Plant regeneration from cotyledon protoplasts of wild Medicago species

Plant Science, 48 (1987) 107--112

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Elsevier Scientific Publishers Ireland Ltd.

PLANT REGENERATION FROM COTYLEDON PROTOPLASTS OF WILD MI~DICAGO SPECIES

D.M. GILMOUR, M.R. DAVEY and E.C. COCKING

Plant Genetic Manipulation Group, Department of Botany, University Park, Nottingham NG 7 2RD (U.K.) (Received July 10th, 1986) (Revision received October 3rd, 1986) (Accepted October 3rd, 1986)

Plant regeneration from cotyledon protoplasts was obtained in Medicago difalcata, M. faicata, M. glutinosa, M. hemicycla and M. varia, using three methods of protoplast culture. Co-cultivation of freshly isolated cotyledon protoplasts with protoplasts from an albino M. sativa (alfalfa, lucerne) cell suspension was beneficial in promoting division. Plant regeneration occurred at low frequency via free floating embryoids in M. falcata, M. glutinosa and M. hemicycla, and at higher frequencies in all species via an intervening callus phase.

Key words: cotyledon protoplasts; nurse culture; albino protoplasts; somatic embryogenesis; plant regeneration ; wild Medicago species.

Introduction Although Medicago sativa L. (alfalfa, lucerne) is one of the most productive forage legumes [1], its agronomic value could be further improved by genetic manipulation, including hybridization with other Medicago sp. [2,3]. However, lack of information on crossing relationships within the genus has hindered introduction of wild germplasm [4]. Somatic hybridization has been suggested as a m e t h o d for transferring useful traits from wild species into the cultivated crop [5]. Indeed, this approach has already been used in somatic hybridization of M. sativa with M. falcata [6]. Other in vitro genetic manipulations used in recent years, some of which employ protoplast technology [7], include transformation by AgroAbbreviations: KP8, K8, Kao (1977) protoplast and cell culture media respectively [29]; MS, Murashige and Skoog (1962) medium [20]; UM, Uchimiya and Murashige (1974) medium [22 ] ; 2,4-D, 2,4-dichlorophenoxyacetic acid; NAA, 1-naphthalene acetic acid; BAP, 6-benzylamino purine.

bacterium tumefaciens [8] and micro-injection of plasmid DNA [9]. A major limitation in all of these somatic cell techniques is the lack of protoplast systems which divide and regenerate into plants [10]. To date, the cultivated crop (alfalfa) has been regenerated from protoplasts derived from leaves [11--15], seedling' cotyledons [16], seedling roots [14,17] and cell suspension cultures [14,15]. In addition, M. falcata has been regenerated from leaf protoplasts [6] and M. arborea from leaf and root protoplasts [29]. This report extends studies on cotyledon protoplasts in the genus Medicago, and describes plant regeneration from M. difalcata Sinsk. (4×), M. falcata L. (4×), M. glutinosa Bieb. (4×), M. hemicycla Grossh. (2X), and M. varia Martyn (4×). These wild species are normally found in north temperate zones [18] and harbour characteristics including frost resistance, (M. falcata, M. hemicycla), wilt resistance and tolerance to high precipitation (M. hemicycla), and a subterranean crown (M. glutinosa), which

0618-9452/87/$03.50 © Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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could be of agronomic value if transferred to cultivated alfalfa [19]. Materials and methods

(1) Seeds and sterilisation Seeds of five wild Medicago populations obtained from All-Union of Plant Industry, 44 Herzen Street, Leningrad, U.S.S.R., and of alfalfa (M. sativa cv. "Europe"; Sinclair McGill Ltd., Boston, U.K.) were treated with 0.1% w/v mercuric chloride and 0.1% w/v sodium lauryl sulphate for 15 min, followed by 20% v/v 'Domestos' bleach {Lever Bros., U.K.) for 30 rain. Seeds were washed with five changes of sterile tap water and sown on the surface of 50-ml aliquots of agar solidified {0.6% w/v; Sigma) hormonefree Murashige and Skoog {1962) based medium with 3.0% w/v sucrose (MSO), contained in screw-capped glass jars (6 oz Powder Round; Beatson Clarke and Co. Ltd., Rotherham, U.K.). 40--50 seedlings were grown in each jar at 25°C, under continuous fluorescent light (2000 lux). (2) Protoplast isolation Cotyledons were excised from 4--7-dayold seedlings, cut into l-ram strips, and plasmolysed for 1 h in a salts solution [21] with 13% w/v mannitol (CPW 13M). Plasmolysed material was incubated in an enzyme solution of 2% w/v Rhozyme (Rohm and Haas Co. Ltd., Philadelphia, U.S.A.), 2% w/v Meicelase (Meiji Seika Kaisha Ltd., Tokyo, Japan) and 0.03% Macerozyme R10 (Yakult Biochemicals Co, Ltd., Nishinomiya, Japan) in CPW 13M, 30 cotyledon pairs per 10 ml of enzyme solution. Enzyme incubation was for 16 h at 25°C on a rotary shaker (30 rev./min). Protoplast8 were released by gently squeezing the cotyledon strips, the enzyme-protoplast mixture filtered (64 pm nylon mesh), and the protoplasts pelleted (100 g, 8 min). Protoplasts were washed by centrifuging through CPW 13M (80 g, 6 rain) followed by KP8 protoplast culture medium (2 changes), resuspended in fresh KP8, and counted.

(3) Albino nurse culture A spontaneously occurring albino mutant was identified amongst normal green alfalfa seedlings, and grown in isolation. The stem and cotyledons of 3-week-old seedlings were cut transversely to give explants 5 mm in thickness, and the latter incubated on agar-solidified {0.6% w/v; Sigma) UM medium containing 2.0 mg 1-1 2,4-D and 0.25 mg 1-1 kinetin at 25°C under continuous fluorescent light (700 lux). Albino seedling explants produced pale-brown callus after 4 weeks, which was maintained by subculture to fresh UM medium every 3--4 weeks. Approximately 1 g of friable callus was used to establish a cell suspension in a 250 ml Erlenmeyer flask, containing 30 ml of liquid UM medium incubated at 25°C on a rotary shaker (60 rev./min). The suspension was subcultured every 7 days by adding 10 ml packed volume of cells to 40 ml of fresh liquid UM medium. Cells were harvested 3 days after transfer and protoplasts isolated using the same enzyme mixture and procedure as that described for seedling cotyledon protoplasts (2--3 g fresh weight of cells per 20 ml of enzyme mixture). (4) Protoplast culture Freshly isolated cotyledon protoplasts were cultured in the dark, under the following conditions: Ca) At a density of 5 × 104/mlin 5 6 X 1 4 mm plastic dishes (A/S Nunc, Kamstrap, Roskilde, Denmark), each dish containing 2.0 ml of liquid KP8 medium. (b) At a density of 5 × 104/ml in drops of a mixture of equal volumes of 1.2% agarose (FMC Colloids, Rockland, ME 04841, U.S.A.) and double strength KP8 medium. 80--100 drops, approximately 200 gl in volume, were allowed to gel on the bottom of 9-cm diameter plastic dishes (Sterilin Ltd., Middx., England), before bathing in 6.0 ml of liquid KP8 medium. (c) 1.0-ml aliquot~ of cotyledon protoplasts in liquid KPS, at a density of 5 × 104/ml, were added to 2.0 ml volumes of M. sativa

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albino cell suspension protoplasts. The albino nurse protoplasts were cultured at a density of 5 × 104/ml at 24°C in the dark for 1--2 days prior to addition of cotyledon protoplasts from the wild Medicago species. The plasmolyticum level in the cultures reduced gradually by diluting the protoplast culture medium with 1.0 ml (a and c) or 2.0-ml (b) aliquots of a mixture of protoplast (KP8) and cell culture media (K8) after 7, 14 and 21 days. The proportions of protoplast to cell culture media in the mixtures were 2 :1, 1 :1 and 0 :1, respectively at the three dilution stages [23]. Plating efficiencies (number of dividing protoplasts expressed as a percentage of the total protoplast population) were estimated 7 days after protoplast isolation.

(5) Plant recovery One week after the last dilution step (day 28 o f culture), colonies were transferred to agar solidified (0.6% w/v; Sigma) K8 medium. 2--3 weeks later, protoplast~ierived callus tissues were sub-cultured to UM agar medium to induce embryogenesis. Somatic embryoids developed 4--6 weeks after transfer to UM medium, and these formed plantlets when placed on agar solidified (0.8% w/v; Sigma) MS medium with 0.05 mg1-1 NAA and 0.5 mg1-1 BAP (MSD4). R o o t development of regenerants was stimulated by transfer to hormone-free MS agar medium (0.8% w/v; Sigma) (MSO). Free floating embryoids were maintained in liquid culture and after the last dilution step, cultures were further diluted at weekly intervals with 1.0-ml aliquots of hormonefree liquid MSO medium. Six weeks after isolation, protoplast-derived globular embryoids were removed from liquid culture and placed on K8 agar medium. Germinated e m b r y o s were transferred to agar solidified MSO medium and grown in a similar way to plants regenerated via an intervening callus phase. When plantlets were 8--10 cm in height

and had developed an adequate r o o t system they were transferred to soft-less c o m p o s t and placed in a mist propagator to maintain humidity for 3--4 weeks. Plants were grown to maturity under greenhouse conditions. Results Seeds sown in batches of approximately 50 produced sterile seedlings from which the cotyledons could be excised and used for protoplast isolation w i t h o u t further sterilisation treatment. Jars containing surface sterilised seed could be stored at 4°C in the dark for 3 weeks prior to germination without reduction in protoplast yield or quality. Germination varied between species and ranged from almost 100% in M. varia to 30% in M. difalcata. Cotyledons which' reached maturity later than 7 days following incubation of seeds at 25°C were discarded. Protoplast yield (average from at least two experiments), ranged from 1.2 × 103 per cotyledon pair for M. falcata to 2 × 103 per pair for M. glutinosa and M. hemicycla (Table I). Cellular debris was eliminated b y slow centrifugation in KP8 culture medium, any debris remaining in suspension. The albino M. sativa cell suspension yielded

I. Comparison of protoplast yields and plating efficiencies (after 7 days in culture) of cotyledon protoplasts from wild Medicago sp. plated in liquid medium, agarose beads and co-cultivated with albino alfalfa nurse protoplasts. Table

Species

M. difalcata M. falcata M. glutinosa M. hemicycla M. varia

Average protoplast yield/pair cotyledons

% Plating efficiency ± S.D. Liquid Agarose culture beads

Nurse culture

xl0 s x103 ×10 s x103

44+4 3727 31~4 49~5

45~4

77-+4 61±7 65_+ 62± '

1.7 ×10 s

3824

21±4

81±5

1.25 1.2 2.0 2.0

52-+5 58i5 38+7

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1.75 × 106 protoplasts per g fresh weight, when cell clumps were harvested at midexponential phase (3--4 days after subculture) prior to enzyme incubation. Although more than 107 albino nurse culture protoplasts were plated, no green colonies were detected. This enabled colonies derived from seedling cotyledon protoplasts which had been mixed with albino nurse protoplasts, to be identified by their yellow-green pigmentation. Cotyledon protoplasts from each wild Medicago sp. exhibited a range of division frequencies according to the method of culture (Table I), the use of an albino nurse culture stimulating protoplast division especially in M. varia. In this culture system cotyledon protoplasts showed a two-fold increase in frequency of division compared to protoplasts plated in liquid culture without nurse cells. Colonies derived from cotyledon protoplasts showed a reduction in survival when transferred directly from liquid culture to U M medium, than when K 8 agar medium was used as an intervening stage. This step was found to be essential in promoting growth of colonies into small calli. Free floating embryos appeared in culture at a low frequency in three species; M. hemicycla at 0.004% of plated protoplasts, and in the case of M. falcata and M. glutinosa, at less than 0.001% of plated protoplasts. Somatic embryogenesis through an intervening callus phase gave higher rates of regeneration. Of the 2--3 m m diameter calli placed on U M agar medium, 46% proliferated and became embryogenic in M. hemicycla, 32% in M. glutinosa, 24% in M. falcata, 23% in M. varia and 8 % in M. difalcata (Table II). Embryogenic calli normally developed between 1 and 5 globular or jar shaped embryoids after 3--5 weeks on UM. When incubated on M S D 4 medium, embryoids developed into plantlets with a single unifoliate leaf, alternate trifoliate leaves, and root development occurring following transfer to M S O agar medium.

Table II. Embryogenic potential of protoplasts and protoplast-derived calli of wild Medicago sp.

Species

Total no. of protoplasts cultured

% of % of protoplasts protoplastwhich derived developed calli into free embryofloating genic on embryoids U M agar medium

M. difalcata M. falcata M. glutinosa M. hemicycla M. varia

1.3 2.0 1.2 1.8 2.3

0 < 0.001 < 0.001 0.004 0

x x x x x

10' 106 104 106 106

8 24 32 46 23

Discussion Protoplast yields from wild Medicago sp. were considerably lower than those previously obtained from seedling cotyledon protoplasts of the cultivated crop. Thus, 2 X 103 protoplasts per cotyledon pair were obtained from M. hemicycla and M. glutinosa, compared with 3.2 X 104 for M. sativa cv. "Europe" [16]. Despite the low yields, cotyledons have a number of advantages as a source of protoplasts. Seedlings require minimal space for growth, and environmental influences which may affect reproducibility of seedling growth and protoplast yield are minimized [16]. In addition, cotyledon protoplasts are less sensitive to the conditions of isolation than leaf mesophyll tissue, crude enzyme mixtures can be used, and high division frequencies are obtained [23]. Such protoplasts provide a useful system for genetic manipulations, including somatic hybridization, as colourless protoplasts from etiolated seedlings can be fused with green protoplasts facilitating heterokaryon identification [16]. Agarose has been used to improve protoplast culture in Nicotiana sp. and Hyoscyamus muticus [24,25], Lycopersicon esculentum, Crepis capillaris, Brassica rapa and Petunia hybrida [24], Daucus carota [25]

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and M. sativa [ 2 6 ] . T h e p r e s e n t s t u d y has s h o w n t h a t agarose is also beneficial t o division o f c o t y l e d o n protoplasts from wild Medicago sp. c o m p a r e d t o c u l t u r e in liquid m e d i u m . H o w e v e r , the m o s t e f f i c i e n t culture m e t h o d is t h a t involving c o - c u l t i v a t i o n o f freshly i s o l a t e d p r o t o p l a s t s w i t h dividing albino cell s u s p e n s i o n n u r s e p r o t o p l a s t s . P r o t o p l a s t s o f the M. sativa albino c u l t u r e m a y also p r o m o t e division o f p r o t o p l a s t s f r o m Medicago species o t h e r t h a n t h o s e described here, or possibly protoplasts of o t h e r legumes a n d n o n - l e g u m e s . P r o t o p l a s t s f r o m albino cell s u s p e n s i o n s have b e e n u s e d t o nurse h e t e r o k a r y o n s o f n o n - l e g u m e s , isolated b o t h m a n u a l l y [27] a n d b y a u t o m a t e d cell sorting [ 2 8 ] . A l b i n o alfalfa p r o t o plasts s h o u l d be p a r t i c u l a r l y useful in nursing Medicago h e t e r o k a r y o n s a n d t h o s e Medicago protoplasts modified by microinjection [9], w h e r e t h e availability o f a l i m i t e d n u m b e r o f cells d e m a n d s e f f i c i e n t c u l t u r e m e t h o d s . Acknowledgements Miss M. M c L e l l a n is a c k n o w l e d g e d f o r advice o n t h e agarose p l a t i n g t e c h n i q u e . T h e a u t h o r s t h a n k Mrs. J. R a y n e r a n d Mr. D. Wilson f o r t e c h n i c a l assistance. D.M. G i l m o u r was s u p p o r t e d b y a g r a n t f r o m the Biomolecular Engineering Programme o f t h e E.E.C. References 1 T. McCoy and K. Walker, Alfalfa, in: P.V. Ammirato, D.A. Evans, W.R. Sharp and Y. Yamada (Eds.), Handbook of Plant Cell Culture, Vol. 3, Crop Species, Collier Macmillan Publishers, London, 1983, p. 171. 2 D.K. Barnes, E.T. Bingham, R.P. Murphy, O.J. Hunt, D.F. Beard, W.H. Skrdla and L.R. Teuber, Alfalfa germplasm in the United States: genetic vulnerability, use, improvement and maintenance. USDA/ARS Technical Bulletin 1571, 1977. 3 S. Arcioni, M.R. Davey, A.V.P. dos Santos and E.C. Cocking, Z. Pflanzenphysiol., 106 (1982) 105. 4 T. McCoy and L.Y. Smith, Can. J. Genet. Cytol., 26 (1984) 511.

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