Plant Science, 52 (1987) 91-97 91 Elsevier Scientific Publishers Ireland Ltd. SEPARATION OF INTACT AND DAMAGED PLANT-PROTOPLASTS BY USING A CELL-SORTER EQUIPPED WITH A TWO-CHANNEL PIEZO VALVE CHAMBER M. BROMOVA and U.C. KNOPF* Agrogen-Foundation, P.O. Box 21, CH 1701 Freiburg (Switzerland) (Received February llth, 1987) (Revision received May 18th, 1987) (Accepted May 18th, 1987) Protoplasts were prepared from a suspension culture of Solanum tuberosum var. Bintje. A mixture of intact and damaged protoplasts was prepared, stained, respectively, with fluorescein diacetate (FDA) and Evans Blue. Subsequently the FDA- stained protoplasts were selected and sorted using a new sorting principle and device. Further analysis showed that 75% of the protoplasts remained intact using this sorting procedure. Key words: protoplast; flow-cytometry; cell-sorting; Solanum tuberosum Introduction A number of techniques have been devel- oped and applied during the past years which enable us to select plant-protoplasts, plant- cells, and, where morphogenesis is possible, en- tire plants with specific genetical, biochemical, physiological or physical characteristics as well. After the development of several genetical and biochemical selection procedures, the in- terests have also been directed towards the de- velopment of combined mechanical, optical and electronical systems which allow the auto- mated selection of plant protoplasts and plant cells. Several authors have discussed the results, problems and perspectives on flow sorting of plant-protoplasts (for review, see for example Refs. 2, 7 and 15). Protoplast sorting involves the passage of cells through a flow chamber containing a channel or orifice with a diameter in the range of 100 #m. The resultant fluid stream intersects *To whom correspondence should be addressed. the focus of a light beam resulting from a high pressure mercury lamp or a laser. The cells, in passing through the focus absorb light, and, if they contain fluorochromes, will subsequently re-emit the light in the form of fluorescence. This fluorescence is detected by the use of wave-length specific filters and one or more photomultipliers. The resultant DC-voltage signals are converted into binary equivalents and are either stored in the memory of a mi- croprocessor for further analysis or directed to the sorting device. Most of the sorting devices constructed so far have relied on the principle of electrostatic deflection of charged droplets created by the oscillation of a piezoelectric crystal. This prin- ciple was first used by Fulwyler [5] for the sep- aration of relatively small particles, i.e. white blood cells. Attempts to sort relatively large and fragile particles with these devices, in par- ticular also plant protoplasts have encoun- tered difficulties [15] because of limitations (orifice size, necessity for a conductive sheath fluid of low viscosity) and the need for modifi- cations of commercially available instruments [2,8,19]. In the past, cells have been sorted based on a number of different parameters. 0618-9452/87/$03.50 ((~ 1987 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
S E P A R A T I O N O F I N T A C T A N D D A M A G E D P L A N T - P R O T O P L A S T S BY U S I N G A C E L L - S O R T E R E Q U I P P E D W I T H A T W O - C H A N N E L P I E Z O V A L V E CHAMBER
M. BROMOVA and U.C. KNOPF* Agrogen-Foundation, P.O. Box 21, CH 1701 Freiburg (Switzerland)
(Received February llth, 1987) (Revision received May 18th, 1987) (Accepted May 18th, 1987)
Protoplasts were prepared from a suspension culture of Solanum tuberosum var. Bintje. A mixture of intact and damaged protoplasts was prepared, stained, respectively, with fluorescein diacetate (FDA) and Evans Blue. Subsequently the FDA- stained protoplasts were selected and sorted using a new sorting principle and device. Further analysis showed that 75% of the protoplasts remained intact using this sorting procedure.
A number of t echn iques have been devel- oped and applied dur ing the past years which enable us to select p lant-protoplas ts , plant- cells, and, where morphogenes i s is possible, en- t i re p lants wi th specific genet ical , b iochemical , physiological or physical cha rac te r i s t i c s as well.
Af ter the deve lopment of several genet ica l and b iochemica l se lec t ion procedures , the in- te res t s have also been d i rec ted towards the de- ve lopment of combined mechanica l , opt ical and e lec t ron ica l systems which al low the auto- mated se lec t ion of p lant pro toplas ts and p lan t cells.
Severa l au tho r s have discussed the results , problems and perspect ives on flow sor t ing of p lant -pro toplas ts ( for review, see for example Refs. 2, 7 and 15).
P ro top la s t sor t ing involves the passage of cells t h r ough a flow chamber con t a in ing a channe l or orifice wi th a d iamete r in the range of 100 #m. The r e su l t an t fluid s t ream in tersec ts
*To whom correspondence should be addressed.
the focus of a l ight beam resu l t ing from a h igh pressure mercu ry lamp or a laser. The cells, in passing t h r o u g h the focus absorb light, and, if they con ta in f luorochromes, will subsequent ly re-emit the l ight in the form of f luorescence. This f luorescence is de tec ted by the use of wave- length specific filters and one or more photomul t ip l ie rs . The r e su l t an t DC-vol tage signals are conver t ed in to b inary equiva len ts and are e i the r s tored in the memory of a mi- c roprocessor for f u r t h e r analys is or d i rec ted to the sor t ing device.
Most of the sor t ing devices cons t ruc ted so far have rel ied on the pr inciple of e lec t ros ta t i c def lect ion of charged drople ts c rea ted by the osci l la t ion of a p iezoelect r ic crystal . This prin- ciple was first used by Fu lwyle r [5] for the sep- a ra t ion of re la t ive ly small part ic les , i.e. whi te blood cells. At tempts to sor t r e la t ive ly large and fragi le par t ic les wi th these devices, in par- t i cu la r also p lan t pro toplas ts have encoun- tered difficulties [15] because of l imi ta t ions (orif ice size, necess i ty for a conduc t ive shea th fluid of low viscosi ty) and the need for modifi- ca t ions of commerc ia l ly ava i lab le in s t rumen t s [2,8,19]. In the past, cells have been sor ted based on a n u m b er of di f ferent parameters .
0618-9452/87/$03.50 ((~ 1987 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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These include the position within the cell cycle, surface antigen expression, cell size, metabolite content, and combinations of these parameters [3,7,11,13,14]. In this paper we report sorting of intact and damaged proto- plasts using a new sorting principle and device [6], a selective stain (FDA) for intact proto- plasts [18] and a counterselect ive stain (Evans Blue) for the damaged protoplasts [9].
Materials and methods
In vitro cultivated plant cells: for our experi- ments we used a suspension culture of the potato var. Bintje ST1. The culture was estab- lished a few years ago in our laboratories from shoot tips and subsequently regularly trans- ferred and maintained on a rotary shaker in the dark at 28"C.
Preparation and staining of isolated proto- plasts: 100 ml of 4-day-old suspension cul ture was centrifuged at 50 x g for 5min. The supernatant was removed, the cells were resuspended in medium P1 (CaC12 1480 mg/1; KH2PO 4 27.2mg/1; KNO3 101mg/1; MgSO4" 7H20 246mg/1; CuSO4"5H20 0.025mg/1; KI 0.16 mg/1; mannitol 13%, pH 4.5) and preplas- molysed for 1 h at 28'~C in the dark. The cells were recentr ifuged and resuspended in 100 ml of aqueous enzyme solution containing 13% mannitol, 750 mg cellulase Onozuka (Yakult, Honsha Co. Ltd., Tokyo, Japan), 300 mg pecti- nase Agrozyme I (Agrogen Promotion, P.O. Box 21, CH-1701 Freiburg, Switzerland) and 0.01% fluorescein diacetate (FDA), [12] (Serva Fein- biochemica, Heidelberg, F.R.G). The solution was incubated for 4 ~ h at 28°C in the dark.
Thereaf ter the protoplasts were filtrated through a nylon mesh 60pm in size, cen- trifuged and washed twice with medium P1 and passed through a nylon mesh (pore size: 40 pm). Thereaf ter the concentra t ion of the protoplasts was adjusted to 104-105/ml. The ex- tent and quali ty of staining with FDA was de- termined using an Olympus fluorescence microscope equipped with an exciting filter IF490, a dichroic mirror B (DM500 + 0515), and a barr ier filter O515.
Sizing of protoplasts: the protoplasts were photographed including meshes with different pore sizes of known diameter.
Preparation of a mixture of intact and dam- aged protoplasts: dead protoplasts were pre- pared by heating the protoplasts for 10 min at 8ffC. An equal amount of heat t reated and un- t reated FDA stained protoplasts was mixed in a 1 : 1 ratio, followed by adding to 2 ml of this mixture of protoplasts, 0.2 ml of Evans Blue (10 mg/20 ml aqueous solution of 0.6 M sor- bitol) [9]. The protoplasts stained dark blue were counted after different time intervals us- ing a light microscope.
Flow-sorting: for the analysis of the stained protoplasts we used a PCOSS I system (Agro- gen Promotion, P.O. Box 21, CH-1701 Freiburg, Switzerland) consisting of a flow-cytometer equipped with a high pressure mercury lamp, an exciting filter BG12, two dichroic mirrors (460 and 590) and a barr ier filter GG 550 as well as a cell-sorter equipped with a two channel flow-chamber (channel sizes: 100 × 200 ttm and 100 × 100 ttm, respectively) containing a Piezo valve and a 16-bit computer with the necessary soft-ware. Before use, the flow cytometer was calibrated, using nuclei of tobacco cells pre- pared as described previously [10]. For elec- tronic selection and sorting of protoplasts we used P1 as sheath-flow medium.
For purposes of analysis and for establish- ment of sort windows, 1-dimensional 256 chan- nel histograms of the emission intensity were accumulated at a flow rate of 200 ce l l s . s ' The sorting parameters were so chosen that we eliminated all the blue stained protoplasts while selecting the FDA stained protoplasts. Sorting of protoplasts was done routinely at room temperature.
After sorting, the protoplasts were concen- t rated by centr ifugation at 100 × g using a table centrifuge and resuspended in medium P1 for further analysis.
Determination of the intactness of the sorted protoplasts: in order to see to what ex- tent the protoplasts used in our experiments were damaged by the sorting procedure, we added Evans Blue to the sorted protoplasts as
above, and determined the percentage of blue and fluorescent protoplasts after 2 time intervals.
In order to check whether any protoplasts did indeed burst during the sorting procedure and which we were not able to detect with either of the dyes mentioned above, we added Hoechst 33342 (Serva Feinbiochemica, Heidel- berg, F.R.G.) as described by Puite and Ten Broeke [16] to the sorted protoplasts as well as to the waste solution before centrifugation. Both solutions were checked thereafter under the fluorescent microscope in order to see whether any free nuclei could be detected.
R esu l t s
The results are shown in Figs.1 and 2. The protoplasts used in these experiments had a size of 25 pm to 60 ttm. Ninety-five percent of the protoplasts used had a size of less than 40 pm. Figure la shows a 1 : 1 mixture of white (living) protoplasts and blue (dead) protoplasts previously heated and stained with Evans Blue before sorting. Figure 2 shows a typical his- togram of potato protoplasts stained with FDA and Evans Blue obtained after flow-cytometric analysis, as well as the sorting window chosen to select exclusively the FDA stained, white (living) protoplasts (i.e. channels 53-69). Six- teen percent of the cells were selected and sorted. As to the recovery rate we were able for example to recover 2054 ( = 89.8%) of 2287 ( = 100%) sorted protoplasts. Figure lb shows the potato protoplasts after electronic analysis and sorting. In our experiments we never found more than 0.5% blue protoplasts after elec- tronic selection and sorting. Figure lb shows that the sorted protoplasts were of all sizes. Although protoplasts of 60 #m were rare in our mixture, the results showed that they could also be sorted.
We were interested in determining to what extent our electronic selection and sorting pro- cedure caused damage to the plant-protoplasts. The results are shown in Fig. lc and d. Figure lc and ld show that some protoplasts were damaged (therefore being stained blue or show-
93
ing no FDA-fluorescence) after the sorting pro- cedure. The quanti tat ive analysis of the sorted protoplasts 30 min after sorting and collection showed that about 23% were damaged. This re- sult did not change significantly when the test with Evans Blue was redone 6 h after sorting and collection. Analysis of the Hoechst 33342 stained sorted protoplast solution and the pro- toplast solution collected from the waste chan- nel showed that we could rarely detect free nuclei in either one of the sorted or the waste protoplast solutions.
D i s c u s s i o n
Flow cytometry has been extensively ap- plied to analysis and separation of mammalian cells [1,11,14]. Therefore the sorting devices and instruments have been constructed partic- ularly around animal cells, generally not tak- ing into account the specific properties of other cells such as plant protoplasts. However, plant protoplasts generally have diameters substan- tially greater than those of cultured animal cells. Furthermore they are generally much more fragile than animal cells and the mainte- nance of their viability includes specific re- quirements as to the (osmotic) properties of the isolation medium. Therefore, previous reports on flow sorting of plant protoplasts included attempts to modify either the plant system, the instruments or both [4,7,8,15]. In this paper we report about the successful application of a new principle and device [6] for sorting plant protoplasts: the particle flow and sheath fluid flow are created by using a slight vacuum (840 mbar) in the flow system. About 2/3 of the sheath fluid surrounding the particle stream is deviated constantly into the waste channel (Fig. 3) which contains a piezo valve. About 1/3 of the fluid goes to the sorting channel (Fig. 3). Whenever a protoplast has to be selected, the piezo valve is activated. This creates a partial reduction of the diameter of the waste channel on one side and a pressure wave on the other. Both events cause a small amount of fluid con- taining the slected protoplast(s) to be directed into the sorting channel instead of into the
94
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Fig. 2. Typical histogram of a mixture of intact and damaged potato protoplasts stained with, respectively, FDA and Evans Blue. The figure shows the sorting window chosen (channels 53~69) in order to eliminate the Evans Blue stained protoplasts. In this experiment 20 749 cells were selected (sorted) from a total of 132 169.
waste channel. The entire event lasts a maxi- mum of 40 ps and the initial flow situation is restored within the same time interval. This principle allows the gentle sorting of up to 1000 plant-protoplasts/s, al though we often use the sorter at a lower sorting frequency. The fact that this sorting principle does not rely on the creation of pressure for creating a particle flow, nor an orifice and the creation of small droplets, nor the use of a conductive sheath fluid and the induction of a tension for charg- ing the droplets for their electrostatic deflec- tion, has made it particularly attractive for its use in sorting fragile plant protoplasts of different sizes. If, as in our case, the entire sort- ing procedure is operated in a closed, i.e. gas- free system, it allows the sorting of plant protoplasts with a recovery of at least up to 75% of intact protoplasts. Because of the differ- ential fragility of protoplasts from different sources, it is difficult to compare this result with those reported by others [3,7] using other protoplast sources and a sorting device based on the droplet-electrostatic deflection method. For practical purposes and further work our result is very encouraging, especially because
97
sorting of plant protoplasts can be done with- our system without having to undergo many compromises as to the culture (flow) media and the plant cell system.
