An improvement of tomato protoplast culture for rapid plant regeneration

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1 Plant Cell, Tissue and Organ Culture 42: , (~) 1995 Kluwer Academic Publishers. Printed in the Netherlands. An improvement of tomato protoplast culture for rapid plant regeneration Monzur Hossain 1, Shigeru Imanishi & Hiroaki Egashira Section of Bioprocess Engineering, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata, 997 Japan. 1Department of Botany, Rajshahi University 6205, Bangladesh Received 11 October 1994; accepted in revised form 5 April 1995 Key words: Lycopersicon esculentum, Solanaceae Abstract Tomato mesophyll protoplasts were cultured in TM2 medium containing 5.7 #M a-naphthaleneacetic acid and 2.4 #M benzyladenine and were incubated either in stationary culture or on an orbital shaker at strokes per min, in combination with interval addition of fresh medium. The effects of stationary and shaking conditions on the growth of the colonies and their subsequent shoot organogenesis were significantly different. The cultures maintained in stationary condition without adding fresh medium accumulated a thin membranous layer on the medium surface and whitish substance in the medium that seemed to precede cell browning and premature colony death. Mild shaking conditions along with the reduction of colony density by one half by dividing the contents of one dish into two dishes, after adding 2 ml of fresh medium on the 4th day and further addition of fresh medium (0.5 ml) on the 8th day of plating, provided optimal conditions for colony growth and suppressed thin layer and whitish substance accumulation. Ten-day-old colonies raised through this protocol regenerated shoots rapidly (within days after initial plating) after transfer to regeneration medium (MS medium with 2.8/zM zeatin riboside, #M gibberellic acid, 4% sucrose and 1% type VII agarose) directly bypassing the callus phase. Abbreviations: BA-benzyladenine, GAa-gibberellic acid, IAA-indoleacetic acid, MS-Murashige & Skoog (1962) medium, NAA-a-naphthaleneacetic acid, SPM-stroke per min, GLM-General Linear Models, 2,4-D - 2,4- dichlorophenoxyacetic acid Introduction Protoplast manipulation in tomato (Lycopersicon esculentum Mill.) is a valuable tool for the introduction of desirable traits of the sexually incompatible wild species through somatic cell fusion or for direct gene transfer (Lefrancois et al. 1993). These techniques, however, require efficient, simple and repeatable methods for protoplast culture and plant regeneration. Several reports on plant regeneration from tomato protoplasts have been published (Morgan & Cocking 1982; Koblitz & Koblitz 1982; Shahin 1985, Niedz et al. 1985; Tan et al. 1987; Sakata et al. 1987; Bellini et al. 1990; Patil et al. 1994). Each method has been efficient with specific genotypes; however, none yielded satisfactory results under our conditions. Genotype-specific culture response and the browning of the colonies dur- ing the initial phase of culture are possible causes of low regeneration ability of tomato protoplasts. Several methods, such as preconditioning of donor plants, modifcation of protoplast isolation process, modification of culture media, or manipulation of culture conditions, have been described for improving tomato protoplast culture (Shahin 1985; Sakata et al. 1987; Bellini et al. 1990; Patil et al. 1994). The dilution of initial cultures with fresh medium has been used by several researchers. The effect of dilution on colony growth, however, has not been critically evaluated in previous reports. In addition, various authors have reported the formation of a thin layer on the medium surface during the early incubation period of tomato protoplast culture followed by accumulation of a whitish substance in the medium during the initial phase of culture. These

2 142 substances seemed to have an adverse effect on colony growth. The present study was designed for the systematic evaluation of the effect of different culture conditions and dilution of initial cultures with fresh medium at definite intervals on colony growth and subsequent plant regeneration from the tomato mesophyll protoplast. Materials and Methods Protoplast isolation and culture Lycopersicon esculentum cvs. 'Ohgata Zuiko' and 'Kyoryoku Tako' were used as plant materials. Aseptic cultivation of young seedlings and isolation of protoplasts were carried out according to Imanishi et al., (1991) with a slight modification. Seeds were dipped in 70% ethanol for 3 min and sterilized with 20% Antiformin solution (Wako Chemical, sodium hypochlorite, available chlorine 5%) containing 0.01% Tween 20 for 25 min. After washing 3 times with sterilized deionized water, the seeds were sown in 200 ml conical flasks (25 seeds/flask) containing 50 ml of growth-regulator-free 1/3 strength MS medium, 10 g 1-1 sucrose and 7 g 1-1 agar and incubated at 25 C in low light intensity (15 #mol m-2s -1, 16-h photoperiod). Three days later, the germinating seeds were transferred into 300 ml flasks (10 seedlings/flask) containing growth-regulator-free MS medium, 30 g 1 -l sucrose and 8 g 1-1 agar. The seedlings were initially incubated for two days at 25 C, 50 #mol m-2s -l, 16- h photoperiod, day-light fluorescent illumination and transferred to a growth chamber with 80 #mol m-2s- 1, 20 C, 16-h photoperiod and 70% relative humidity. Protoplasts were isolated from cotyledons of 9-10 dayold-seedlings. Cotyledons were notched, floated abaxially on CPW salts (Frearson et al. 1973) in 9% mannitol (CPW9M) solution and incubated in the dark for 1 h. CPW9M solution was replaced with 25 ml (for 1 g tissue) filter-sterilized enzyme solution (ph 6.0) contained 2.5% (w/v) Meicelase P (Meiji Seika, Japan), 0.25% (w/v) Mecerozyme R10 (Yakult Biochemical, Japan), 0.15% (w/v) Driselase (Kyowa Hakko Kogyo, Japan), 5.3 mm (w/v) morpholinoethanesulfonic acid, 0.5% (w/v) polyvinylpyrrolidone, 9% mannitol and CPW salts. The tissues were incubated in the dark at 26 C for 15 h. Enzyme solution was carefully pipetted off and tissues were squeezed in CPW9M solution to release the protoplasts. The undigested tissues were removed by passing the suspension through a 100 #m stainless steel mesh. The suspension was centrifuged at 60xg for 4 min. The pellet was resuspended in CPW in 21% sucrose (CPW21S) solution and centrifuged at 60xg for 8 rain. The protoplasts were collected from the top of the CPW21S solution and were washed twice with TM2 washing solution (Shahin 1985). The protoplasts were counted using a haemocytometer and their viability was confirmed using fluorescein diacetate (Larkin 1976) before culture. The protoplasts were cultured at a density of 5 x 104/ml in 60x 15 mm plastic Petri dishes each conraining 2 ml filter sterilized TM2 medium (Shahin 1985) contained 5.7 #M NAA and 2.4 #M BA. Different combinations and concentrations of BA + NAA, BA + 2,4-D, zeatin riboside + NAA, zeatin riboside + 2,4 -D or BA + NAA + 2,4 -D were also tested for protoplast division and growth. The growth regulator components in the present study were not critical to protoplast division and a wide range of auxins and cytokinins could be used. NAA at 5.7 #M with BA at 2.4 #M was used in the present study because this combination was found the most suitable for tomato cotyledon protoplast culture in our conditions. The cultures were incubated in the dark at 25 C for 3 days, and then transferred to 50 #mol m-2s -1, 16-h photoperiod, daylight fluorescent illumination. Maintenance of cultures Cultures were maintained either in a stationary condition (protocols 1-4) or on an orbital shaker at strokes per min (SPM) from the 4th day of culture (protocols 5-8). The procedures for the stationary and shake cultures without adding any fresh medium were designated as protocols 1 and 5, respectively. In protocols 2 and 6, 0.5 ml fresh TM2 medium without growth regulators was added to the cultures on the 4th day. Similarly, in protocols 3 and 7, the content of one culture dish was divided into two dishes after adding 2.0 ml fresh medium on the 4th day. In addition to this, 0.5 ml of the fresh medium was again added to the cultures on the 8th day in protocols 4 and 8, respectively. Plant regeneration Ten-day-old colonies raised through the different protocols were transferred directly onto regeneration medium. The regeneration medium consisted of MS salts, 1% agarose (type VII), 4% sucrose, 2.8 #M zeatin riboside and I/~M GA3. Zeatin riboside and

3 143 Table 1. The effect of various culture conditions on colony growth, and the accumulation of thin layer and whitish substance in the culture media in protoplasts culture of tomato (combined means for two cultivars). Culture Culture Addition of Repli- Plating Colony size protocols conditions fresh medium cations efficiency Intensity of (Diameter, ram) a (ml on the) 1 st 2nd Thin layer Whitish substance 1 Stationary very high very high " very high very high " high high " moderate moderate Shaker(25-30 spm) high moderate " very high moderate " low low " none low 0.67 adifference between two eultivars for colony diameter were not significant (p>0.05, F-0.18, df-1, 29). GA3 were filter sterilized and added to medium after autoclaving. Old medium from the culture was carefully removed with a Pasteur pipette prior to transfer of the colonies. The colonies were washed once with 5 ml growth-regulator-free TM2 and resuspended in 4 ml of the same medium. The colonies were then transferred, with a wide mouth pipette, onto the surface of regeneration medium 0.5 ml colony suspensions per 60 x 15 mm culture dish). Excess liquid medium was removed from the regeneration medium surface such that colonies remained half covered with liquid medium. The cultures were incubated at 25 C in a 70 #mol m-: s h photoperiod of cool white fluorescent illumination. Unless stated otherwise, all media were adjusted to ph 5.8 and sterilized by autoclaving for 20 min at 120 C and 108 kpa. Data recording Plating efficiency (number of protoplast-derived colonies/total number of protoplast plated x 100), a subjective rating of the thin layer and whitish substance accumulation, and colony size were determined on the 10th day of culture. The degree of accumulation of the thin layer and whitish substance was described as 'trace' when they could only slightly detected in the culture dishes, 'moderate' when a readily visible accumulation, and 'high' and 'very high' when easily recognized by the naked eye. For colony diame- Table 2. ANOVA for analysis of variance for colony diameter. Analysis was carried out from pooled data of two cultivars. Sources of variation d.f. m.s. F Culture condition (S) "** Culture dilution (D) "** SxD n.s. Error ***Significant at 0.01% level ter at least 100 randomly selected colonies/dish from 3 dishes/lieatment/replication were measured. Each treatment consisted of at least 4 replica culture dishes and each experiment was repeated at least three times. The experiments were analyzed as completely random designs. Analysis of variance for colony diameter was done accarding to the GLM procedure (General Linear Model) of the SAS (1989). Results and discussion Protoplasts in all culture protocols started to divide after 3 days of culture. A transparent, membranous, layer formed on the medium surface after the 3rd or 4th day, preceding the formation of a macroscopic, opaque, whitish substance observed from the 6th day of

4 144 stationary culture when no fresh medium was added. In protocols 1 and 2, the whitish substance increased dramaticauy on the 7th and 8th day and, at the same time, colonies often stopped their vigorous growth, became brown, and declined within 2 or 3 days. The thin layer and whitish substance probably resulted from metabolic byproducts of the rapidly growing cells under unfavorable conditions, such as high density, poor oxygen, or after nutrient depletion. In the shake culture of protocol 8, the whitish substance was either undetected or present in trace amounts. There was no difference between the two cultivars in the intensity of the accumulation of the thin layer and whitish substance. The plating efficiency, the intensity of thin layer and whitish substance and colony size at the 10th day of culture are shown in Table 1. The analysis of variance indicates that the difference between two cultivars in colony diameter was not significant (p>0.05) whereas differences between shake and stationary cuitures and among 4 kinds of culture manipulation were highly signifcant (Table 2). Maximum colony growth was observed when the cultures were maintained on a shaker from the 4th day of incubation and when colony density was decreased by dividing the content of one dish into two after adding 2 ml fresh medium on the 4th day, and further addition of 0.5 ml fresh medium on the 8th day after plating (protocol 8). No reports about the effect of agitation on protoplast culture have been found except for that of Ochatt et al., (1988) which demonstrated that shake culture benefited colony development of colt cherry protoplasts. Our result is the first indication of the beneficial effects of shake culture on tomato protoplast culture. Different methods, such as dilution, reduction of colony density, and washing the protoplasts-derived colonies with fresh medium have been employed to encourage shoot regeneration from tomato mesophyll protoplasts (Shahin 1985; Sakata et al. 1987). In these reports, however, the effects of such culture procedures on colony growth and shoot regeneration were not critically examined. Moreover, fresh medium used for dilution of culture in previous reports on tomato protoplast culture usually contained plant growth regulators. In the present study, growth-regulator-free TM2 or TM2 containing the same growth regulators as the initial plating medium were used for dilution. Growthregulator-free dilution medium in this study was more effective in sustaining normal colony growth and subsequent plant regeneration (data not shown). The formation of the thin layer and whitish substance generally decreased inversely with colony size Fig. 1. Plant regeneration from cotyledon protoplasts of tomato through organogenesis. (A) Small green calli with shoot primordia, 5 days after transferring onto MS regeneration medium supplemented with 2.8 #M zeatin riboside and #M GA3. Arrows indicate shoot primordia (bar = 0.4 mm). (B) Shoots proliferating from small calli, 20 days after the initial plating of protoplasts (bar = 1.5 mm). (C) Shoots proliferating from protoplast derived calli 28 days after initial plating (bar = 2.0 mm).

5 145 Table 3. The effect of different culture conditions on shoot regeneration from protoplasts colonies of two tomato cultivars. The colonies were transferred onto regeneration medium 10 days after protoplast isolation. Cultivars Culture Culture Ohgata Zuiko Kyoryoko Toko protocols conditions % ealli % green Days to % ealli % green Days to becoming calli shoot becoming enili shoot green regenerat- im'fiation a green regenerat- initiation ing tug 1 Stationary 0(1251) b 0 0 0(1041) " 0(1308) 0 0 0(1068) " 0(1398) 0 0 0(841) " e t (1340) (974) 5 Shaker 0(1530) 0 0 0(1340) 0 0 (25-30 spin) 6 " 0(1234) 0 0 0(939) " I (1447) (1046) 8 " , (1496) (1200) afrom the day of initial plating. bvalues in parentheses are total number of colonies in 3 experiments. CValues are + standard error. Statistical analysis for data given in percentage was carried out from angular transformed values, Incubation period 15 days. (Table 1). Shake culture, periodic addition of fresh medium, and division of the contents of one dish into two almost completely suppressed the formation of the thin layer and whitish substance. Concomitantly cell browning was reduced and vigorous growth of colony cell was observed. The different culture protocols also affected the subsequent morphogenic response of the colonies on regeneration medium (Table 3). The colonies raised by protocols 1, 2, 3, 5 and 6 failed to form green calli and regenerate. On the other hand, the colonies raised by protocols 4, 7 and 8 continued to grow, became green and subsequently regenerated shoots. However, the colonies raised in shake culture showed higher organogenic potential than those raised in stationary culture. In protocol 8, protoplasts continued to divide rapidly to form compact colonies most of which developed organized meristemoid-like-structures within 8-10 days. These colonies continued active growth, became green, and developed bud primordia within 5 days after transfer onto regeneration medium (Fig. 1A). Shoot buds appeared within days after initial plating (Fig. 1B). In previous reports (Morgan & Cocking 1982; Shahin 1985; Niedz et al., 1987; Patil et al. 1994), a three-step culture method --protoplast to colony formation, colony to callus formation, and callus to shoot induction--was employed for plant regeneration from tomato protoplasts. The time required for shoot induction (from the date of initial culture) was generally greater than in the present study. Shahin (1985) reported shoot induction 27 days after initial culture; Sakata et al. (1987), 48 days; Tan et al. (1987), 90 days; Koblitz & Koblitz (1982), 100 days; Niedz etal. (1985), 110 days and Patil etal. (1994), 42 days. The present study shows that shortening of shoot induction period from tomato protoplasts was possible by shake culture, by bypassing a separate phase for callus development (Fig. 1C). Similar rapid shoot regeneration from cotyledon protoplasts has also been confrmed for cv. 'Sekaiichi' (Egashira et al. 1993). The excised shoot buds continued to develop on transfer to MSagar medium containing 2% sucrose and 0.6 #M IAA. Regenerated plants were successfully transferred to the greenhouse.

6 146 Preconditioning of donor plants grown in the green house and in a controlled environment employed in previous studies was not required in the present study. The suppression of the thin layer and whitish substance by maintaining cultures on a shaker along with dilution of initial cultures and reduction of cell density at definite intervals, are considered to be critical factors that lead to vigorous colony growth and rapid shoot regeneration. Acknowledgements We wish to thank Professor Y. Sasaki, Kyoto University, for valuable suggestion for the statistical analysis of data. We also thank Dr. H. Suzuki and Ms. A. Matsumoto for technical help. References Bellini C, Chupean MC, Gervais M, Vastra G & Chupcau Y (1990) Importance of myoinositol, calcium and ammonium for the viability and division of tomato (Lycopersicon esculentum) protoplast. Plant Cell T~ss. Org. Cult. 23:27-37 Frearson EM, Power JB, & Cocking EC (1973) The isolation, culture and regeneration of Petunia leaf protoplasts. Dev. Biol. 33: Egashira H, Hossain M & Imanishi S (1993) Effect of ammoniumion on shoot regeneration from protoplast colony Japan J. Breed. 43(Suppl.1) 35. (in Japanese) Imanishi S, Egashira H, I-Iirono N & Satoh K ( 1991 ) An alternative to 'peeling' method for the isolation of leaf protoplast of Lycopersicon and Solanum. Bull. Yamagata Univ. Agri. Sci. 11(2): , (in Japanese) Koblitz H & Koblitz D (1982) Experiments on tissue culture in the genus Lycopersicon Miller. Mesophyll protoplasts regeneration to plant inlycopersicon esculentum ev. 'Nadja '+. Plant Cell Rep. 1: Larkin P (1976) Purification and viability determination of plant protoplasts. Planta 128: Lefrancois C, Chupean Y & JP Bourgin JP (1993) Sexual and somatic hybridization in the genus Lycopersicon. Theor. Appl. Genet. 86: Morgan A & Cocking EC (1982) Plant regeneration from protoplast of Lycopersicon esculentum Mill. Z. Pflanzenphysiol. 106: Murashige T & Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plant 15: Niedz RP, Rutter SM, Handley LW & Sink KC (1985) Plant regeneration from leaf protoplasts of six tomato cultivars. Plant Sci. 39: Ochatt SJ, Chand PK, Rech EL, Davey MR & Power JB (1988) Electroporation-mediated improvement of plant regeneration from colt cherry (Prunus avium pseudocerasus) protoplast. Plant Sci. 54: Patti RS, Davey MR & Power JB (1994) Highly effcient plant regeneration from mesophyll protoplasts of Indian field coltivars of tomato (Lycopersicon esculentura). Plant Cell Tiss, Org. Cult. 36:255'-258 Sakata Y, Nishio T & Takayanagi K (1987) P)ant regeneration from mesophyll protoplasts of tomato cv. Ponderosa. J. Japan. Soc. Hort. SCI. 56: SAS Institute Inc. (1989) SAS users guide: Statistics. Gary, North Carolina Shahin, EA (1985) Totipotency of tomato protoplasts. Theor. Appl. Genet. 69 : Tan MM, Rietveld EM, Van Marrewij~ GAM & Kool AJ (1987) Regeneration of leaf mesophyll protoplast of tomato cultivars (L. esculentum): factors important for efficient pmtoplast culture and plant regeneration. Plant Cell Rep. 6: