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Plant Regeneration from Strawberry (Fragaria x Ananassa) Mesophyll Protoplasts
J. PlantPhysiol. Vol. 133. pp. 375-377(1988}
Short Conlnlunication
Plant Regeneration from Strawberry (Fragaria x Ananassa) Mesophyll Protoplasts MARIE NYMAN
and
ANITA WALLIN
Department of Physiological Botany of the University of Uppsala, Box 540, S-751 21 Uppsala, Sweden Received April 4, 1988 . Accepted May 13, 1988
Summary Leaf mesophyll protoplasts were enzymatically isolated from in vitro-grown plants of Fragaria x ananassa Duch. (strawberry). A reproducible high yield of protoplasts was obtained when leaves from plants grown on hormone-free medium with low sugar concentration were treated with an enzyme solution containing 1.0 % (w/v) Cellulysin and 0.05 % (w/v) Pectolyase. When cultured in a modified 8p-medium containing 1.0 mg/l 2,4-D and 0.5 mg/l BA, the protoplasts retained their cells walls, started to divide and formed cell colonies. Plants were regenerated on solid MS-medium with addition of 0.2 mg/l NAA and S.0-10.0mg/1 BA.
Key words: Fragaria x ananassa, plant regeneration, protoplast isolation. Abbreviations: BA = N6-benzyladenine, 2,4-D = 2,4-dichlorophenoxy acetic acid, IBA butyric acid, MS = Murashige and Skoog (1962), NAA = naphthalene acetic acid.
Introduction In vegetatively propagated species with strong heterozygosity and high ploidy levels, unconventional breeding methods such as somatic hybridization, direct gene transfer and induction of somaclonal variation are of special interest. In strawberry breeding, where it is desirable to introduce specific traits, i.e. winter hardiness, earliness and disease resistance, into already established cultivars, these techniques have a potential value. Prerequisites for the practical use of protoplasts in attempts to change a plants genome are efficient procedures for isolation and for plant regeneration. To our knowledge, there is only one brief report on this subject concerning strawberry (Binding et al. 1981), and as far as we know, this is the first report on plant regeneration using leaf tissue as source material.
Material and Methods Plant material
Leaf material was taken from shoots of two in vitro-cultured strawberry cultivars, Sengana and Canoga. The shoots were grown © 1988 by Gu:stav Fischer Verlag, Stuttgart
Indole-3-
on shoot-inducing medium, a modified MS-medium containing 4 % glucose, 1.5 mg/I BA and 0.75 mg/I IBA. Young shoots were transferred to hormone-free medium with different sugar concentrations (Fig. 5) 2-3 weeks before protoplast isolation.
Protoplast isolation and purification
Leaves were cut into pieces in a preplasmolysis solution containing 0.3 M sorbitol and 0.05 M CaCh· H 2 0 (Carlberg et al. 1983). After one hour, the solution was replaced by cell wall degrading enzymes dissolved in medium K3 (Menzel et al. 1981) with DAM sucrose. Five different enzymes, Cellulysin (Calbiochem-Behring), Cellulase «Onozuka RIO" (Serva), Macerase (Calbiochem-Behring), Macerozyme RIO (Serva) and Pectolyase (Sigma), were used in various concentrations (Table 1) and incubation took place overnight (15 -18 h) in darkness at 25°C. Released protoplasts were separated from the undigested material by filtering through a 53/Lm nylon sieve. The protoplast suspension was diluted 1: 1 with CPW 16 (Banks and Evans 1976) and centrifuged at 100g for 5 minutes. Floating protoplasts were collected from several centrifuge tubes and 2 ml of the collected suspension was poured on top of 8 ml CPW 16 and centrifuged as above. Finally the centrifugation procedure was repeated in W5 (Menzel et al. 1981) to pellet the protoplasts.
376
MARIE NYMAN and ANITA WALLIN
Protoplast culture and plant regeneration Freshly isolated protoplasts were cultured at a density of 50,000 protoplasts/ml in a modified 8p-medium (Glimelius et al. 1986) containing O.4M glucose, 1.0 mg/I 2,4-D and 0.5 mg/I BA. After three days in culture, the auxin concentration and protoplast density was
Fig. 1: Fig. 2: Fig. 3: Fig. 4:
lowered by diluting the culture medium with modified 8p-medium lacking 2,4-D. When cell colonies had been formed after 3 -4 weeks, the liquid culture was poured onto semi-solid 8p-medium containing 1 % sucrose, 0.2 M mannitol, 1.0 mg/I BA, 0.1 mg/I NAA and 0.2 % agarose. As the cell colonies reached a size of about 2 - 3 mm in diameter, they were transferred to plant regeneration medium, a
Strawberry shoots growing on shoot-inducing medium. Freshly isolated mesophyll protoplasts. Cell division. Protoplast derived strawberry plant growing on hormone-free medium.
Plants from Fragaria x ananassa protoplasts
377
Table 1: Yield of protoplasts from leaves of plants (cv. Sengana) cultured on hormone-free medium with 1 % sucrose. Enzyme combinations
..
Yield of protoplasts/gram fresh weight. (X±S.E.) 1.0 % Cellulysin/O.5 % Macerase 6.1 x 105 (± 5.0 x 105) 1.0% Cellulysin/0.1 % Macerase 3.8x 105 (±3.0x 105) 1.0% Cellulase Rl0/0.l % Macerozyme RIO 2.2 x 105 (± 1.3 x 105) 1.0 % Cellulysin/O.l % Pectolyase 18.8 x 105 (H.O X 105) 1.0% Cellulysin/O.OS % Pectolyase 23.6 x 105 (±7.1 X 105)
20
r. .2' GI
~ 15
r.
1/1
~
E tel
t;, 10
"1/1
ti
.!!!
Q.
o
£
5
1% S
1% G 2% G 3% G 4% G
Fig. 5: Yield of protoplasts from leaves of plants cultured in vitro on medium with different sugar concentrations. S = sucrose, G = glucose. Cell walls were digested overnight at 25°C in 1.0% (w/v) Cellulysin and 0.05 % (w/v) Pectolyase dissolved in medium K3 with O.4M sucrose. 0 = cv. Sengana ~ = cv. Canoga. Bars represent the mean ± S.E. calculated from four independent experiments. In each experiment, 500 protoplasts were counted. MS-medium with 2% sucrose, 0.2mg/l NAA, 5.0-10.0mg/l BA and 0.6 % agarose.
Results and Discussion Culturing strawberry shoots on BA-containing medium resulted in rapid shoot production, confirming results reported by Boxus (1974). Shoots grown on shoot-inducing medium had short petioles, more or less folded leaves and no roots (Fig. 1). Under these culture conditions, the plant material only sporadically yielded protoplasts after enzyme treatment. When the shoots were transferred to hormone-free medium they developed into plants with unfolded leaves, extended growth of the petioles and root growth, but still the protoplast yield was very low. However, by reducing the sugar concentration in the culture medium, the protoplast yield was greatly increased (Fig. 5). To obtain leaves suitable for protoplast preparation, young shoots were transferred from shoot-inducing medium to fresh hormone-free medium with low sugar concentration (1 % sucrose or glucose) 2-3
weeks before isolation. A reproducible high yield of protoplasts was obtained only when Pectolyase was included in the enzyme solution (Table 1). Protoplasts re-synthesized cell walls within 24 hours and started to divide two days later. In many species, plant regeneration is the limiting step in protoplast techniques. This seems not to be the case with strawberries, which in our experiments have shown a great tendency to regenerate plants from callus. After four weeks on regeneration medium, the first shoots were visible on the surface of the calli. These shoots were then easily rooted by transfer to hormone-free medium (Fig. 4). Preliminary experiments indicate that shoot formation occurs only on medium containing high levels of BA (5.0-10.0 mg/l). On these media shoots were obtained from 70 -75 % of the calli initially transferred to regeneration medium. The most important of the remaining problems in our work on strawberry protoplasts is the poor survival of the protoplasts during the first days after isolation. Work is however in progress which ensures greater survival of the protoplasts and thereby enable their use in strawberry breeding. Acknowledgements We wish to thank The Swedish University of Agricultural Sciences, Division of Fruit Breeding, Balsgard, for providing us with plant material. This work was supported by grants from the Swedish National Board for Technical Development.
References BANKS, M. S. and P. K. EVANS: Plant Sci. Lett. 7, 409-416 (1976). BINDING, H., R. NEHLS, R. KOCK, J. FINGER, and G. MORDHORST: Z. Pflanzenphysiol. 101, 119-130 (1981). Boxus, P.: J. Hort. Sci. 49, 209-210 (1974). CARLBERG, I., K. GLIMELIUS, and T. ERIKSSON: Plant Cell Report 2, 223-225 (1983). GLIMELIUS, K., M. DJUPSJOBACKA, and H. FELLNER-FELDEGG: Plant Science 45, 133-141 (1986). MENZEL, L., F. NAGY, Zs. R. KIZZ, and P. MALIGA: Theor. Appl. Genet. 59, 191-195 (1981). MURASHIGE, T. and F. SKOOG: Physiol. Plant. 15, 473 -497 (1962).
Short Conlnlunication
Plant Regeneration from Strawberry (Fragaria x Ananassa) Mesophyll Protoplasts MARIE NYMAN
and
ANITA WALLIN
Department of Physiological Botany of the University of Uppsala, Box 540, S-751 21 Uppsala, Sweden Received April 4, 1988 . Accepted May 13, 1988
Summary Leaf mesophyll protoplasts were enzymatically isolated from in vitro-grown plants of Fragaria x ananassa Duch. (strawberry). A reproducible high yield of protoplasts was obtained when leaves from plants grown on hormone-free medium with low sugar concentration were treated with an enzyme solution containing 1.0 % (w/v) Cellulysin and 0.05 % (w/v) Pectolyase. When cultured in a modified 8p-medium containing 1.0 mg/l 2,4-D and 0.5 mg/l BA, the protoplasts retained their cells walls, started to divide and formed cell colonies. Plants were regenerated on solid MS-medium with addition of 0.2 mg/l NAA and S.0-10.0mg/1 BA.
Key words: Fragaria x ananassa, plant regeneration, protoplast isolation. Abbreviations: BA = N6-benzyladenine, 2,4-D = 2,4-dichlorophenoxy acetic acid, IBA butyric acid, MS = Murashige and Skoog (1962), NAA = naphthalene acetic acid.
Introduction In vegetatively propagated species with strong heterozygosity and high ploidy levels, unconventional breeding methods such as somatic hybridization, direct gene transfer and induction of somaclonal variation are of special interest. In strawberry breeding, where it is desirable to introduce specific traits, i.e. winter hardiness, earliness and disease resistance, into already established cultivars, these techniques have a potential value. Prerequisites for the practical use of protoplasts in attempts to change a plants genome are efficient procedures for isolation and for plant regeneration. To our knowledge, there is only one brief report on this subject concerning strawberry (Binding et al. 1981), and as far as we know, this is the first report on plant regeneration using leaf tissue as source material.
Material and Methods Plant material
Leaf material was taken from shoots of two in vitro-cultured strawberry cultivars, Sengana and Canoga. The shoots were grown © 1988 by Gu:stav Fischer Verlag, Stuttgart
Indole-3-
on shoot-inducing medium, a modified MS-medium containing 4 % glucose, 1.5 mg/I BA and 0.75 mg/I IBA. Young shoots were transferred to hormone-free medium with different sugar concentrations (Fig. 5) 2-3 weeks before protoplast isolation.
Protoplast isolation and purification
Leaves were cut into pieces in a preplasmolysis solution containing 0.3 M sorbitol and 0.05 M CaCh· H 2 0 (Carlberg et al. 1983). After one hour, the solution was replaced by cell wall degrading enzymes dissolved in medium K3 (Menzel et al. 1981) with DAM sucrose. Five different enzymes, Cellulysin (Calbiochem-Behring), Cellulase «Onozuka RIO" (Serva), Macerase (Calbiochem-Behring), Macerozyme RIO (Serva) and Pectolyase (Sigma), were used in various concentrations (Table 1) and incubation took place overnight (15 -18 h) in darkness at 25°C. Released protoplasts were separated from the undigested material by filtering through a 53/Lm nylon sieve. The protoplast suspension was diluted 1: 1 with CPW 16 (Banks and Evans 1976) and centrifuged at 100g for 5 minutes. Floating protoplasts were collected from several centrifuge tubes and 2 ml of the collected suspension was poured on top of 8 ml CPW 16 and centrifuged as above. Finally the centrifugation procedure was repeated in W5 (Menzel et al. 1981) to pellet the protoplasts.
376
MARIE NYMAN and ANITA WALLIN
Protoplast culture and plant regeneration Freshly isolated protoplasts were cultured at a density of 50,000 protoplasts/ml in a modified 8p-medium (Glimelius et al. 1986) containing O.4M glucose, 1.0 mg/I 2,4-D and 0.5 mg/I BA. After three days in culture, the auxin concentration and protoplast density was
Fig. 1: Fig. 2: Fig. 3: Fig. 4:
lowered by diluting the culture medium with modified 8p-medium lacking 2,4-D. When cell colonies had been formed after 3 -4 weeks, the liquid culture was poured onto semi-solid 8p-medium containing 1 % sucrose, 0.2 M mannitol, 1.0 mg/I BA, 0.1 mg/I NAA and 0.2 % agarose. As the cell colonies reached a size of about 2 - 3 mm in diameter, they were transferred to plant regeneration medium, a
Strawberry shoots growing on shoot-inducing medium. Freshly isolated mesophyll protoplasts. Cell division. Protoplast derived strawberry plant growing on hormone-free medium.
Plants from Fragaria x ananassa protoplasts
377
Table 1: Yield of protoplasts from leaves of plants (cv. Sengana) cultured on hormone-free medium with 1 % sucrose. Enzyme combinations
..
Yield of protoplasts/gram fresh weight. (X±S.E.) 1.0 % Cellulysin/O.5 % Macerase 6.1 x 105 (± 5.0 x 105) 1.0% Cellulysin/0.1 % Macerase 3.8x 105 (±3.0x 105) 1.0% Cellulase Rl0/0.l % Macerozyme RIO 2.2 x 105 (± 1.3 x 105) 1.0 % Cellulysin/O.l % Pectolyase 18.8 x 105 (H.O X 105) 1.0% Cellulysin/O.OS % Pectolyase 23.6 x 105 (±7.1 X 105)
20
r. .2' GI
~ 15
r.
1/1
~
E tel
t;, 10
"1/1
ti
.!!!
Q.
o
£
5
1% S
1% G 2% G 3% G 4% G
Fig. 5: Yield of protoplasts from leaves of plants cultured in vitro on medium with different sugar concentrations. S = sucrose, G = glucose. Cell walls were digested overnight at 25°C in 1.0% (w/v) Cellulysin and 0.05 % (w/v) Pectolyase dissolved in medium K3 with O.4M sucrose. 0 = cv. Sengana ~ = cv. Canoga. Bars represent the mean ± S.E. calculated from four independent experiments. In each experiment, 500 protoplasts were counted. MS-medium with 2% sucrose, 0.2mg/l NAA, 5.0-10.0mg/l BA and 0.6 % agarose.
Results and Discussion Culturing strawberry shoots on BA-containing medium resulted in rapid shoot production, confirming results reported by Boxus (1974). Shoots grown on shoot-inducing medium had short petioles, more or less folded leaves and no roots (Fig. 1). Under these culture conditions, the plant material only sporadically yielded protoplasts after enzyme treatment. When the shoots were transferred to hormone-free medium they developed into plants with unfolded leaves, extended growth of the petioles and root growth, but still the protoplast yield was very low. However, by reducing the sugar concentration in the culture medium, the protoplast yield was greatly increased (Fig. 5). To obtain leaves suitable for protoplast preparation, young shoots were transferred from shoot-inducing medium to fresh hormone-free medium with low sugar concentration (1 % sucrose or glucose) 2-3
weeks before isolation. A reproducible high yield of protoplasts was obtained only when Pectolyase was included in the enzyme solution (Table 1). Protoplasts re-synthesized cell walls within 24 hours and started to divide two days later. In many species, plant regeneration is the limiting step in protoplast techniques. This seems not to be the case with strawberries, which in our experiments have shown a great tendency to regenerate plants from callus. After four weeks on regeneration medium, the first shoots were visible on the surface of the calli. These shoots were then easily rooted by transfer to hormone-free medium (Fig. 4). Preliminary experiments indicate that shoot formation occurs only on medium containing high levels of BA (5.0-10.0 mg/l). On these media shoots were obtained from 70 -75 % of the calli initially transferred to regeneration medium. The most important of the remaining problems in our work on strawberry protoplasts is the poor survival of the protoplasts during the first days after isolation. Work is however in progress which ensures greater survival of the protoplasts and thereby enable their use in strawberry breeding. Acknowledgements We wish to thank The Swedish University of Agricultural Sciences, Division of Fruit Breeding, Balsgard, for providing us with plant material. This work was supported by grants from the Swedish National Board for Technical Development.
References BANKS, M. S. and P. K. EVANS: Plant Sci. Lett. 7, 409-416 (1976). BINDING, H., R. NEHLS, R. KOCK, J. FINGER, and G. MORDHORST: Z. Pflanzenphysiol. 101, 119-130 (1981). Boxus, P.: J. Hort. Sci. 49, 209-210 (1974). CARLBERG, I., K. GLIMELIUS, and T. ERIKSSON: Plant Cell Report 2, 223-225 (1983). GLIMELIUS, K., M. DJUPSJOBACKA, and H. FELLNER-FELDEGG: Plant Science 45, 133-141 (1986). MENZEL, L., F. NAGY, Zs. R. KIZZ, and P. MALIGA: Theor. Appl. Genet. 59, 191-195 (1981). MURASHIGE, T. and F. SKOOG: Physiol. Plant. 15, 473 -497 (1962).