) following application of gibberellin A J . Plant Cell PhysioL 24 (7), 1269-1279 (1983). COGLlATII, D. H. and R- SANCHEZ: lnflucncia del fitocromo sobre eI crecimiento foliar en Taraxacum offlCinale. Phyton 42 (2), 191-199 (1982). COGUATTI, D. H. and M. R. GUITMAN: Some observations on the effect of gibberellic acid on the shape of Tar=um officinale leaf primordia. j. Plant PhysioL 115, 97 -103 (1984). ENGELKE, A. L.. G. H. HAMzi, and F. SKOOG: Cytokinin-gibberellin regulation of shoot development and leaf form in tobacco plantlets. Amer. j. Bot. 60 (6),491-495 (1973). FELDMAN, L. J. and E. H. CUTIER: Regulation of leaf form in Centaurea solstitialis L. II. The developmental potentialities of excised leaf primordia in sterile culture. Bot. Gaz. 131, 39-49 (1970). FRANCK, D. H.: Comparative morphology and early leaf histogenesis of adult and juvenile leaves of Darlingtonia californica and their bearing on the concept of heterophylly. Bot. Gaz. 1.17 (1),20-34 (1976). FUt.U1, H., S.lMAKAW.... and T. TAMURA: Effects of growth regulators and sugar on the growth of apple ap;cal ,hoot. J. Japan_ Soc. Hart. Sci. 49 (4),549-556 (1981). HALPERIN, W.: Organogenesis at the shoot apex. Ann. Rev. Plant Physiol. 29, 239-272 (1978), HILTON, J. R. and H, SMITH: The presence of phytochrome in purified barley etioplast and its in vitro regulation of biologically active gibberellin levels in etioplast. Planta 148, 312-318 (1980). HUSSEY, G.: The mode of origin of a leaf primordium in the shoot apex of the pea (Pisum sativum).J. Exp. Bot. 23, 675-682 (1972). L ... RSON, P. R.: Development and organization of the primary vascular system in Populus deltoides acco,ding to phyllotaxy_ Am. J. Bot. 62, 1084-1099 (1975)_ LYNDON, R. F.: Rates of cell division in the shoot apical meristems of Pisum. Ann. Bot. 34, 1-17 (1970). MUII..ASHIGE, T. and F. SKOOG: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473 - 497 (1962). SANCHEZ, R. A. and D. H. COGUATII; The interaction between phytochrome and white light irradiance in the control of leaf shape in Taraxacum officinale. Bot. Gaz. 136 (3), 281-285 (1975). V ARNELL, R. J. and I. K. VASIL: Experimental studies of the shoot apical meristem of seed plants. I. Morphological and cytochemical effects of IAA applied to the exposed meristem of Lu· pinus albus. Amer. J. Bot. 65 (1), 40-46 (1978). W ... REING, P. f. and I. D. 1- PHILLIPS: The control of growth and differentiation in plants. Pergamon Press. 39 (1975).
]. Plant Plrysiol. Vol 122. pp. 413-421 {1986}