Plant Cell Reports
Plant Cell Reports (1987) 6: 379- 381
© Springer-Verlag 1987
Reproduction of the beet cyst nematode Heterodera schachtii Schm. on transformed root cultures of Beta vulgaris L. H. Paul, C. Zijlstra, J. E. Leeuwangh, F. A. Krens, and H.J. Huizing Foundation for Agricultural Plant Breeding, P.O. Box 117, NL-6700 AC Wageningen, The Netherlands Received Mai 5, 1987 / Revised version received June 30, 1987 - Communicated by H. L6rz
ABSTRACT
METHODS AND MATERIALS
Transformed hairy root cultures of Beta vulgaris L., grown in petri-dishes, were inoculated with a suspension of surface-sterilized larvae of the beet cyst nematode Heterodera schachtii Schm. Larvae developed into both male and female adults. Juvenile larvae were hatched from the newly-formed cysts, indicating that fertilization had occurred. Results of a glasshouse test showed that H.sehachtii did not lose Its pathogenicity after being cultured on transformed roots. This technique can be developed further for the mass-propagation of sedentary nematodes and for the in vitro storage of isolates.
Plant material~ induction and growth of transformed root cultures
INTRODUCTION In the last few decades many techniques have been developed for the in-vitro culturing of nematodes. Migratory nematodes can be maintained on suitable callus tissues. Examples are the culture of Ditylenchus dipsaci and Aphelenchoides ritzemabosi on alfalfa callus (Krusberg, 1961). Sedentary nematodes, like the root-knot and cyst nematodes, need curse cells or giant cells (syncytia) for feeding and can only grow on tissues in which such cells can be induced, e.g. excised roots. Culturing of the beet cyst nematode Heterodera sehaehtii has been reported on excised roots of Beta vulgaris (Johnson and Viglierchio, 1969; Moriarty, 1964) and on excised Brassica napus roots (HOller, 1978). Sedentary nematodes cannot as yet be cultured on completely undifferentiated callus tissues (Jones, 1980). Tissue-cultured nematodes can be used to study various aspects of their biology and morphology. Monoxenic cultures of Ditylenchus dipsaei and Meloidogyne ineognita have been used in the selection of resistant breeding material (Bingefors and Bingefors, 1976; Nelson and Kao, 1986). At this moment, in vitro techniques are far more used For the migratory nematodes than for the sedentary nematodes, although the latter are very important. This is due to the laborious work with excised roots and the more eemplex life-cyc]e of these nematodes. The development of practical systems for culturing sedentary nematodes however is of high signiFicance (Eriksson, 1980; Krusberg and Babineau, ]977). This paper deals with the reproduction of H.schaehtii on transformed root c u l L u r ~ o f Beta vulgaris and possible applications of this technique are discussed.
Offprint requests to: H. Paul
After mechanical removal of the seedcoats, seeds of Beta vulgaris L. ev "Regina" were surface-sterilized by subsequent transmission through 70% (v/v) ethanol (few seconds) and l~ (w/v) of a sodium hypochlorite solution (15 min.). Following extensive rinsing with sterile distilled water, the seeds were transferred to glass tubes containing MS (Murashige and Skoog, 1962) medium (Flow Laboratories, U.K.) at half strength without phytohormones, supplemented with 3% (w/v) sucrose and 0,8% (w/v) Difco bactoagar (designated as ½MS), at pH 5.8 prior Lo autoclaving. The glass tubes were incubated at 22 °C, 16h light (2000 lux). Germination occurred within one week. For inoculation experiments two weeks old seedlings with well-developed cotyledons were used. The cotyledons were cut into pieces of approximately ¼ cm2 and inoculated in a l:lO or l:lO0 dilution of an overnight culture of Agrobacterium rhizogenes LBA 9402, grown in YMB medium (Hooykaas et al., 1977). After 5 minutes the segments were collected and blotted dry with sterile filter paper. Incubation was continued on ½MS medium in peLri-dishes. After two days the segments we[e transferred to ~MS i medium containing 200 mg 1 -~ cefotaxime (Calbiochem, U.S.A.) in order to suppress bacterial growth. Roots were collected three weeks after inoculation. The transferred nature of the hairy roots was investigated by testing for the presence of agropine and mannopine, according to Peerbolte et al. (1985) Root E~ltures were maintained on ½MS medium with 200 mg 1 cefotaxime by monthly transfer of young root tips. For the nematode inoculation experiments well-establish~d root cultures grown on 1/2MS medium with 200 mg 1-- cefotaxime were used. Nematode sterilization and inoculation Cysts of Heterodera schachtii Schm. were collected from infested sugarbeet plants grown in the glasshouse and placed in a ZnCl~-solution(lmM). Twice a week hatched juvenile larvae-were collected and stored at 4°C in a refrigerator. Prior to inoculation the larval suspension was poured onto a Sartorius membrane filter(pore width 5 ~m) and washed with a O.02~(w/v) HgCl^-solution for at least 3 minutes and then extensively washed with sterile distilled water. Additionally the larvae were ~e'slwspe~ed in a O.05%(w/v)
380 tetracycline-solution (Sigma). Larvae were kept in the tetracycline-solution for at least 15 minutes. The sterilization procedure is a modification of the procedures as described by MOller (1970, 1978) and 's Jacob and Van Besooyen (1984). Small droplets of the larvae suspension were deposited on the roots in the petri-dishes by means of a micropipeLte. After inoculation the petri-dishes were sealed wiLh parafilm and stored at 22 % . Root culLures were examined under a Zeias SV-8 stereomicroscope.
A
_
M-
In-vitro produced cysts were placed in the ZnCI~solution for hatching. Seedlings of Beta vulgar~s L., grown in the glasshouse in silver sand on a nutrient solution, were inoculated with the hatched larvae according to Lubberts and Toxopeus (1982), to score for cyst production in proof of pathogenicity.
RESULTS
Root growth and confirmation of the transformation
Inoculations of cotyledons of Beta vulgaris with A. rhizogenes resulted in fast growing root material (Tig. i). After transfer of the roots to medium without phytohormones growth continued. Many lateral roots were formed and dense growth of root hairs was observed. The presence of agropine and mannopine was demonstrated and confirmed the transformed nature of the roots (Tig. 2).
1
2
3
4
Fig. 2 Electrophoretic analysis of extracts of Beta vu]oaris plant material; l~ne ]: agropine/ /mannopine standard, lane 2: leaves (untransformed), lane 3: roots (untransformed), lane 4: transformed roots. A = agropine; M = mannopine.
Reproduction of Heterodera schachtii and test for pathogenicity Approximately two weeks after inoculation of the roots with the larvae, both elongated male and swollen female nematodes (cysts) were observed in the petri-dishes. The male nematodes became free-living , while Lhe cysts remained attached to the roots. Root cultures had turned brownish in the meantime, except for the young root tips (Fig. 3, 4). After four weeks - some time is required for the embryogenesis of the larvae - cysts were removed from the hairy roots and placed in the ZnCl^-solution for z hatching. Viable larvae were hatched from the cysts, indicating that fertilization of the females by the males had occurred. The in-vitro produced larvae proved to be pathogenic. After inoculation of seedlings of Beta vulgaris grown in &he glasshouse with the in-vitro produced larvae, new cysts were formed on the rootlets. DISCUSSION
Fig. 1 Induction of hairy roots on cotyledons of B. vulgaris L. after indculation with A. rhizogenes LBA 9402. Photograph taken 21 days after inoculation.
Hairy root cultures of Beta vulgaris L. proved to be a suitable substrate for the beet cyst nematode Heterodera schachtii Schm. Apparantly, the larvae, added to the roots under in-vitro conditions, were able to induce syncytia for feeding and developed into adults, iq spite of the fact that morphology and physiology of the rootsystem is influenced by transformation. Furthermore, results of the glasshouse test showed that H. schaehtii did not lose its pathogenicity after being cultured on transformed roots. Comparable results were obtained by Yaeoub and Tepfer (1987), who reported on the reproduction of the
381
~g. 3 Transformed root culture of B. vulgaris, used in the inoculation experiments. Photograph taken 33 days after inoculation.
very easy. The roots can be subcultured by transFerring young root tips to fresh media without phytohormones. Since many lateral roots are formed, the entire root surface of these cultures is very large. It is therefore expected, that with this system ultimately even more cysts can be produced than on excised roots, which do not show extensive lateral root formation. Many factors are involved in the production of larvae of Heterodera sehachtii on transformed root cultures, like age and surface of the culture, inoculation density, cyst size, sex ratio and fertilization rate. Also the conditions for embryogenesis and hatching are important. In this communication the basic techniques are described. Research will be continued to optimize production circumstances. The production of several nematode speeims has been commercialized already (Nelson and Kao, 1986). The system described here might offer the possibility for commercialization of the production of H. schachtii. The larvae can be used in conventional breeding programs on nematode resistance in sugarbeet and erueiferous crops. Since cysts can be stored at low temperatures, transformed root cultures might also be useful for the storage of nematode isolates. Furthermore, the procedure of hairy root formation and subsequent nematode development on these roots, can be applied in assaying beet material obtained after genetic manipulation for nematode resistance. This is currently under investigation in our laboratory. It is of interest to extend the research mentioned here to other plant-nematode combinations. AKNOWLEDGMENTS We are grateful to the group of Prof. Sehilperoort (Leiden, The Netherlands) for kindly sgpplying Agrobacterium rhizogenes LBA 9402 and to J. Temp~ (Paris, France) For supplying the sgropine/mannopine standard. REFERENCES
Fig. 4 Cyst of the beet cyst nematode H. sehaehtii on a transformed root of B. vulgaris. Photograph taken 33 days after inoculation. fungus Polymyxa betae on transformed B. vulgaris roots. MOller (1978) cultured H. sehachtii in-vitro on exeised 8rassiea napus roots for use in in-vitro tests. Several hundreds of cysts were produced per petridish. Petri-dishes could be stored at low temperatures as inoculum stock and were used when necessary. However, this method appeared to be very costly and in his later work in-vivo produced surface-sterilized larvae were used (MUller, 1985). Once established, culturing of transformed roots is
Bingefors S, Bingefors S (1976) Swedish J. Agrie. Res. 6:13-17 Eriksson KB (1980) EPPO Bull. lO: 371-378 Hooyksas PJJ, Klapwijk PM, Nuti MP, Schilperoort RA, R6rsch A (1977) J. Gen Mierobiol. 98:477-484 s Jacob JJ, Van Besooyen J (1984) Nematol. Dept. Agric. Univ. Wageningen (19877pp~) Johnson RN, Viglierchio DR Nematologica 15: 129-143 Jones MGK (1980) In: Ingram DS, Helgeson JP (eds) Tissue Culture Methods for Plant Pathologists, 81ackwe11 Scientific, Oxford, pp 161-166 Krusberg LR (1961) Nematolagiea 6:181-200 Krusberg LR, Babineau DE (1977) In: Sharp WR, Larsen PO, Paddock EF, Raghaven V (eds) Ohio State Univ. Press, Colombus, pp 401-419 Lubberts JH, Toxopeus H (1982) Zaadbelangen 36:66-69 Moriarty F (1964) Parasitology 54:289-293 MUller J (1970) Nematologica 16:154-155 M8ller J (1978) Rev. N6matol. l: 47-52 MO11er J (1985) Vortr. Pflanzenz. 9 : 5 9 - 6 9 Murashige T, Skoog F (1962) Physiol. Plant 15:473-497 Nelson Cg, Kao JC (1986) In: Agrobiotee "86, Convention Proceedings, 8ologna 29/5-106 1986 Peerbolte R, Krens FA, Mans RMW, Floor M, Hoge JHC, Wullems GJ, Sehilperoort RA (1985) Plant Mol. Biol. 5:235-246 Yaeoub A, Tepfer D (1987) In: Proceedings of the 50th Winter Congress fIRS, Brussels 11-12 febr. 1987, p 307-308
Plant Cell Reports (1987) 6: 379- 381
© Springer-Verlag 1987
Reproduction of the beet cyst nematode Heterodera schachtii Schm. on transformed root cultures of Beta vulgaris L. H. Paul, C. Zijlstra, J. E. Leeuwangh, F. A. Krens, and H.J. Huizing Foundation for Agricultural Plant Breeding, P.O. Box 117, NL-6700 AC Wageningen, The Netherlands Received Mai 5, 1987 / Revised version received June 30, 1987 - Communicated by H. L6rz
ABSTRACT
METHODS AND MATERIALS
Transformed hairy root cultures of Beta vulgaris L., grown in petri-dishes, were inoculated with a suspension of surface-sterilized larvae of the beet cyst nematode Heterodera schachtii Schm. Larvae developed into both male and female adults. Juvenile larvae were hatched from the newly-formed cysts, indicating that fertilization had occurred. Results of a glasshouse test showed that H.sehachtii did not lose Its pathogenicity after being cultured on transformed roots. This technique can be developed further for the mass-propagation of sedentary nematodes and for the in vitro storage of isolates.
Plant material~ induction and growth of transformed root cultures
INTRODUCTION In the last few decades many techniques have been developed for the in-vitro culturing of nematodes. Migratory nematodes can be maintained on suitable callus tissues. Examples are the culture of Ditylenchus dipsaci and Aphelenchoides ritzemabosi on alfalfa callus (Krusberg, 1961). Sedentary nematodes, like the root-knot and cyst nematodes, need curse cells or giant cells (syncytia) for feeding and can only grow on tissues in which such cells can be induced, e.g. excised roots. Culturing of the beet cyst nematode Heterodera sehaehtii has been reported on excised roots of Beta vulgaris (Johnson and Viglierchio, 1969; Moriarty, 1964) and on excised Brassica napus roots (HOller, 1978). Sedentary nematodes cannot as yet be cultured on completely undifferentiated callus tissues (Jones, 1980). Tissue-cultured nematodes can be used to study various aspects of their biology and morphology. Monoxenic cultures of Ditylenchus dipsaei and Meloidogyne ineognita have been used in the selection of resistant breeding material (Bingefors and Bingefors, 1976; Nelson and Kao, 1986). At this moment, in vitro techniques are far more used For the migratory nematodes than for the sedentary nematodes, although the latter are very important. This is due to the laborious work with excised roots and the more eemplex life-cyc]e of these nematodes. The development of practical systems for culturing sedentary nematodes however is of high signiFicance (Eriksson, 1980; Krusberg and Babineau, ]977). This paper deals with the reproduction of H.schaehtii on transformed root c u l L u r ~ o f Beta vulgaris and possible applications of this technique are discussed.
Offprint requests to: H. Paul
After mechanical removal of the seedcoats, seeds of Beta vulgaris L. ev "Regina" were surface-sterilized by subsequent transmission through 70% (v/v) ethanol (few seconds) and l~ (w/v) of a sodium hypochlorite solution (15 min.). Following extensive rinsing with sterile distilled water, the seeds were transferred to glass tubes containing MS (Murashige and Skoog, 1962) medium (Flow Laboratories, U.K.) at half strength without phytohormones, supplemented with 3% (w/v) sucrose and 0,8% (w/v) Difco bactoagar (designated as ½MS), at pH 5.8 prior Lo autoclaving. The glass tubes were incubated at 22 °C, 16h light (2000 lux). Germination occurred within one week. For inoculation experiments two weeks old seedlings with well-developed cotyledons were used. The cotyledons were cut into pieces of approximately ¼ cm2 and inoculated in a l:lO or l:lO0 dilution of an overnight culture of Agrobacterium rhizogenes LBA 9402, grown in YMB medium (Hooykaas et al., 1977). After 5 minutes the segments were collected and blotted dry with sterile filter paper. Incubation was continued on ½MS medium in peLri-dishes. After two days the segments we[e transferred to ~MS i medium containing 200 mg 1 -~ cefotaxime (Calbiochem, U.S.A.) in order to suppress bacterial growth. Roots were collected three weeks after inoculation. The transferred nature of the hairy roots was investigated by testing for the presence of agropine and mannopine, according to Peerbolte et al. (1985) Root E~ltures were maintained on ½MS medium with 200 mg 1 cefotaxime by monthly transfer of young root tips. For the nematode inoculation experiments well-establish~d root cultures grown on 1/2MS medium with 200 mg 1-- cefotaxime were used. Nematode sterilization and inoculation Cysts of Heterodera schachtii Schm. were collected from infested sugarbeet plants grown in the glasshouse and placed in a ZnCl~-solution(lmM). Twice a week hatched juvenile larvae-were collected and stored at 4°C in a refrigerator. Prior to inoculation the larval suspension was poured onto a Sartorius membrane filter(pore width 5 ~m) and washed with a O.02~(w/v) HgCl^-solution for at least 3 minutes and then extensively washed with sterile distilled water. Additionally the larvae were ~e'slwspe~ed in a O.05%(w/v)
380 tetracycline-solution (Sigma). Larvae were kept in the tetracycline-solution for at least 15 minutes. The sterilization procedure is a modification of the procedures as described by MOller (1970, 1978) and 's Jacob and Van Besooyen (1984). Small droplets of the larvae suspension were deposited on the roots in the petri-dishes by means of a micropipeLte. After inoculation the petri-dishes were sealed wiLh parafilm and stored at 22 % . Root culLures were examined under a Zeias SV-8 stereomicroscope.
A
_
M-
In-vitro produced cysts were placed in the ZnCI~solution for hatching. Seedlings of Beta vulgar~s L., grown in the glasshouse in silver sand on a nutrient solution, were inoculated with the hatched larvae according to Lubberts and Toxopeus (1982), to score for cyst production in proof of pathogenicity.
RESULTS
Root growth and confirmation of the transformation
Inoculations of cotyledons of Beta vulgaris with A. rhizogenes resulted in fast growing root material (Tig. i). After transfer of the roots to medium without phytohormones growth continued. Many lateral roots were formed and dense growth of root hairs was observed. The presence of agropine and mannopine was demonstrated and confirmed the transformed nature of the roots (Tig. 2).
1
2
3
4
Fig. 2 Electrophoretic analysis of extracts of Beta vu]oaris plant material; l~ne ]: agropine/ /mannopine standard, lane 2: leaves (untransformed), lane 3: roots (untransformed), lane 4: transformed roots. A = agropine; M = mannopine.
Reproduction of Heterodera schachtii and test for pathogenicity Approximately two weeks after inoculation of the roots with the larvae, both elongated male and swollen female nematodes (cysts) were observed in the petri-dishes. The male nematodes became free-living , while Lhe cysts remained attached to the roots. Root cultures had turned brownish in the meantime, except for the young root tips (Fig. 3, 4). After four weeks - some time is required for the embryogenesis of the larvae - cysts were removed from the hairy roots and placed in the ZnCl^-solution for z hatching. Viable larvae were hatched from the cysts, indicating that fertilization of the females by the males had occurred. The in-vitro produced larvae proved to be pathogenic. After inoculation of seedlings of Beta vulgaris grown in &he glasshouse with the in-vitro produced larvae, new cysts were formed on the rootlets. DISCUSSION
Fig. 1 Induction of hairy roots on cotyledons of B. vulgaris L. after indculation with A. rhizogenes LBA 9402. Photograph taken 21 days after inoculation.
Hairy root cultures of Beta vulgaris L. proved to be a suitable substrate for the beet cyst nematode Heterodera schachtii Schm. Apparantly, the larvae, added to the roots under in-vitro conditions, were able to induce syncytia for feeding and developed into adults, iq spite of the fact that morphology and physiology of the rootsystem is influenced by transformation. Furthermore, results of the glasshouse test showed that H. schaehtii did not lose its pathogenicity after being cultured on transformed roots. Comparable results were obtained by Yaeoub and Tepfer (1987), who reported on the reproduction of the
381
~g. 3 Transformed root culture of B. vulgaris, used in the inoculation experiments. Photograph taken 33 days after inoculation.
very easy. The roots can be subcultured by transFerring young root tips to fresh media without phytohormones. Since many lateral roots are formed, the entire root surface of these cultures is very large. It is therefore expected, that with this system ultimately even more cysts can be produced than on excised roots, which do not show extensive lateral root formation. Many factors are involved in the production of larvae of Heterodera sehachtii on transformed root cultures, like age and surface of the culture, inoculation density, cyst size, sex ratio and fertilization rate. Also the conditions for embryogenesis and hatching are important. In this communication the basic techniques are described. Research will be continued to optimize production circumstances. The production of several nematode speeims has been commercialized already (Nelson and Kao, 1986). The system described here might offer the possibility for commercialization of the production of H. schachtii. The larvae can be used in conventional breeding programs on nematode resistance in sugarbeet and erueiferous crops. Since cysts can be stored at low temperatures, transformed root cultures might also be useful for the storage of nematode isolates. Furthermore, the procedure of hairy root formation and subsequent nematode development on these roots, can be applied in assaying beet material obtained after genetic manipulation for nematode resistance. This is currently under investigation in our laboratory. It is of interest to extend the research mentioned here to other plant-nematode combinations. AKNOWLEDGMENTS We are grateful to the group of Prof. Sehilperoort (Leiden, The Netherlands) for kindly sgpplying Agrobacterium rhizogenes LBA 9402 and to J. Temp~ (Paris, France) For supplying the sgropine/mannopine standard. REFERENCES
Fig. 4 Cyst of the beet cyst nematode H. sehaehtii on a transformed root of B. vulgaris. Photograph taken 33 days after inoculation. fungus Polymyxa betae on transformed B. vulgaris roots. MOller (1978) cultured H. sehachtii in-vitro on exeised 8rassiea napus roots for use in in-vitro tests. Several hundreds of cysts were produced per petridish. Petri-dishes could be stored at low temperatures as inoculum stock and were used when necessary. However, this method appeared to be very costly and in his later work in-vivo produced surface-sterilized larvae were used (MUller, 1985). Once established, culturing of transformed roots is
Bingefors S, Bingefors S (1976) Swedish J. Agrie. Res. 6:13-17 Eriksson KB (1980) EPPO Bull. lO: 371-378 Hooyksas PJJ, Klapwijk PM, Nuti MP, Schilperoort RA, R6rsch A (1977) J. Gen Mierobiol. 98:477-484 s Jacob JJ, Van Besooyen J (1984) Nematol. Dept. Agric. Univ. Wageningen (19877pp~) Johnson RN, Viglierchio DR Nematologica 15: 129-143 Jones MGK (1980) In: Ingram DS, Helgeson JP (eds) Tissue Culture Methods for Plant Pathologists, 81ackwe11 Scientific, Oxford, pp 161-166 Krusberg LR (1961) Nematolagiea 6:181-200 Krusberg LR, Babineau DE (1977) In: Sharp WR, Larsen PO, Paddock EF, Raghaven V (eds) Ohio State Univ. Press, Colombus, pp 401-419 Lubberts JH, Toxopeus H (1982) Zaadbelangen 36:66-69 Moriarty F (1964) Parasitology 54:289-293 MUller J (1970) Nematologica 16:154-155 M8ller J (1978) Rev. N6matol. l: 47-52 MO11er J (1985) Vortr. Pflanzenz. 9 : 5 9 - 6 9 Murashige T, Skoog F (1962) Physiol. Plant 15:473-497 Nelson Cg, Kao JC (1986) In: Agrobiotee "86, Convention Proceedings, 8ologna 29/5-106 1986 Peerbolte R, Krens FA, Mans RMW, Floor M, Hoge JHC, Wullems GJ, Sehilperoort RA (1985) Plant Mol. Biol. 5:235-246 Yaeoub A, Tepfer D (1987) In: Proceedings of the 50th Winter Congress fIRS, Brussels 11-12 febr. 1987, p 307-308
