Tissue Culture of Some Arboreal Leguminous Plants

LKrome Jnemorial Umtltuie

Proc. Fla. State Hort. Soc. 105:271-273. 1992.

TISSUE CULTURE OF SOME ARBOREAL LEGUMINOUS PLANTS

Mohamed-Yasseen, Y. and T. L. Davenport (Morton, 1990). Its bright orange flowers make it one of University of Florida, TREC the most striking and gorgeous of the tropical trees (Bailey, 18905 SW 280 St., Homestead, FL 33031 1916). This report describes in vitro shoot regeneration from different explants derived from seedlings of W. E. Splittstoesser tamarind, carob and royal poinciana. University of Illinois, Dept. of Horticulture 1103 W. DornerDr., Urbana, IL 61801 Materials and Methods

Additional index words, tamarind, carob, royal poinciana, Seed germination. Seeds of tamarind (Tamarindus indica micropropagation. L.), carob (Ceratonia siliqua L.) and royal poinciana (Delonix regia) were collected from trees grown in Miami. Carob Abstract. The Leguminosae is the third largest family of flow seeds were obtained from a tree which has been noted for ering plants. Many subtropical legume trees are still largely its flavorful pathogen-free fruits, an uncommon charac unimproved. Tissue culture offers a good alternative for plant teristic for carob under the humid climate of South improvement over classical breeding approaches. Tamarind Florida. Seeds of tamarind and carob were extracted from (Tamarindus indica L), carob (Ceratonia siliqua L), and mature pods, cleaned with water and detergent, surface royal poinciana (Delonix regia) are three subtropical legumes sterilized in 70% ethanol for 3 min, and soaked in 2 g/liter cultivated for their edible fruits or for ornamental purpose. A benomyl for 30 min. At this stage, seeds were screened, protocol for in vitro shoot regeneration was developed from and those which developed wrinkled seed coats were elimi aseptically germinated seedlings. Shoot tips, stem nodes, nated. Seeds with smooth seed coats were incubated in cotyledonary nodes, and cotyledons were cultured in 0.78% NaCIO (v/v) for 15 min and rinsed threetimes in Murashige and Skoog media supplemented with combina sterile distilled water. Seeds of royal poinciana were sur tions of thidiazuron and indolebutyric acid or benzyladenine face sterilized in 1.5% NaCIO for 15 min. The seed coat and naphthaleneacetic acid. Multiple shoots were obtained was carefully trimmed at the proximal or distal part of the after four weeks culture under long day photoperiod. This seed or left intact for comparison, then surface-sterilized system may have application for improvement of these sub in 0.1% NaCIO for 10 min and rinsed three times in sterile tropical legume trees. distilled water. All seeds were germinated in Murashige and Skoog (1962) medium (MS) supplemented with 30 g/ Tissue culture offers a good alternative for crop im liter sucrose and 8 g/liter agar (Bacto-agar, Difco). MS provement over conventional breeding methods (Pierik, medium was adjusted to pH 5.7 with In KOH before au- 1987). Although many herbaceous species have been toclaving at 1.6 kg cm-2 for 20 min at 121C. All cultures studied extensively through tissue culture, arboreal species were incubated at 28C in a growth chamber providing an have received little attention. Tamarind (Tamarindus indica 18 h photoperiod with cool white fluorescent lights, 40 L.), carob (Ceratonia siliqua L.), and royal poinciana (Delonix l^1 regia) are trees belonging to the family Leguminosae. All Explant preparation. Explants were taken from tamarind three species are subtropical, grow well in dry soils, and do seedlings whichwere germinated in MS medium and had not require special care. They are cultivated for their edi developed the first epicotyledonary node. Seedlings of ble fruits, or for ornamental or forage purposes. The carob and royal poinciana were used at an earlier stage tamarind fruit is the source of a well-known refreshing before developing epicotyledonary nodes. Explants were drink. It also has medicinal proprieties as an antibacterial derived from the following tissue from all three type of and is a good source ofthiamine and niacin (Alian et al., seedlings: cotyledons, cotyledonary and epicotyledonary 1983). Fruits and twigs of tamarind are used in many ways, nodes from tamarind seedlings, shoot tips and cotyledons principally as food flavoring, and the seeds are a source of from carob seedlings; shoot tips, cotyledons and cotyledo starch (Anonymous, 1971; Bueso, 1980). Carob is another nary nodes from royal poinciana seedlings. Cotyledons evergreen, drought-tolerant tree which is grown mostly for were cut at the connection with the hypocotyl as described its pods. Free from caffeine and theobromine, they are for soybean (Mohamed-Yasseen and Splittstoesser, 1990). used as a substitute for chocolate (Winer, 1980). Carob In some instances cotyledons were either left intact or di requires a dry climate during fruit maturation, however, vided into three longitudinal or transverse sections. which makes its economic value doubtful in South Florida Cotyledonary nodes of royal poinciana were prepared by (Grainger and Winer, 1980). Royal poinciana, a rapidly- shortening the epicotyle and hypocotyl near the cotyle growing, deciduous tree, is a popular ornamental in all dons, slicing the remainder ofboth of the epicotyle and tropical regions as well as in South Florida and the Keys hypocotyle longitudinally, and discarding about half of the cotyledon. Regeneration protocol. Explants of all three species were Florida Agricultural Experiment Stations Journal Series No. N-00693. placed in culture media abaxial side down. Tamarind and

Proc. Fla. State Hort. Soc. 105: 1992. 271 Table 1. Shoot regeneration from shoot tips and cotyledonary nodes of royal poinciana cultured in MS supplemented with growth regulators.

Number of shoots per explant Supplements Shoot tip Cotyledonary node

8.8 ftM BA + 0.01 |xm NAA 2.2 a 4.8 a 13.3 m-m BA + 0.01 fjiM NAA 0.9 b 2.3 b zMean separation within column by Duncan's multiple range test at P = 0.05. carob explants were cultured in MS medium supple mented with 30 g/liter sucrose, 8 g/liter agar (Bacto-agar, Difco), and either 0.15 |xM thidiazuron (TDZ), with 1.2 fJiM indolebutyric acid (IBA); or 6.6 |xm benzyladenine (BA), with 0.2 |xm napthaleneacetic acid (NAA). Explants of royal poinciana were cultured in MS medium supple mented with 30 g/liter sucrose, 8 g/liter agar, and either 8.8 jjlm BA with 0.01 |xm NAA or 13.3 |xm BA with 0.01 |iM NAA. Regenerated shoots were transferred to MS medium supplemented with 0.1 |am indolebutyric acid with or without 1 g/liter activated charcoal for further growth. All media were adjusted to pH 5.7 with In KOH after adding growth regulators and before autoclaving at 1.6 kg cm-2 for 20 min at 121C. All cultures were incubated at 28C in a growth chamber providing an 18 h photoperiod with cool white fluorescent lights (40 jimole-nr^s-1). Data were recorded after 4-5 weeks in culture.

Fig. 2. Typical shoots regenerated from carob shoot tip explant.

Results and Discussion

Seeds of tamarind and carob have relatively hard seed coats. Virtually all seeds which developed wrinkled seed coats after surface sterilization and incubation in 2 g/liter benomyl for 30 min showed signs of bacterial infection after culture in MS medium. Wrinkled seed coats may re sult from damage which facilitates infection by microor ganisms and reduces seed vigor (Mohamed-Yasseen and Splittstoesser, 1990; 1991). Thus, only seeds with smooth seed coats were further considered for germination. Seeds of royal poinciana take from 2-3 years to germinate (Mor ton, 1990). Seeds trimmed at the proximal or distal side germinated within one week in culture, but those left intact did not germinate during the 12 weeks of the experiment. Callus formation and shoot regeneration were differ ent depending on genotype and type of explant. Cotyle dons of tamarind and royal poinciana formed callus and did not regenerate shoots. Cotyledons of carob which were left intact regenerated multiple shoots at the proximal side. More than ten shoots were arranged along the proximal side of each cotyledon suggesting that they originated from the cotyledon tissues and not from the cotyledonary node. Carob cotyledons which were cut in three longitudinal or transverse sections formed callus and did not regenerate any shoots. Cotyledonary nodes and epicotyledonary nodes of tamarind produced multiple shoots. More shoots Fig. 1. Typical shoot regenerated from tamarind shoot tip explant. were regenerated from cotyledonary nodes (about 5 shoots

272 Proc. Fla. State Hort. Soc. 105: 1992. Shoot cultures were established and increased from seedling explants of three legume trees. Shoots regener ated from tamarind (Fig. 1), carob (Fig. 2), and royal poin ciana explants (Fig. 3) were vigorous in most cases and thus amenable to rooting which is still in progress.

Literature Cited

Alian A., E. El Ashweh, and N. Eid. 1983. Antibacterial properties of some Egyptian non-alcoholic beverage with special reference to tamarind. Egypt. J. Food. Sci. 11:109-114. Anonymous. 1971. Manufacture of tamarind seed starch. Siet Institute. Hyderabad. India. Bailey, L. H. 1916. The standard cyclopedia of horticulture, Vol. V. Mao Millan, New York. Bueso, C. E. 1980. Soursop, tamarind, and chironja. pp. 375-406. In: S. Nagy and P. E. Shaw (eds.). Tropical and subtropical fruits. Composi tion, properties and uses. AVI, Westport, Conn. Grainger, A. and N. Winer. 1980. A bibliography oiCeratonia siliqua, the R Q carob tree. Intl. Tree Crops J. 1:37-47. Kopp, M. S. and K. Nataraja. 1990. In vitro plantlet regeneration from shoot tip cultures of Tamarindus indica L. Indian J. For. 13:30-33. Fig. 3. Typical shoots regenerated from cotyledonary node explant of Mohamed-Yasseen, Y. and W. Splittstoesser. 1990. Regeneration of soy royal poinciana. bean [Glycine max (L.) Merr.] from the seedling apex; stem node, cotyledonary node and cotyledons. Plant Growth Regulat. Soc. Amer. per explant) than from epicotyledonary (about 3 shoots 18:203-210. per explant). Shoot tips of tamarind often produced one, Mohamed-Yasseen, Y. and W. Splittstoesser. 1990. The role of the onion or at best, 2 shoots. Shoot tips of royal poinciana produced (Allium cepa L.) seed coat in aging and ultrastructral changes in root tip of low vigor seedling. Proc. Plant Growth Regulat. Soc. Amer. about 2 shoots per explant (Table 1), and shoot tips of 17:10-15. carob produced about 5 shoots per explant (Fig. 2). Kopp Mohamed-Yasseen, Y. and W. Splittstoesser. 1991. Scanning electron and Nataraja (1990) obtained only one shoot from each microscopic studies on onion {Allium cepa L.) seeds and their relation shoot tip explant of tamarind; Sebastian and McComb to viability. Interamerican Soc. Trop. Hort. 37:(In press). (1986), using zeatin, found that carob shoot tips produced Morton, J. 1990. Trees, shrubs, and flowers for Florida landscaping na tive and exotic. Fla. Dept. of Agr. and Consumer Services. multiple shoots. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth There were no significant differences in shoot number and bioassay with tobacco tissue cultures. Physiol. Plant. 15:473-497. when using media containing TDZ with IBA or BA with Pierik, R. L. M. 1987. In vitro culture of higher plants. Martinus Nijhoff, NAA. Increasing BA concentrations reduced the number Dordrecht, The Netherlands. Sebastian, K. T. and J. A. McComb. 1986. A micropropagation system (Table 1) and length (data not shown) of shoots regener for carob. Scientia Hort. 28:127-131. ated from shoot tips and cotyledonary nodes of royal poin Winer, N. 1980. The potential of the carob {Ceratonia siliqua). Intl. Tree ciana. Crops J. 1:15-26.

Proc. Fla. State Hort. Soc. 105:273-275. 1992.

PRODUCTION OF FRESH MARKET HERBS IN WEST-CENTRAL FLORIDA

A. A. Csizinszky 1988-89) and spring (Mar.-Jun. 1989). Plants were grown Gulf Coast Research and Education Center without pesticides and received 87N, 38P and 72K Ib/acre. IFAS, University of Florida Anise and dill had a higher plant survival rate and yield with Bradenton, FL 34203 a single harvest in winter 1988-89 than with multiple har vests in spring 1989. Infestation of chervil by aphids Additional index words. Full-bed mulch, anise, Pimpinella (Aphididae) and borage by lepidopterous larvae in spring anisum, borage, Borago officinalis, chervil, Anthriscum cere- 1989 rendered these herbs non-marketable. folium, dill, Anethum graveolens, fennel, Foenicum vulgare, elemental concentration. In the United States, herbs for culinary use are the fastest-growing segment of specialty vegetables (Agricul Abstract. Production of anise, Pimpinella anisum L, borage, tural Outlook, 1989; Miller and Harper, 1990). For exam Borago officinalis L, chervil, Anthriscum cerefolium (L.) ple, the domestic and imported shipments of herbs in Hoffm., dill, Anethum graveolens L, and fennel, Foenicum creased from 11 thousand cwt in 1985 to 312 thousand cwt vulgare Mill. var. azoricum (Mill.) Thell. was investigated on in 1991 (Annual Shipment Information, 1991; Vegetables fine sandy soil with a full-bed mulch system and furrow irri and Specialties, 1991). The greatest demand for herbs is gation. Studies were conducted during winter (Dec-Jan. during the November to May market season (Calderin, 1989). Climatic conditions in west central Florida, mild Florida Agricultural Experiment Station Journal Series No. N-00680. temperatures and adequate rainfall, should be favorable

Proc. Fla. State Hort. Soc. 105: 1992. 273