Journal of Experimental Botany, Vol. 51, No. 348, pp. 1237-1242,
July 2000
© 2000 Oxford University Press
Original papers |
Efficient intergeneric fusion of pea (Pisum sativum L.) and grass pea (Lathyrus sativus L.) protoplasts
INRA, Centre de Recherches de Dijon, Unité de Recherches en Génétique et Amélioration des Plantes, Laboratoire de Physiologie et Culture in Vitro, 21110 Bretenières, France
Received 17 November 1999; Accepted 21 March 2000
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
|---|
Large numbers of viable protoplasts of pea (Pisum sativum) and grass pea (Lathyrus sativus) were efficiently and reproducibly obtained and, for the first time, fused. Different procedures for fusion were compared, based either on electrofusion (750, 1000, 1250 or 1500 V cm-1), or on the use of macro or micro-methods with a polyethylene glycol (PEG 6000 or PEG 1540), or a glycine/high pH solution. Over 10% of viable heterokaryons were obtained, with PEG as the most efficient and reproducible agent for protoplast fusion (>20% of viable heterokaryons). Both the division of heterokaryons and the formation of small calluses were observed.
Key words: Protoplast fusion, Pisum sativum L., Lathyrus sativus L., grain legumes.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Pea (Pisum sativum L.) is an important protein-rich crop, and is increasingly used as an animal feed, with France as the main producer in Europe. New varieties are being developed to adapt this grain legume to various abiotic and biotic stresses. One way of increasing the genetic variability of peas is by somatic hybridization, which can help to introduce new characters from other species or genera. In this context, the grass pea (Lathyrus sativus L.) is a wild relative of pea that possesses several interesting agronomic traits that might be useful for P. sativum, especially in terms of disease resistance (Campbell, 1997
). As these species are cross-incompatible, the only way to obtain such intergeneric hybrids is protoplast fusion and somatic hybridization. Several papers have been published on protoplast isolation from pea tissues (Lehminger-Mertens and Jacobsen, 1989, 1993; Puonti-Kaerlas et al., 1992; Ochatt et al., 2000a). Most authors reported callus regeneration, some obtained shoots (Puonti-Kaerlas and Eriksson, 1988; Lehminger-Mertens and Jacobsen, 1989; Böhmer et al., 1995) and, more rarely, whole plants were regenerated either via embryogenesis (Lehminger-Mertens and Jacobsen, 1993) or organogenesis and embryogenesis (Ochatt et al., 2000a). Conversely, for Lathyrus sp. calluses were obtained from L. odoratus L. protoplasts (Razdan et al., 1980), but not even protoplast isolation has been reported for L. sativus. On the other hand, polyethylene glycol was used for the fusion of Pisum protoplasts (Kao et al., 1974; Kao and Michayluk, 1974; Constabel and Kao, 1974), but not electrofusion. In this study, a range of chemical and electrical fusion methods has been tested, to select the most efficient and reproducible one. |
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Plant material
Young (3–5 cm) shoots from embryo axes of Pisum sativum cv. Frisson and of a white-seeded Lathyrus sativus genotype were used as the source of material for protoplast isolation. Dry seeds were surface-sterilized and imbibed overnight as reported elsewhere (Ochatt et al., 2000b
). Embryo axes were then excised as described previously (Lehminger-Mertens and Jacobsen, 1993) and cultured in hormone-free B5 medium (Gamborg et al., 1968) with 10 mM NH4Cl, 3% sucrose and 0.6% agar (Böhmer et al., 1995). For germination, plates with 10 axes each were placed at 24/22 °C, with a 16/8 h (light/dark) photoperiod of 90 µE m-2 s-1 from cool white fluorescent tubes.
Standard protoplast isolation
Protoplasts were isolated as described previously (Ochatt et al., 2000a). Briefly, epicotyls were finely chopped and plasmolysed for 1 h in 10 cm3 CPW medium (Frearson et al., 1973) with 9% (Pisum) or 13% (Lathyrus) mannitol. Tissues were digested overnight on a continuous rotary shaker in an enzyme solution based on LP* medium (modified from Lehminger-Mertens and Jacobsen, 1989) with 72 g l-1 of myo-inositol containing 2% Macerozyme R-10, 5% Fluka Cellulase (from Trichoderma viride) and 0.1% Pectolyase Y-23 for Pisum, but 3% Macerozyme R-10, 4% Cellulase Onozuka RS and 0.2% Pectolyase Y-23 for Lathyrus. Protoplasts were sieved (40 µm) and centrifuged successively at 35 g (5 min, 10 °C) and 70 g (5 min, 10 °C). Each pellet was resuspended in 250 mm3 of the appropriate plasmolyticum. Pellets were mixed together and were finally layered on top of 7 cm3 of CPW solution plus 21% sucrose, and spun at 80 g (10 min, 10 °C). The protoplast density was determined and the viability evaluated with fluorescein diacetate (FDA) under UV light (B1 IF 420–485 filter) as described earlier (Widholm, 1972).
Isolation of protoplasts for fusion
For chemical fusion:
Plasmolysis, digestion and the two first centrifugation steps were identical to those of the standard isolation protocol but, before the third centrifugation, pellets were stained with FDA (Lathyrus) or Rhodamine B isothiocyanate (Pisum). Both staining solutions were prepared by adding 150 mm3 from a stock (of 5 mg FDA or 30 mg Rhodamine per cm3 of acetone) to 7 cm3 of plasmolyticum, from which five drops (approximately 150 mm3) were added to the pellets with Pisum or Lathyrus protoplasts, prior to floating them on a sucrose gradient as above. Under UV light, protoplasts stained with FDA fluoresced yellow-green while those stained with Rhodamine B fluoresced red. Density and viability were evaluated as described previously.
For electrofusion:
All steps were identical to the isolation procedure for chemical fusion, but all solutions were devoid of salts, i.e. mannitol at 9% (w/v) for Pisum but at 13% (w/v) for Lathyrus, were used as plasmolytica. The pellets were resuspended in an electroporation solution consisting of 6 mM MgCl2, 200 mM MgSO4, 0.5 M mannitol, and 3 mM MES modified from that of Rech et al. (Rech et al., 1987) and the protoplasts were floated on a 21% (w/v) sucrose-containing CPW solution (Power and Davey, 1990). Density and viability were evaluated as described above.
Protoplast fusion
Macromethod:
Equal volumes of stained protoplasts of each species were mixed (1 : 1, v/v) with the fusion solution (Table 1) and spun at 100 g (10 min, 25 °C). The pellet was resuspended in a 2 : 1 vol. of washing solution prepared by adding 0.74% (w/v) CaCl2.2H2O to CPW 13 M (Power and Davey, 1990) and centrifuged as before. Fused and rinsed protoplasts were finally resuspended (1 : 1, v/v) in washing solution, and the percentage of heterokaryons formed was determined by counting the protoplasts that fluoresced both green and red under UV light.
|
Micromethod:
Small volumes (about 150 mm3 with 105 protoplasts) of stained protoplasts of each partner were dispensed in the centre of culture wells and, after 20 min, were mixed (1 : 1) with the fusing agent (Table 1
); 10 min later, the fusion solution was replaced by a 2 : 1 volume of washing solution. After 10 min, this solution was removed and all wells were filled to 1 cm3 with culture medium. The efficiency of heterokaryon formation was assessed as above.
Electrofusion
Aliquots (400 mm3) of a 1 : 1 (v/v) mixture of stained protoplasts of each species were dispensed into the cuvettes of an Electro Cell Manipulator ECM®6OO (BTX, California) with electrodes 1 mm apart. Three pulses at 750, 1000, 1250 or 1500 V cm-1 were delivered at 10 s intervals by discharging a 75 µF capacitor. Protoplasts were collected, the percentage of heterokaryons produced was determined under UV light, and they were placed in culture medium.
Culture
Protoplasts were cultured at 105 cm-3, either in media based on LP* medium with 60 g l-1 myo-inositol plus 0.2 mg l-1 picloram and 0.5 mg l-1 kinetin (LP*60), or 0.1 mg l-1 picloram and 0.5 mg l-1 thidiazuron (LP*Tdz) or in media based on KM (Kao and Michayluk, 1975) medium with 0.1 mg l-1 2,4-D, 0.2 mg l-1 zeatin and 1 mg l-1 NAA (KP), 0.1 mg l-1 2,4-D, 0.2 mg l-1 thidiazuron and 1 mg l-1 NAA (KPTdz); 0.1 mg l-1 picloram, 0.2 mg l-1 zeatin and 1 mg l-1 NAA (Kpic) or 0.1 mg l-1 picloram, 0.2 mg l-1 thidiazuron and 1 mg l-1 NAA (KpicTdz). After 1 week, a dilution was performed with the same medium and, as soon as the majority of cells had regenerated their wall, weekly dilutions were carried out with culture medium containing 30 g l-1 myo-inositol (instead of 60 g l-1) for LP*-based media and 20 g l-1 sucrose and 10 g l-1 glucose (instead of 250 mg l-1 and 100 g l-1, respectively) for KM-based media.
Experiments were repeated at least twice, with a minimum of three replicated dishes per fusion treatment. Results were expressed as the percentage of dividing heterokaryon-derived cells, and were statistically analysed by ANOVA (P=0.05).
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Isolation and culture
With the isolation protocol used, more than 90% of viable protoplasts were obtained (Fig. 6
) for both pea and Lathyrus. Such a high viability is a prerequisite for success with their subsequent fusion. A density of 2.0±0.2x106 protoplasts g-1 FW of digested tissues, sufficient for culture at the initial plating density of 1x105 protoplasts cm-3, was consistently obtained.
|
This is the first report of the successful isolation of large numbers of highly viable protoplasts from tissues of L. sativus. In the past, only L. odoratus had been studied in this context (Razdan et al., 1980
). The enzyme mixture used was close to one already employed for pea (Lehminger-Mertens and Jacobsen, 1989), but the cellulase used in the current study was weaker, Onozuka RS instead of Onozuka YC. Previous work in this laboratory has shown the latter to cause reduced viability with only a marginal improvement of yield, for freshly isolated grass pea protoplasts. For both species, the best rate of division and microcallus formation was observed from non-fused, stained protoplasts on KP medium. Kpic medium did not sustain proliferation of a large number of colonies with Lathyrus but could, nevertheless, be used with protoplasts of both genotypes. Medium LP*60 seems adequate for Pisum protoplasts although the number of microcalli produced was less important than with medium KP. However, LP*60 medium caused bursting and death of Lathyrus protoplasts within a few days. Thidiazuron-containing media were comparable to medium LP*60 for pea protoplasts, but the Lathyrus protoplasts were strongly plasmolysed in such media, and extensive bursting occurred resulting in death.
These results differ from those reported for leaf protoplasts of L. odoratus, that were cultured at a lower density on a B5 modified medium (Razdan et al., 1980). Likewise, for pea protoplasts, only Puonti-Kaerlas and Eriksson (Puonti-Kaerlas and Eriksson, 1988) and the authors (Ochatt et al., 2000a) had used a KM-based medium, while all other reports dealing with pea protoplasts generally preferred various modifications of LP* medium as described previously (Lehminger-Mertens and Jacobsen, 1989).
KP medium was chosen for culture of the heterokaryons as based on responses from non-fused protoplasts, it gave the best results for both parents. Within 3 d, the division of heterokaryon-derived cells was observed, for both chemical and electrical fusion methods and, 2 weeks later, small cell colonies were formed.
Chemical fusion
Three different agents were used for chemical fusion of protoplasts (glycine, PEG 6000, PEG 1540; see Table 1). Fusion was possible with all three agents (Figs 1, 2), but glycine was statistically the least efficient, with about 10% of heterokaryons produced. In addition, with glycine, the formation of crystals and agglomerations of debris that entrapped the heterokaryons and curtailed their subsequent development was always observed. Rinsing the fused protoplasts twice or using a fresh glycine solution each time failed to prevent these phenomena. No difference was detected between the macro and micro-method. Used alone, this agent (High pH/Ca2+) did not appear to be very efficient, but it had been noticed (Kao and Michayluk, 1974; Kao et al., 1974) that, coupled with a PEG-treatment, glycine permitted an increased heterokaryon formation.
|
|
Fusion with PEG was most efficient, with about 20% of heterokaryons produced (Figs 1
, 3). Statistically, no differences were detected between macro and micro-methods. Also, PEG 6000 was as efficient as PEG 1540. Chand et al. (Chand et al., 1988) with Solanum viarum (+) S. dulcamara, and Kao and Michayluk (Kao and Michayluk, 1974) with Vicia hajastana (+) Pisum sativum observed similar results. However, the use of PEG 1540 involves a second rinsing and could thereby decrease the density of protoplasts. For both PEG solutions, micro-methods seemed technically more suitable, because of the absence of centrifugation, which consistently damaged the protoplasts and decreased their density. Viability was not measured after fusion, but the division of heterokaryons was apparent for micro-methods using PEG solutions ( . 5), showing them not to be toxic. In this respect, the lack of division for protoplast-derived cells fused using macro-methods is likely to be due to a reduced cell density following the repeated centrifugations needed for rinsing the fused protoplasts. The non-toxic nature of PEG at the concentration and duration used had already been observed (Kao and Michayluk, 1974).
|
|
Electrofusion
Although protoplast fusion was possible for all voltages tested (Figs 1, 4), and was coupled with an efficiency of heterokaryon formation that increased with the voltage applied, no statistical differences appeared between the various electrofusion treatments. Thus, the production of heterokaryons at 1500 V cm-1 was best, but large variations between successive experiments have been observed, probably reflecting the difference of protoplast quality between several independent isolations. Electrofusion induces heavy mechanical shocks (due to micropore formation) and, although the Ca2+ ions present in the solution for electrofusion protect the membranes, protoplasts can explode after fusion. Interest was therefore attached to the re-evaluation of protoplast (and heterokaryon) viability following electrofusion, as shown in Fig. 6. This figure clearly shows the efficiency and reproducibility of the isolation protocol, with nearly negligible variations in the viability of freshly isolated protoplasts for both genotypes. In this context, the isolation protocols for each genotype were identical during all successive experiments, but small differences (age of material, exact duration of digestion, time between two centrifugations, etc.) still exist and cannot be entirely suppressed. These factors could influence protoplast quality and can thus explain the variations observed in the efficiency of electrofusion and in the subsequent viability of the fused protoplasts. Consequently, despite having observed sustained proliferation from electrofused protoplasts, the use of a chemical micro-method with PEG 6000 as fusing agent should be preferred. This is in line with data by Chand et al. (Chand et al., 1988), with protoplasts of Nicotiana tabacum and Solanum dulcamara, but contrasts those observed by Bates (Bates, 1985) with N. tabacum and N. plumbaginifolia, who obtained 19% of heterokaryons with electrofusion, versus 10% with PEG 8000 treatment.
|
This is the first report on the isolation and culture of viable protoplasts of Lathyrus sativus L. Also, for the first time the fusion of protoplasts of pea and grass pea is described. In addition, although chemical versus electrofusion have been frequently contrasted in the past, this has not been the case for comparisons of macro- and micro-methods for chemical fusion, nor had electrofusion ever been tested with Pisum protoplasts before. The strategies detailed here allowed the development of heterokaryons which reproducibly underwent cell division to give small colonies (Table 2
).
|
Whole plant regeneration from such, heterokaryon-derived microcalluses will take at least 12–15 months (Böhmer et al., 1995
; Ochatt et al., 2000a; Puonti-Kaerlas and Eriksson, 1988), but will permit the creation of genetic novelties including interesting agronomic traits, in terms of stress tolerance and rusticity from Lathyrus, and with respect to grain quality from Pisum.
Note added in proof
While this manuscript was in litteris, data were reported on the isolation of protoplasts from leaves and cell suspensions of one grass pea accession (McCutchan et al., 1999), but these failed to undergo sustained division during culture.
|
Acknowledgments |
|---|
Technical support by L Jacas and C Pontécaille is gratefully acknowledged. These experiments are part of FAO/IAEA Research Agreement No. 10420RO.
|
Notes |
|---|
1 To whom correspondence should be addressed. Fax: +33 3 8063 3263. E-mail: ochatt{at}epoisses.inra.fr
|
Abbreviations |
|---|
MES, 2-N-morpholinoethanesulphonic acid; NAA,
-naphthaleneacetic acid; PEG, polyethylene glycol; 2,4-D, 2,4-dichlorophenoxyacetic acid.. |
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Bates GW.1985. Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana. Planta 165, 217–224.
Böhmer P, Meyer B, Jacobsen H-J.1995. Thidiazuron-induced high frequency of shoot induction and plant regeneration in protoplast-derived pea callus. Plant Cell Reports 15, 26–29.
Campbell CG.1997. Grass pea, Lathyrus sativus L. Rome: Gatersleben/IPGRI.
Chand PK, Davey MR, Power JB, Cocking EC.1988. An improved procedure for protoplast fusion using polyethylene glycol. Journal of Plant Physiology 133, 480–485.
Constabel F, Kao KN.1974. Agglutination and fusion of plant protoplasts by polyethylene glycol. Canadian Journal of Botany 52, 1603–1606.
Frearson EM, Power JB, Cocking EC.1973. The isolation, culture and regeneration of Petunia leaf protoplasts. Developmental Biology 33, 130–137.[Web of Science][Medline]
Gamborg OL, Miller RA, Ojima K.1968. Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Results 50, 151–158.
Kao KN, Constabel F, Michayluk MR, Gamborg OL.1974. Plant protoplast fusion and growth of intergeneric hybrid cells. Planta 120, 215–227.
Kao KN, Michayluk MR.1974. A method for high-frequency intergeneric fusion of plant protoplasts. Planta 115, 355–367.
Kao KN, Michayluk MR.1975. Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta 126, 105–110.
Lehminger-Mertens R, Jacobson H-J.1989. Plant regeneration and organogenesis from pea protoplasts. In Vitro Cellular and Developmental Biology—Plant 25, 571–574.
Lehminger-Mertens R, Jacobsen H-J.1993. Regeneration of plants from protoplasts of pea (Pisum sativum L.). In: YPS Bajaj, ed. Biotechnology in agriculture and forestry, Vol. 22. Plant protoplasts and genetic engineering III. Berlin, Heidelberg: Springer-Verlag, 97–104.
McCutchan JS, Larkin PJ, Stoutjesdijk PA, Morgan ER, Taylor WJ.1999. Establishment of shoot and suspension cultures for protoplast isolation in Lathyrus sativus L. SABRAO Journal of Breeding and Genetics 31, 43–50.
Ochatt SJ, Mousset-Déclas C, Rancillac M.2000a. Fertile pea plants regenerate from protoplasts when calluses have not undergone endoreduplication. Plant Science (in press).
Ochatt SJ, Pontécaille C, Rancillac M.2000b. The growth regulators used for bud regeneration and shoot rooting affect the competence for flowering and seed set in regenerated plants of protein peas. In Vitro Cellular and Developmental Biology—Plant 36, (in press).
Power JB, Davey MR.1990. Protoplasts of higher and lower plants. Isolation, culture and fusion. In: Pollard JW, Walker JM, eds. Methods in molecular biology Vol. 6. Plant cell and tissue culture. New Jersey: The Humana Press, 237–259.
Puonti-Kaerlas J, Eriksson T.1988. Improved protoplast culture and regeneration of shoots in pea (Pisum sativum L.). Plant Cell Reports 7, 242–245.
Puonti-Kaerlas J, Ottoson A, Eriksson T.1992. Survival and growth of pea protoplasts after transformation by electroporation. Plant Cell, Tissue and Organ Culture 30, 141–148.
Razdan MK, Cocking EC, Power JB.1980. Callus regeneration from mesophyll protoplasts of sweet pea (Lathyrus odoratus L.). Zeitschrift für Pflanzenphysiologie 96, 181–183.
Rech EL, Ochatt SJ, Chand PK, Power JB, Davey MR.1987. Electro-enhancement of division of plant protoplast-derived cells. Protoplasma 141, 169–176.
Widholn JM.1972. The use of fluorescein diacetate and phenosaphranine for determining the viability of cultured cells. Stain Technology 47, 189–194.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  R. Zhang, S.-F. Hwang, B. D. Gossen, K.-F. Chang, and G. D. Turnbull A Quantitative Analysis of Resistance to Mycosphaerella Blight in Field Pea Crop Sci., January 22, 2007; 47(1): 162 - 167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Zhang, S.-F. Hwang, K.-F. Chang, B. D. Gossen, S. E. Strelkov, G. D. Turnbull, and S. F. Blade Genetic Resistance to Mycosphaerella pinodes in 558 Field Pea Accessions Crop Sci., October 2, 2006; 46(6): 2409 - 2414. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||