Plant Cell, Tissue and Organ Culture 43: 223-228, 1995. 223 @ 1995 Kluwer Academic Publishers, Printed in the Netherlands.

Callus induction and culture from explants of Erysimum scoparium in a growth regulator-free medium

Juan Felipe Ptrez-Francts, Francisco Valdts & Raquel Martin Departamento de Biologla Vegetal, ( Fisiolog[a Vegetal), Universidad de La Laguna, 38206 La Laguna, Tenerife, Spain

Reveived 28 April 1993; accepted in revised form 31 July 1995

Key words: Brassicaceae, growth regulator-autonomous callus, habituated callus, hormone autotrophy

Abstract

Calli from cotyledon, hypocotyl and radicle explants of Er).simum scoparium (Brassicaceae) were induced and cultured using a MS medium without growth regulators. Calli obtained from cotyledon or hypocotyl explants showed a higher growth rate while radicle-derived calli exhibited poor growth. Cotyledon-derived calli were compact and green-colored while hypocotyl- and radicle-derived calli were friable and with creamy-green pigmentation. Histological analysis was carried out during culture and the development of abundant vascular elements and meristematic nodules were found. Subculture on a growth regulator-free medium during 10 months did not ecrease callus growth.

Introduction

For the induction of callus, an explant is normally cul- tured on a medium containing growth-regulating sub- stances. However, some excised tissues produce callus in the absence of exogenous growth regulators (Yeo- man & Macleod, 1977; Masuda et al., 1977; Bayer, t982). Typically, this is a wound-reaction where the cells at the cut surface are induced to undergo mitoses leading to callus formation, i.e. a process of wound- healing (Kahl, 1983). On the other hand, some cul- tures previously induced in the presence of exogenous growth regulators can habituate to grow in the absence of such substances. Habituation has been frequently reported in various plant tissues cultured in vitro (Gau- theret, 1959; Meins, 1989). As well tumor tissues and differentiated explants of tumorous plants can grow well on growth regulator-free media (Bayer, 1982).

Erysimum scoparium (Brouss. ex Willd.) Wettst., Brassicaceae (Hansen & Sunding, 1993) is an endem- ic species from Tenerife (Canary Islands) that pro- duces several cardiac glycosides (Gonz~ilez & Luque Escalona, 1975). The main aim of this work is to study metabolite yield of in vitro cultures of this plant. Research to study the induction, growth and differen-

tiation of calli from E. scoparium explants cultured on a growth regulator-free medium is summarized in this report.

Materials and methods

Seeds from a wild plant of E. scoparium (El Por- tillo, Tenerife) were surface-sterilized in 3% calci- um hypochlorite for 15 rain, washed three times in autoclaved distilled water and germinated aseptical- ly in Petri dishes containing wet filter paper, at 25 °C in darkness. Hypocotyl, cotyledon and radicle explants were excised from 10-day-old aseptically- grown seedlings and cultured under light for callus initiation. Woundless and wounded whole seedlings with their roots were also used in an experiment using growth regulator-free medium and medium with 2,4-dichloropenoxyacetic acid 2,4-D. Seedlings were wounded by small incisions with a scalpel on the cotyledon or hypocotyl surface before culturing. Explants were placed on 20 ml medium in culture tubes (24 x 160 mm). Each tube contained one explant and a minimum of 40 cultures were used for each treatment. Experiments were repeated at least twice.

224

The following culture media were examined: A medium containing Murashige & Skoog (1962) inor- ganic salts and vitamins supplemented with 30 g 1-1 sucrose and 0.8% agar (No. A-7002, Sigma Chemical Co.) without growth regulators (MSO) and a Gamborg et al., (1968) medium without growth regulators (B50). To compare callus growth on medium with and without growth regulators a MSO medium supplemented with 4.52 I.tM 2,4-D and 0.046 gM kinetin was used (MSH in Fig. 1). This medium produced optimal callus for- mation.

Calli were removed from primary explants after 35 days and subcultured every 4 weeks on fresh medi- um. For each subculture, an inoculum of unorganized pieces of 173 4- 37 mg fresh weight and 17.9 4- 3.7 mg dry weight was used.

Cultures were incubated at 25 4- 2 °C under contin- uous light of 60 gmol m -2 s- l using fluorescent tubes (Ozon Daylight LF 36w/62).

Callus tissues for light microscopy were fixed in formalin-acetic acid-ethyl alcohol (5:5:90) (Johansen, 1940). Fixed tissues were dehydrated in an ethanol series, embedded in paraffin (Paraplast Plus, Sher- wood Medical, Athy, Ireland), and sectioned with a microtome at 10 I.tm. These sections were stained in safranin, and counter stained with fast green. Other samples were first fixed in 3% glutaraldehyde in 0.0125 M cacodylate buffer pH 7.4 and then post-fixed for 2h with 1% osmium tetroxide in 0.1 M cacodylate buffer pH 7.4. The specimen was then dehyrated in an ethanol series and embedded in Spurr resin. Sections 1 gm in thickness were cut on an ultramicrotome (C. Reichert OmV3) with glass knives and secondarily stained with toluidine blue/borax (I: 1).

Results and discussion

Callus induction on a growth regulator-free medium

Callus cultures were successfully induced on a growth regulator-free medium using all explant types of E. scoparium (hypocotyl, cotyledon and radicle).These calli were predominantly initiated at the cut edges of the explants. Callus cultures derived from cotyledon explants were compact and green-colored while calli from hypocotyl and radicle were friable and exhibit- ed a creamy-green pigmentation. On the other hand, calli derived from radicles presented a great hetero- geneity.

Table 1 shows that MSO medium was better than B50 for callus induction: cotyledon explants cultured on MSO medium responded significantly better to form callus tissue than those cultured on B50 medium. The percentage of callus induction also varied depending on the type of explant. Hypocotyl explants exhibited a higher percentage of callus induction (Table 1 ).

Whole seedlings of E. scoparium cultured on a growth regulator-free medium did not produce callus. In this case, it was necessary to add auxin (0.9 gM 2,4-D), as a regulatory stimulus, for callus induction. However, callus formation on a growth regulator-free medium was observed at the cut surface after whole seedlings were wounded. It is known that relatively small cuttings can produce large calli at the cut sur- face in the absence of growth regulators (Yeoman & Macleod, 1977). Plant hormones are present in higher quantities after wounding and are involved in cell pro- liferation at the wound site (Rosenstock & Kahl 1978; Kahl 1983). Therefore, excision alone can initiate a wound response and the induction of cell division may produce a callus.

Callus growth on a growth regulator-free medium

Calli derived from the different explant types were capable of growing on growth regulator-flee medium for ten months through successive subcultures. Calli obtained from cotyledon or hypocotyl explants showed a higher growth rate while radicle-derived calli exhibit- ed very poor growth. On the other hand, MSO medium produced higher growth rates than B50 medium.

Callus cultures derived from hypocotyl explants were propagated for ten months on a growth regulator- free medium (MSO) and its growth capacity was main- tained (Fig. 1). The same figure also shows that callus grew approximately at the same rate on MSO medium as on a medium containing 2,4-D and kinetin (MSH) except at the first subculture where significant differ- ences (at p < 0.01) were observed. Cotyledon-derived calli presented a very similar growth response (results not shown).

Surival of primary calli cultured on a growth regulator-free medium

Survival of primary calli differed on MSO and B50 after 35 days of culture. In addition, significant differ- ences in survival between calli induced from different explant types were observed (Table 1). MSO medi- um resulted in lower survival in primary calli derived

225

Table 1. Percentage of callus induction and survival of primary cultures derived from cotyle- don (cot.), hypocotyl (hyp.) and radicle (rad.) explants of E. scoparium cultured on two growth regulator-free media

% Callus induction % Surviving cultures 3

Medium cot. hyp. tad. cot. hyp. rad.

MSO 33.34-2.01,2 46.94-5.2 15.64-1.0 80.24-1.0 69.84-3.1 15.64-5.2

B50 3.14-1.0 19.84-3.1 5.24-5.0 26.0-I-5.0 71.9+3.1 44.84-3.1

I The number of cultures per treatment ranged from 40 to 48 for each experiment. 2 Values represent the mean of two experiments 4- standard deviation. 3 Percentage of surviving cultures during callus induction were recorded after 35 days in culture

2 5

O

O

"-i- I - -

© c r

(.9

2 0

15

10

5

-1"- -

I I I I I I

O 1 2 3 4 5 6 7 8

-A- MS0

-*- M S H

I 1

9 1 0

S U B C U L T U R E N U M B E R Fig. 1. Growth curve of E. scoparium callus cultures on growth regulator-free medium (MSO) and on MSH medium during subculture. Calli were removed from hypocotyl explants after 35 days and unorganized pieces were subcultured every 4 weeks on fresh medium. W = final weight, Wo = starting weight. Data points are mean values (with bars indicating standard deviation) obtained from 48 callus pieces and they are not statistically different using an analysis of variance. With the exception of the first subculture differences were significant at p < 0.01.

226

Fig, 2. Micr~graphs ~f secti~ns (cut at ~ ~m) ~btained fr~m hyp~c~ty~derived ca~us ~f E~ sc~parium gr~wing ~n MS~ medium during the ~th subculture and stained with toluidine blue and borax, (a) Unorganized peripheral region of callus after 10 days of subculture. Large and highly vacuolated cells (*) are observed, Note extracetlular secretions (at arrowheads) surrounding some cells. Bar = 100 p.m, (b) Peripheral region of callus after 15 days of subculture where some cells have undergone periclinal divisions (at arowhead) producing cell files. P = peripheral zoner Bar = 50 ~tm. (e) Meristemoid (M) detected near the suface of a 25-day old callus. Bar = 50 p.m. (d) Two nodular structures (N) with vascular elements also observed at the periphery of the same callus described in C. Bar= 50 ram. (e) Nodule with vascular elements (at arrowhead) arising on the surface of a 25-day-old callus. Bar = 50 p.m. (39 Meristematic nodule (N) with very densely stained cells containing abundant starch at the surface (P) of callus after 20 days of subculture, Unstained vacuolated cells at the periphery of the nodule are also observed. Bar = 50 ~tm,

from radicle explants, whereas B50 medium resulted in higher mortality in those callus cultures derived from cotyledons.

Differentiation

Primary calli (from cotyledon or hypocotyl explants) frequently developed green nodules on their surface when excised and cultured on MSO medium. Each month, undifferentiated regions of calli were isolat- ed and subcultured onto fresh growth regulator-free medium. On the other hand, friable and undifferentiat- ed calli were only produced when callus explants were cultured on a MS medium supplemented with 2,4-D and kinetin (data not presented).

An examination of long-term callus cultures induced and maintained on MSO medium revealed that during the first 10 days of each subculture, a con- siderable lack of organization was detected (Fig. 2a). Hypertrophical and meristematic cells were observed in calluses. Sometimes, extracellular secretions sur- rounding cell clusters and single cells were found in the area external to calli.

In several regions it can be seen that some cells have undergone periclinal divisions producing files of cells (Fig. 2B). Many nodules that arose from groups of small, densely cytoplasmic cells (meristemoids) at or near the tissue surface were detected in such regions (Fig. 2c). Frequently, vascular elements were visible in these structures (Fig. 2d), while gradients of vascular differentiation could be found in the callus tissue on the surface of growth-regulator-autonomous calli (Fig. 2e). The starch content in the nodular cells was high, and frequently, many cells located in the center of the nodule were intensely stained in those histological sections treated with toluidine blue/borax (1:1) (Fig. 2f).

Cells with characteristics of embryogenic cells were occasionally observed in the external regions of calli (Fig. 3a). These cells showed central large nuclei with prominent enlarged nucleoli and little- vacuolised cytoplasm with abundant starch grains. Clumps derived from these cells frequently presented some physical isolation due to the presence of extra- cellular secretions. This isolation differentiates these structures from other types of meristemoids frequently found in calli (Fig. 2c). Several cell clumps appeared to be degenerated (Fig. 3b) while some compact glob- ular structures were observed near embryogenic cell groups (Fig. 3c).

227

Fig. 3. Micrographs of sections cut at I p.m (a and b) and at 10 ~tm (c) from hypocotyl-derived calli induced and subcultured on growth regulator-free medium during the 10th subculture. (a) Vacuolated cells and meristematic cell groups (e) are present at the surface of a 25-day-old callus stained with toluidine blue and borax. The cell isolation due to extracellular secretions is visible (at arrowhead). {b) Globular structure (Pe) at the surface of the callus described in a. The arrowheads show the gelification of walls around the clusters which seem to be necessary for the subsequent isolation of these structures. Nu = nucleolus, = degenerated cell clumps. (c) Globular structure (Ge) at the surface of a 30-day-old callus stained with safranin and fast green. Bar = 50 p.m.

228

Our his tological analysis shows a great variabili ty

in the morpho logy o f induced calli g rowing on M S O

medium. It is clear that the cells o f these call i are

affected by a very he terogeneous environment .

Acknowledgements

The authors thank Dr. Patricia Kishi and Dr. Francisco

Garcfa for the rev iew of manuscript .

References

Bayer MH (1982) Genetic Tumors: Physiological aspects of tumor formation in interspecies hybrids In: Kahl G & Schell JS (eds) Molecular Biology of Plant Tumors (pp 33--67),Academic Press, New York

Gamborg OL, Miller RA & Ojima K (I968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 151-158

Gautheret R (1959) La culture des tissus v6g6taux, Masson Paris Gonzgtlez AG & Luque Escalona M (1975) Nuevos glic6sidos car-

di~icos de Cheiranthus scoparius Brouss. Ann. Quim. 71: 97- 103

Hansen A & Sunding P (1993) Flora of Macaronesia. Checklist list of vascular plants (4 revised edition). Sommerfeltia 17 (p. 70)

Johansen DA (1940) Plant microtechnique, McGraw-Hill Book Co. New York (p,41)

Kahl G (1983) Wound repair and tumor induction in higher plants. In: Akazawa T & hnasei H (eds) The New Frontiers in Plant Biochemistry' (pp 193-216). Japan Scientif. Soc. Press, Tokyo and Martinus Nijhoff/Dr. W. Junk PUN., The Hague

Masuda K, Koda Y & Okazawa Y (1977) CaIlus formation and embryogenesis of endosperm tissues of parsley seed cultured on hormone-free medium. Physiol. Plant. 41:135-138

Meins F (1989) Habituation: Heritable variation in the requirement of cultured plant cells for hormones. Annu. Rev. Genet. 23: 395-408

Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobbaco tissue cultures. Physiol. Plant. 15: 473-497

Rosenstock G & Kahl G (1978) Phytohormones and the regulation of cellular processes in aging storage tissues. In: Kahl G (ed) Biochemistry of Wounded Plant Tissues (pp 623-671 ). Walter de Gruyter & Co., Berlin

Yeoman MM & Macleod AJ (1977) Tissue (callus) cultures Tech- niques. In: Street He (ed) Plant Tissue and Cell Culture 2nd edition (pp 31-59 ). Blackwell Scientfic Publ., Oxford