Plant Cell Reports
Plant Cell Reports (1996) 15:350-354
9 Springer-Verlag1996
Predictive correlates of shoot regeneration from potato protoplast culture S. N. R. Barr, L.A. Payne, M. E B. Dale, and M.d. Wilkinson Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK Received 21 February 1995/Revised version received 12 June 1995 - Communicated by M. R. Davey
Summary. Adaptation of protoplast regeneration systems for use on untested or recalcitrant potato genotypes can be a time-consuming exercise. Callus growth and xylogenesis were evaluated as early correlates of shooting potential to shorten this process. Callus growth was of limited value for predicting organogenesis but a linear relationship was observed between xylogenesis and shooting frequency. Increases in xylem content above a minimum threshold corresponded with increases in Shooting frequency. The predictive value of the relationship was tested using a simple protocol modification (the culture of calli on a filter paper base). Calli on filter paper produced more xylem elements and shoots than those plated directly on medium. The potential of xylem content as a predictive test of shooting frequency is discussed.
Introduction It is well established that protoplasts isolated from potato clones (Solanum tuberosum L.) vary in their ability to regenerate plants (Bingham et al. 1975; Wenzel 1979; Moilers et al. 1992). There have been many reports in which protocols were found to be productive on some genotypes but ineffectual when applied to others (e.g. Shepard 1982; Foulger and Jones 1986). This variability means that, as yet, there is no genotype independent regeneration system and modifications are generally required to adapt a protocol for application to new genotypes (Dai et al. 1987; Taylor and Secor 1988). This is a time-consuming process that effectively limits the number of genotypes used and reduces the practical value of the technology for research and breeding purposes. Recalcitrant genotypes may fail to produce shoots or else form shoots at such low frequencies that meaningful comparisons between treatments are impossible. The Correspondence to: M. J. Wilkinson
identification of characteristics that correlate with shooting frequency early in the regeneration process would shorten the time required for protocol adaptation and also provide useful data on recalcitrant genotypes. Taylor and Secor (1990) reported that shoot initiation from protoplast-derived potato calli is positively correlated to vigorous callus growth. Subsequently, the authors concluded that average callus diameter may provide an important, non-destructive tool for determining the regeneration potential of protoplastderived calli (Taylor and Secor 1992). Xylem elements are frequently observed in plant callus cultures and many authors have made use of this to study the physiological mechanisms controlling xylogenesis (for review see Seagull and Falconer 1991). George and Sherrington (1984) suggested xylem elements in organogenic callus cultures may represent an early stage in the development of shoot meristems. There have been several reports, indeed, where shoot or embryo formation has been associated with the appearance of xylem elements in callus cultures (e.g. Chen and Galston 1967; Cassells 1979). In the present work, callus growth dynamics and xylogenesis are evaluated as predictive measures of the shooting potential of isolated protoplasts from a range of Solarium genotypes.
Materials and Methods Plant material: Solanum tuberosum L cultivars Brodick,Pentland
Squire and MaxisPiper andthe SCRIbreedingcloneG176B/102were suppliedfromthepotatocollectionbasedat the ScottishCropResearch Institute (SCRI),Dundee,UK. SeedsamplesofS. stoloniferum (CPC 2618) wereobtainedfromthe CommonwealthPotatoCollection,also held at SCRI. Explant material: Plant material (sprouted shoots fxom tubers or
germinatedseedlings)weresurfacesterilizedby bathingin 70% (v/v) ethanol for 30s followed by immersion in 10% (v/v) commercial bleach solution(Domestos,LeverIndustrial, Merseyside,UK) for 15
351 rain and rinsing three times in sterile distilled water (5 rain each). Explants were cultured on half strength Murashige and Skoog (1962) basal medium, with the additions of Coleman et al. (1991) and maintained at 22"C with a 16h photoperiod (160 p2xa-Zs-]).
Protoplast isolation and culture: Young leaves taken from 3 to 4 week cultures were pretreated for 16h by soaking them in the dark at 4"C in conditioning medium A of Shepard and Totten (1977). PYotoplasts were isolated, cultured and (where appropriate) dectrofused according to the method of Fonlger and Jones (1986), as modified by Coleman et at. (1991). old
central sections of each series.
Results
Callus growth The growth rates of calli cultured on medium C were essentiaLLysimilar for all genotypes examined (Fig. 1). 1.8. 1.6, 1,4
Protoplasts were adjusted to a density of 1Xl0 s m1-1 through the addition of freshly made VKCL medium containing 0.1% carbenicillin and incubated in the dark at 22"C for 7d to induce ceLL division. Suspensions were diluted to a density of 2X104 mi-a with VKCL medium and transferred to one compartment of a split plate. Medium 9 of Shepard and Totten (1977) had been aliquoted into the adjacent, connected section of the plate. The split plates were sealed and cultured for 12-18d with a 16h photoperiod (35pEru-Is -~) at 22"C to allow microcallus formation. Microoalli were transferred either directly onto agar-solidified medium C of Shepard and Totten (1977) or else onto a sterile filter paper support rested on top of the medium. Plates were incubated for 25-30d (22"C, 16h photoperiod at 351zEm-Zs-~) to allow colony development. After this period, calli were transferred onto medium D (Shepard and "rotten 1977) containing lmgl -] zeatin and 0.1mgl -] IAA to encourage development of shoot primordia. Calli grown directly on media were moved between plates individually using a sharp scalpel to minimize ceLLdamage (Shahin 1984). Otherwise, the filter paper was used as a base for callus transfer. Calli were suboultured onto fresh medium D every 4 weeks until shoot primordia were observed. They were then transferred to SP medium containing 0.25mgl q BAP and 0.1 mgl -~ GA3 (Shepard and Totten 1977) to promote shoot extension. Two to four independent protoplast isolations were made for each of the genotypes examined. Each isolation produced sufficient microcalli for 5-10 plates of medium C. Where appropriate, these were subdivided further into equal subsamples for comparative purposes.
Measurement of callus growth and xylogenesis: Callus growth and xylogenesis was monitored during development on medium C. Mean diameter of twenty randomly selected calli from each plate were measured at 2-6d intervals on a calibrated dissecting microscope (X20 magnification). Two methods of measuring xylem frequency were used: xylem element counts from (a) squashed calli or Co) transverse sections. (a) Callus squashes: Total xylem number in calli of cvs Marls Piper, Pentland Squire and Brodick, the clone G176b/102 and ~ stoloniferum (CPC 2618) were counted from squash preparations. Ten representative caLLi (i.e. those of mean diameter for the plate + SE) were softened in 5N HCI for 5h at 60"C. They were then washed briefly in distilled water (30s) before being mounted, tapped out and squashed on a glass slide in 1% w/v aceto-orcein. The number of xylem dements was counted on a calibrated compound microscope (X400 magnification). (b) Callus sections: Protoplast-derived microcalli of cv. Pentland Squire and GI76BI102 and the fusion calli between ev. Brodick and G176/102 were transferred either to medium C on a filter paper base or else pipetted directly onto the medium. Xylem frequency and the distribution of xylem elements in each plate were recorded from transverse sections of five representative calli. Calli containing shoot initials were also sectioned. Calli to be sectioned were pretreated for 24h at 5"C in an embedding solution containing 30% sucrose, 15% Cryo-M-Bed (Bright Instrument Co., Huntingdon, UK) and 1% gumarabic (BDH Laboratory Supplies, Merck Ltd., Lutterworth, UK). Serial sections of embedded calli were made using a freezing microtome. Frequency of xylem elements in each callus (expressed as the number of elements per mm z) was recorded as the mean number in the five
t.2
=
0.8-
0.60,4 1
4~S.stoloniferam
02
~
;0
;s
2'0
2's
Time (Days)
Fig. 1. Callus growth on medium-C, Mean diameter (ram) of protoplast-derived calli of cv. Penfland Squire, cv. Maxis Piper, cv. Brodick, G176B/102 and S. stoloniferum (CPC 2618) during culture on medium C (Shepard and "rotten 1977)
Variation was observed in the period of linear growth and this led to significant differences between genotypes in the mean diameter of calli after 25d of culture. Cultivar Pentland Squire usually possessed the largest calli at this stage and G176B/102 the smallest. Differences between genotypes were significant (P
Plant Cell Reports (1996) 15:350-354
9 Springer-Verlag1996
Predictive correlates of shoot regeneration from potato protoplast culture S. N. R. Barr, L.A. Payne, M. E B. Dale, and M.d. Wilkinson Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK Received 21 February 1995/Revised version received 12 June 1995 - Communicated by M. R. Davey
Summary. Adaptation of protoplast regeneration systems for use on untested or recalcitrant potato genotypes can be a time-consuming exercise. Callus growth and xylogenesis were evaluated as early correlates of shooting potential to shorten this process. Callus growth was of limited value for predicting organogenesis but a linear relationship was observed between xylogenesis and shooting frequency. Increases in xylem content above a minimum threshold corresponded with increases in Shooting frequency. The predictive value of the relationship was tested using a simple protocol modification (the culture of calli on a filter paper base). Calli on filter paper produced more xylem elements and shoots than those plated directly on medium. The potential of xylem content as a predictive test of shooting frequency is discussed.
Introduction It is well established that protoplasts isolated from potato clones (Solanum tuberosum L.) vary in their ability to regenerate plants (Bingham et al. 1975; Wenzel 1979; Moilers et al. 1992). There have been many reports in which protocols were found to be productive on some genotypes but ineffectual when applied to others (e.g. Shepard 1982; Foulger and Jones 1986). This variability means that, as yet, there is no genotype independent regeneration system and modifications are generally required to adapt a protocol for application to new genotypes (Dai et al. 1987; Taylor and Secor 1988). This is a time-consuming process that effectively limits the number of genotypes used and reduces the practical value of the technology for research and breeding purposes. Recalcitrant genotypes may fail to produce shoots or else form shoots at such low frequencies that meaningful comparisons between treatments are impossible. The Correspondence to: M. J. Wilkinson
identification of characteristics that correlate with shooting frequency early in the regeneration process would shorten the time required for protocol adaptation and also provide useful data on recalcitrant genotypes. Taylor and Secor (1990) reported that shoot initiation from protoplast-derived potato calli is positively correlated to vigorous callus growth. Subsequently, the authors concluded that average callus diameter may provide an important, non-destructive tool for determining the regeneration potential of protoplastderived calli (Taylor and Secor 1992). Xylem elements are frequently observed in plant callus cultures and many authors have made use of this to study the physiological mechanisms controlling xylogenesis (for review see Seagull and Falconer 1991). George and Sherrington (1984) suggested xylem elements in organogenic callus cultures may represent an early stage in the development of shoot meristems. There have been several reports, indeed, where shoot or embryo formation has been associated with the appearance of xylem elements in callus cultures (e.g. Chen and Galston 1967; Cassells 1979). In the present work, callus growth dynamics and xylogenesis are evaluated as predictive measures of the shooting potential of isolated protoplasts from a range of Solarium genotypes.
Materials and Methods Plant material: Solanum tuberosum L cultivars Brodick,Pentland
Squire and MaxisPiper andthe SCRIbreedingcloneG176B/102were suppliedfromthepotatocollectionbasedat the ScottishCropResearch Institute (SCRI),Dundee,UK. SeedsamplesofS. stoloniferum (CPC 2618) wereobtainedfromthe CommonwealthPotatoCollection,also held at SCRI. Explant material: Plant material (sprouted shoots fxom tubers or
germinatedseedlings)weresurfacesterilizedby bathingin 70% (v/v) ethanol for 30s followed by immersion in 10% (v/v) commercial bleach solution(Domestos,LeverIndustrial, Merseyside,UK) for 15
351 rain and rinsing three times in sterile distilled water (5 rain each). Explants were cultured on half strength Murashige and Skoog (1962) basal medium, with the additions of Coleman et al. (1991) and maintained at 22"C with a 16h photoperiod (160 p2xa-Zs-]).
Protoplast isolation and culture: Young leaves taken from 3 to 4 week cultures were pretreated for 16h by soaking them in the dark at 4"C in conditioning medium A of Shepard and Totten (1977). PYotoplasts were isolated, cultured and (where appropriate) dectrofused according to the method of Fonlger and Jones (1986), as modified by Coleman et at. (1991). old
central sections of each series.
Results
Callus growth The growth rates of calli cultured on medium C were essentiaLLysimilar for all genotypes examined (Fig. 1). 1.8. 1.6, 1,4
Protoplasts were adjusted to a density of 1Xl0 s m1-1 through the addition of freshly made VKCL medium containing 0.1% carbenicillin and incubated in the dark at 22"C for 7d to induce ceLL division. Suspensions were diluted to a density of 2X104 mi-a with VKCL medium and transferred to one compartment of a split plate. Medium 9 of Shepard and Totten (1977) had been aliquoted into the adjacent, connected section of the plate. The split plates were sealed and cultured for 12-18d with a 16h photoperiod (35pEru-Is -~) at 22"C to allow microcallus formation. Microoalli were transferred either directly onto agar-solidified medium C of Shepard and Totten (1977) or else onto a sterile filter paper support rested on top of the medium. Plates were incubated for 25-30d (22"C, 16h photoperiod at 351zEm-Zs-~) to allow colony development. After this period, calli were transferred onto medium D (Shepard and "rotten 1977) containing lmgl -] zeatin and 0.1mgl -] IAA to encourage development of shoot primordia. Calli grown directly on media were moved between plates individually using a sharp scalpel to minimize ceLLdamage (Shahin 1984). Otherwise, the filter paper was used as a base for callus transfer. Calli were suboultured onto fresh medium D every 4 weeks until shoot primordia were observed. They were then transferred to SP medium containing 0.25mgl q BAP and 0.1 mgl -~ GA3 (Shepard and Totten 1977) to promote shoot extension. Two to four independent protoplast isolations were made for each of the genotypes examined. Each isolation produced sufficient microcalli for 5-10 plates of medium C. Where appropriate, these were subdivided further into equal subsamples for comparative purposes.
Measurement of callus growth and xylogenesis: Callus growth and xylogenesis was monitored during development on medium C. Mean diameter of twenty randomly selected calli from each plate were measured at 2-6d intervals on a calibrated dissecting microscope (X20 magnification). Two methods of measuring xylem frequency were used: xylem element counts from (a) squashed calli or Co) transverse sections. (a) Callus squashes: Total xylem number in calli of cvs Marls Piper, Pentland Squire and Brodick, the clone G176b/102 and ~ stoloniferum (CPC 2618) were counted from squash preparations. Ten representative caLLi (i.e. those of mean diameter for the plate + SE) were softened in 5N HCI for 5h at 60"C. They were then washed briefly in distilled water (30s) before being mounted, tapped out and squashed on a glass slide in 1% w/v aceto-orcein. The number of xylem dements was counted on a calibrated compound microscope (X400 magnification). (b) Callus sections: Protoplast-derived microcalli of cv. Pentland Squire and GI76BI102 and the fusion calli between ev. Brodick and G176/102 were transferred either to medium C on a filter paper base or else pipetted directly onto the medium. Xylem frequency and the distribution of xylem elements in each plate were recorded from transverse sections of five representative calli. Calli containing shoot initials were also sectioned. Calli to be sectioned were pretreated for 24h at 5"C in an embedding solution containing 30% sucrose, 15% Cryo-M-Bed (Bright Instrument Co., Huntingdon, UK) and 1% gumarabic (BDH Laboratory Supplies, Merck Ltd., Lutterworth, UK). Serial sections of embedded calli were made using a freezing microtome. Frequency of xylem elements in each callus (expressed as the number of elements per mm z) was recorded as the mean number in the five
t.2
=
0.8-
0.60,4 1
4~S.stoloniferam
02
~
;0
;s
2'0
2's
Time (Days)
Fig. 1. Callus growth on medium-C, Mean diameter (ram) of protoplast-derived calli of cv. Penfland Squire, cv. Maxis Piper, cv. Brodick, G176B/102 and S. stoloniferum (CPC 2618) during culture on medium C (Shepard and "rotten 1977)
Variation was observed in the period of linear growth and this led to significant differences between genotypes in the mean diameter of calli after 25d of culture. Cultivar Pentland Squire usually possessed the largest calli at this stage and G176B/102 the smallest. Differences between genotypes were significant (P
