Abstract Caryopsis culture of a minor millet (Paspalumscrobiculatum L. cv. PSC 1) on N6 medium supplementedwith high concentrations of thidiazuron (TDZ, 11.25 µMand 22.5 µM), a phenylurea derivative known to simulatecytokinin action, resulted in the formation of multipleshoots from the base of the seedling. This is the first timethat multiple-shoot formation by a seedling cultured onTDZ without a callus interphase has been reported in gra-minaceous crop plants. The presence of a cytokinin, 6-benzylaminopurine (BAP), at low or high concentrationsfailed to evoke any morphogenic response. The presenceof the auxin 2,4-dichlorophenoxyacetic acid (2,4-D,4.5 µM) either alone or with BAP (4.5 µM) resulted in theformation of embryogenic callus from the base of theseedlings, which subsequently differentiated into somaticembryos. The combination of TDZ and the auxin (4.5 µM,2,4-D) in the medium stimulated the differentiation ofshoot buds in embryogenic callus cultures. This effect ofTDZ, noted for the first time in a monocotyledonousplant, was evident in terms of a significant increase in thefrequency of shoot-bud formation in embryogenic calluscultures and occurred only at a high concentration of TDZ(11.25 µM). This requirement for a high concentration ofTDZ for the induction of multiple shoots from culturedseedlings or shoot buds in an embryogenic callus cultureof a monocot is contrary to its effect at low concentrationsin dicotyledonous plants. Complete plantlets, derived ei-ther from somatic embryos or shoot buds, could be regen-erated on hormone-free basal medium or on basal mediumfortified with activated charcoal (0.5%). Following a grad-ual acclimatization in a culture room, these regenerantssurvived on transfer to soil and ultimately set seed.

Keywords Caryopsis · Minor millet (Paspalum scrobiculatum L.) · Shoot regeneration · Somatic embryo

Abbreviations BAP: 6-Benzylaminopurine 2,4-D: 2,4-Dichlorophenoxyacetic acid IAA: Indole-3-acetic acid P-CIB: P-Chloroisobutyric acid TDZ: Thidiazuron

Introduction

For the in vitro regeneration of graminaceous crops, em-bryonic tissues such as unemerged inflorescences andimmature embryos are commonly used as totipotent ex-plants that are capable of somatic embryogenesis(Bhaskaran and Smith 1990). As an alternative, the cul-ture of mature caryopses (Krans et al. 1982; Marouskyand West 1990; Torello et al. 1984) also results in theproduction of somatic embryos.

An emerging trend in the tissue culture of dicotyledo-nous plants is the utilization of TDZ, a non-purine phenyl-urea derivative, for the induction of multiple shoots (Beattie and Garrett 1995; Malik and Saxena 1992) andthe direct differentiation of somatic embryos (Gill andSaxena 1992; Iantcheva et al. 1999). In monocots, save fora solitary report on Alstromeria (Lin et al. 1997), themorpho-regulatory effect of this compound remains to beevaluated. In the investigation reported here, we employedcaryopsis culture of Paspalum scrobiculatum L. and ob-tained TDZ-induced multiple-shoot formation on a seed-ling, an occurrence hitherto unreported in a monocot cropplant. We also observed the promotion of shoot-bud dif-ferentiation by TDZ on embryogenic callus cultures forthe first time. Our results are compared with the shoot-in-ducing capacity of a highly potent cytokinin, BAP.

Materials and methods

The genus Paspalum Linn. comprises 355 species that are widelydistributed throughout the tropics and sub-tropics of both hemi-spheres. P. scrobiculatum is currently cultivated for grazing andhay production and is often an introduced species for pasture andrange management. In Asia, P. scrobiculatum has been domesti-cated as a cereal grain (Jarret et al. 1998).

Communicated by H. Lörz

Vikrant · A. Rashid (✉ )Department of Botany, University of Delhi, Delhi-110007, Indiae-mail: arashid_du@hotmail.com

Present address:Vikrant, Department of Plant Molecular Biology, University of Delhi South Campus, Delhi-110021, India

Plant Cell Rep (2002) 21:9–13DOI 10.1007/s00299-002-0466-8

CELL BIOLOGY AND MORPHOGENESIS

Vikrant · A. Rashid

Induction of multiple shoots by thidiazuron from caryopsis culturesof minor millet (Paspalum scrobiculatum L.) and its effect on theregeneration of embryogenic callus cultures

Received: 10 September 2001 / Revised: 12 March 2002 / Accepted: 26 March 2002 / Published online: 31 May 2002© Springer-Verlag 2002

Seeds of P. scrobiculatum L. cv. PSC 1 were procured from theUniversity of Agricultural Sciences, Bangalore, India and washedthoroughly in a 2% (v/v) detergent solution of Teepol in distilledwater for 15 min. The sterilization procedure consisted of an etha-nol (70%, w/v) treatment for 1 min, followed by a treatment withHgCl2 (0.1%, w/v) for 5 min. The seeds were then rinsed repeat-edly with sterile distilled water and inoculated aseptically on N6mineral medium (Chu et al. 1975) or on medium supplementedwith various concentrations of 2,4-D (4.5, 9.0, 18.0 and 22.5 µM),TDZ (0.01, 0.1, 1.0, 4.5, 11.25 and 22.5 µM) and BAP (4.5, 11.25and 22.5 µM) either alone or in combination.

The nutrient medium contained (2%, w/v) sucrose that was so-lidified with 0.8% (w/v) agar. The pH of the medium was adjustedto 5.8 prior to autoclaving at 120°C for 15 min. Cultures wereraised in 15-ml screw cap vials and maintained at 25±2°C undercontinuous illumination (fluorescent lighting; 15 µmol m–2 s–1).For every treatment at least 20 replicate cultures were raised, andthe values given (Figs. 2, 3) are the means of four independent ex-periments.

Results and discussion

Mature caryopses cultured on hormone-free medium ger-minated readily to form seedlings (Fig. 1a) that eventual-ly developed into normal plants.

Effect of TDZ on seedlings

When the nutrient medium was fortified with a low con-centration of TDZ (0.01, 0.1, 1.0 µM), seed germinationwas quite normal. However, following the culture ofcaryopses on media containing higher concentrations ofTDZ (11.25 and 22.5 µM) seed germination was relative-ly slow, with a poor seedling development. These seed-lings, 3–4 weeks after culture initiation, exhibited agradual degeneration of the main shoot, followed by adifferentiation of multiple shoots (Fig. 1b) from the baseof the seedling. A maximal frequency of 40% of the cul-tures (Fig. 2) exhibited multiple-shoot formation at ahigh concentration of TDZ (22.5 µM). This morphogenicresponse, direct multiple-shoot formation, was also ob-served, but at a reduced level, on medium supplementedwith a low concentration of TDZ (4.5 µM). Multiple-shoot formation on TDZ-treated caryopses has not previ-ously been reported for a graminaceous crop plant.

TDZ, originally designed as a defoliant, is a phenyl-urea derivative that simulates cytokinin activity in vitroby promoting the growth of cytokinin-dependent calluscultures of Phaseolus lunatus (Mok et al. 1982) and Gly-cine max (Thomas and Katterman 1986). TDZ was alsoobserved to stimulate the expansion of radish cotyledonsand induce adventitious shoots on tobacco leaf discs(Thomas and Katterman 1986). The association betweenTDZ-induced responses and endogenous plant growthregulators of dicotyledonous plants has been documentedby means of the quantification of their profiles. TDZ promotes the synthesis and accumulation of purines (Capelle et al. 1983) and also alters cytokinin metabolism(Mok et al. 1982). Phenylurea also inhibits cytokinin oxi-dase (Laloue and Fox 1989). TDZ was able to stimulatesprouting in potato only following a reduction in the level

of abscisic acid (Li and Yang 1988). Since TDZ is in-volved in cytokinin metabolism, we decided to explorethe effect of cytokinin on multiple-shoot formation.

Effect of BAP on seedlings

Caryopses were cultured on media containing differentconcentrations of BAP (4.5, 11.25, 22.5 µM). Followinga normal process of germination, the seedlings thatformed failed to show any change in morphogenesis. Wetherefore concluded that TDZ was effective on its own ininducing multiple shoots on caryopses during culture.However, the concentration of TDZ that was effectivewas higher than that generally employed for the regener-ation of dicotyledonous plants. With respect to its effica-cy in the propagation of dicotyledonous plants, herba-ceous as well as woody, TDZ is highly effective at a level very much lower than that required by BAP (Huetteman and Preece 1993). This effectiveness of TDZin promoting regeneration in dicots at levels lower thanthose required by a cytokinin needs to be explained. Inthe present investigation on a monocotyledonous plant,we found BAP to be ineffective, and multiple-shoot for-mation by TDZ was possible only at higher concentra-tions. This implies that the two plant groups, monocoty-ledons and dicotyledons, differ in their requirement forTDZ and in their response to cytokinin.

Effect of TDZ on embryogenic cultures

Since TDZ was effective in inducing multiple shoots oncaryopsis cultures, we investigated its effect on calluscultures capable of somatic embryogenesis. The inclu-sion of 2,4-D to the nutrient medium at various levels(4.5, 9.0, 18.0, 22.5 µM) resulted in a remarkable inhibi-tion of seed germination, particularly at higher concen-trations of 2,4-D. Two-week-old seedlings cultured onmedium supplemented with 2,4-D exhibited an initialslight proliferation at the base, which was followed bythe formation of compact and friable types of calli within4 weeks of culture initiation. The compact calli turnedembryogenic within 6 weeks and differentiated into so-matic embryos (Fig. 1c) in about 30% of the culturesmaintained at a low concentration of 2,4-D (4.5 µM). Athigher concentrations of 2,4-D (9.0 µM and 18.0 µM),both friable and compact calli formed somatic embryosin over 50% of the cultures. These somatic embryoswere typical in being cup-shaped with a prominent scute-llum-like structure. In 6-week-old cultures, the somaticembryos in rare instances germinated in situ.

When the caryopses were cultured on a medium sup-plemented with various concentrations of TDZ (4.5,11.25, 22.5 µM) and 2,4-D (4.5 µM), a reduction in thefrequency of seed germination in comparison to the con-trol was followed by profuse callus formation at the baseof the seedling as well as from developing roots in 4- to 5-week-old cultures. Following 5–6 weeks of culture, many

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Fig. 1a–e Paspalum scrobi-culatum, caryopsis culture. a Two-week-old seedling onhormone-free, basal medium.Bar 1.0 cm. b Six-week-oldseedling on medium supple-mented with 11.25 µM TDZ,with multiple-shoots from itsbase. Bar 1.0 mm. c Five-week-old seedling on mediumsupplemented with 4.5 µM2,4-D, with callus, and its dif-ferentiation into numerous so-matic embryos (arrow). Bar1.0 mm. d Seven-week-oldcaryopsis culture on mediumsupplemented with 4.5 µMTDZ and 4.5 µM 2,4-D, show-ing profuse callusing and a fewshoot buds (arrow) at the basalpart of seedling. Somatic em-bryos at other parts of the cal-lus are also evident. SE Somat-ic embryos, SH shoot-buds (arrow). Bar 1.0 mm. e Forma-tion of plantlets from somaticembryos after transfer of a 6-week-old embryogenic cul-ture to hormone-free medium.Bar 1.0 mm

shoot buds had differentiated on the callus formed at thebasal portion of the seedling, whereas on other parts of thecompact callus, somatic embryos also appeared along withshoot buds (Fig. 1d). The frequency of the cultures withan embryogenic callus and differentiating shoot buds onthe callus depended on the concentration of TDZ in themedium. At an equimolar concentration of TDZ and 2,4-D (4.5 µM), the frequency of embryogenic callus andshoot-bud formation was minimal (Fig. 3). However, fol-lowing an increase in the concentration of TDZ(11.25 µM), a maximum of 37% of the cultures had shoot-bud-bearing calli. At higher concentrations of TDZ(11.25 µM and 22.5 µM) and the same concentration of2,4-D (4.5 µM), the compact embryogenic calli failed todifferentiate into somatic embryos and instead differentiat-ed into shoot buds. This is the first report of TDZ havingthis effect in a monocot. There has been only one previousinvestigation on a monocot, Alstroemeria, in which TDZ(10 µM) together with indole-3-butyric acid (0.5 µM) in-duced shoot regeneration from leaf explants (Lin et al.

1997). Also of significance is the induction of shoot budsby TDZ on recalcitrant bean (Malik and Saxena 1992) andclover (Beattie and Garrett 1995) plants and severalwoody plants (Huetteman and Preece 1993). In addition todisplaying a cytokinin-like activity, it has been suggestedthat TDZ modulates endogenous levels of auxin. Howev-er, it remains to be resolved whether it has auxin activityor is involved in auxin metabolism (Lu 1993).

TDZ at very low levels induces direct somatic embry-ogenesis in geranium (Qureshi and Saxena 1992) and al-falfa (Iantcheva et al. 1999). Somatic embryogenesisalso occurs in watermelon (Compton and Gray1992) andmuskmelon (Gray et al. 1992) on medium containingTDZ and 2,4-D. TDZ replaces the auxin-cytokinin re-quirement for high-frequency somatic embryogenesis ofgeranium (Visser et al. 1992) and peanut (Murthy et al.1995). In somatic embryogenesis of Geranium (Visser etal. 1992) and Arachis (Murthy et al. 1995) TDZ stimu-lated de novo synthesis of auxin by augmenting the levelof IAA and its precursor, tryptamine. In Geranium, the

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Fig. 2 P. scrobiculatum cary-opsis culture: frequency of cultures with multiple shootson medium with different concentrations of TDZ or BAP

Fig. 3 P. scrobiculatum, cary-opsis culture: frequency of cultures with somatic embryosand shoot buds on medium supplemented with 2,4-D anddifferent concentrations ofTDZ or BAP

auxin inhibitor P-CIB reduced the level of IAA and try-ptamine, but the level of auxin in tissue treated withTDZ and P-CIB remained elevated.

In this study, shoot regeneration via shoot-bud forma-tion from embryogenic callus cultures was possible at high-er concentrations of TDZ. In an earlier investigation on amonocot TDZ was also effective in inducing shoots at ahigh concentration (Lin et al. 1997). As for the promotionof regeneration by TDZ, an increase in the level of ethyl-ene (Yip and Yang 1986) occurs in response to TDZ treat-ment of seedlings. In wilted wheat leaf, 10 µM TDZ pro-duced more ethylene than 1 mM BAP, indicating that TDZis more effective in “stress-ethylene” production than a cy-tokinin. This indicates that the possibility that TDZ- stimu-lated shoot regeneration is an ethylene-mediated response.

Effect of BAP on embryogenic cultures

Although BAP was ineffective in inducing multipleshoots on the caryopses, we decided to study its effect onthe differentiation of embryogenic callus during culture.Poor callusing at the base of the seedlings was followedby the differentiation of somatic embryos within 5 weeksof culture initiation in about 20% of the cultures (Fig. 3)when caryopses were cultured at an equimolar (4.5 µM)level of 2,4-D and BAP. These embryogenic calli, how-ever, failed to differentiate into shoot buds. Calli that de-veloped when exposed to high concentrations of BAP(11.25 µM and 22.5 µM) together with a low concentra-tion of 2,4-D (4.5 µM) remained undifferentiated; thesecultures gradually turned brown and necrosed.

Regeneration of plants

When 6- to 7-week-old embryogenic callus cultures weretransferred to hormone-free basal medium, the differenti-ated somatic embryos 'germinated' to form plantlets within1 week of transfer (Fig. 1e). Multiple-shoot formation atthe base of the seedling or shoot buds that differentiatedfrom embryogenic callus after transfer to hormone-freebasal medium or medium fortified with activated char-coal (0.5%) formed completely rooted plantlets within8–9 weeks of culture. These plantlets were gradually ac-climatized in a culture room and transferred to soil, wheremore than 70% of the regenerants flowered and set seed.

Acknowledgements We are grateful to Dr. A. Seetharam, Univer-sity of Agricultural Sciences, Bangalore, India for the generoussupply of seeds. Vikrant is also grateful to CSIR, New Delhi forthe award of a Junior and Senior Research Fellowship.

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