Plant Cell Reports

Plant Cell Reports (t983) 2:101-104

© Springer-Verlag 1983

Prolific Plant Regeneration from Protoplast-Derived Tissues of Lotus corniculatus L. (Birdsfoot Trefoil) P.S.Ahuja, S. Hadiuzzaman *, M. R. Davey and E. C. Cocking Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, University Park, Nottingham NG7 2RD, UK

Received March 25, 1983

ABSTRACT Protoplasts isolated enzymatically from seedling roots, hypocotyls and cotyledons of £o¢~s eovn~e~;a#~s L. produced callus which underwent p r o l i f i c shoot regeneration. The r a p i d i t y and ease of recovering plants from protoplast-derived tissues makes t h i s forage legume an a t t r a c t i v e experimental system f o r genetic manipulation. INTRODUCTION ~ o ~ eom~#e~;a~s L. (birdsfoot t r e f o i l ) is a high tannin containing bloat-safe t e t r a p l o i d legume which is grown for pasture in Eastern Canada and the United States. In B r i t a i n i t is widely d i s t r i b u t e d and able to grow on s o i l s too poor to support legumes such as ~ i ~ o ~ m p ~ a ~ s e L. (red clover) and ~ed~eaFo ~#~va L. (lucerne), but is now seldom grown as a forage crop. Even though t h i s plant does not hold an important place in B r i t i s h A g r i c u l t u r e , or in temperate a g r i c u l t u r e in general, Rogers (1975) suggested i t would be wise to continue t e s t i n g in the United Kingdom new v a r i e t i e s r e s u l t i n g from the North American breeding programmes. Tomes (1979) established tissue cultures f o r m u l t i p l i c a t i o n of Canadian v a r i e t i e s of ~.eo~n~e~a~s L. including both normal and dwarf genotypes (Tomes & Peterson, 1981), and emphasised the a t t r a c t i o n of aseptic methods in terms of space considerations and in preventing loss of genotypes f r e q u e n t l y encountered in f i e l d nurseries during inclement weather. He found maintenance of cultures at reduced temperatures of 24oc to be p a r t i c u l a r l y u s e f u l , the Cultured shoots having a high survival rate on t r a n s f e r to compost. The regeneration c a p a b i l i t y of £o¢~ cultures has been exploited for the i s o l a t i o n of variants such as those r e s i s t a n t to 2,4-dichlorophenoxyacetic acid (2,4-D), which f i n d a p p l i c a t i o n under f i e l d conditions (Swanson & Tomes, 1980). However, t h i s v a r i a t i o n a r i s i n g from c a l l u s and c e l l suspensions is r e s t r i c t e d to changes w i t h i n the plant genome, and suitable protoplast systems are essential to e x p l o i t i n t e r s p e c i f i c and i n t e r g e n e r i c gene flow. This report discusses the establishment of various protoplast systems suitable f o r the genetic manipulation of somatic c e l l s of ~o¢~s eormie~Za¢~ L. together with the conditions f o r plant regeneration.

MATERIALS AND METHODS Pot-grown and s t e r i l e seedlings - Seeds of £o#~s comnie~;a¢ms cv Leo and Maitland were obtained from

* On leave from Department of Botany, University of Dacca, Bangladesh

the Department of Crop Science, U n i v e r s i t y of Guelph, Ontario, Canada NIG 2Wl, and sown in s o i l - l e s s compost. Seedlings were maintained in a c o n t r o l l e d environment cabinet (Santos et a l , 1980). Other seed samples were surface s t e r i l i s e d in I0% v/v Domestos bleach (Lever Bros. UK) f o r 20 m and washed with 5 changes of s t e r i l e tap water. Seeds were germinated on agar s o l i d i f i e d (0.8% w/v; Sigma) Murashige and Skoog (1962) based medium (MS) containing 3% sucrose, but lacking growth regulators, and incubated in the dark or in the l i g h t (2,000 l u x , d a y l i g h t f l u o r e s cent tubes) at 23±2°C. Callus and suspension cultures - Callus was induced on aseptic seedling hypocoty'l explants using agar s o l i d i f i e d (0.8%; Sigma) Uchimiya and Murashige (1974) medium (UM). Cultures were maintained under d i f f u s e l i g h t (800 l u x ) . Cell suspensions were i n i t i a t e d by t r a n s f e r of callus to 50 ml a l i q u o t s of UM l i q u i d medium, with a g i t a t i o n on a rotary shaker (80 rpm, 800 l u x , 18°C). Suspensions were t r a n s f e r red every 8 days. Protoplast i s o l a t i o n - Expanded leaves were excised from 30 to 50-day-oTd plants and s t e r i l i s e d using I0% v/v Domestos for 20 mimPeeled l e a f l e t s , with t h e i r lower epidermis removed, were plasmolysed for I h by placing the exposed mesophyll in contact with CPW salts solution (Frearson et a l , 1973) containing 13% w/v mannitol. Roots, hypocotyls and cotyledons were excised from 2 to 7 - d a y - o l d aseptic seedlings, cut into 0.5 mm transverse sections and plasmolysed in a s i m i l a r manner. In some cases seedlings were maintained at 4oc f o r 48 h in the dark p r i o r to use. The solution used to plasmolyse l e a f l e t s was replaced with one of eight enzyme mixtures (see Table l , Results), while plasmolysed sections of roots, hypocotyls and cotyledons were incubated in enzyme mixtures B and E (Table l ) . Enzyme incubation was at 28°C f o r 6 to 16 h in the dark without a g i t a t i o n . Protoplasts were released by gently squeezing the plant material. Suspension c e l l s were used 7 days a f t e r sub-culture, and incubated in enzyme mixture E (Table l ) for 16 h with a g i t a t i o n on a rotary shaker (80 rpm). Protoplasts were passed successively through nylon sieves of pore sizes 64 um and 20 um and p e l l e t t e d by c e n t r i f u g a t i o n (80 x g, 5 m~nl. Preparations were washed by f u r t h e r c e n t r i f u g a t i o n through two changes of CPW salts solution with 13% w/v mannitol, followed by two changes of protoplast c u l ture medium. Protoplast c u l t u r e - Protoplasts were cultured at l.O x lO 5 ml -! in 40 x 12 mm and 54 x 14 mm Nunclon dishes (A/S Nunc, Kamstrup, DK-4000 Roskilde, Denmark) containing 1.5 ml and 4.0 ml r e s p e c t i v e l y

102 of l i q u i d KM8P medium (Kao and Michayluk, 1975) or protoplast c u l t u r e medium (Kao, 1977). In some experiments, isolated protoplasts were maintained in culture medium at 4°C for 48 h in the dark before p l a t i n g . The media were d i l u t e d at i n t e r v a l s of 5 days with 0.5 ml or 1.0 ml volumes (depending on the size of culture vessel) of KM8P or protoplast culture medium previously mixed with c e l l culture medium A (Kao & Michayluk, 1980) in the proportions of 3:1, 2:1, I : I , and 1:2 volume:volume. F i n a l l y , protoplasts were maintained in c e l l culture medium alone. Cultures were incubated in the dark for 72 h (27oc), and subsequently under a l i g h t i n t e n s i t y of 800 lux ( d a y l i g h t fluorescent tubes) at 25°C. Protoplast p l a t i n g e f f i c i e n c y (number of d i v i d i n g protoplasts expressed as a percentage of the t o t a l protoplast population) was recorded a f t e r 7 days of c u l t u r e . Cell colonies were transferred to agar s o l i d i f i e d (0.8% w/v; Sigma ) MS based medium containing 0.2, 0.5 or 1.0 mg L - i of 6-benzylaminopurine (BAP) or zeatin for regeneration. Regenerated shoots were transferred to hormone-free MS agar medium f o r root induction, and p l a n t l e t s potted in s o i l - l e s s compost, hardened o f f and maintained in the greenhouse. RESULTS AND DISCUSSION A mixture of Cellulase RIO, Macerozyme RIO and Rhozyme HP 150 (enzyme mixture A; Table I ) previously employed f o r the routine i s o l a t i o n of mesophyll protoplasts of Mediaago sativa L. (Kao and Michayluk, 1980), Trifolium repens L. and Onobrychis viciifolia Scop. (Ahuja et a l , 1983), was i n e f f e c t i v e in releasing protoplasts from Lotus l e a f l e t s . The mixture was also i n e f f e c t i v e f o l l o w i n g the addition of Driselase (mixture B). Protoplast release necessitated the use of P e c t o l y t i c enzyme (mixtures G and H), but such protoplasts were r e l a t i v e l y unstable during subsequent culture. Protoplasts from seedling roots, hypocotyls and cotyledons were obtained r e a d i l y using enzyme mixtures B and E (Table l ) y i e l d s being higher with the former s o l u t i o n . Enzyme mixture E, consisting of Meicelase, Rhozyme and Macerozyme was tested since i t proved to be useful in e a r l i e r studies on the isol a t i o n of protoplasts from seedling roots (Xu et a l , 1981, 1982a,b,c) and cotyledons (Lu et a l , 1982) of a number of plants. The optimum age a f t e r sowing in Lotus for maximum protoplast release varied with the explant used as source material (Table 2), preventing the simultaneous i s o l a t i o n of protoplasts from d i f f e r e n t explants of the same batch of seedlings. Cell suspensions were a convenient source of material, 3.0 - 4.0 X lOb protoplasts being obtained from the contents of a single 250 ml c u l t u r e vessel (approx 15 g fresh weight) 6 to 7 days a f t e r t r a n s f e r of the suspension c e l l s to fresh medium. P u r i f i c a t i o n of a l l protoplast preparations was d i f f i c u l t because of the small size of the isolated protoplasts (15 to 20 vm diameter), e l i m i n a t i n g the use of sucrose f l o t a t i o n as a method of removing c e l l u l a r debris (Power & Davey, 1980). Mesophyll protoplasts showed a low p l a t i n g e f f i c i e n c y (