Plant Cell, Tissue and Organ Culture 31: 47-50, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Somatic embryogenesis and high frequency plantlet regeneration in callus cultures of Thevetia peruviana

Anjani Kumar Department of Botany, North-Eastern Hill University, Shillong 793 014, India

Received 10 July 1991; accepted in revised form 16 March 1992

Key words: embryoids, leaf callus, medicinal tree

Abstract

Plantlet regeneration through somatic embryogenesis has been achieved in the apocynaceous medicinal tree Thevetia peruviana L. Calluses obtained by culturing young leaf discs on MS medium containing 9 IxM 2,4-dichlorophenoxyacetic acid and 4.6 IxM kinetin, when subjected to reduced levels of the growth regulators followed by higher cytokinin treatment, produced numerous somatic embryos. Somatic embryos developed into complete plantlets on a medium devoid of growth regulators. An average of 40-50 plantlets were obtained from 50 mg of embryogenic callus. Survival of transplants was 60% under glasshouse conditions.

Introduction

Thevetia peruviana L. (Apocynaceae) is a plant of medicinal importance since it contains two cardiac active compounds, peruvoside and thevetin, in its latex. Peruvoside, a cardiac glycoside, has a relatively high therapeutic index compared to digoxin and has already been intro- duced into modern medicine and marketed in Germany under the trade name 'Endocordin' (Bhakuni 1990). Thevetin, a cardiac glucoside, gives rise to theverisin on hydrolysis. Both theverisin and thevetin have digitalis-like action on the heart indicating their possible clinical application. Thus the plant selected for the pres- ent investigation offers an excellent scope for biotechnological applications. Efficient plant re- generation from cell and tissue cultures, prefer- ably through somatic embryogenesis, is one of the constituents of biotechnology and has be- come a pre-requisite for any in vitro manipula- tion (Eapen & George 1989). Earlier Dasgupta & Datta (1987) reported regeneration of plant- lets by culturing cotyledonary explants from ma-

ture seeds. This report describes high frequency plant regeneration through somatic em- bryogenesis in Thevetia peruviana using foliar explants from mature trees.

Materials and methods

Juvenile leaves collected from young branches of mature Thevetia peruviana trees (12-15 years of age) growing in the Civil Hospital campus, Shil- long and Forest Range Office, Nongpoh (Meghalaya), were washed thoroughly with Teepol (detergent) solution under running tap water for 30 min. After several rinses with dis- tilled water (5-7 times), the leaves were surface disinfested with 10% sodium hypochlorite (v/v) for 5min and then washed thoroughly with sterile double-distilled water. Leaf discs were punched from the leaf lamina aseptically with a sterile cork borer (5 mm diameter). A single disc was inoculated on solidified Murashige and Skoog (MS) (Murashige & Skoog 1962) medium

48

containing 3% sucrose, 0.8% agar (Qualigens, bacteriological grade) and supplemented with 9.01xM 2,4-dichlorophenoxyacetic acid (2,4-0) and 4.62 ~xM kinetin in combination, for callus induction. Initially, the discs were cultured in 25 × 150mm culture tubes containing 15 ml of the medium each. After 15 days proliferating discs were transferred to 100 ml flasks containing 30ml of the medium. Non-absorbant cotton plugs, with aluminium foil wrap at the top, were used as closure of the culture vessels. The pH of the culture medium was adjusted to 5.8 prior to autoclaving at 121°C and 98 kPa for 20 min.

Approximately 50 mg of the induced calluses, broken into tiny pieces (2-3 mm), were sub- cultured on MS medium containing 4.5 IxM 2,4-0 + 0.46 IxM kinetin. From this medium, healthy, growing, organised-looking callus pieces were subsequently transferred to medium supple- mented with 0.45 I~M 2,4-D and 9.26 IxM kinetin or 5.95 p,M 2-isopentyl adenine (2-iP). Callus with clusters of embryoids were transferred to medium without any growth regulator for plant- let regeneration. All the cultures were incubated at 24 +-2°C under cool-white fluorescent light (40 ~mol m-2 s- L) with a 16-h photoperiod. For each t reatment 20 replicates were taken and the experiment was repeated twice.

Specimens for scanning electron microscopy (SEM) studies were placed in 2% (v/v) glutaral- dehyde (0.2 M sodium cacodylate, pH7 .3 ) for 2 h, washed in 0.05 M sodium cacodylate buffer ( p H 7 . 3 ) and post fixed in 2% (v/v) osmium tetroxide (0.2 M sodium cacodylate, pH 7.3) for 2 h at room temperature. Tissue samples were washed in buffer, dehydrated in graded ethanol dried up to critical point in CO 2 , affixed on aluminium stubs with silver paint and coated with gold palladium in a fine coat Ion Sputter JFC-1100. The samples were examined under a scanning electron microscope, Jeol JSM-35-CF, at accelerating voltage between 10-15 Kv.

Plantlets of 4-5 cm in size, with 2-3 leaves and one or more developed roots, were removed from cultures and transferred to pots with auto- claved soil :sand (1:1) mixture for acclimatiza- tion. Acclimatization was accomplished by cov- ering the pots with a transparent polythene cover and maintaining them in a growth chamber for two weeks.

Results and discuss ion

Friable greenish white callus was obtained from leaf discs cultured on MS + 9.0pxM 2,4-D + 4.6 IxM kinetin within 30 days. The growth reg- ulator effect in callus induction in cultured leaf discs was similar regardless of age of the tree and genotype. No differences among the genotypes ( three trees sampled from each locality) were observed in callus proliferation and embryogenic capabilities (data not given). Initially, the growth of the callus was observed to be slow, but sub- culturing of calluses broken into 2-3 mm pieces, to MS medium containing a reduced growth regulator level (4.5 txM 2,4-o + 0.46 txM kinetin) accelerated the growth rate and produced numerous highly organised structures on the sur- face. A similar response on lowering the growth regulator level has been observed previously by Guimaraes et al. (1988) in Cyphomandra be- tacea. Concentrations above 4.5 IxM 2,4-0 re- duced callus growth and organisation of calluses and cultures subjected to 22.5 ixM 2,4-0 failed to survive. Healthy and organised looking calluses, when transferred to the medium fortified with high concentrations of cytokinins (9.26 txM kinetin or 5.95 IxM 2-iP) and a low concentration of 2,4-0 (0.45 txM), grew rapidly and produced clusters of embryoids of various stages within 30-45 days (Fig. 1). The embryoids of different stages could be separated from the cluster and germinated. Embryogenic calluses when ex-

Fig. I. Several embryoids developing on callus grown on MS medium supplemented with 5.95 IxM 2-iP and 0.45 ~M 2,4-o. (Bar represents 1 cm)

49

Fig. 2. Scanning electron micrograph (SEM) of an em- bryogenic callus showing a well-developed embryoid resem- bling typical dicot embryo.

amined with the scanning electron microscope revealed embryoids of different stages that re- semble a typical dicotyledonous embryo (Fig. 2). The calluses undergoing embryogenesis looked like aggregates of embryoids. MS medium sup- plemented with 2-iP and 2,4-D produced a higher number of somatic embryos compared to kinetin and 2,4-D (Table 1). Similar embryogenic poten- tial of calluses subjected to high levels of cyto- kinins and low levels of auxin has been reported for crownvetch (Coronilia varia) (Mariotti & Arcioni 1983), and finger millet (Eleusina caracana) (Eapen & George 1989). Aggregated somatic embryoids on transfer to MS basal medium devoid of growth regulators, regener- ated into complete plantlets with well-developed roots without showing any secondary callusing from the embryoids as observed occasionally (Mariotti & Arcioni 1983). The regenerants were easily isolated and planted in pots for acclimati- zation. At the same time embryoids separated from the cluster were transferred to growth reg-

Fig. 3. Potted regenerant growing under glasshouse. (Bar represents 1 cm)

ulator-free medium for germination and recovery of plants. However, rate of germination was noted to be 80% (approx.) and the plantlets obtained from separated embryoids had a poorly developed root system. These plantlets needed to be maintained on ½-strength MS medium with 2% sucrose and no growth regulators for two weeks in the dark for the development of strong viable roots.

From 50 mg of embryogenic callus, an average of 40-50 plantlets was obtained. Of the 50 plants that were subjected to acclimatization, 60% sur- vived under glasshouse conditions. The plants grew well in the pots and looked similar to plants in their natural environment (Fig. 3). There were no differences among plantlets in mor- phology.

Somatic embryogenesis offers the fastest meth- od of in vitro multiplication but has been re- ported only in a few hardwood species (von Arnold & Wallin 1988). This report clearly de- monstrates high frequency somatic em- bryogenesis and plant regeneration in callus tis- sue of foliar origin in agreement with a similar

Table I. Frequency of somatic embryogenesis in leaf callus cultures of Thevetia peruviana.

Treatments Calluses Cultures showing Average number of subjected somatic somatic embryos

embryogenesis per callus (%) +S.E.

MS + 2,4-D (0.45 p.M) 60 86 + kinetin (9.26 p.M)

MS + 2,4-D (I).45 I.tM) 48 + 2-iP (5.95 ~M)

88

54_+6

93+_8

50

repor t in C o f f e a (P ie rson et al. 1983). The single cell or igin of somat ic embryos in tissue cul ture ( H a l p e r i n 1969; B a c k s - H u s e m a n n & Re ine r t 1970; K o n a r et al. 1972; McWil l iam et al. 1974; Haccius 1978) makes it eminen t ly sui ted for mu- t an t se lect ion and o ther genet ic man ipu la t ions of the plant . Select ion of favourable mu tan t s at the cel lular level is one of the most impor t an t pos- sibili t ies offered by tissue cul ture (Scowcroft 1977; T h o m a s et al. 1979). However , the possibi- lity of genet ic var iabi l i ty a n d / o r somaclona l vari- a t ion has b e e n emphas ized in the clones der ived f rom callus cul ture (Lark in & Scowcroft 1981) and these var iants may be uti l ized for the selec- t ion of improved cell lines.

Acknowledgements

The au thor thanks Professor P ramod T a n d o n , D e p a r t m e n t of Bo tany , N E H U and the Head , Reg iona l Sophis t icated I n s t r u m e n t a t i o n Cent re , N E H U for provid ing labora tory and SEM

facilities, respectively.

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