PHYSIOL. PLANT. 56: 324-328. Copenhagen 1982 The isolation, culture and division of protoplasts from citrus cotyledons David W. Burger and Wesley P. Hackett Burger, D. W. and Hackett, W. P. 1982. The isolation, culture and division of protoplasts from citrus cotyledons. - Physiol. Plant. 56i 324—328. High yields (2.3 X 10^ to 1.3 X 10' protoplasts/g.f.wt.) of isolated protoplasts were obtained from cotyledons ot Citrus sinensis (L.) Osb. 'Valencia'. Osmotic potential of the medium and enzyme concentrations were important in obtaining high viability of preparations as indicated by FDA fluorescence. Adding malt extract to a Murashige-Tucker basal medium increased plating efficiencies somewhat, but not the rate or duration of cell division. However, modifying the NAA and kinetin concentration optimized platiog efficiencies (up to 20%) of protoplasts and also the rate or duration of cell division. The highest plating efficiency and number of cells per colony were obtained on a defined medium containing NAA (15 \>M) and kinetin (4.6 (xM). Coincidence of percentage protoplast viability after 13 days (assessed by FDA fluorescence) with plating efficiency after 21 days indicates that FDA fluorescence is an accurate indicator of citrus protoplast viability. Additional key words - Cellulysin, Macerase, malt extract, plating efficiency, protoplast viability, protoplast yield, Valencia. D. W. Burger {reprint requests and permanent address), Texas A <& I Univ. Citrus Center, P.O. Box 2000, Weslaco, TX 78596, USA; W. P. Hackett, Dept. of Environmental Horticulture, Univ. of California, Davis, USA. Introduction The successful use of protoplasts to study any problem depends on the ability to isolate large numbers of viable protoplasts. The definition of important parameters affecting protoplast yield and viability is the first step toward this goal and is the focus of this paper. Osmoticum concentration and cell wall digestive enzyme concentrations are regarded as important paratneters affecting protoplast yield and viability (Coltnan and Mawson 1978, Kirby and Cheng 1979). Proper osmotic conditions are required to provide adequate plasmolysis, thus minimizing hydrolytic enzyme damage to membranes and reducing membrane damage from over-plasmolysis or membrane distension (Tribe 1955). Enzyme concentrations and/or times of exposure to the enzyme can influence protoplast release as well as influence the degree of damage to the cell membrane (Cassells and Badass 1976, Okuno and Furusawa 1977). It is possible that glycoprotein constituents ofthe cell membrane may be subject to attack if left exposed to digestive enzymes too long or to high concentrations of enzymes. Plating density has been found to be important in other protoplast systems (Durand 1979, Nagata and Takebe 1970, Nehls 1978, Power et al. 1976) and malt extract has been found to increase organogenic events in citrus tissue cultures (Kochba and Spiegei-Roy 1973). Plating density and malt extract concentration were varied here in an attempt to inerease the incidence of cell division. In the experiments described, factors required for isolating a large number of viable protoplasts, culture conditions and techniques necessary to achieve high plating efficiencies of citrus cotyledon protoplasts were investigated. Cotyledons were chosen as a tissue source of protoplasts based on their accessibility and their inherent potential for the regeneration of adventitious organs (D. W. Burger, 1980, Thesis, Univ. of Califor- Received 26 November, 1981; revised 12 June, 1982 324 0031-9317/82/110324-05 $03.00/0 © 1982 Physiologia Plantarum Ptiysiot. Plant. 56. t!>82 nia, Davis, USA). These results are important steps in developing a whole plant-to-protoplast-to-whole plant system. with Cellulysin at concentrations of 1, 2, 3, and 4% and Macerase at concentrations of 0.1, 0.3, 0.5, and 1.0%. Cotyledon segments were exposed to all treatments for Abbreviations — BAP, benzylaminopurine; HDA, fluorescein a total of 4 h in 0.6 M mannitol. Yield and viability diacetate; ME, malt extract; NAA, naphthaleneacetic acid; measurements were taken each honr. MT, Murashige and Tucker. Eaeh mannitol concentration (0.4 M to 0.65 M) tested was used for both the isolation and culture of the protoplasts for 13 days. Enzyme concentrations used lor Materials and methods isolation were 3% Cellulysin and 0.3% Macerase. Seeds from fruit of Citrus sinensis (L.) Osbeck 'Valencia' were used in all experiments. Mature fruit was har- Plating efficiency and optimum plating density. Plating vested and stored in a refrigerator at 3—4''C for at least efficiency, the percentage of isolated protoplasts under21 days before seeds were extracted. going division, was estimated after three weeks in culture. At the time of plating, random fields of protoplasts Protoplast isolation. The seeds were disinfested under in the agar were- marked by etching a circle 1.2 mm in aseptic conditions by a 1-3 s dip in 95% ethanol fol- diameter around the area of interest on the plastic petri lowed by flaming in an alcohol lamp. The seed coat was dish. The number of protoplasts in each field was removed and the cotyledons excised, weighed, sliced counted. After three weeks, the number of dividing cells into small pieces (less than 2 x 2 mm), and placed in a in each field was determined. The plating efficiency was 60 X 15 mm plastic petri dish containing a basal calculated as the number of dividing cells (cell colonies) Murashige and Tucker (MT) medium (Murashige and divided by the number of protoplasts plated x 100. This Tucker 1969) containing mannitol as osmoticum. The method of determination was designated the pre-desolution was removed after 45-60 min and replaced teimined field method. The isolated protoplasts, 95 ± with the same medium containing the cell wall hydroly- 5% viable, were cultured at densities ranging from 1 x tic enzymes Cellulysin and Macerase (Calbiochem). The 10* • ml-' to 1 X 10* • m|-' in MT medium solidified with petri dish containing the cotyledon pieces and enzyme 0.5% Taiyo agar. solution was rotated at ca. 70 rpm in the dark on a horizontal shaker. After 30—60 min the enzytne solution Malt extract, NAA, and kinetin effects on plating effiwas replaced with identical fresh solution and the tissue ciency. Protoplasts were cultured in an agar medium was incubated on the shaker for an additional 120-200 containing various concentrations of malt extract, min in the dark. The resulting solution was then passed naphthaleneacetic acid (NAA), and/or kinetin. The through a 37-[xm nylon filter to separate protoplasts number of cells per colony was determined by microsfrom cell debris and aggregates. The filtrate containing copic observation of all the colonies. intact protoplasts was centrifuged at ca. lOOg for five min. The supernatant was discarded and the pellet was resuspended in 1-2 ml of MT culture medium. This Results and discussion washing sequence was repeated twice. The final protoplast suspension was brought to a known volume and Optimum mannitol concentration for yield and viablitjaliquots were taken to measure protoplast yield and Protoplast yield was greatest when 0.6 M mannitol was viability. The protoplasts were cultured in hanging used (Fig. 1). Lower mannitol concentrations (0.4 M drops or suspended in an equal volume of MT medium and 0.45 M) caused bursting. The results of a viability containing molten 1% Taiyo agar (37-^0°C) in the dark at 25°C. Yield and viability determinations. Protoplast counts were made with a hemacytometer. Viability was assessed using fluorescein diacetate (FDA) as a test of membrane integrity and internal diesterase activity (Widholm 1972). After 2-5 min in 0.01% (w/v) FDA in culture medium, protoplasts were observed under ultraviolet light using a Zeiss standard photomicroscope equipped with epi-fluorescent attachments. The percent viability was calculated as the number of protoplasts fluorescing green per total number of intact protoplasts existing at day-0 x 100. Determination of optimum enzyme and mattnitol concentrations. A 4 X 4 factorial experiment was designed Ptiysiol. POant. 56. 1982 •0.4 0.4S 5 0.55 C c0ncentrati{in tM) Fig. 1. Effect of mannitol concentration on the yield of protoplasts from 'Valencia' cotyledons. Murashige-Tucker basai medium used in all solutions. Enzyme solution-3% Cellulysin, 0.3% Macerase. Bars represent ± SD from the mean of 5 replicates. 325 ConcBntratiam MannitsI M H M Oi.SS M 0.60 M o.es M Q'.4O 0.4 S O.S0 ~ A £a m D • O Tab. 1. Viable and total protoplasts (x 10^) released per g fresh weight from 'Valencia' cotyledons in various concentrations of Cellulysin and Macerase. Means of 5 replicates separated by Duncan's New Multiple Range Test, 5% level. Tabulated values for viable and total were statistically analyzed separately. Total or viable values followed by the same ietter(s) are not significantly different. Proto% Macerase plasts g drop culture Fig. 2. Time course study of viability of 'Valencia' cotyledon protoplasts isolated and cultured in various mannitol concentrations. A Murshige-Tucker basal medium was used in all solutions. Enzynae solutions contained 3% Cellulysin, 0.3% Macerase. Bars represent ± SD from the mean of 5' replicates. 0.1 0.3 0.5 1.0 viable total viable total viable total viable total % Ceilulysin 1 1.8 1.8 2.9 2.9 1.5 1.5 3.4 5.4 2 bi hi ghi hi 4.7 cde 4.7 fgh 5.3 cd 6.0 efg b 3.3 efg 4.2 ghi fgh 3.1 fgh fgh 7.8 defg 3 4 9.4 b 9.4 bcde 13.0 a 14.7 a 4.9 cde 12.6 ab 2.3 ghi 12.0 abc 9.2 b 10.5 bed 5.7 c 8.4 cdef 4.3 def 1].] abed 0 i 11.4 abed time course study tising FDA are shown in Fig. 2. Mannitol at 0.6 M gave the highest viability throughout the time course of the experiment. The decrease in viability after one day in the 0.4 M mannitol treatment was due to protoplast bursting. The 0.45 M or 0.5 M mannitol treatments supported protoplast viability longer than 0.4 M mannitol, but in both treatments protoplasts eventually lost all viability (Fig. 2). Although bursting of protoplasts was not observed, the numbers of protoplasts in these treatments dimitiished rapidly just prior to final loss of viability. This observation suggested a deterioration of the membrane over time due to osmotic pressure in the culture medium, insufficient plasmolysis of the tissue during isolation resulting in enzymeinduced injury, or to some other unknown factor. Results in Fig. 2 demonstrate that the viability of protoplasts measured immediately after isolation may not necessarily reflect the ability of the protoplasts to survive in culture over a period of time. It is likely that varying numbers of protoplasts are injured to some degree due to the duration or composition of the isolation treatment. Therefore, at the end of an isolation period, each treatment consists of a population of protoplasts of variable quality because protoplasts have been exposed to the digestion conditions for varying lengths of time. As a result, it is important to follow protoplast viability over a period of time to obtain a clearer picture of their response to isolation and culture conditions. enzyme combination gives a graphic illustration of toxic effects of the enzyme (note the lower right corner of Tab. 1). The critical Macerase concentration with regard to viability is 0.3%. At concentrations of 0.5% and greater, increasing concentrations of Celiulysin were particularly detrimental to protoplast viability. These results do not show the basis of decreased viability frotn exposure to super-critical enzyme concentrations. Viability measurements with FDA depend on membrane integrity and internal diesterase activity. Internal diesterase activity would have to be depressed indirectly since enzyme molecular size prevents direct contact with internal constituents. It is most likely that the digestive enzyme's deleterious effects act primarily on membrane integrity either by impeding membrane functions directly or as a result ofthe deleterious effects of associated impurities. At each enzyme digestion duration for which data were taken (every hour for 4 h), the same enzyme concentrations (3% Cellulysin and 0.3% Macerase) resulted in the highest yield and viability. Higher concentrations of the two enzymes caused loss of viability and lower concentrations did not liberate sufficient numbers of protoplasts. Optimum enz^ime concentrations Cellulysin at 3% and Macerase at 0.3% was the best enzyme combination for high yield and viability (Tab. 1). Effects of enzyme concentrations on yield and viability were different. As the concentration of enzymes increased, so did the yield of protoplasts. However, as the concentration of either enzyme was increased (Tab. 1), a point was reached where toxic effects of the enzyme solution were detected by decreases in viable protoplasts per gram fresh weight. Comparing the yield of protoplasts to the yield of viable protoplasts at each The maximum plating efficiency (ca 5%) was obtained at 1 X 10* protoplasts • m r ' (Fig. 3). Vardi et al. (1975) found that 'Shamouti' orange protoplasts from cell suspension cultures divided and formed colonies best (% efficiency) at a culture density of ca 1 x 10^ cells • ml~'. A major difference between the plating efficiencies reported by Vardi et al. (1975) and those reported here is the method of calculating efficiencies. Vardi and coworkers calculated plating efficiencies by microscopically scoring twenty random fields of plated protoplasts 326 Plating density Physiol. Plant. 56, 1982 Tab. 2. Protoplast plating efficiencies using two methods of determination and cells per colony as affected by malt extract additions to a Murashige-Tucker basal medium. Tabulated values are means of no less than 3 replicates. Mean separation witbin a column by Duncan's New Multiple Range Test, 5% level. Plating efficiency values for each method followed by the same letter are not significantly different. Malt (mg/l) 0 100 500 1000 Cells per colony 7.1 9.9 8.4 8.8 Plating efficiency Random field method 21.6 b 20.9 b 13.7 b 39.0 a Pre-determined field method • 5.1 b 4.9 b 5.1 b 8.4 a not significant 10* S«10 10 5»tO 10° 2). This treattnent increased the number of protoplasts undergoing division, but did not increase the rate of division as shown by the "cells per colony" data. Table 2 also shows the difference in two methods of calculaFig. 3. Effect of culture density on the plating efficiency of 'Valencia' cotyledon protoplasts isolated and cultured in a ting plating efficiencies. The random field method (as Murashige-Tucker basal medium containing 0.6 M mannitol. found in the literattu"e) exaggerated the plating effiProtoplasts were plated in tbe sanae medium containing 0.5% ciency in citrus preparations. Even though malt extract Taiyo agar. Protoplasts isolated using 3% Celulysin and 0.3% Macerase. Bars represent ± SD from the mean of not less than slightly increased protoplast plating efficiency and can three replicates. increase organogenic events in citrus tissue cultures (Kochba and Spiegel-Roy 1973), it was eliminated from four weeks after plating. This method has a major subsequent experiments because it is an undefined subdrawback in that the total number of protoplasts stance and therefore complicates the analysis of factors counted after four weeks is not an accurate estimate of controlling cell wall reformation and cell division. the total number of protoplasts plated since some have Plating efficiencies were significantly increased when surely burst. The burst protoplasts would not be the hormone levels of the MT basal medium were alcounted, thus plating efficiency estimates would be tered (Tab. 3). The protoplasts in the MT basal medium high. However, the random field method may be an (Treatment A) had a plating efficiency of 5.1%, which accurate estimate of plating efficiency in systems where is consistent with the results of the previous experiment. mortality rate is low. The method reported here takes These indicate that kinetin is limiting colony formation into account all protoplasts plated since the total in the MT basal medium (Treatment A) since when its number in a field is counted at the time of plating. level was raised to 2.3 [iM or 4.6 ^M with the same level Kao and Michayluk (1975) found that Vicia protop- of NAA (30 (iM, Treatments B and C) the plating effilasts developed poorly on defined media at low culture ciency increased threefold. When the kieetin:NAA densities (1-1000 protoplasts • ml"'). The culture den- ratio was increased further (Treatment D), the plating sity could be lowered if undefined components such as coconut water were added to the medium, suggesting that this low density phenomenon was due to diffusion Tab. 3. Effects of NAA (^lAf) and kinetin (\iM) in a of necessary metabolites from the cells to the medium, Murasbige-Tucker basal medium on plating efficiencies of 'Valencia' cotyledon protoplasts. Plating efficiencies were dedepleting the cells to levels too low to survive. These termined by the pre-determined field method. Mean separaresuits of Kao and Michayluk and our subsequent re- tion within a column by Duncan's New Multiple Range Test, sults with malt extract and growth regulators suggest 5% level. Values in columns followed by tbe same letter are that optimal plating densities for citrus cotyledon pro- not significaBtly different. toplasts may be lower than those reported here if malt Plating Treatment NAA:Kinetin Cells per extract or growth regulators at optimal concentrations colony efficiency are included in the medium. Culture density Cprotoplasts/ml} Malt extract, NAA and kinetin effects on plating efficiency' Plating efficiency was increased by supplementing a basal MT medium with 1000 mg- ]"' malt extract (Tab. 22 Pliysiol. Plant. 56, 1982 A B C D E 30:0.09 30:2.3 30:4.5 15:4.6 2.7:4.6 • 7.1 b 5.8 b 5.6 b 15.2 a 2.2 c 5.1 c 14.0 b 13.8 b 19.8 a 2.5 c 327 efficiency increased to nearly 20 %. This was the highest plating efficiency observed in any of the treatments. Results of Treatment E suggested that there was a threshold level of NAA required, or that the kinetin: NAA ratio was super-optimal. Treatment D not only increased numbers of cells undergoing division, it also increased the rate or duration of division (cells per colony). These results indicate that atixin and cytokinin levels are very important for initiating and sustaining division of cells derived from citrus cotyledon protoplasts. Hormone species and levels have been shown to be important in protoplast development. Power et al. (1976) found that leaf protoplast plating efficiency in Petunia was maximized in a Murashige and Skoog medium containing 11—27 \iM NAA and 1.6—3.2 uM BAP, very similar to the results reported here. Dudits et al. (1976) found that carrot protoplasts divided best in a Kao medium (Kao and Michayluk 1975) containing 1 X iO-^M NAA and 5 x lO'^'M zeatin, an auxin: cytokinin ratio of 2:1. The highest plating efficiency observed here (20%) is interesting in that it closely coincides with the percentage of the total cotyledon protoplasts that were viable (fluorescing green) after 13 days (Fig. 2). This fact argues in favor of using FDA as an accurate indicator of the viability of citrus protoplasts. It is also of interest in that the highest plating efficiency and cells per colony were obtained on a defined medium with quite specific levels of NAA and kinetin. Conclusion Citrus cotyledons liberate adequate numbers of high quality protoplasts which will reform a celi wall and divide in defined culture media. Callus cultures derived from protoplast colonies have yet to be induced to become organogenic. This is the remaining, important event currently under study, which must occur if this system is to be useful in following reproductive maturation at the cellular level. References Cassells, A. C. & Barlass, M. 1976. Environmentally induced changes in the cell walls of tomato leaves in relation to cell and protoplast release. - Physiol. Plant. 37: 239-246. Colman, B. & Mawson, B. T. 1978. The role of plasmolysis in tbe isolation of photosynthetically active leaf mesophyll cells. - Z . Pflanzenpbysiol. 86: 331-338. Dudits, D., Kao, K. N., Constabel, F. & Gamborg, O. L. 1976. Embryogenesis and formation of tetraploid and hexaploid plants from carrot protoplasts. - Can. .J. Bot. 54 (10): 1063-1067. Durand, J. 1979. High and reproducible plating efficiencies of protoplasts isolated from in vitro grown haploid Nicotiana sylvestris Spegax. et Comes. — Z. Pfianzenphysiol. 93: 283-295. Kao, K. N. & Michayluk, M. R. 1975. 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