Conditions leading to flower formation on excised Begonia fragments cultured in vitro

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1 Plant & Cell Physio!., 9, (1968) Conditions leading to flower formation on excised Begonia fragments cultured in vitro F. RINGE and J. P. NITSCH Botanisches Institut, Justus Liebig-Universitat, Giessen, Germany and Laboratoire de Physiologie Pluricellulaire, C. N. R. S., Gif-sur-Yvette, France 1 (Received May 27, 1968) Fragments of leaves, petioles and floral stalks of several species and varieties of Begonia have been cultured in vitro on denned media. The presence of a cytokinin, adenine and an auxin was found necessary for optimum bud formation, especially on petioles and floral stalks. Flower buds could be produced on leaves and floral stalks, but not on petioles. They were of the male type. As early as 1892 SACHS (1) reported that cuttings taken from flowering begonias regenerated plants which flowered much earlier than plants regenerated from begonias which were still vegetative. This observation led him to postulate the existence of a special flower-forming principle. In recent years, it has been possible to obtain the de novo formation of flower buds on fragments of stems, leaves and roots cultured in vitro (see review articles 2-5). Using such aseptic techniques, we have tried to get some information about the conditions under which flower buds can be induced to form on excised fragments of leaves, stems, petioles and floral stalks of various species and varieties of Begonia. Materials and methods Downloaded from at Penn State University (Paterno Lib) on March 6, 216 Plant material The species and varieties used were Begonia x cheimantha "Gloire de Lorraine" and "Regent", B. teuscheri x coccinea "Corallina de Lucerna" It is a real pleasure to express our gratitude to Professor Dr. von DENFFER who gave the initial impetus for this piece of research and granted a leave of absence to Dr. F. RINGE in order to originate the in vitro culture of Begonia species in the Laboratoire de Physiologie Pluricellulaire at Gif-sur-Yvette. 639

2 64 F. RINGE and J. P. NITSCH Vol. 9 (1968) (abbreviated "Lucerna"), B. socotrana, B. sutherlandii, B. "Comte de Miribel" (abbreviated "Miribel"), B. evansiana and B. multiflora. In all cases, the stock plants were in flower when the explants were taken from them. Preparation of the explants Upon excision, the cut surfaces of the fragments were sealed by dipping in molten paraffin. They were sterilized with a filtered suspension of 5 % calcium hypochlorite in water for 1-15 min and washed twice with sterile, distilled water. Segments approximately 1 mm in length were then prepared from the sterilized stem or petiole material and planted horizontally on agar media under aseptic conditions. In the case of leaves, disks mm in diameter were excised with a sterile cork borer. Media After an initial experiment with B. socotrana, in which the macroelements of the formulae of MURASHIGE-SKOOG and KNOP were tested, the following basal medium was adopted for all subsequent work (in mg/1): Macro-elements (KNOP): Ca(NO 3 ) 2-4H 2 O (5), KNO 3 (5), MgSO,-7H 2 O (5), KH 2 PO 4 (5). Minor elements: MnSCV4H 2 O (25), H 3 BO 3 (1), ZnS(V7H 2 O (1), KI (1), Na 2 MoCV2H 2 O (.25), CuSO<-5H 2 O (.25), CoCl 2-6H 2 (.25) plus 5 ml of a solution containing 7.45 g/1 of the disodium salt of ethylenediaminetetraacetic acid (EDTA) and 5.57 g/1 of FeSO4-7H 2 O. Organic addenda : glycine (2),»zyo-inositol (1), nicotinic acid (5), pyridoxine hydrochloride (.5), thiamine hydrochloride (.5), folic acid (.5) and biotin (.5). To this basal medium were added varying proportions of sucrose, a cytokinin, an auxin, adenine and other organic substances. The ph of the medium was adjusted to 5. with HC1 or NaOH before adding.8 % Difco Bacto-agar and autoclaving for 15 min at 5- C (that is a pressure of 14.2 lbs/sq. in.). Environment of the cultures All the cultures were subjected to a daily illumination of 16 hours, except those of B. socotrana which received only 1 hours of light per day. The combination of fluorescent and incandescent lamps used gave an illumination of about 7 ft. c. outside the culture tubes, which were closed with "Bellco" stainless steel, non-transparent caps. The temperature reached 28 C during the light period and decreased to 2 C during the dark period. Downloaded from at Penn State University (Paterno Lib) on March 6, 216 Scoring of the results Generally, the quantitative results are given in terms of the percentage of cultures producing vegetative or floral buds. In the latter case, a

3 Flower formation on Begonia fragments in vitro 641 culture producing at least one flower bud is recorded as flowering, even though vegetative buds may also be present. Experimental results Conditions leading to the initiation of buds The first problem to be solved was the stimulation of bud production on the explants. While Begonia rex is well-known for its potentialities of neoformation, other species of Begonia, such as some of the ones employed in our experiments, do not produce buds readily. At first, it seemed that both an auxin and a cytokinin had to be added to the medium in order to cause bud formation. This was the case, for example, with petioles of B. evansiana (Fig. 1). No bud whatsoever was produced when benzyladenine alone was added to the basal medium, but profuse bud formation occurred when both benzyladenine and phenylacetic acid were present together. Similar results were obtained with other species and with other organs such as leaf disks, as shown in Table 1. The inactivity of the cytokinin when used alone was rather unexpected since cytokinins are well-known to promote bud formation on isolated leaf disks of B. rex (6, 7). On the other hand, we observed, as a rule, a slight but definite promotive effect of auxins when added to the basal medium without cytokinin. The effect of auxin was studied further, in Table 1 Effect of a cytokinin and an auxin, alone or in combination, on the formation of buds on different types of Begonia explants Leaf disks: Begonia multiflora Begonia evansiana Intensity of bud formation medium +^ + Au ffi"'" ± Downloaded from at Penn State University (Paterno Lib) on March 6, 216 Petioles: Begonia multiflora Begonia evansiana The number of crosses indicates the intensity of the response in each series. ±= very slight bud formation. Benzyladenine (1" 6 M) in all cases. * Phenylacetic acid (1"' M). ' Indolyl-3-acetic acid (5.7X1" 7 M). 6 Phenylacetic acid (1" 6 M).

4 642 F. RINGE and J. P. NITSCH Vol. 9 (1968) combination with a cytokinin (see for instance Fig. 1 and Table 1). Thus, in the case of floral stalks, increasing concentrations of indolyl-3-acetic acid (IAA) resulted in an increasing percentage of cultures producing buds (Table 2). A comparison of three cytokinins (Table 3) showed that benzyladenine was the most effective one in causing bud formation : at 1" 5 M not only did all the cultures produce buds, but these buds formed at both extremities and were larger and better developed than in the other series. Table 2 Effect of IAA upon bud formation in segments of floral stalks cultured in vitro IAA cone Cultures producing buds (96) (/ig/1) B. "Lucerna" B. "Miribel" ' 17 1, The media contained 4 % sucrose, benzyladenine (5X1" 7 M) and adenine (3X1" 4 M). Six replicate cultures in each case. " 5 replicate cultures only. Table 3 Effect of three cytokinins on bud formation in segments of petioles of Begonia sutherlandii grown in vitro in the presence of IAA Cone. Number Cultures (M) Qf cultures with buds ^ None 82 Kinetin Kinetin Benzyladenine Benzyladenine Benzyladenine 1" 6 1" 5 1" 7 1" 6 1" " Downloaded from at Penn State University (Paterno Lib) on March 6, 216 Isopentenyladenine Isopentenyladenine Isopentenyladenine io- 7 1" 6 1" 5 The media contained 2 % sucrose, IAA (lmg/1) and adenine (4mg/l). after weeks of culture. " Largest buds Results

5 Flower formation on Begonia fragments in vitro 643 Fig. 1. Combined effects of an auxin and a cytokinin in promoting bud formation in petiole sections of Begonia evansiana cultured in vitro. 1 = basal medium alone ; 2 + benzyladenine (1~ 6 M) ; 3 = + phenylacetic acid (1~ 4 M) ; 4 = + benzyladenine + phenylacetic acid. Picture taken after 6 weeks of culture. Adenine 1"' 3 x 1-* Table 4 Effect of adenine upon bud formation in the presence of a cytokinin and an auxin Molar concentrations of : BA 5 x 1"' // // IAA 5.7 x 1"' // * Number of cultures Cultures producing buds (%) 83 1 Downloaded from at Penn State University (Paterno Lib) on March 6, 216 1"' 3 x 1-«5 x 1"' n // 5.7 x 1" 6 M ir 91 1 The media contained A% sucrose in all cases. BA = benzyladenine ; IAA = indolyl- 3-acetic acid. Segments of floral stalks of B. "Lucerna". Results after 1 weeks of culture.

6 644 F. RINGE and J. P. NITSCH Vol. 9 (1968) IAA Fig. 2. Necessity of the incorporation of IAA for bud formation in petiole sections of Begonia evansiana. Left column : without IAA ; right column : with IAA (1 ftg/l). The cytokinins used were : K = kinetin (3X1~ 6 M), B = benzyladenine (2.5x1"' M), I = isopentenyladenine (2.5xlO" 7 M). Picture taken after 8 weeks of culture. The necessity of an auxin in the medium along with the cytokinin was clear with all the tested cytokinins, as shown by Fig. 2 for the case of petiole sections of B. evansiana. At the concentrations used, the cytokinins evoked bud formation only when IAA was present also. Nevertheless, the necessity of adding an auxin probably depends upon the level of natural auxins in the explants, for buds were obtained without the incorporation of an auxin into the medium in the case of petioles of other species such as B. sutherlandii and, sometimes, B. "Lucerna". Finally as has been shown in the case of Plumbago indica (8) and other species (9), the addition of adenine to a medium already supplemented with a cytokinin and an auxin further increased bud formation (Table 4). Thus, optimal bud formation seems to occur when the basal medium is supplemented with three growth factors: (1) an auxin, (2) a cytokinin and (3) adenine. Downloaded from at Penn State University (Paterno Lib) on March 6, 216

7 Flower formation on Begonia fragments in vitro 645 Conditions leading to the formation of flower buds Flower buds were obtained much more readily on certain organs than on others. For example, except for one exception with B. socotrana, flower buds did not form on explants excised from petioles, even under conditions which readily lead to flower formation in explants from floral stalks (Table 5). In the case of leaf explants of B. socotrana, the floral buds arose on very much reduced shoots originating near the cut edges of the disks (Fig. 3). Buds forming on the petiolar stump have been vegetative, whether the explant had been planted upside-down, as in Fig. 3, or right side-up. Auxins are necessary for the production of flower buds as shown in Table 5. It can be seen from this table that petiolar explants reach 1 % bud formation with an IAA concentration of 1 /tg/1, whereas Downloaded from at Penn State University (Paterno Lib) on March 6, 216 Fig. 3. Inflorescence of Begonia socotrana produced at the edge of a leaf disk planted upside-down on a medium containing 2 % sucrose, benzyladenine (2/xg/l) and IAA (1/ig/l). Picture taken after 15 weeks of culture under short days of 1 hrs.

8 646 F. RINGE and J. P. NITSCH Vol. 9 (1968) Table 5 Effect of the origin of the explant upon its capacity for producing floral buds IAA cone. (Mg/D Petioles : 1 1 1, Floral stalks : Q 1 1 1, Number of explants % Explants producing : Veg. buds Q Floral buds Media containing 2% sucrose, benzyladenine (5X1"'M) and adenine (3x1 4 M). Sections of equal lengths excised from B. "Lucerna". Results after 3 months of culture. Q: & Downloaded from at Penn State University (Paterno Lib) on March 6, 216 Fig. 4. Effect of auxin upon the formation of flower buds on explants taken from floral stalks of Begonia "Lucerna". Media containing 2 % sucrose, benzyladenine (5X1" 7 M), adenine (3X1" 4 M) and the following IAA concentrations: 1 =, 2 = 1/ig/l, 3 = 1/xg/l, 4 = l,>g/l. Picture taken after 3 months of culture.

9 Flower formation on Begonia fragments in vitro 647 Table 6 Effect of adenine upon the formation of flower buds Adenine cone, (M) H IAA cone. (MX 5.7) 1"? lfl" 6 Number of cultures % Veg. buds Q Cultures with : Floral buds 1-4 // 3 x 1~ 4 // 1"' 1" 6 io-' 1' 6 Explants from floral stalks of Begonia "Lucerna". Media containing 4 ; and benzyladenine (5x1"'M). Results after 9 weeks of culture. Fig. 5. Effect of the concentration of adenine upon the formation of buds on floral stalks of Begonia "Lucerna". The media contained A% sucrose, benzyladenine (5 x 1"' M) and two levels of IAA : 5.7 x 1"' M (A) and 5.7 x 1" 6 M(B). Horizontally: = no adenine, 1 = 1" 4 M, 2=3 x 1" 4 M adenine. Picture taken after 9 weeks of culture. If 87 73; 8 17 If 1 92 Downloaded from at Penn State University (Paterno Lib) on March 6, 216 it takes 1 times more auxin for explants taken from floral stalks to reach a percentage of This aspect of the cultures is given in Fig. 4. One can see that, under these experimental conditions, flower buds arise directly on the explants.

10 648 F. RINGE and J. P. NITSCH Vol. 9 (1968) In addition to auxin and a cytokinin, adenine is very important for the formation of flower buds on floral stalk explants, as can be seen in Table 6. Thus, when the IAA concentration is 5.7 x 1" 7 M, most of the cultures in this experiment produce only vegetative buds when the adenine concentration is 1' 4 M. When this concentration is raised to 3x1"'M, however, all the cultures produce flower buds. This aspect of the cultures is given in Fig. 5. Discussion A new case of the in vitro expression of thefloweringstate Among the reported cases of flower formation in vitro, NITSCH (3, 4) has clearly distinguished two fundamentally different situations. In the first one, explants are taken from plants which are completely vegetative, such as photoperiodic species maintained under non-inductive conditions. The induction of flowering, then, occurs in the test tube. Such is the case, for example of the long-day species Cichorium intybus (1) and the short-day Plumbago indica () or Streptocarpus nobilis (). The second physiological situation comprises instances in which the explants are taken from plants which are flowering already: in this case best results are usually obtained with fragments from the inflorescence, as in Nicotiana tabacum (13), Cichorium intybus (3,14,15), Lunaria annua (3,16) etc. Here one does not deal, any more with the induction of flowering in vitro, but rather with the expression of a floral state which is already inscribed in the tissues. The case of the various species of Begonia, which is presented here, falls into the second category, since the explants have been taken from plants which were already in flower. Begonias, however, never develop a terminal flower at the summit of the primary axis (17); the shoot apex remains rather indefinite as well as the apices of the leafy side-branches. Flowering occurs in a sympodial manner, the inflorescences arising in most Begonia species in the axils of the normal foliage leaves which are produced by the terminal meristems of the main axis and the leafy sidebranches. These apices continue to produce new foliage leaves and hence remain strictly vegetative during the whole life history of the plant. The inflorescences, which are dichasial flower clusters, are spatially separated from the vegetative regions. They arise in the axils of foliage leaves and develop a system of branches, bracts and flowers independent from the vegetative system (18). When planted in vitro on the same nutrient medium, fragments of petioles and fragments of the basal internode of the dichasial floral system (partial florescence with reference to TROLL (19)) behave differently in the case of B. "Lucerna", even when both are taken out of the same region of the plant: the petioles produce only vegetative buds, while floral stalks (that is to say, the internode Downloaded from at Penn State University (Paterno Lib) on March 6, 216

11 Flower formation on Begonia fragments in vitro 649 below the first prophyll of the primary flower of the dichasial partial florescence) produce flower buds (Table 5). In other words, the capacity for the production of either vegetative or flower buds must somehow be inscribed in cells which are capable of neoformation. Possibly, the activation of the corresponding genes might be involved in these complex processes. Results pertaining to bud formation As far as bud formation is concerned, the present investigation has provided results which generalize certain points which had been demonstrated with other plant material. Thus, the general necessity of supplying both an auxin and a cytokinin for bud formation, already shown by SKOOG and MILLER (2) in tobacco cultures, has been established for explants of several Begonia species (see Table 1 and Figs. 1 and 4). HEIDE (21) found similar results with leaf cuttings of B. semperflorens and B. socotrana x tuberhybrida "President", but his experiments, as well as those of BIGOT (7), stress mostly the importance of cytokinins. In the present investigation it was found that no bud formation occurred on leaf disks of B. multiflora or B. evansiana, on petiole segments of the same species (Table 1), on floral stalks of B. "Lucerna" or B. "Miribel" (Table 2) unless auxin was incorporated also into the medium. In these species, therefore, auxin probably has a role to play in the budding process. Another component of the substance complex which favours bud formation is adenine. Although this substance was early found to promote bud formation (see, for example, 22, 23), its synergistic effect with cytokinins has been recognized only recently (9,2,24). In the case of floral stalks of B. "Lucerna", no buds at all were obtained, unless adenine was present in the medium, in addition to an auxin and a cytokinin (Table 4). Results pertaining to flower formation Previous results obtained with Cichorium intybus (1), Nicotiana tabacum (13) and Plumbago indica (8,) had shown that auxins added to the medium were inhibitory to flowering. In the present case, two auxins, IAA and phenylacetic acid (a weak auxin, but used at a high concentration) not only did not inhibit flowering but actually promoted it (Fig. 4). In fact, as can be seen in Table 5, it takes more IAA to obtain a high percentage of cultures forming floral buds than cultures forming vegetative buds. This unexpected result may be due to an extremely low auxin content in the floral stalks. Since, in the species just cited, no bud formation whatsoever occurred without auxin, it may be that the auxin effect is actually indirect; i. e., that auxin does not stimulate flowering as such in Begonia explants, but that it only helps in keeping them alive until they organize meristems capable of further development. Such an indirect effect might also account for another instance in which auxin has been found to promote flowering, namely that of excised leaf disks of Stfeptocarpus nobilis (). Downloaded from at Penn State University (Paterno Lib) on March 6, 216

12 65 F. RINGE and J. P. NITSCH Vol. 9 (1968) Another factor which turned out to stimulate flowering in vitro has been adenine. Thus, with 1~ 4 M of this purine, most of the buds formed on floral stalk explants of B. "Lucerna" were vegetative, whereas they were all flowering when a concentration three times higher was used (Table 6). The effect of adenine in promoting flowering has already been shown in another instance, that of Plumbago indica. The exact role of adenine is not yet clear, but there is a high specificity of this substance among other purines and pyrimidines (25). Fig. 6. Aspect of a flower produced on a segment of floral stalk of B. "Lucerna" showing the stamens. A : lateral view. B: front view. The medium contained 2 % sucrose, benzyladenine (33 pg/\), IAA (1 fig/l) and adenine (4 rag/1). Finally, it should be mentioned that, although explants have been taken from various regions of the Begonia partial florescence (the internode below the first prophyll of a male or female flower, the internode above the second prophyll of male flowers, or even parts of the ovary of female flowers), only male flowers have been observed in vitro (Fig. 6). In some cases, the flowers were reduced to stamens with only rudimentary petals. The study of factors responsible for the development of male or female flowers will be the subject of another investigation. B Downloaded from at Penn State University (Paterno Lib) on March 6, 216 We wish to thank the "Studienstiftung des deutschen Volkes" and the "Stiftung Volkswagenwerk" for the grant given to one of us (F. R.). We are indebted to Miss B. NORREEL for the photographs of Fig. 6.

13 Flower formation on Begonia fragments in vitro 651 References ( 1) SACHS, J Physiologische Notizen. Flora, 75, 1-3. ( 2) NlTSCH, J. P Phytohormones et genese des bourgeons vegetatifs et floraux. In Les Phytohormones et I'Organogenese. Congres et Coll. Univ. de Liege, p ( 3) NITSCH, J. P L'induction de la floraison in vitro: resultats et perspectives. Mim. Soc. Bot. France, in press. (4) NlTSCH, J. P Towards a biochemistry of flowering and fruiting: the contribution of the "in vitro" technique. Proc. XHth Int. Hort. Congress, 3, (5) NITSCH, J. P., C. NITSCH, L. M. E. ROSSINI, F. RINGE and H. HARADA L'induction florale in vitro. In Cellular and Molecular Aspects of Floral Induction. Liege, in press. (6) SCHRAUDOLF, H. and J. REINERT Interaction of plant growth regulators in regeneration processes. Nature, 184, ( 7) BIGOT, C Action de trois adenines substitutes sur l'apparition de neoformations sur des explantats foliaires de Begonia. Bull. Soc. Bot. France, 3, (8) NlTSCH, C. and J. P. NlTSCH, The induction of flowering in vitro in stem segments of Plumbago indica L. II. The production of reproductive buds. Planta (Berl.), 72, (9) NITSCH, J. P., C. NITSCH, L. M. E. ROSSINI and D. Bui DANG HA The role of adenine in bud differentiation. Phytomorphology, in press. (1) PAULET, P. and J. P. NlTSCH, La nfeoformation de fleurs sur cultures in vitro de racines de Cichorium intybus L.: 6tude physiologique. Ann. Physiol. V6g., 6, () NlTSCH, J. P. and C. NITSCH Neoformation de fleurs in vitro chez une espfece de jours courts: Plumbago indica L. ibid., 7, () ROSSINI, L. M. E. and J. P. NITSCH Induction de la floraison in vitro chez une plante de jours courts, Streptocarpus nobilis. C. R. Acad. Sci., Paris, Ser. D 263, (13) AGHION-PRAT, D Neoformation de fleurs in vitro chez Nicotiana tabacum L. Physiol. Vig., 3, (14) MARGARA, J Comparaison in vitro du developpement de bourgeons de la tige florig^ne de Cichorium intybus L. et de revolution de bourgeons neoformfes. C. R. Acad. Sci., Paris, 26, (75) HARADA, H Effects of photoperiod on the formation of flower buds by flower stalk sections of Cichorium intybus in tissue culture. Bot. Mag. Tokyo, 79, 9-3. (16) PlERIK, R. L. M Regulation of morphogenesis by growth regulators and temperature treatment in isolated tissues of Lunaria annua L. Proc. Kon. Ned. Akad. van Wetenschappen, C, 68, (17) IRMSCHER, E Begoniaceenstudien. Bot. Jb., 78, (18) RlNGE, F Beitrage zur Kenntnis der Morphologie von Begonia semperflorens gracilis. Diss. Naturwiss. Fak. Univ. Giessen. (19) TROLL, W Die Infloreszenzen. Typologie und Stellung im Aufbau des Vegetationskorpers. Teil I. VEB Gustav Fischer, Jena. (2) SKOOG, F. and C. O. MILLER Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp. Soc. Exp. Biol.,, Downloaded from at Penn State University (Paterno Lib) on March 6, 216

14 652 F. RINGE and J. P. NITSCH Vol. 9 (1968) Reprinted in Molecular and Cellular Aspects of Development Edited by E. BELL. p Harper and Row, New York. (22) HEIDE, O. M Interaction of temperature, auxins, and kinins in the regeneration ability of Begonia leaf cuttings. Physiol. Plant., 18, (22) SKOOG, F. and C. TSUI Chemical control of growth and bud formation in tobacco stem segments and callus cultured in vitro. Amer. J. Bot., 35, (23) JACQUIOT, C Action du meso-inositol et de l'adenine sur la formation de bourgeons par le tissu cambial d' Ulmus campestris cultive in vitro. C. R. Acad. Set., Paris, 233, (24) NITSCH, C. and J. P. NITSCH The induction of flowering in vitro in stem segments of Plumbago indica L. I. The production of vegetative buds. Planta (Berl.), 72, (25) NITSCH, C Induction in vitro de la floraison chez une plante de jours courts: Plumbago indica L. Ann. Sci. Nat., erne Sir., Bot., 19, Downloaded from at Penn State University (Paterno Lib) on March 6, 216