Plant Cell Reports
Plant Cell Reports (1996) 15:597-600
9 Springer-Verlag1996
Effect of culture methods on the regeneration of albino rice (Oryza sativa L.) plantlets Masayoshi Tsukahara *, Takayasu Hirosawa **, and Haruko Murayama Nursery Technology Inc., 3377 Kitsuregawa, Tochigi 329-14, Japan * Present address: Kirin Brewery Co., Ltd., 1-13-5 Fukuura, Kanazawa, Yokohama 236, Japan ** Present address: Kirin BreweryCo., Ltd., 3377 Kitsuregawa, Tochigi 329-14, Japan Received 24 November 1994/Revisedversion received 24 August 1995 - Communicated by A. Komamine
Ab~ra~ In our study, we investigated the effects of regeneration conditions on both green and albino rice plants (Oryza sativa L.). The regeneration frequency of an albino cell line was compared to a normal cell line obtained from mature seed under two kinds of culture conditions; namely, the static culture on semi-solid regeneration medium and the suspension culture in liquid regeneration medium. The albino cell line, from which only albino plantlets were regenerated, was induced from the albino leaf segments. There were no significant differences in the regeneration frequencies between normal and albino calli on the semisolid regeneration medium. On the other hand, the frequency of regeneration of albino calli was significantly lower than that of the control specifically in the liquid regeneration medium.
Key words: Albino, Liquid regeneration medium, sativa L.,
Plant regeneration,
Oryza
Semi-solid regeneration
medium
Introduction The occurrence of somaclonal variation in plants regenerated in tissue culture, which has been reported in many plant species and extensively reviewed (Larkin and Scowcroft 1981; Scowcroft 1985; Karp 1991), has been considered as a new and novel source of variation for crop improvement (Shepard et aI. 1980). On the other hand, clonal mass propagation in valuable plants, such as for F1 hybrids and transgenic plants, is based on the high efficiency of the tissue culture process (Vasil 1990). The variations observed during the tissue culture process may pose serious problems for elonal propagation and genetic transformation. Therefore, both crop improvement and mass propagation, it is necessary, to analyze and control the occurrence of somaclonal variation (Karp 1989). Correspondence to: M. Tsukahara
Recently, RFLP analysis has been employed to determine the occurrence and extent of somaclonal variation (Muller et al. 1990). However, this method is associated with several limitations such as the selection of probes and the length of time for detection (Brown 1991). On the other hand, the production of albino plants is one of the most frequent and conspicuous manifestations of somaclonal variation (Brown 1991). Since albino plants have been frequently observed in many species of regenerated plants and have been easily detected in vitro, this character could be used as a simple marker for the occurrence of variations in vitro. The following factors have been reported to influence the emergence of albino plants: genotype and physiological status of the anther donor plants (Bullock et al. 1982; Reiffers and Freirs 1990), developmental stage of microspores (Chen and Lin 1976), culture temperature for callus induction (Ouyang et al. 1983; Huang 1984), cold pretreatment of anther (Genovesi and Magill 1979), light intensity during culture (Bjornstad et al. 1989; Zieglar et al. 1990), floating culture of anther (Zhou and Konzak 1989), callus selection (Heszky 1989), growth regulator combination (Rout and Sarma 1991), addition of amino acids (Zhu et aL 1990) and sucrose concentration in combination with growth regulators (Clampham 1973). In those studies, the factors listed above affected not only the regeneration of albino plants but also that of green plants. The objective of this report is to study the factors affecting the occurrence of somaclonal variation. More specifically, we focused our study on factors that influence the regeneration frequency of albino plantlets using two regeneration methods: semi-solid regeneration medium and liquid regeneration medium.
Materials and Methods Plant materials
Mature seeds of rice (Oryza sativa L. cv. Sasanishiki)
598 were used throughout this study for the induction of a normal cell line. Albino planflets regenerated from the normal cell line on the semi-solid regeneration medium were used as explants for the induction of the albino cell line. Induction of callus and initiation o f suspension culture The seeds were dehusked and surface-sterilized with 10% sodium hypochlorite for 30 min and then rinsed three times with sterile distilled water. Fifteen sterilized seeds were placed on the callus induction medium that contained the inorganic salts and vitamins of N6 medium (Chu et al. 1975) supplemented with 12 mM proline, 0.1 g/1 casein acid hydrolysate (CH), 4 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D), 10 g/l sucrose, 30 g/1 sorbitol, 5 mM 2-(Nmorpholino)ethanesulfonic acid (MES) and 2 g/1 gellan gum (Kelco Co.). The pH of the medium was adjusted to 5.8 with KOH, and gellan gum was added prior to autoclaving at 121~ for 15 min. The calli which were formed in the scntellar region of the seeds were removed and transferred onto fresh callus induction medium and incubated for an additional 14 days. Suspension cultures were initiated by transferring approximately 1 g of callus to 500-ml Erlenmeyer flasks containing 100 ml of subculture medium. This subculture medium was the same as the callus induction medium except gellan gum. The cell suspension was cultured on a rotary shaker at 80 rpm in darkness and was subcultured at 7-day-intervals. At the time of the subculture, the cell clusters were broken into smaller clusters and cell clusters that passed a nylon mesh with a pore size of 1 mm were subcultured. The cell suspension was subcultured at least 6 times prior to use in regeneration studies. Albino callus was induced from 5-10 mm leaf segments of regenerated albino plantlets. Methods of initiation for the albino suspension culture were the same as those for the normal cell line. Plantlet regeneration Methods for plantlet regeneration on the semi-solid medium and in the liquid medium were the same as those described by Tsukahara and Hirosawa (1992) and Tsukahara et al. (submitted), respectively. After 7 days of suspension culture in a fresh medium, cell clusters were collected on a nylon mesh with a pore size of 1 ram. The collected cell clusters were rinsed three times with 2,4-Dfree subculture medium. The average size of the cell cluster was approximately 1-1.5 mm in diameter. A total of 20 mg of cells was transferred to a 9 cm plastic Petridish containing 25 ml of the semi-solid regeneration medium or to 300-ml Erlenmeyer flasks containing 25 ml of the liquid regeneration medium. The semi-solid regeneration medium contained inorganic salts and vitamins of MS medium (Murashige and Skoog 1962) supplemented with 2 g/1 CH, 2 rag/1 1-Naphthaleneacetic acid (NAA), 1 mg/1 kinetin, 30 g/1 sucrose, 30 g/1 sorbitol,
5 mM MES and 4 g/l geUan gum. The liquid regeneration medium contained the inorganic salts and vitamins of N6 medium supplemented with 12 mM proline, 0.1 g/l CH, 10 g/1 sucrose, 30 g/1 sorbitol, 5 mM MES, 0.1 mg/1 NAA and 0.1 rag/1 kinetin. The pH of the media was adjusted to 5.8 with KOH and the media were autoclaved at 121~ for 15 rain. Six replicated plates and/or flasks were prepared for each experiment. The cultures were incubated following a regular cycle of fluorescent light (16h, 30~ followed by darkness (8h, 25~ Flasks were cultured on a rotary shaker at 80 rpm with 70 mm strokes. Plates were cultured statically. After incubation for 6 weeks, the number of regenerated green and albino plantlets was counted.
Results and discussion
In previous studies, we had developed two methods of regeneration using either semi-solid or liquid media (Tsukahara and Hirosawa 1992, Tsukahara et al. submitted). These studies as well as this current study focused on plant regeneration on either semi-solid or liquid media. The results of this study showed that the total number of green and albino plants were about the same for regenerated plants grown on or suspended in semi-solid or liquid media, respectively (Table 1). Similar results were observed from our previous studies (Tsukahara and Hirosawa 1992, Tsukahara et al. submitted). However, when comparing the spontaneous emergence of albino plants, we found that there was about a one-third reduction in the number of albino plants regenerated in the liquid regeneration medium compared to those on the semi-solid medium (Table 1). However, the difference in the regeneration frequency was not significant due to uncertainty in the data. In addition, it was difficult to study the effects of culture conditions on the albino regeneration because the frequency of spontaneous emergence of albino was significantly low. To analyze the difference in the regeneration frequency of albino plants between the two regeneration methods and to distinguish the factors that affect the regeneration of albino plants from those that affect the regeneration of green plants, further study was needed using an albino cell line. An albino cell line was induced from leaves of albino plantlets that had spontaneously regenerated froma
Table 1. Regeneration frequency of green and albino plants from normal cell lines on either semi-solid or liquid media. Regeneration Number of green Number of albino medium plants plants Semi-solid medium 67.4+ 19.4 a 3.8+2.9 Liquid medium 71.7_+26.6 1.2__.0.8 aData are means_+ SE (n=6).
599 normal cell line. The albino cell line regenerated only albino plantlets. There was no significant difference in the appearance and growth rate of the subcultures, and in the regeneration frequency on the semi-solid regeneration medium between normal and albino cell lines. However, fwhen the normal and albino cell lines were regenerated in the liquid medium, we found about a one-tenth reduction in the number of regenerated plants from the albino cell line compared to those plants from the normal cell line (Table 2). By using the albino cell line, it was clearly demonstrated that only the liquid culture condition affected the regeneration of albino plants, even though those two kinds of culture conditions did not show significant differences in the frequency of the regeneration of the plants from normal cell line. To examine whether the above phenomenon is specific to these albino cell lines or not, we also used other albino cell lines induced from regenerated albino plants of two rice varieties, Akihikari and Akiyutaka. Similar results were also observed in these albino cell lines (data not shown), however, we only mentioned the results of albino plants induced by tissue culture. As mentioned earlier, it is generally recognized that medium components affect the regeneration of albino. The reason for a one-tenth reduction in albino frequency may be due to the differences in the nutrient composition of the semi-solid or liquid media. For example, the concentration of plant growth regulators, and of sucrose, and/or of other vital nutrients varied for each medium. Hence, to examine whether albino plant regeneration was dependent on media composition, we regenerated both normal and albino cell lines on liquid medium and liquid medium plus gellan gum. The liquid medium was prepared as outlined in the materials section of this report. Hence, the only difference between the above media was the addition of gellan gum. The media containing gellan gum will be referred to as "solidified liquid media". However, the normal cell line was the control. The number of regenerated plants of the liquid medium and the solidified liquid medium was compared. As shown in Table 3, fewer plants regenerated from the albino cell line than from the normal line. This result was
observed for both liquid and solidified liquid media. However, the number of albino plants were significantly fewer in the liquid medium than in the solidified liquid medium. Therefore, the reduction in the number of regenerated albino plants observed here was not due to differences in the nutrient components between the two regeneration media used in this study. A similar test was conducted on the semi-solid medium Liquefied "semi-solid" medium was prepared and the regeneration frequency of albino plants were compared with albino plants on semi-solid medium. Liquefied "semisolid" media was prepared from the same stock solution as semi-solid media but lacking gellan gum. For this test, no regenerated plants were observed from both normal and albino cell lines in the liquefied "semi-solid" medium. As mentioned earlier, environmental conditions, such as light intensity and temperature, affecting the regeneration of albino was also reported. As the static culture in Petri-dishes was used for semi-solid medium and suspension culture in flasks for liquid medium, there were many different factors in environmental conditions, such as the rate of gas exchange and humidity. The rate of oxygen supplement might be lower in cultures of the liquid medium than those of the semi-solid medium. Albino plants were assumed to be sensitive to the oxygen concentration because they could not depend on the photosynthesis for ATP supply and had to depend on oxygen respiration. Lower oxygen concentration inhibited the regeneration regardless of cell lines on the semi-solid medium (Table 4). In addition, cultures in darkness did not affect the frequency of regeneration of both cell lines (data not shown). These results suggested that the reduction of albino plants in the liquid medium could not be explained by the above assumption. We have also examined the effects of environmental conditions such as, light intensity, culture temperature and initial pH of medium on the regeneration of both green and albino plants on the semi-solid regeneration medium. However, these factors affected the regeneration of both green and albino plants in a similar manner (data not shown). On the other hand, a series of studies reported by Vassal et al. (Swedlund and Vasil 1985; Shenoy and Vasil
Table 2. Comparison between the number of plants regenerated on either semi-solid or liquid media using normal and albino cell lines. Cell line Number of regenerated plants semi-solid liquid medium medium Normal cell line 1 59.3__.17.5a 44,3 +_11.7 Albino celllinel 57.5+ 8.3 6.1+_ 4.8
Table 3. Comparison between the number of plants regenerated on either solidified liquid or liquid media using normal and albino cell lines. Cell line Number of regenerated plants solidified liquid medium liquid mediums Normal cell line 33.0+_3.6b 20.0+_5.3 Albino cell line 11.7+_0.6 0.7+_0.6 aSolidified medium was obtained after addition of 4 g/1 gellan gum to the liquid medium. Other culture conditions was same as those of the semi-solid medium. bData are means+_SE (n=6).
Normal cellline 2 72.5+ 5.1 Albino cell line 2 61.0+_ 12.5 ~Data are means+_SE (n=6).
108.7+_20.5 0.6+_ 0.3
600 Table 4, Effects of 02 concentrations on the number of plants regenerated on the semi-solid medium using normal and albino cell lines. Cell line 02 concentration (%)a 5 10 20 Normalcellline 2.3_+1.9 b 12.1_+8.5 42.3_+4.5 Albino cell line 1.8_+0.2 20.1_+7.7 51.8_+5.8 aO2 concentrations were controlled by the addition of nitrogen gas to air. bData are means_+ SE (n=6).
1992; Chowdhury and Vasil 1993) has shown that no variations at the cytological, biochemical and molecular levels were observed among the plants derived from somatic embryogenesis in several plant species. By comparing somatic embryogenesis versus organogenesis, they suggested that the occurrence of variations depended on the regeneration process. It is possible that the reduction of regeneration of albino plants may be due to differences in the regeneration process. However, based on the microscopic observation of the regeneration process, it appears that both green and albino plants were regenerated via somatic embryogenesis in the methods we developed. In the present study, we observed a reduction in the regeneration frequency of albino plants in the liquid medium. Our results suggested that this phenomenon was due to physical factors associated with the liquid culture instead of nutrient components of the medium. It still remains to be determined why only the regeneration frequency of albino plants decreased in the liquid medium. The analysis of the mechanisms that affects the regeneration of albino plants may enable to control the occurrence of not only albino but also other variations in regenerated plants.
Chu CC, Wang CC, Sun CS, Hsu C, Yin KC, Chu CY, Bi FY (1975) Sci Sin 18:659-668 Clapham D, Edwards J, Portlock P (1973) Z Pflanzenzuchtg 69:142-155 Genovesi AD, Magill CW (1979) Crop Sci 19:662-664 Heszky LE, Binh DQ, Kiss E, Gyulai G (1989) Plant Cell Rep 8:174-177 Huang B, Dunwell JM, Powell W, Hayter AM, Wood W (1984) Z Pflanzenzuchtg 92:22-29 Karp A (1985) Newsletter 58:2-11 Karp A (1991) In: Oxford Surveys Plant Molecular and Cell Biology. vol 7 Oxford University Press, Oxford, pp 1-58 Larkin PJ, Scowcroft WR (1981) Theor Appl Genet 60:197-214 Muller E, Brown PHT, Hartke S, Lorz H (1990) Theor Appl Genet 80:673-679 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Ouyang JW, Zhou SM, Jia SE (1983) Theor Appl Genet 66:101109 Reiffers I, Freire AB (1990) Plant Cell Tissue Organ Cult 21:165-170 Rout JR, Sarma NP (1991) Euphytica 54:155-159 Scowcroft WR (1985) In: Genetic Flux in Plant. Springer-Verlag Wien, New York, pp 217-245 Shenoy VB, Vasil IK (1992) Theor Appl Genet 83:947-955 Shepard JF, Bidney D, Shahin E (1980) Science 208:17-24 Swedlund B, Vasil 1I((1985) Theor Appl Genet 69:575-581
References Bjomstad A, Opsahl-Ferstad HG, Aasmo M (1989) Plant Tissue Organ Cult 17:27-37
Tsukahara M, Hirosawa T (1992) Bot Mag Tokyo 105:227-233 Cell
Bullock WP, Baenziger PS, Schaeffer GW, Bottino PJ (1982) Theor Appl Genet 62:155-159 Brown PHT (1991) Newsletter 66:14-25 Chen CC, Lin MH (1976) Bot Bull Acad Sin 17:18-24 Chowdhury MKU, Vasil IK (1993) Theor Appl Genet 86:181188
Tsukahara M, Hirosawa T, Kishine S (submitted) Vasil IK (1990) Bio/Technology 8:296-301 Zhou H, Konzak CF (1989) Crop Sci 29:817-821 Zhu M, Xu A, Yuan M, Huang C, Yu Z, Wang L, Yu J (1990) Plant Cell Tissue Organ Cult 22:201-204 Ziegler G, Dressler K, Hess D (1990) Plant Breeding 105:40-46
Plant Cell Reports (1996) 15:597-600
9 Springer-Verlag1996
Effect of culture methods on the regeneration of albino rice (Oryza sativa L.) plantlets Masayoshi Tsukahara *, Takayasu Hirosawa **, and Haruko Murayama Nursery Technology Inc., 3377 Kitsuregawa, Tochigi 329-14, Japan * Present address: Kirin Brewery Co., Ltd., 1-13-5 Fukuura, Kanazawa, Yokohama 236, Japan ** Present address: Kirin BreweryCo., Ltd., 3377 Kitsuregawa, Tochigi 329-14, Japan Received 24 November 1994/Revisedversion received 24 August 1995 - Communicated by A. Komamine
Ab~ra~ In our study, we investigated the effects of regeneration conditions on both green and albino rice plants (Oryza sativa L.). The regeneration frequency of an albino cell line was compared to a normal cell line obtained from mature seed under two kinds of culture conditions; namely, the static culture on semi-solid regeneration medium and the suspension culture in liquid regeneration medium. The albino cell line, from which only albino plantlets were regenerated, was induced from the albino leaf segments. There were no significant differences in the regeneration frequencies between normal and albino calli on the semisolid regeneration medium. On the other hand, the frequency of regeneration of albino calli was significantly lower than that of the control specifically in the liquid regeneration medium.
Key words: Albino, Liquid regeneration medium, sativa L.,
Plant regeneration,
Oryza
Semi-solid regeneration
medium
Introduction The occurrence of somaclonal variation in plants regenerated in tissue culture, which has been reported in many plant species and extensively reviewed (Larkin and Scowcroft 1981; Scowcroft 1985; Karp 1991), has been considered as a new and novel source of variation for crop improvement (Shepard et aI. 1980). On the other hand, clonal mass propagation in valuable plants, such as for F1 hybrids and transgenic plants, is based on the high efficiency of the tissue culture process (Vasil 1990). The variations observed during the tissue culture process may pose serious problems for elonal propagation and genetic transformation. Therefore, both crop improvement and mass propagation, it is necessary, to analyze and control the occurrence of somaclonal variation (Karp 1989). Correspondence to: M. Tsukahara
Recently, RFLP analysis has been employed to determine the occurrence and extent of somaclonal variation (Muller et al. 1990). However, this method is associated with several limitations such as the selection of probes and the length of time for detection (Brown 1991). On the other hand, the production of albino plants is one of the most frequent and conspicuous manifestations of somaclonal variation (Brown 1991). Since albino plants have been frequently observed in many species of regenerated plants and have been easily detected in vitro, this character could be used as a simple marker for the occurrence of variations in vitro. The following factors have been reported to influence the emergence of albino plants: genotype and physiological status of the anther donor plants (Bullock et al. 1982; Reiffers and Freirs 1990), developmental stage of microspores (Chen and Lin 1976), culture temperature for callus induction (Ouyang et al. 1983; Huang 1984), cold pretreatment of anther (Genovesi and Magill 1979), light intensity during culture (Bjornstad et al. 1989; Zieglar et al. 1990), floating culture of anther (Zhou and Konzak 1989), callus selection (Heszky 1989), growth regulator combination (Rout and Sarma 1991), addition of amino acids (Zhu et aL 1990) and sucrose concentration in combination with growth regulators (Clampham 1973). In those studies, the factors listed above affected not only the regeneration of albino plants but also that of green plants. The objective of this report is to study the factors affecting the occurrence of somaclonal variation. More specifically, we focused our study on factors that influence the regeneration frequency of albino plantlets using two regeneration methods: semi-solid regeneration medium and liquid regeneration medium.
Materials and Methods Plant materials
Mature seeds of rice (Oryza sativa L. cv. Sasanishiki)
598 were used throughout this study for the induction of a normal cell line. Albino planflets regenerated from the normal cell line on the semi-solid regeneration medium were used as explants for the induction of the albino cell line. Induction of callus and initiation o f suspension culture The seeds were dehusked and surface-sterilized with 10% sodium hypochlorite for 30 min and then rinsed three times with sterile distilled water. Fifteen sterilized seeds were placed on the callus induction medium that contained the inorganic salts and vitamins of N6 medium (Chu et al. 1975) supplemented with 12 mM proline, 0.1 g/1 casein acid hydrolysate (CH), 4 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D), 10 g/l sucrose, 30 g/1 sorbitol, 5 mM 2-(Nmorpholino)ethanesulfonic acid (MES) and 2 g/1 gellan gum (Kelco Co.). The pH of the medium was adjusted to 5.8 with KOH, and gellan gum was added prior to autoclaving at 121~ for 15 min. The calli which were formed in the scntellar region of the seeds were removed and transferred onto fresh callus induction medium and incubated for an additional 14 days. Suspension cultures were initiated by transferring approximately 1 g of callus to 500-ml Erlenmeyer flasks containing 100 ml of subculture medium. This subculture medium was the same as the callus induction medium except gellan gum. The cell suspension was cultured on a rotary shaker at 80 rpm in darkness and was subcultured at 7-day-intervals. At the time of the subculture, the cell clusters were broken into smaller clusters and cell clusters that passed a nylon mesh with a pore size of 1 mm were subcultured. The cell suspension was subcultured at least 6 times prior to use in regeneration studies. Albino callus was induced from 5-10 mm leaf segments of regenerated albino plantlets. Methods of initiation for the albino suspension culture were the same as those for the normal cell line. Plantlet regeneration Methods for plantlet regeneration on the semi-solid medium and in the liquid medium were the same as those described by Tsukahara and Hirosawa (1992) and Tsukahara et al. (submitted), respectively. After 7 days of suspension culture in a fresh medium, cell clusters were collected on a nylon mesh with a pore size of 1 ram. The collected cell clusters were rinsed three times with 2,4-Dfree subculture medium. The average size of the cell cluster was approximately 1-1.5 mm in diameter. A total of 20 mg of cells was transferred to a 9 cm plastic Petridish containing 25 ml of the semi-solid regeneration medium or to 300-ml Erlenmeyer flasks containing 25 ml of the liquid regeneration medium. The semi-solid regeneration medium contained inorganic salts and vitamins of MS medium (Murashige and Skoog 1962) supplemented with 2 g/1 CH, 2 rag/1 1-Naphthaleneacetic acid (NAA), 1 mg/1 kinetin, 30 g/1 sucrose, 30 g/1 sorbitol,
5 mM MES and 4 g/l geUan gum. The liquid regeneration medium contained the inorganic salts and vitamins of N6 medium supplemented with 12 mM proline, 0.1 g/l CH, 10 g/1 sucrose, 30 g/1 sorbitol, 5 mM MES, 0.1 mg/1 NAA and 0.1 rag/1 kinetin. The pH of the media was adjusted to 5.8 with KOH and the media were autoclaved at 121~ for 15 rain. Six replicated plates and/or flasks were prepared for each experiment. The cultures were incubated following a regular cycle of fluorescent light (16h, 30~ followed by darkness (8h, 25~ Flasks were cultured on a rotary shaker at 80 rpm with 70 mm strokes. Plates were cultured statically. After incubation for 6 weeks, the number of regenerated green and albino plantlets was counted.
Results and discussion
In previous studies, we had developed two methods of regeneration using either semi-solid or liquid media (Tsukahara and Hirosawa 1992, Tsukahara et al. submitted). These studies as well as this current study focused on plant regeneration on either semi-solid or liquid media. The results of this study showed that the total number of green and albino plants were about the same for regenerated plants grown on or suspended in semi-solid or liquid media, respectively (Table 1). Similar results were observed from our previous studies (Tsukahara and Hirosawa 1992, Tsukahara et al. submitted). However, when comparing the spontaneous emergence of albino plants, we found that there was about a one-third reduction in the number of albino plants regenerated in the liquid regeneration medium compared to those on the semi-solid medium (Table 1). However, the difference in the regeneration frequency was not significant due to uncertainty in the data. In addition, it was difficult to study the effects of culture conditions on the albino regeneration because the frequency of spontaneous emergence of albino was significantly low. To analyze the difference in the regeneration frequency of albino plants between the two regeneration methods and to distinguish the factors that affect the regeneration of albino plants from those that affect the regeneration of green plants, further study was needed using an albino cell line. An albino cell line was induced from leaves of albino plantlets that had spontaneously regenerated froma
Table 1. Regeneration frequency of green and albino plants from normal cell lines on either semi-solid or liquid media. Regeneration Number of green Number of albino medium plants plants Semi-solid medium 67.4+ 19.4 a 3.8+2.9 Liquid medium 71.7_+26.6 1.2__.0.8 aData are means_+ SE (n=6).
599 normal cell line. The albino cell line regenerated only albino plantlets. There was no significant difference in the appearance and growth rate of the subcultures, and in the regeneration frequency on the semi-solid regeneration medium between normal and albino cell lines. However, fwhen the normal and albino cell lines were regenerated in the liquid medium, we found about a one-tenth reduction in the number of regenerated plants from the albino cell line compared to those plants from the normal cell line (Table 2). By using the albino cell line, it was clearly demonstrated that only the liquid culture condition affected the regeneration of albino plants, even though those two kinds of culture conditions did not show significant differences in the frequency of the regeneration of the plants from normal cell line. To examine whether the above phenomenon is specific to these albino cell lines or not, we also used other albino cell lines induced from regenerated albino plants of two rice varieties, Akihikari and Akiyutaka. Similar results were also observed in these albino cell lines (data not shown), however, we only mentioned the results of albino plants induced by tissue culture. As mentioned earlier, it is generally recognized that medium components affect the regeneration of albino. The reason for a one-tenth reduction in albino frequency may be due to the differences in the nutrient composition of the semi-solid or liquid media. For example, the concentration of plant growth regulators, and of sucrose, and/or of other vital nutrients varied for each medium. Hence, to examine whether albino plant regeneration was dependent on media composition, we regenerated both normal and albino cell lines on liquid medium and liquid medium plus gellan gum. The liquid medium was prepared as outlined in the materials section of this report. Hence, the only difference between the above media was the addition of gellan gum. The media containing gellan gum will be referred to as "solidified liquid media". However, the normal cell line was the control. The number of regenerated plants of the liquid medium and the solidified liquid medium was compared. As shown in Table 3, fewer plants regenerated from the albino cell line than from the normal line. This result was
observed for both liquid and solidified liquid media. However, the number of albino plants were significantly fewer in the liquid medium than in the solidified liquid medium. Therefore, the reduction in the number of regenerated albino plants observed here was not due to differences in the nutrient components between the two regeneration media used in this study. A similar test was conducted on the semi-solid medium Liquefied "semi-solid" medium was prepared and the regeneration frequency of albino plants were compared with albino plants on semi-solid medium. Liquefied "semisolid" media was prepared from the same stock solution as semi-solid media but lacking gellan gum. For this test, no regenerated plants were observed from both normal and albino cell lines in the liquefied "semi-solid" medium. As mentioned earlier, environmental conditions, such as light intensity and temperature, affecting the regeneration of albino was also reported. As the static culture in Petri-dishes was used for semi-solid medium and suspension culture in flasks for liquid medium, there were many different factors in environmental conditions, such as the rate of gas exchange and humidity. The rate of oxygen supplement might be lower in cultures of the liquid medium than those of the semi-solid medium. Albino plants were assumed to be sensitive to the oxygen concentration because they could not depend on the photosynthesis for ATP supply and had to depend on oxygen respiration. Lower oxygen concentration inhibited the regeneration regardless of cell lines on the semi-solid medium (Table 4). In addition, cultures in darkness did not affect the frequency of regeneration of both cell lines (data not shown). These results suggested that the reduction of albino plants in the liquid medium could not be explained by the above assumption. We have also examined the effects of environmental conditions such as, light intensity, culture temperature and initial pH of medium on the regeneration of both green and albino plants on the semi-solid regeneration medium. However, these factors affected the regeneration of both green and albino plants in a similar manner (data not shown). On the other hand, a series of studies reported by Vassal et al. (Swedlund and Vasil 1985; Shenoy and Vasil
Table 2. Comparison between the number of plants regenerated on either semi-solid or liquid media using normal and albino cell lines. Cell line Number of regenerated plants semi-solid liquid medium medium Normal cell line 1 59.3__.17.5a 44,3 +_11.7 Albino celllinel 57.5+ 8.3 6.1+_ 4.8
Table 3. Comparison between the number of plants regenerated on either solidified liquid or liquid media using normal and albino cell lines. Cell line Number of regenerated plants solidified liquid medium liquid mediums Normal cell line 33.0+_3.6b 20.0+_5.3 Albino cell line 11.7+_0.6 0.7+_0.6 aSolidified medium was obtained after addition of 4 g/1 gellan gum to the liquid medium. Other culture conditions was same as those of the semi-solid medium. bData are means+_SE (n=6).
Normal cellline 2 72.5+ 5.1 Albino cell line 2 61.0+_ 12.5 ~Data are means+_SE (n=6).
108.7+_20.5 0.6+_ 0.3
600 Table 4, Effects of 02 concentrations on the number of plants regenerated on the semi-solid medium using normal and albino cell lines. Cell line 02 concentration (%)a 5 10 20 Normalcellline 2.3_+1.9 b 12.1_+8.5 42.3_+4.5 Albino cell line 1.8_+0.2 20.1_+7.7 51.8_+5.8 aO2 concentrations were controlled by the addition of nitrogen gas to air. bData are means_+ SE (n=6).
1992; Chowdhury and Vasil 1993) has shown that no variations at the cytological, biochemical and molecular levels were observed among the plants derived from somatic embryogenesis in several plant species. By comparing somatic embryogenesis versus organogenesis, they suggested that the occurrence of variations depended on the regeneration process. It is possible that the reduction of regeneration of albino plants may be due to differences in the regeneration process. However, based on the microscopic observation of the regeneration process, it appears that both green and albino plants were regenerated via somatic embryogenesis in the methods we developed. In the present study, we observed a reduction in the regeneration frequency of albino plants in the liquid medium. Our results suggested that this phenomenon was due to physical factors associated with the liquid culture instead of nutrient components of the medium. It still remains to be determined why only the regeneration frequency of albino plants decreased in the liquid medium. The analysis of the mechanisms that affects the regeneration of albino plants may enable to control the occurrence of not only albino but also other variations in regenerated plants.
Chu CC, Wang CC, Sun CS, Hsu C, Yin KC, Chu CY, Bi FY (1975) Sci Sin 18:659-668 Clapham D, Edwards J, Portlock P (1973) Z Pflanzenzuchtg 69:142-155 Genovesi AD, Magill CW (1979) Crop Sci 19:662-664 Heszky LE, Binh DQ, Kiss E, Gyulai G (1989) Plant Cell Rep 8:174-177 Huang B, Dunwell JM, Powell W, Hayter AM, Wood W (1984) Z Pflanzenzuchtg 92:22-29 Karp A (1985) Newsletter 58:2-11 Karp A (1991) In: Oxford Surveys Plant Molecular and Cell Biology. vol 7 Oxford University Press, Oxford, pp 1-58 Larkin PJ, Scowcroft WR (1981) Theor Appl Genet 60:197-214 Muller E, Brown PHT, Hartke S, Lorz H (1990) Theor Appl Genet 80:673-679 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Ouyang JW, Zhou SM, Jia SE (1983) Theor Appl Genet 66:101109 Reiffers I, Freire AB (1990) Plant Cell Tissue Organ Cult 21:165-170 Rout JR, Sarma NP (1991) Euphytica 54:155-159 Scowcroft WR (1985) In: Genetic Flux in Plant. Springer-Verlag Wien, New York, pp 217-245 Shenoy VB, Vasil IK (1992) Theor Appl Genet 83:947-955 Shepard JF, Bidney D, Shahin E (1980) Science 208:17-24 Swedlund B, Vasil 1I((1985) Theor Appl Genet 69:575-581
References Bjomstad A, Opsahl-Ferstad HG, Aasmo M (1989) Plant Tissue Organ Cult 17:27-37
Tsukahara M, Hirosawa T (1992) Bot Mag Tokyo 105:227-233 Cell
Bullock WP, Baenziger PS, Schaeffer GW, Bottino PJ (1982) Theor Appl Genet 62:155-159 Brown PHT (1991) Newsletter 66:14-25 Chen CC, Lin MH (1976) Bot Bull Acad Sin 17:18-24 Chowdhury MKU, Vasil IK (1993) Theor Appl Genet 86:181188
Tsukahara M, Hirosawa T, Kishine S (submitted) Vasil IK (1990) Bio/Technology 8:296-301 Zhou H, Konzak CF (1989) Crop Sci 29:817-821 Zhu M, Xu A, Yuan M, Huang C, Yu Z, Wang L, Yu J (1990) Plant Cell Tissue Organ Cult 22:201-204 Ziegler G, Dressler K, Hess D (1990) Plant Breeding 105:40-46
