Centre of Advanced Study, Department of Botany, University of Calcutta, 35 Ballygunge. Circular Road, Calcutta-700019, India. Received 27 July 1988; ...
Plant Cell, Tissue and Organ Culture 19: 71-75, 1989. © 1989 Kluwer Academic Publishers. Printed in the Netherlands. Short communication
Regeneration through somatic embryogenesis from anther explants of Scilla indica (Roxb.) Baker BIPASHA C H A K R A V A R T Y & S U M I T R A SEN Centre of Advanced Study, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Calcutta-700019, India Received 27 July 1988; accepted in revised form 15 March 1989 Key words: Scilla indica, Liliaceae, anther culture, somatic embryogensis Abstract. Somatic embryogenesiswas induced from anther calli of Scilla indica (Roxb.) Baker
grown on Murashige & Skoog's medium supplemented with napthaleneacetic acid and coconut milk. The calli developed embryos in hormone-free MS medium. These embryos germinated into plantlets capable of transfer to field conditions. A histological study revealed the involvement of a group of cells in the initiation of embryogenesis.Embryos with distinct root and shoot apices were observed.
Introduction
Somatic embryogenesis from zygotic embryo explants within the Gramineae is well documented, while reports of the phenomenon within monocots exclusive of this family are rare [10, 12]. Protocols for micropropagation have been successfully developed for many species of the family Liliaceae, but somatic embryogenesis has only been reported in Allium sativum [1], species of Asparagus [11], Hemerocallis [7] and Urginea indica [5]. In Scilla indica of the family Liliaceae, regeneration from leaf-tip explants through organogenesis has previously been reported [2]. In the present report, development of somatic embryos from callus tissues derived from anthers and their potential for plant regeneration is described.
Materials and methods
Bulbs of Scilla indica (Roxb.) Baker collected from the western coastal belts of India (20°N latitude) were replanted in the experimental garden of the Department of Botany, University of Calcutta. Unopened flower buds (before pollen grain formation) excised from young inflorescences were
72 washed with 5°,/0Teepol (wetting agent) sterilized in 0.1% mercuric chloride for 15 min and rinsed in sterile distilled water several times. The green anthers with filaments attached were excised from the buds aseptically and inoculated (3 to 4 anthers per tube) abaxially into culture tubes (150mm x 25mm) plugged with non-absorbent cotton wool and containing 20ml of Murashige & Skoog's (MS) [9] basal medium supplemented with 2 mg 1-1 2,4-dichlorophenoxyacetic acid (2,4-D), 15% (v/v) coconut milk (green coconut obtained from local market and filtersterilized) and 3% (w/v) sucrose. The medium was solidified with 0.6% (w/v) bacteriological-grade agar (BDH) and the pH adjusted at 5.6. The cultures were grown at a temperature of 25 _ 1 °C with a maximum relative humidity of 55% under Philips fluorescent day-light tubes emitting 32 × 108#mols-~m -2 for a 16h photoperiod followed by 8h darkness. After two months growth, the callus cultures were transferred to MS medium containing 15% coconut milk and 2 mgl -~ napthaleneacetic acid (NAA). For histological studies, the callus tissues were fixed overnight in FAA (formalin:acetic acid:ethanol, 9:0.5:0.5) dehydrated by passing through tertiary butyl alcohol series [6] and embedded in paraffin at 52-54°C. Microtome sections were cut at a thickness of 15 #m and stained with tannic acid and iron chloride [4]. Ten replicates were used for each experiment and the experiment repeated twice.
Results and discussion
Initiation of callusing occurred within fifteen days from surface of anther explants after rupture of anther wall on MS medium supplemented with 2mg1-1 2,4-D and 15% (v/v) coconut milk. About 87% of the anthers responded and profuse calli were formed within two months. The calli were pale-yellow, compact and continued to grow by repeated transfers onto fresh medium. The callus presumably developed from somatic tissues of the anther containing the anther-wall, filaments and connective i.e. from sporophytic tissues. Two-month-old callus on transfer to MS medium containing 15% (v/v) coconut milk and 2 mg1-1 NAA initiated friable callus with greening of surfaces and appearance of nodular structures after 20-25 days. Microscopic examination of a squash preparation of the callus tissue showed groups of small, thin-walled cells rich in cytoplasm and with large nuclei. These cells were interspersed with large, thick-walled cells (Fig. 1).
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Fig. l, Squash preparation of callus tissue showing embryogenic (E) and non-embryogenic cells (N). Fig, 2. Origin of somatic embryos from callus. Fig, 3. Germination of somatic embryo into plantlet with root and shoot. Fig. 4. Section through callus tissue showing two embryos originating from surface of callus. Fig. 5. Section through embryo with root and shoot apices at opposite ends. The root meristem (R) can be seen clearly.
74 These friable calli on transfer to hormone-free MS medium with 3% sucrose and without coconut milk resulted in the formation of somatic embryos after 30 days. About 62% of the calli showed embryogenesis. The embryos were of different shapes and sizes and in different stages of development (Fig. 2). The somatic embryos germinated in MS basal medium with 3% sucrose and without coconut milk showing the emergence of shoots and roots within 15-20 days (Fig. 3). Some of the germinating embryoids showed abnormal, hypertrophied structures in early stages of development but all of them became normal plantlets in later stages of growth and were morphologically similar to plants growing in the field. The germinated somatic embryos were transferred to MS medium with half-strength MS salts and 1.5% sucrose which promoted the further development of individual plantlets and formation of bulblets. About 70% of the plantlets survived on transfer to soil conditions and developed into normal plants. The origin of somatic embryos was confirmed by histological observations. The embryos were observed to be composed of a small group of cells, which developed into a globular mass (Fig. 4) that later detached from the main callus tissue. The embryos had distinct root and shoot apices in later stages (Fig. 5). All the embryos were associated with a surrounding zone of collapsed necrotic cells. Additional embryos were formed by repeated transfer of the embryogenic calli on basal media without hormones. The calli maintained their embryogenic potential for more than 12 months. The results show that S¢illa indica can be propagated by somatic embryogenesis in addition to organogenesis [2]. The former method is superior to the latter since the time taken for regeneration of complete plantlets is less (70 to 75 days) and the number of plants regenerating was 70 to 80 per explant. Since the cultural conditions and nutrient media provided were more or less similar in the two processes, the factor determining the occurrence of somatic embryogenesis is the explant used, viz. young anthers in pollen mother cell stages. Somatic embryogenesis from callus, derived from anther explants has been reported in other species as well [8]. In the present report, increase in the sucrose concentration [3] or pre-chilling treatment of anthers were not necessary for initiating embryogenesis.
Acknowledgements The authors thank Prof. A.K. Sharma, Programme Coordinator, Centre of
75 Advanced Study, for facilities provided. The first author is thankful to CSIR, New Delhi, for financial assistance.
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