Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Plant Cell, Tissue and Organ Culture 27: 65-69, 1991. (~) 1991 Kluwer Academic Publishers. Printed in the Netherlands.

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora G.R. Rout, B.K. Debata & P. Das* Regional Plant Resource Centre, Bhubaneswar-751012 (Orissa) India (*requests for offprints)

Received 30 October 1990; accepted in revised form 15 May 1991

Key words: callus, in vitro, L-proline, Rosa hybrida cv. Landora, somatic embryogenesis Abstract

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30g 1-1 sucrose (½ MS) and supplemented with 2.2. /xM BA, 5.4/zM NAA and 2.2-9.0/zM 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1 MS + 2.2/xM BA + 0.05/~M NAA + 0.3/xM GA 3 + 200-800 mg 1-~ L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8 --- I°C for 4 days germinated on 1 MS + 2.2/zM BA + 0.3/xM GA 3 + 24.7/xM adenine sulphate.

Abbreviations: BA-benzyladenine, NAA-a-naphthaleneacetic acid, 2,4-D-2,4-dichlorophenoxyacetic acid, GA 3 -gibberellic acid

Introduction

In vitro propagation protocols have been developed for many rose cultivars using shoot-tip and axillary-bud explants (Rout et al. 1989; Skirvin et al. 1990). Earlier attempts to induce organogenesis from callus cultures of roses were unsuccessful (Khosh-khui & Sink 1982). Recently, adventitious shoot differentiation in callus cultures originated from leaf, stem internode and zygotic embryo segments has been demonstrated (Tweedle et al. 1984; Lloyd et al. 1988; Burger et al. 1990). Somatic embryogenesis has the potential to be an efficient method for plant multiplication and improvement. This paper reports on the successful induction of somatic embryogenesis in Rosa hybrida cv. Landora.

Materials and methods

Young non-flowering shoots, measuring 15-

20 cm in length, were cut from pot-grown plants of Rosa hybrida cv. Landora. The 5-7 cm apical portion from each shoot was discarded. The remaining portion with attached leaves was thoroughly rinsed in a 2% (v/v) solution of 'Labolene' detergent (Glindia, India) and then surface disinfested in 0.1% (w/v) mercuric chloride solution for 25 min followed by four 2 min rinses in sterile distilled water. Leaves and stem internodes were cut into smaller segments (0.5 cm x 1.0 cm and 0.3-0.5 cm respectively) and aseptically inoculated onto semisolid nutrient medium. The abaxial surface of leaf segments remained in contact with medium. The basal nutrient medium consisted of Murashige & Skoog (1962) mineral salts, vitamins and organics of half strength and 30 g 1-I sucrose (½ MS) supplemented with 0.89 or 2.2/xM BA in factorial combinations with 5.4/xM NAA and 2.2, 5.4, 9.0/xM 2,4-D. Callus cultures initiated from leaf explants on ½MS medium containing 2.2/zM BA + 5.4/zM

66 NAA + 2.2/xM 2,4-D and in stem explants on basal ½ MS medium containing 2.2/xM BA + 5.4/zM NAA + 9.0/zM 2,4-D. Twenty-five cultures were used per treatment. The friable callus derived from leaf and stem explants were transferred 1 MS medium plus 2.2/zM BA, 0.05/zM NAA, 0.3/xM GA 3 and 0-800 mg 1-1 L-proline for induction of somatic embryogenesis. Basal medium was supplemented with 2.2/xM BA + 0.3/xM GA 3 +24.7/~M adenine sulphate for germination of the somatic embryos. The pH of all media was adjusted at 5.7 before adding 8 g 1-~ agar (Glindia, India), autoclaving at 121°C (1.06 kg- cm -2) for 15 min. Cultures were grown in 25 mm x 150 mm glass culture tubes (Borosil, India), with 25ml nutrient medium per tube, which were capped with cotton plugs and incubated at 25-+ 2°C in a growth room, either in continuous dark for callusing or under cool white, fluorescent light (50/x mol m -2 s -1) 16h per day for induction of somatic embryogenesis. Callus was subcultured at 4-week intervals onto fresh callusing medium with similar composition. In experiments on somatic embryo germination, the embryogenic cultures were stored in a refrigerator at 8 -+ I°C for 2-7 days prior to separation and inoculation of the somatic embryos on regeneration medium. For histological examinations, the embryogenic calluses at different developmental stages were fixed in FAA (formalin/glacial acetic acid/ ethanol, 5 : 5 : 9 0 v / v ) for 48h, dehydrated through graded ethanol-xylol series and embedded in paraffin wax. The tissues were sectioned at 10/xm and stained with 0.1% (w/v) solution of toludine blue.

Results and discussion

Leaf segments expanded on all media after 2-3 days of inoculation. Depending on the auxin and cytokinin concentrations of culture media, calluses were initiated from cut surfaces and margins as well as along the petiole and midrib after 7-12 days of inoculation. Subsequently, small masses of callus also appeared at different points distributed on the adaxial surface of the leaf explant. Callus formation was not observed on the abaxial surface of the leaf explants. Calluses

were formed on cut ends of stem internodes after 15-20 days of culture on media containing growth regulators. BA (0.89 or 2.2/xM) alone in the culture medium failed to stimulate callusing on any of the leaf or internode explants (Table 1). NAA and 2,4-D at all concentrations stimulated callus formation on both types of explants. The highest frequency (92%) of callus formation, however, was recorded on leaf segments when cultured on ½MS + 2.2/zM BA + 5.4 # M NAA + 2.2/xM 2,4-D. Similarly, ~ MS + 2.2/xM BA + 5.4/xM + 9.0 ~ M 2,4-D proved to be the most effective medium combination for callus development on internode explants were 76% of the explants responded positively (Table 1). Initial callus on leaf explants was greenish, hard and nodular whereas that on internodes was whitish and compact. Regardless of the origin, the primary callus rapidly proliferated into friable, whitish to pale yellow callus upon subculturing on the respective callusing medium. In order to induce somatic embryogenesis, pieces (250 rag-400 rag) of rapidly proliferating friable callus were removed from the respective callusing medium after the second subculture and placed on induction medium with either 0, 200, 400, 600 or 800 mg 1 1 of L-proline (Table 2). On a medium devoid of L-proline the friable

Table 1. Effect of BA, N A A and 2,4-D on callus development in cultured leaf and stem internode explants of Rosa hybrida cv. Landora. ½MS + Growth regulators ( # M)

Callus development after 4 weeks (Percentage ±S.E. means)"

BA

NAA

2,4-D

Leaf

Internode

0 0.89 2.2 0.89 2.2 0.89 0.89 0.89 0.89 2.2 2.2 2.2

0 0 0 5.4 5.4 0.0 5.4 5.4 5.4 5.4 5.4 5.4

0 0 0 0 0 2.2 2.2 5.4 9.0 2.2 5.4 9.0

0 0 0 30.7±0.3 44.0±0.8 34.7±0.5 72.0±0.5 65.3±0.3 57.3±0.3 92.0±0.5 74.7±0.5 60.0±0.5

0 0 0 20.0±0.5 22.7±0.3 24.0±0.5 34.7±0.5 53.3±0.5 68.0±0.5 40.0±0.9 58.7±0.7 76.0±0.8

Mean of three repeated experiments. Each treatment consisted of 25 replicates. a

67 Table 2. Embryogenic response of leaf and stem internode calluses of Rosa hybrida cv. Landora upon transfer from respective callusing medium onto induction medium (½ MS +2.2/zM BA +0.05/zM NAA+0.3/~M GA3 + L-proline concentrations as specified). L-proline Callus source (mgl -l) Leaf Internode Cotyledonary stage Percentage cultures Cotyledonary s t a g e Percentage cultures somatic embryo/ forming embryogenic somatic embryo/ forming embryogenic embryogeniccallusb callusa embryogeniccallusb callusa

0 200 400 600 8~

0 17.8 28.9 57.8 40.0

0 4.1±0.3 6.3±0.4 11.9±0.4 8.4±0.6

0 0 15.6 46.7 24.4

0 0 2.9±0.5 5.5±0.3 4.3±0.2

(15 replicates/treatment) a Measured after the first four weeks of culturing on induction medium. b Mean ---standard error of 3 repeated experiments after second four weeks of culturing on the induction medium.

callus slightly increased in volume and gradually hardened. Somatic embryogenesis was not observed in these cultures even after maintaining them for up to six months through four-week transfers to fresh medium of similar composition. Callus readily differentiated into two types of tissues after 3 - 4 weeks of culture on all media with L-proline. These cultures were composed of white, friable, non-embryogenic sectors and light green, highly globular, embryogenic sectors (Fig. 1A). For embryogenic callus initiation, stem int e r n o d e callus had a higher threshold requirement for L-proline than leaf callus. However, 600 mg 1-1 of L-proline in the induction medium was found to be optimum for embryogenic callus formation on calluses originated from both types of explants (Table 2). After culturing for up to four weeks on induction medium, embryogenic callus was separated from the rest and cultured on fresh induction medium. Within two weeks, various structures appeared on the embryogenic callus that were typical of globular, heart-shaped and cotyledonary stage somatic embryos in morphology (Fig. 1B). Medium with 600mg 1-1 of L-proline proved to be the best for somatic e m b r y o production from both leaf and stemderived calluses. It was observed that frequency of somatic e m b r y o formation on leaf callus was higher than that from stem callus (Table 2). Some of the somatic embryos were morphologically normal showing distinct cotyledons and radicles. The embryos were loosely attached

to the m o t h e r callus with a short suspensor-like structure at the basal end. In addition to embryos with normal morphology, other exhibited anomalies in shape and structure. Some embryos had only one cotyledon, while others had two or more equal or unequal cotyledons, often fused together to render a cup shape to the embryo. Vitrification of embryos also occurred as described for many other species (John 1986). Somatic embryos also arose from the basal region of other embryos in clusters of 2 to 4 (Fig. 1C), indicating a clear case of secondary somatic embryogenesis (Maheswaran & Williams 1986; A m m i r a t o 1987). Cotyledonary stage somatic embryos failed to germinate on the induction medium and many reverted to callusing. The embryogenic callus mass was also transferred to L-proline deficient medium (½ MS + 2 . 2 / z M B A + 0.05/.tM N A A + 0.05/~M GAa) after 2 - 3 induction passages on different Lproline rich media. On this medium, fewer abnormalities were observed and callusing of the embryos minimised. More somatic embryos developed, which was due to secondary somatic embryogenesis as mentioned before. Continuous secondary embryogenesis was achieved by subculturing the developing embryogenic mass at 4-week intervals on flesh medium without Lproline and the cultures have been maintained for a period up to 16 months without any loss in the regenerative capacity. T o p r o m o t e germination and development

68

Fig. 1. A - E . Somatic embryogenesis in Rosa hybrida cv. Landora (A) Non-embryogenic (ne) and embryogenic (e) tissue development on stem callus. (B) An embryogenic leaf callus showing numerous somatic embryos (arrows) at various stages of development. (C) The embryogenic dumps showing secondary somatic embryos (arrows). (D) Germination of cotyledonary stage somatic embryos on ~ MS + 2.2/.tM BA + 0.3/~M G A 3 + 24.7 ~M adenine sulphate, Note the formation of a black callus mass instead of roots at the radicular end of the germinating embryos. (E) A later-stage somatic embryo showing cotyledons (c), radicle (r) and the provascular system (v). (Scale bars = 1 cm in A, B, 1 mm in C, 0.5 mm in D and 100 p,m in E).

into plantlets, somatic embryos were individually separated from mother cultures and inoculated onto a regeneration medium comprising of ½ MS + 2.2/xM BA + 0.3/.~M GA 3 + 24.7/xM a d e n i n e s u l p h a t e . A f t e r 1 0 - 1 5 d a y s of i n o c u l a t i o n u n d e r light, t h e w h i t e s o m a t i c e m b r y o s

turned greenish but did not germinate and develop into plantlets. However, upon storing the e m b r y o g e n i c c u l t u r e s for 4 d a y s at 8 +- 1°C p r i o r to s e p a r a t i o n a n d r e c u l t u r e o f t h e c o t y l e d o n a r y s t a g e s o m a t i c e m b r y o s o n fresh r e g e n e r a t i o n medium under illuminated conditions, about

69 12% of the embryos elongated and formed leaves (Fig. 1D). Cotyledonary stage embryos had provascular strands (Fig. 1E). There was feeble callusing at the base of the somatic embryos that was apparently from the suspensor cells. This callus subsequently stopped growth and rapidly blackened. Root formation on the embryos could not be achieved. Experiments concerning enhancement of embryo to plantlet conversion are presently under progress. This investigation demonstrates induction of somatic embryogenesis on callus derived from both leaf and stem internode segments of Rosa hybrida cv. Landora on a medium containing BA, NAA, GA 3 and L-proline. The results of this study indicate that induction of somatic embryogenesis in callus cultures of rose is dependent on the presence of L-proline in the culture medium. L-proline and other amino acids have been shown to stimulate a similar response in other species (Rangaswamy 1986; Ammirato 1986; Radojevic 1988). In this study, it was further shown that the presence of L-proline is essential only for induction of somatic embryogenesis during the initial culture passage. Once induced, somatic embryogenesis and secondary somatic embryogenesis can be retained for long period without decline in a manner somewhat similar to that observed in cassava (Szabados et al. 1987) and also grape (Gray & Mortensen 1987). Conditions must be refined in order to achieve a greater initiation rate of embryogenic cultures as well as higher embryo germination rates and plant recovery in roses.

Acknowledgement The authors wish to acknowledge the financial assistance from the Department of Environment, Government of India, New Delhi for this investigation.

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