Plant Cell, Tissue and Organ Culture33: 39-44, 1993. 1993 KluwerAcademic Publishers. Printedin the Netherlands.

Direct root tip conversion of Catasetum into protocorm-like bodies. Effects of auxin and cytokinin
Sandra Colli ~ & Gilberto B. Kerbauy* ~Department of Animal and Plant Biology, Londrina State University, 86.100, Londrina, PR, Brazil; Department of Botany, University of S~o Paulo, P.O. Box 11461 05422-970, S~o Paulo, SP, Brazil (* requests for offprints) Received 8 October 1991; accepted in revised form 13 October 1992

Key words: Catasetum fimbriatum, orchid, regeneration, root bud

Abstract

Root apex conversion of Catasetum fimbriatum into protocorm-like bodies (PLBs) can occur in the absence of any added plant growth regulator. The presence of exogenous auxins in media drastically reduced the number of PLBs formed; on the other hand the concentrations of these auxins used greatly increased the process of callus formation. No effect on the mean number of root tip conversions into PLBs was observed with chlorogenic acid. However, this process was significantly increased in one of the concentrations used of p-coumaric acid. BA did not have any effect on callus formation, but caused marked acceleration in the process of root tip conversion and on the mean number of PLBs formed. PLB formation observed in the absence of any exogenous growth substance seemed to reflect a disruption in the interactions between the excised roots and the rest of the plants. The presence of light decreased the process of conversion.

Abbreviations: BA-6-benzylaminopurine, IAA-indole-3-acetic acid, IBA-indolebutyric acid, 2,4-0-2,4dichlorophenoxyacetic acid, PLB-protocorm-like body
Introduction

Root bud formation takes place predominantly in mature parts of the root system. According to Peterson (1975), bud regeneration in the region of root apical meristem could be considered a relatively rare process in higher plants. In the orchid family the direct conversion of root tip cells into protocorm-like bodies (PLBs) under natural conditions was described in Neottia nidus-avis by Champagnat (1971). However, this process has been also observed in vitro with root-tip segments of Catasetum (Kerbauy 1984a) and other allied epiphytic genera studied in this laboratory, and in Cyrtopodium (S~inchez 1988). Orchid PLBs were considered to be true somatic embryos by Morel (1974).

Despite the major changes which must occur during the conversion of meristematic root cells into PLB cells, the process as a whole in Catasetum can be considered relatively fast and easily achieved (Kerbauy 1984a) when compared to other patterns of orchid root-tip regeneration in vitro (Kerbauy 1984b, 1988, 1991). Basically, PLB formation in root tips of Catasetum involves a direct conversion of the apices (Kerbauy 1984a), while in most other orchid species studied a callus stage has proved to be a 'sine qua non' condition for in vitro regeneration (Stewart & Button 1978; Kerbauy 1984b, 1988, 1991). These noteworthy features of Catasetum roots seem to represent an interesting and useful system for certain approaches on plant cell differentiation studies. The present paper investi-

40 gate the influence of auxin, cytokinin and other substances which interfere with endogenous IAA oxidase activity, on direct conversion of isolated root tip segments of Catasetum into PLBs. at 25-+ IC. The experimental design was randomly with three replicates for each treatment. The data were submitted to analysis of variance and the mean of each treatment was compared with the mean of the control group, according to bilateral Dunnett's test, significant at 5% and 1% levels. A transformation of data by V ~ + 0.5 was necessary before statistical analysis to render treatment variances homogeneous.

Material and methods

Root-tip segments about 0.5 cm long, obtained from seedlings of Catasetum fimbriatum (Morren) Lindl. (Orchidaceae) at four months of age (6-8 cm in height) were used as explants. Seedlings were obtained from mature seeds germinated asymbiotically in vitro on Knudson C medium (1946). Prior to inoculation seeds were disinfected for 20min in 20% v/v commercial bleach (1% available chlorine), then rinsed twice with sterile distilled water. Knudson C medium was modified by the addition of 60 g 1- t ripe banana pulp, 1 g1-1 activated charcoal, 0.8% w/v agar, and by substituting ferric sulfate with 27.8 mg 1-I Fe-EDTA. Cultures were maintained at 26--2C with 16-h fluorescent light at 40 Ix mol m -2 s -I. The basal medium (BM) used for root tip culture was Linsmaier & Skoog (1965). The pH was adjusted to 5.8 before the addition 0.8% agar. As auxin sources IAA, IBA and 2,4-D were used at concentrations of 0.5, 1.0, 2.0, 4.0 and 8.0 mg 1-1. The cytokinin used was BA at 0.5, 2.0 and 8.0 mg 1-~. Phenolic substances (0.015, 0.15, 1.5 and 15 mgl-L), such as chlorogenic acid and paracoumaric acid, both with modulatory effects on IAA-oxidase activity (Schaeffer et al. 1967; Lee et al. 1982), were used in order to verify effects of endogenous auxin on root-tip conversion. They were membrane filter-sterilized and added to the autoclaved medium after cooling to approximately 40C. After addition of the growth regulators, the media were dispensed into 125 ml Erlenmeyer flasks and autoclaved for 15 min at 120. The flasks were sealed with perforated rubber stoppers. Glass tubes were used to facilitate the obtainment of photos of cultures grown in the former flasks. Some of the cultures were maintained at 26---2C with 16-h fluorescent light at 40 ~ mol m -2 s -~ and some incubated in the dark

Results

Auxins
Variance analysis of results regarding to PLB formation showed a significant interaction (p < 0.01) to each of the three auxins used and the presence and absence of light. Every IAA concentration promoted a significant interaction ( p < 0 . 0 5 ) in both environmental conditions, whereas with IBA and 2,4-9 significant differences (p <0.01) were found only in the auxinfree media. By comparison with control, the three auxins displayed, as a whole, a significant inhibitory effect on root tip conversion. A strong inhibition of root tip conversion independent of light conditions was indued by 2,4-D, while in the presence of IAA and IBA this inhibitory effect took place predominantly in darkness (Table 1). All concentrations of I A A / I B A and light/ dark conditions showed significant mutual interaction in the process of caius formation (p < 0.05). However, no significant interaction with either environmental condition was found when 2,4-D was used; in this case, callus formation depended basically on 2,4-0 concentrations. 2,4-9 was the auxin with the most prominent effect on callus formation, whereas, IAA was the least effective (Table 2).

Phenolic compounds
No significant interaction ( p < 0 . 0 5 ) was observed between light/dark conditions and the presence of chlorogenic acid or p-coumaric acid (Table 3). However, if only the concentrations of the two substances are taken into account, a significant stimulation of PLB formation was ob-

41 Table I. Influence of different concentrations of IAA, IBA and 2,4-D on the mean number of PLB formed in root rip segments of C. fimbriatum cultured in vitro during 30 days. Auxin concentration (mgl -l) Mean number of PLBs formed in root-tip segments subjected to light or dark and the influence of auxins IAA Light None 0.5 1.0 2.0 4.0 8,0 2.04 0.88** 1.46 "s 1.39 Ns 2.1i r~s 2.59 r4s Dark 3,18 1.56"* 0,88** 0,71"* 1.00"* 0,71"* Light 2.04 1.17 Ns 0.88 ~s 0.7I Ns 0.71 r~s 0,88 Ns IBA Dark 3,18 1,00' 0.71" 0.71"* 0,88** 0.88** Light 2.04 0.88** 0.71"* 0.71"* 0.71"* 0.71"* 2,4-D Dark 3.18 0.71"* 0.71"* 0.71"* 0.71"* 0.71"*

~s,

Not significant according to bilateral Dunnett's test. *'** Significantly different from control at 5% and i % levels respectively, according to bilateral Dunnett's test. Table 2. Effect of different concentrations of IAA, IBA and 2,4-0 on the mean number of callus formed in root tip segments of C. fimbriatum cultured in vitro during 30 days. Auxin concentration (mg1-1 ) Mean number of calli formed in root-tip segments subjected to light or dark and the influence of auxins IAA Light None 0.5 1.0 2.0 4.0 8.0 0.71 0.71Ns 1.17 "s 1.64" 0.88 Ns 0.88 Ns Dark 0,71 1,86"* 2.59** 3.43** 3,88** 4.02** Light 0.71 2.90** 2.80** 3.81"* 3.18"* 1.72" IBA Dark 0.71 3.46** 3.51"* 3.84** 4.06** 3.12"* 2,4-D ~ 0.71 4.11"* 3.91"* 3.91"* 2.53** 0.99 Ns

Ns, Not significant according to bilateral Dunnett's test. *'** Significantly different from control at 5% and 1% levels respectively, according to bilateral Dunnett's test. Without interaction among light, dark and 2,4-D concentrations; the values represent the means including light and dark results. Table 3. Effect of chlorogenic acid and p-coumaric acid at different concentrations on the rate on the mean number of PLB formed in root tip segments of C. fimbriatum cultured in vitro during 30 days. Substances added to media chlorogenic acid p-coumaric acid mg 1-j None 2.87 2.87 mg 1-1 0.015 3.02 Ns 2.98 ns mg 1-1 0.15 2.90 Ns 2.76 ns mg 1-1 1.5 2.91 "s 2.82 r~s mg 1-1 15 2.48 Ns 3.62*

Ns, Not significant according to bilateral Dunnett's test. * Significantly different from control at 5% level according to bilateral Dunnett's test. served in the presence of the greatest concent r a t i o n o f p - c o u m a r i c a c i d ( 1 5 m g 1-1, s e e T a b l e 3). Cytokinin The presence of BA induced a strong inhibitory e f f e c t o n r o o t e l o n g a t i o n . A f t e r 10 d a y s o f i n c u bation significant interaction correlation with root-tip conversion was observed among treatments with BA concentrations and the two light conditions. If light/dark conditions are not taken into account, then the root-tip conversion process proves to be stimulated by two concert-

42
Table 4, Effect of different concentrations of BA on the mean number of PLB formed in the root tip segments of C. fimbriatum cultured in vitro after 10, 20 and 30 days.
BA (mgl -~ ) Incubation duration 10 days ~ Light None 0.5 2.0 8,0 1.02 1.75" 1.70 Ns 1.89"* 1.29 3.75** 3.67** 2.70** 20 days Dark 1.93 2.11 ~s 1.97 Ns 3~76"* Light 1.29 3.87** 4.18"* 3.I7"* 30 days Dark 2.67 2.34 ~s 2.41 ~s 3.76**

Ns, Not significant according to bilateral Dunnett's test. * * * Significantly different from control at 5% and 1% levels respectively, according to bilateral Dunnett's test. Without interaction among light, dark and BA concentrations: the values represent the means including light and dark results.

trations of BA (0.5 and 8.0 mg 1-~). Significant interactions ( p < 0.01) were observed only after twenty and thirty days of culture (Table 4), when PLB formation was more favorably stimulated by the presence of light. In this respect, it is important to highlight that this stimulatory effect of BA/light conditions on root conversion is somewhat opposite to the inhibitory light effect found with hormone-free media (Tables 1 and 4). Regardless of the BA concentrations used,

and following root-tip conversion, PLBs presented two basic patterns of development, one of them represented by direct plant formation (Fig. 1) and the other by a secondary PLB regeneration, giving rise, as a consequence, to clusters of new PLBs (Fig. 2). In spite of the stimulatory effects of BA on root tip conversion, it is interesting to note that this process can also occur in media devoid of any of the substances used in this study. Moreover, it may also be emphasized that no PLB was formed at the tips of attached roots, either in the presence or absence of growth regulators. As a rule, the process of conversion only occurs when root apices are isolated from donor plants. Such behavior in vitro has been routinely observed in

Fig. I. Plant regenerated directly from a root tip meristem ol C. fimbriaturn. Arrow shows mature part of original explant
after 90 days of culture.

Fig. 2. Mass of PLBs formed from a root tip meristem of C. firnbriatum cultured in BA added medium after 90 days of
culture.

43 this laboratory in other Catasetum species, as well as in allied genera. medium, irrespective of the presence or absence of light. Cytokinins have also shown stimulatory effects on lateral root bud formation in several non-orchidaceous plant species (Lazzeri & Dunwell 1984; Kunieda & Kerbauy 1986; Eapen & Gill 1986). Of particular interest, is the relatively high frequency of root conversion obtained with BA in the presence of light. In general, according to what we have frequently observed in this laboratory, root apices of Catasetum and allied genera, when cultured in conditions of darkness, display a more pronounced effect in the conversion process than under light. However, this pattern of behavior in virto seems to be considerably modified by the presence of BA in the medium. The effectiveness of exogenous cytokinins in root bud formation of non-orchidaceous plants was also observed in the presence of light (Kefford & Caso 1972; Montaldi 1972). The high inhibitory effect of BA on the longitudinal growth of the explants has also been detected in root apices of other orchidaceous plants cultured in vitro (Kerbauy 1984b, 1988, 1991). The high frequency of root-tip conversion observed in the absence of exogenously added growth regulators appears to be a consequence of a disruption in the interactions between the explant and the rest of the donor plant. In this sense it was already observed that full strength Linsmaier & Skoog medium (1965), devoid of any growth substance, stimulated more intensive root-tip conversion in Cataseturn pileatum, than other less concentrated media (Kraus 1986). Thus, in conclusion, these results suggest that the direct conversion of roots into PLBs following root isolation, would reflect profound biochemical/physiological alterations in the root apices of C. fimbriatum.

Discussion
In a broad context, the effects of exogenous auxins on the process of root-tip conversion in C. fimbriatum were represented basically by a significant suppression of the number of PLBs formed, together with a conspicuous increase in callus formation. These marked effects of auxins on the callus inducing process have also been widely observed in roots of non-orchidaceous plants (Torrey 1958; Eliasson 1961; Kefford & Caso 1972; Lazzeri & Dunwell 1984). In some cases 2,4-D has proved to be the best auxin for callus formation in orchid roots (Kerbauy 1991). Root-tips of some orchid species have been considered generally recalcitrant to form callus or PLB in vitro; such is the case, for example, with Epidendrurn, Oncidium and Cattleya plants (Stewart & Button 1978; Kerbauy 1984b; 1991). To investigate the role of endogenous auxins in the process of root-tip conversion in C. timbriatum, chlorogenic acid and p-coumaric acid were used, two substances involved in modulatory effects on IAA oxidase activity (Schaeffer et al. 1967; Lee et al. 1982). Chlorogenic acid, an inhibitor of IA oxidase (Schaefer et al. 1967), did not have any significant effect on the frequency of root tip conversion. On the other hand, p-coumaric acid, a substance with a stimulatory effect on IAA-oxidase activity (Lee et al. 1982), increased PLB formation significantly in one of the concentrations used. This may suggest that the eventual decrease in endogenous auxin induced by p-coumaric acid might be involved in the process of PLB formation. If it is taken into account that root apices are considered to be important sites of cytokinin synthesis (Torrey 1976), it is plausible to suppose the occurrence of a change in the endogenous auxin-cytokinin synergism provoked by pcoumaric acid. A stimulatory effect of endogenous zeatin riboside on the process of root bud formation in Cichorium intybus was suggested by Bui-Dang-Ha & Nitsch (1970). In this sense it is important to highlight the favorable effects on PLB formation, when BA was added to the

Acknowledgements
This work was supported by FAPESP (Fundaq~o de Amparo ~ Pesquisa do Estado de S~o Paulo) and CNPq (Conselho Nacionat de Desenvolvimento Cientifico e Tecnol6gico). The authors wish to thank Dr. Alasdair G. Burman and Dr. Tiemi Matsuo for critically reading the manuscript and statistical analysis assistance respectively.

44 References
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