Plant Cell, Tissue and Organ Culture 7:257-261 (1986) © Martinus N~ihoff Publishers, Dordrecht - - Printed in the Netherlands

Short communication

Somatic embryogenesis from cell suspension cultures in Carica candamarcens i s

MIGUEL JORDAN*

Laboratorio de Botfirtica, Facultad de Ciencias Biol6gicas, Pontificia Universidad Catdlica de Chile, Casilla 114-D, Santiago, Chile

(Received 30 October 1985; in revised form and accepted 5 May 1986)

Key words: Carica candamarcensis, papaya, somatic embryogenesis, cell suspension cultures

Abstract. Cell suspension cuRures of Carica candamarcensis derived from hypocotyl cam were tested concerning their in vitro embryogenic capacity to improve asexual propagation rates in this species. Somatic embryos developed in culture from cells in suspension or from microcalli. Responses were affected by nutrient media and phy- tohormones used. Best results were obtained by growing the cells in suspension in Nitsch and Nitsch medium containing naphthaleneacetic acid and then plating them upon the same medium containing benzyladenine, or combinations of both hormones.

Introduction

Carica candamarcensis Hook. f. (Carica pubescens, Lerm6 and Koch), the mountain papaya [9] is the only edible species of the Caricaceae family growing in Chile. Papaya has become an important cash crop in the last few years, as fruits and proteolytic enzymes such as papain are required by the pharmaceutical, food and leather curing industries. New uses as oils, and jellyfying properties are being tested [3]. Economic exploitation is ham- pered at present by lack of selected clones, poor responses in asexual pro- pagation and the occurrence of different floral forms affecting productivity.

Conditions to trigger somatic embryogenesis in callus were discussed in a previous report [4]. The present work explores further potentialities shown by suspension culture systems that may be of interest for future breeding work and clonal propagation of this fruit crop.

Materials and methods

Sections of hypocotyls (3 mm long) from plants grown in the greenhouse were cultivated for callus initiation on a Nitsch and Nitsch salts medium (N) [8] containing naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA), both in 1 mg 1-1 as described before [4]. After 30 days, microscopic exami- nation of callus showed no evidence of embryoids or organogenesis. At this

*To whom correspondence should be addressed.

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time fresh weight of calli averaged above 2000 mg. To obtain cell suspension~ 5 pieces (5 mm diam., approx. 60mg each) of these non-embryoforming un-organized calli, were transferred to 50ml Erlenmeyer flasks each con- taining 4 m l of N or modified Gamborg's B5 medium (G) [1] (iron, micro- nutrients and vitamins same as N) with combinations of NAA and BA at 0 or with 1.0mg1-1, and agitated at 120strokes/min (Eberbach reciprocal shaker). The cultures were maintained at 28 °C and 50/aE m -2 s -1 (Philips fluorescent lamps TL 40/54) for 18 hours daily. After 10 days the sus- pensions were examined microscopically for the presence of embryoids and for viability, using 0.1% bromphenol blue [2]. In all of the 8 conditions tested, single cells but no embryoids were detected; viability was always higher than 90%. Then, 2.0 ml of each checked suspension were used to plate 4 glass Petri dishes (5 cm diam.) containing N or G media with 0.5% agarose (Sigma A 6013) and combinations of NAA and BA at either 0 or 1 mg 1-1 . The dishes were maintained at 28 °C in the dark for the first 4 days and then in 18 hours light (100/aE m -2 s -1 ) per day.

Results and discussion

Morphogenic events and growth responses of cell suspensions in the dif- ferent treatments assayed are summarized in Table 1. Several nutrient and hormonal combinations resulted in somatic embryogenesis either in sus- pension cultures or in callus. Most embryoids were obtained by first sus- pending callus in N-medium containing NAA (1 mg 1-1) and plating later on the N-medium with BA (1 mg 1-1). Under these conditions more than 20 yellowish-greenish embryoids were found in each Petri-dish after 8 weeks. In addition, several developing proembryoids were observed under the micro- scope (Figure la). Other treatments also led to embryoids, predominantly in callus tissue, but the frequency was reduced.

The presence of BA (1 mg 1-1) was always necessary to obtain embryos either in the suspension or plating phases. Although most embryos were obtained using N-medium throughout, embryos were also formed in cells

Figure la. Embryogenic calli and embryoids developed from suspended cells after 8 weeks (arrows). Several other embryoids originating throughout the calli tissue are also visible. b. Close-up showing three cell clusters and a proembryolike structure (inset) derived from cell suspension after 2-3 weeks in culture (N-medium). c. Further developmental stage of b, showing embryoid originated from a cell cluster after 3-4 weeks (N-medium). d. Section of a developed embryoid represented in e. f. Embryogenic microcaUi showing some parenchyma cells with meristematie activity. g and h. Cross sections of two embryoids developing inside and on the surface of eaUi. i and j. SEM photographs: embryoids protruding from callus tissue. k. A fully developed embryoid arising through the callus tissue. Bar = 250 ~M

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plated on modified G-medium, when previously suspended in N-medium and vice-versa. In most experiments using G-medium only, proliferation of micro- calli, but no clearly distinguishable embryoids were observed, even after three months.

Figure lb shows an early stage of proembyroid formation as small dense clusters derived from suspended cells. A further stage of development is shown in Figure lc. Adult embryoids 8 weeks old, average size 1 mm are shown in Figure 1 d and 1 e.

Embryogenesis from microcalli occurred inside dense yeUow coloured cell outgrowths. Histological observation showed groups of small meris- tematic cells in the whitish parenchymatic microcalli (Figure If)followed by embryo development within 15 days (Figure lg). In later stages embryoids attached to caUi (Figure lh) and protruding from them were visible (Figure li, j, k).

Results suggest that the composition of the nutrient medium (N) also used to induce somatic embryogenesis in callus cultures [4] and the presence of BA in the last plating phase are favourable conditions to induce embryo- genesis in this species. Although somatic embryogenesis from cell suspensions seems to be easily expressed for C papaya [7] and for C stipulata [5, 6] further studies are necessary to define requirements and enhance embryo formation in C. candamarcensis suspension cultures.

Acknowledgements

The author wishes to thank Carlos Roveraro, Irma Cort6s and Andr6e Goreux for their technical assistance. This work was supported by Catholic Uni- versity funds DIUC 73/84 to Prof. M. Jordan.

References

1. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50:151-158

2. Hanson MR (1984) Manipulation of single cells and protoplasts of plants: Experi- mental protocols, University of Virginia, Dept. Biology, CharlottesviUe VA 22903 USA.

3 Hermosilla J (1984) Papaina activa en Carica candamarcensis Proc. V Reunidn Nacional de Bot~nica, La Serena, Chile.

4. Jord~in M, Cort6s I, Montenegro G (1983) Regeneration of plantlets by embryo- genesis from callus cultures of Carica candarnarcensis. Plant Sci Lett 28:321-326

5. Litz RE, Conover RA (1979) Development of systems for obtaining parasexual Carica hybrids. Proc Fla State Hort Soc 92:281-283.

6. Litz RE, Conover RA (1980) Somatic embryogenesis in cell cultures of Carica stipulata. Hort-Science 15:733-735.

7. Litz RE, Conover RA (1983) High-frequency somatic embryogenesis from Carica suspension cultures. Ann Bot 51:683-686

8. Nitsch JP, Nitseh C (1969) Haploid plants from pollen grains. Science 163:85-87 9. Uphof JCTh (1968) Dictionary of economic plants. Verlag von J Cramer, Lehre,

pp 591.