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Endogenous Levels of Abscisic Acid in Embryogenic Cells, Nonembryogenic Cells and Somatic Embryos of Carrot (Daucus carota L.)
Biochem. Physiol. Pflanzen 188, 343-347 (1992) Gustav Fischer Verlag lena
Short Communication
Endogenous Levels of Abscisic Acid in Embryogenic Cells, Nonembryogenic Cells and Somatic Embryos of Carrot (Daucus carota L.) TOMOHIRO KIYOSUE1, MASATOSHI NAKAJIMA2, ISOMARO YAMAGUCHI2, SHINOBU SATOH 1, HIROSHI KAMADA 1, and HIROSHI HARADA 1 1 Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, and 2 Department of Agricultural Chemistry, The University of Tokyo, Tokyo, Japan Key Term Index: Abscisic acid (ABA), ELISA, embryogenic competence; Daucus carota L.
Summary The levels of endogenous ABA were measured by ELISA in embryogenic cells, nonembryogenic cells and somatic embryos of carrot. Embryogenic cells were estimated to contain a 67.2 times higher level of ABA than non-embryogenic cells, and a 2.5 times higher level than somatic embryos. The correlation between the endogenous level of ABA and embryogenic competence of carrot cultured cells is discussed. Somatic embryogenesis in carrot is well-known as a model system to study various aspects underlying plant early development (NOMURA and KOMAMINE 1986). Somatic embryos of carrot can be readily induced by the transfer of cultured embryogenic cells from auxin-containing medium to auxin-free medium, while non-embryogenic cells cannot form somatic embryos before or after such a transfer (STEWARD et al. 1958; SATOH et al. 1986). Recently, we have characterized an intracellular protein of carrot, ECP 31, which accumulates in embryogenic cells but not in non-embryogenic cells and developed somatic embryos (KIYOSUE et al. 1991). ECP31 was not detected in carrot seedlings, while it began to accumulate when the hypocotyl segments were cultured on auxincontaining medium, and the appearance of ECP31 coincided with visible embryogenic callus formation. Immunohistochemical analysis demonstrated that ECP31 was concentrated in the peripheral regions of embryogenic callus, where the proembryogenic masses possessing high ability to produce somatic embryos were known to originate. From the above results, we postulated that ECP31 functions in the induction and/or maintenance of embryogenic competence. Partial amino-acid sequences of ECP 31 demonstrated that ECP 31 had regions of homology to late-embryogenesis abundant (Lea) protein D 34 of cotton, whose gene Abbreviations: ABA, abscisic acid; Bo, 100% binding; ECP, embryogenic-cell protein; ELISA, enzyme-linked immunosorbent assay; NSE, non-specific binding; PNPP, p-nitrophenylphosphate; THF, tetrahydrofuran
BPP 188 (1992) 5
343
expression has been shown to be induced by exogenous ABA (GALAU et al. 1986; BAKER et aI. 1988; I{JYOSUE et aI. 1992). In this paper, we report the endogenous levels of ABA in embryogenic cells, non-embryogenic cells and somatic embryos and discuss the relationships among ECP31, ABA, and embryogenic competence in carrot. Plant Materials and Cell Cultures Embryogenic cell clusters of carrot, Daucus carota L. cv. US-Harumakigosun, were obtained from 1-month-old nodular calluses which had been formed by culturing segments of 1-week-old hypocotyls on Murashige and Skoog's (MS) agar medium (MURASHIGE and SKOOG 1962) that contained 4.5 X 1O- 6 M 2,4-D. Non-embryogenic cells were obtained from the same plant material as described in SA TOH et al. (1986). Cultures of both embryogenic and non-embryogenic cells were maintained by transfer at intervals of 2 weeks to fresh MS liquid medium supplemented with 4.5 x 1O- 6 M 2,4-D. Somatic embryos were produced by the transfer of embryogenic cells to auxinfree MS medium, as described in SATOH et al. (1986). Extraction and Quantification of ABA Embryogenic cells, non-embryogenic cells or somatic embryos (5 g fr wt. were harvested on the 14th day after the transfer to fresh media. Cells were homogenized in 30 ml of 80 % acetone, and the homogenates were centrifuged at 4,500 x g for 20 min. The supernatant was collected and the precipitate was washed twice with 30 ml of 80% acetone, and then the combined supernatant was concentrated in vacuo at 37°C to remove the acetone. The resulting aqueous solutions were diluted with distilled water up to 30 ml, and adjusted to pH 2.5 with 6 N H 2S04 , then were extracted thrice with ethyl acetate (10, 5, 5 ml), and a further three times with saturated NaHC0 3 solution (10, 5, 5 ml). The NaHC03 phase was acidified to pH 2.5 with 6 N H 2S04 and extracted thrice with ethyl acetate (20, 10, 10 ml). The acidic ethyl acetate extract was dried over anhydrous Na2S04, and concentrated at 25°C in vacuo and analyzed by gel-permeation chromatography (OPC). The dried samples were dissolved in 1 ml of tetrahydrofuran (THF) and filtered through a membrane filter (0.45 ~m in pore size). The filtrate was injected into a Shodex-Pak OPC column (20 mm Ld. X 500 mm, lOA in pore size; Showa Denko K. K., Tokyo) with a precolumn (8 mm i.d. x 500 mm) and eluted with THF at a flow rate of 3.2 ml min-I. Each eluate fraction with the retention time corresponding to ABA was collected and evaporated to dryness at 25°C in vacuo (YAMAGUCHI et al. 1983; MIYAZAKI and FUJII 1991). Quantification of ABA was carried out using an enzyme immunoassay kit (PHYTODETEKABA; Idetek Inc., California, USA) (WEILER 1982). An aliquot (100 ~l) of the sample dissolved at an adequate concentration with the dilution buffer was mixed with 100 ~l of ABA-alkaline phosphatase conjugate (tracer) solution in a small well which had been coated with a monoclonal antibody to 2-cis-( + )ABA, and incubated at 4°C. After a 4 h-incubation, the unbound tracer was washed away with the wash solution, and 200 ~l of p-nitrophenylphosphate (PNPP) (substrate) solution was poured into the well. The reaction of the substrate with the tracer which had coupled to the antibody was then allowed to proceed for 60 min at 37°C. Absorbance at 405 nm was measured with a microplate spectrophotometer (MTP-02; Corona Electric Co., Ltd., Katsuta, Japan). The average absorbances of triplicate standards and samples were used to calculate the percent binding (B/Bo%) and Logit B/Bo as follows (according to the kit manual); 0/ /0
B' d'
m mg
=
Standard or Sample O. D. - NSB O. D. x 100 Bo O. D. - NSB O. D.
. B/Bo = Ln ( Loglt Bo = 100% binding, NSB = Non-specific Binding. 344
BPP 188 (1992) 5
B/B o% ) 100 - BIBo%
2
'0 ED
iii .Cl
0
0 -1
..J
-2 -3
0.01
10
0.1 (+) ABA
(pmol)
Fig. 1. Standard logit-log plot for the ELISA and the points of endogenous levels of ABA in two cultured cells and somatic embryos. Competitive reaction with monoclonal antibody, between ABA-alkaline phosphatase conjugate (tracer) and ABA in samples were carried out in a small well of rnicrotitre plate. Absorbance of yellow substance, which is formed by the reaction of the tracer with PNPP, was measured at 405 nm. Based on the absorbance, Logit (B/Bo) were calculated. Logit (B/Bo) = In {(B/Bo)/( 1 - B/Bo)} where B = binding of tracer in the presence of unlabeled ABA; Bo = binding of tracer in the absence of unlabeled ABA. The points of 200 times dilution of extracts of embryogenic cells and somatic embryos and 20 times dilution of extracts of non-embryogenic cells are indicated with. , .. and ., respectively. Standard solution points are indicated by O . The endogenous levels of ABA were estimated using ELISA . The system gave a good linearity between 30 fmol and 3pmol of ABA in the standard logit-log plot (Fig. 1). The amounts of endogenous ABA calculated from the plot are shown in Table 1. The endogenous level of ABA in embryogenic cells was 67.2 times higher than that in nonembryogenic cells, and 2.5 times higher than that in somatic embryos, per unit fresh weight. Table 1. Abscisic acid contents in two types of cultured cells and somatic embryos. Embryogenic cells, non-embryogenic cells and somatic embryos were analyzed for ABA content at 14 d after the inoculations . ABA contents were calculated from the logit-Iog plot for ELISA (Fig. 1). Values represent the average ± SE of triplicates . ABA Contents
Embryogenic cells Non-embryogenic cells Somatic embryos
(pmol/g fr wt)
(pmol/g dry wt)
86± 11 1.28 ±0.03 34.6±3.1
428±52 12.0±0.3 322 ± 29
The relative endogenous levels of ABA in the above two types of cultured cells and in somatic embryo of carrot coincide with ECP31 levels in those cells and somatic embryos (KIYOSUE et al. 1991), which suggests that the amounts of ECP31 in those cells and organs may be determined by the endogenous levels of ABA. In light of the observation
23
BPP 188 (1992) 5
345
that the accumulation of ECP31 can be enhanced by exogenously applied ABA in somatic embryos (KIYOSUE et al. 1992), ABA appears to be involved in regulation of ECP31 expression. Recently, KAIMOR! and ISHIHARA (1991) reported a drying technique for selection and isolation of embryogenic cells from mixtures of embryogenic and non-embryogenic cells. They found that the drying procedure killed almost all of the non-embryogenic cells but did not kill embryogenic cells of carrot. This could be due to the difference in the endogenous levels of ABA between these two types of cultured cells, which was demonstrated in this paper for the first time, because it is known that ABA-treated plants can tolerate water stress better than non-treated plants (BARTELS et al. 1990). A higher level of ABA has also been reported in many plants during seed maturation and under stress conditions (FINKELSTEIN and CROUCH 1986; PERATA et al. 1990), and in embryogenic callus of Napiergrass, Pennisetum purpureum Schum., a monocot plant (RAJASEKARAN et al. 1987 a). It has also been demonstrated by using fluridone, an inhibitor of carotenoid biosynthesis, that higher levels of endogenous ABA are important to induce embryogenic competence in Napiergrass tissue culture (RAJASEKARAN et al. 1987b). It may also be ture in carrot culture system that a high level of ABA is necessary to induce or maintain embryogenic competence.
Acknowledgements Authors thank Dr. D. MACER for his linguistis suggestions. This research was supported in part by a Grant-in-Aid for Special Research on Priority Areas (No. 03226lO1) from the Ministry of Education, Science and Culture, Japan and a Special Coordination Funds of the Science and Technology Agency of the Japanese Government.
References BAKER, J. C., STEELE, C., and DURE, L. III.: Sequence and characterization of 6 Lea proteins and their genes from cotton. Plant Mol. BioI. 11, 227-291 (1988). BARTELS, D., SCHNEIDER, K., TERSTAPPEN, G., PIATKOWSKI, D., and SALAMINI, F.: Molecular cloning of abscisic acid-modulated genes which are induced during desiccation of the resurrection plant Craterostigma plantagineum. Planta 181,27-34 (1990). FINKELSTEIN, R. R., and CROUCH, M. L.: Rapeseed embryo development in culture on high osmoticum is similar to that in seeds. Plant Physiol. 81, 907-912 (1986). GALAU, G. A., HUGHES, D. W., and DURE, L. III: Abscisic acid induction of cloned cotton late embryogenesis-abundant (Lea) mRNAs. Plant Mol. BioI. 7,155-170 (1986). KAIMORI, N., and ISHIHARA, A.: Selection of embryogenic callus of carrot by drying treatment. Japan. J. Breed. 41, 169-173 (1991). KIYOSUE, T., SATOH, S., KAMADA, H., and HARADA, H.: Purification and immunohistochemical detection of an embryogenic cell protein in carrot. Plant Physiol. 95, lO77-lO83 (1991). KIYOSUE, T., NAKAYAMA, J., SATOH, S., ISOGAI, A., SUZUKI, A., KAMADA, H., and HARADA, H.: Partial amino-acid sequence of ECP31, a carrot embryogenic-cell protein, and enhancement of its accumulation by abscisic acid in somatic embryos. Planta 186, 337 - 342 (1992). MIYAZAKI, A., and FUJII, T.: Distribution ofIAA and ABA in gravistimulated primary roots of Zea mays L. Bot. Mag. Tokyo 104, 309-321 (1991). MURASHIGE, T., and SKOOG, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15,473-497 (1962). 346
BPP 188 (1992) 5
NOMURA, K., and KOMAMINE, A. : Molecular mechanisms of somatic embryogenesis. Oxf. Surv. Plant Mol. BioI 3, 456-466 (1986). PERA TA, P., PICCIARELLI, P., and ALPI, A.: Pattern of variations in abscisic acid content in suspensors, embryos, and integuments of developing Phaseolus coccineus seeds. Plant Physiol. 94, 1776-1780 (1990). RAJASEKARAN, K., HEIN, M . B., DAVIS, G. c., CARNES, M. G., and VASIL, I. K.: Endogenous growth regulators in leaves and tissue cultures of Pennisetum purpureum Schum. J. Plant Physiol. 130, 13-25 (l987a). RAJASEKARAN, K., HEIN, M. B., and VASIL, 1. K.: Endogenous abscisic acid and indole-3-acetic acid and somatic embryogenesis in cultured leaf explants of Pennisetum purpureum Schum. Plant Physiol. 84, 47-51 (l987b). SATOH, S., KAMADA, H., HARADA, H., and FUJII, T.: Auxin-controlled glycoprotein release into the medium of embryogenic carrot cells. Plant Physiol. 81, 931-933 (1986). STEWARD, F. C., MAPES , M. 0 ., and MEARS, K.: Growth and organized development of cultured cells . I. Growth and division of freely suspended cells. Am. J. Bot. 45, 693-703 (1958). WEILER, E. W.: An enzyme-immunoassay for cis-(+)-abscisic acid . Physiol. Plant. 54, 510-514 (1982). YAMAGUCHI, I., FUJISA WA, S., and TAKAHASHI, N. : Systematic ultramicroanalysis of plant growth regulators. In: IUPAC Pestic. Chern . Hum. Welfare Environ. 2, (Ed. MIYAMOTO, J.) Pergamon Press, pp. 145-150, Oxford 1983.
Received February 25, 1992; revised/orm accepted May 29, 1992 Authors' address: Dr. T. KIYOSUE, Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki-ken, 305, Japan .
23*
BPP 188 (1992) 5
347
Short Communication
Endogenous Levels of Abscisic Acid in Embryogenic Cells, Nonembryogenic Cells and Somatic Embryos of Carrot (Daucus carota L.) TOMOHIRO KIYOSUE1, MASATOSHI NAKAJIMA2, ISOMARO YAMAGUCHI2, SHINOBU SATOH 1, HIROSHI KAMADA 1, and HIROSHI HARADA 1 1 Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, and 2 Department of Agricultural Chemistry, The University of Tokyo, Tokyo, Japan Key Term Index: Abscisic acid (ABA), ELISA, embryogenic competence; Daucus carota L.
Summary The levels of endogenous ABA were measured by ELISA in embryogenic cells, nonembryogenic cells and somatic embryos of carrot. Embryogenic cells were estimated to contain a 67.2 times higher level of ABA than non-embryogenic cells, and a 2.5 times higher level than somatic embryos. The correlation between the endogenous level of ABA and embryogenic competence of carrot cultured cells is discussed. Somatic embryogenesis in carrot is well-known as a model system to study various aspects underlying plant early development (NOMURA and KOMAMINE 1986). Somatic embryos of carrot can be readily induced by the transfer of cultured embryogenic cells from auxin-containing medium to auxin-free medium, while non-embryogenic cells cannot form somatic embryos before or after such a transfer (STEWARD et al. 1958; SATOH et al. 1986). Recently, we have characterized an intracellular protein of carrot, ECP 31, which accumulates in embryogenic cells but not in non-embryogenic cells and developed somatic embryos (KIYOSUE et al. 1991). ECP31 was not detected in carrot seedlings, while it began to accumulate when the hypocotyl segments were cultured on auxincontaining medium, and the appearance of ECP31 coincided with visible embryogenic callus formation. Immunohistochemical analysis demonstrated that ECP31 was concentrated in the peripheral regions of embryogenic callus, where the proembryogenic masses possessing high ability to produce somatic embryos were known to originate. From the above results, we postulated that ECP31 functions in the induction and/or maintenance of embryogenic competence. Partial amino-acid sequences of ECP 31 demonstrated that ECP 31 had regions of homology to late-embryogenesis abundant (Lea) protein D 34 of cotton, whose gene Abbreviations: ABA, abscisic acid; Bo, 100% binding; ECP, embryogenic-cell protein; ELISA, enzyme-linked immunosorbent assay; NSE, non-specific binding; PNPP, p-nitrophenylphosphate; THF, tetrahydrofuran
BPP 188 (1992) 5
343
expression has been shown to be induced by exogenous ABA (GALAU et al. 1986; BAKER et aI. 1988; I{JYOSUE et aI. 1992). In this paper, we report the endogenous levels of ABA in embryogenic cells, non-embryogenic cells and somatic embryos and discuss the relationships among ECP31, ABA, and embryogenic competence in carrot. Plant Materials and Cell Cultures Embryogenic cell clusters of carrot, Daucus carota L. cv. US-Harumakigosun, were obtained from 1-month-old nodular calluses which had been formed by culturing segments of 1-week-old hypocotyls on Murashige and Skoog's (MS) agar medium (MURASHIGE and SKOOG 1962) that contained 4.5 X 1O- 6 M 2,4-D. Non-embryogenic cells were obtained from the same plant material as described in SA TOH et al. (1986). Cultures of both embryogenic and non-embryogenic cells were maintained by transfer at intervals of 2 weeks to fresh MS liquid medium supplemented with 4.5 x 1O- 6 M 2,4-D. Somatic embryos were produced by the transfer of embryogenic cells to auxinfree MS medium, as described in SATOH et al. (1986). Extraction and Quantification of ABA Embryogenic cells, non-embryogenic cells or somatic embryos (5 g fr wt. were harvested on the 14th day after the transfer to fresh media. Cells were homogenized in 30 ml of 80 % acetone, and the homogenates were centrifuged at 4,500 x g for 20 min. The supernatant was collected and the precipitate was washed twice with 30 ml of 80% acetone, and then the combined supernatant was concentrated in vacuo at 37°C to remove the acetone. The resulting aqueous solutions were diluted with distilled water up to 30 ml, and adjusted to pH 2.5 with 6 N H 2S04 , then were extracted thrice with ethyl acetate (10, 5, 5 ml), and a further three times with saturated NaHC0 3 solution (10, 5, 5 ml). The NaHC03 phase was acidified to pH 2.5 with 6 N H 2S04 and extracted thrice with ethyl acetate (20, 10, 10 ml). The acidic ethyl acetate extract was dried over anhydrous Na2S04, and concentrated at 25°C in vacuo and analyzed by gel-permeation chromatography (OPC). The dried samples were dissolved in 1 ml of tetrahydrofuran (THF) and filtered through a membrane filter (0.45 ~m in pore size). The filtrate was injected into a Shodex-Pak OPC column (20 mm Ld. X 500 mm, lOA in pore size; Showa Denko K. K., Tokyo) with a precolumn (8 mm i.d. x 500 mm) and eluted with THF at a flow rate of 3.2 ml min-I. Each eluate fraction with the retention time corresponding to ABA was collected and evaporated to dryness at 25°C in vacuo (YAMAGUCHI et al. 1983; MIYAZAKI and FUJII 1991). Quantification of ABA was carried out using an enzyme immunoassay kit (PHYTODETEKABA; Idetek Inc., California, USA) (WEILER 1982). An aliquot (100 ~l) of the sample dissolved at an adequate concentration with the dilution buffer was mixed with 100 ~l of ABA-alkaline phosphatase conjugate (tracer) solution in a small well which had been coated with a monoclonal antibody to 2-cis-( + )ABA, and incubated at 4°C. After a 4 h-incubation, the unbound tracer was washed away with the wash solution, and 200 ~l of p-nitrophenylphosphate (PNPP) (substrate) solution was poured into the well. The reaction of the substrate with the tracer which had coupled to the antibody was then allowed to proceed for 60 min at 37°C. Absorbance at 405 nm was measured with a microplate spectrophotometer (MTP-02; Corona Electric Co., Ltd., Katsuta, Japan). The average absorbances of triplicate standards and samples were used to calculate the percent binding (B/Bo%) and Logit B/Bo as follows (according to the kit manual); 0/ /0
B' d'
m mg
=
Standard or Sample O. D. - NSB O. D. x 100 Bo O. D. - NSB O. D.
. B/Bo = Ln ( Loglt Bo = 100% binding, NSB = Non-specific Binding. 344
BPP 188 (1992) 5
B/B o% ) 100 - BIBo%
2
'0 ED
iii .Cl
0
0 -1
..J
-2 -3
0.01
10
0.1 (+) ABA
(pmol)
Fig. 1. Standard logit-log plot for the ELISA and the points of endogenous levels of ABA in two cultured cells and somatic embryos. Competitive reaction with monoclonal antibody, between ABA-alkaline phosphatase conjugate (tracer) and ABA in samples were carried out in a small well of rnicrotitre plate. Absorbance of yellow substance, which is formed by the reaction of the tracer with PNPP, was measured at 405 nm. Based on the absorbance, Logit (B/Bo) were calculated. Logit (B/Bo) = In {(B/Bo)/( 1 - B/Bo)} where B = binding of tracer in the presence of unlabeled ABA; Bo = binding of tracer in the absence of unlabeled ABA. The points of 200 times dilution of extracts of embryogenic cells and somatic embryos and 20 times dilution of extracts of non-embryogenic cells are indicated with. , .. and ., respectively. Standard solution points are indicated by O . The endogenous levels of ABA were estimated using ELISA . The system gave a good linearity between 30 fmol and 3pmol of ABA in the standard logit-log plot (Fig. 1). The amounts of endogenous ABA calculated from the plot are shown in Table 1. The endogenous level of ABA in embryogenic cells was 67.2 times higher than that in nonembryogenic cells, and 2.5 times higher than that in somatic embryos, per unit fresh weight. Table 1. Abscisic acid contents in two types of cultured cells and somatic embryos. Embryogenic cells, non-embryogenic cells and somatic embryos were analyzed for ABA content at 14 d after the inoculations . ABA contents were calculated from the logit-Iog plot for ELISA (Fig. 1). Values represent the average ± SE of triplicates . ABA Contents
Embryogenic cells Non-embryogenic cells Somatic embryos
(pmol/g fr wt)
(pmol/g dry wt)
86± 11 1.28 ±0.03 34.6±3.1
428±52 12.0±0.3 322 ± 29
The relative endogenous levels of ABA in the above two types of cultured cells and in somatic embryo of carrot coincide with ECP31 levels in those cells and somatic embryos (KIYOSUE et al. 1991), which suggests that the amounts of ECP31 in those cells and organs may be determined by the endogenous levels of ABA. In light of the observation
23
BPP 188 (1992) 5
345
that the accumulation of ECP31 can be enhanced by exogenously applied ABA in somatic embryos (KIYOSUE et al. 1992), ABA appears to be involved in regulation of ECP31 expression. Recently, KAIMOR! and ISHIHARA (1991) reported a drying technique for selection and isolation of embryogenic cells from mixtures of embryogenic and non-embryogenic cells. They found that the drying procedure killed almost all of the non-embryogenic cells but did not kill embryogenic cells of carrot. This could be due to the difference in the endogenous levels of ABA between these two types of cultured cells, which was demonstrated in this paper for the first time, because it is known that ABA-treated plants can tolerate water stress better than non-treated plants (BARTELS et al. 1990). A higher level of ABA has also been reported in many plants during seed maturation and under stress conditions (FINKELSTEIN and CROUCH 1986; PERATA et al. 1990), and in embryogenic callus of Napiergrass, Pennisetum purpureum Schum., a monocot plant (RAJASEKARAN et al. 1987 a). It has also been demonstrated by using fluridone, an inhibitor of carotenoid biosynthesis, that higher levels of endogenous ABA are important to induce embryogenic competence in Napiergrass tissue culture (RAJASEKARAN et al. 1987b). It may also be ture in carrot culture system that a high level of ABA is necessary to induce or maintain embryogenic competence.
Acknowledgements Authors thank Dr. D. MACER for his linguistis suggestions. This research was supported in part by a Grant-in-Aid for Special Research on Priority Areas (No. 03226lO1) from the Ministry of Education, Science and Culture, Japan and a Special Coordination Funds of the Science and Technology Agency of the Japanese Government.
References BAKER, J. C., STEELE, C., and DURE, L. III.: Sequence and characterization of 6 Lea proteins and their genes from cotton. Plant Mol. BioI. 11, 227-291 (1988). BARTELS, D., SCHNEIDER, K., TERSTAPPEN, G., PIATKOWSKI, D., and SALAMINI, F.: Molecular cloning of abscisic acid-modulated genes which are induced during desiccation of the resurrection plant Craterostigma plantagineum. Planta 181,27-34 (1990). FINKELSTEIN, R. R., and CROUCH, M. L.: Rapeseed embryo development in culture on high osmoticum is similar to that in seeds. Plant Physiol. 81, 907-912 (1986). GALAU, G. A., HUGHES, D. W., and DURE, L. III: Abscisic acid induction of cloned cotton late embryogenesis-abundant (Lea) mRNAs. Plant Mol. BioI. 7,155-170 (1986). KAIMORI, N., and ISHIHARA, A.: Selection of embryogenic callus of carrot by drying treatment. Japan. J. Breed. 41, 169-173 (1991). KIYOSUE, T., SATOH, S., KAMADA, H., and HARADA, H.: Purification and immunohistochemical detection of an embryogenic cell protein in carrot. Plant Physiol. 95, lO77-lO83 (1991). KIYOSUE, T., NAKAYAMA, J., SATOH, S., ISOGAI, A., SUZUKI, A., KAMADA, H., and HARADA, H.: Partial amino-acid sequence of ECP31, a carrot embryogenic-cell protein, and enhancement of its accumulation by abscisic acid in somatic embryos. Planta 186, 337 - 342 (1992). MIYAZAKI, A., and FUJII, T.: Distribution ofIAA and ABA in gravistimulated primary roots of Zea mays L. Bot. Mag. Tokyo 104, 309-321 (1991). MURASHIGE, T., and SKOOG, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15,473-497 (1962). 346
BPP 188 (1992) 5
NOMURA, K., and KOMAMINE, A. : Molecular mechanisms of somatic embryogenesis. Oxf. Surv. Plant Mol. BioI 3, 456-466 (1986). PERA TA, P., PICCIARELLI, P., and ALPI, A.: Pattern of variations in abscisic acid content in suspensors, embryos, and integuments of developing Phaseolus coccineus seeds. Plant Physiol. 94, 1776-1780 (1990). RAJASEKARAN, K., HEIN, M . B., DAVIS, G. c., CARNES, M. G., and VASIL, I. K.: Endogenous growth regulators in leaves and tissue cultures of Pennisetum purpureum Schum. J. Plant Physiol. 130, 13-25 (l987a). RAJASEKARAN, K., HEIN, M. B., and VASIL, 1. K.: Endogenous abscisic acid and indole-3-acetic acid and somatic embryogenesis in cultured leaf explants of Pennisetum purpureum Schum. Plant Physiol. 84, 47-51 (l987b). SATOH, S., KAMADA, H., HARADA, H., and FUJII, T.: Auxin-controlled glycoprotein release into the medium of embryogenic carrot cells. Plant Physiol. 81, 931-933 (1986). STEWARD, F. C., MAPES , M. 0 ., and MEARS, K.: Growth and organized development of cultured cells . I. Growth and division of freely suspended cells. Am. J. Bot. 45, 693-703 (1958). WEILER, E. W.: An enzyme-immunoassay for cis-(+)-abscisic acid . Physiol. Plant. 54, 510-514 (1982). YAMAGUCHI, I., FUJISA WA, S., and TAKAHASHI, N. : Systematic ultramicroanalysis of plant growth regulators. In: IUPAC Pestic. Chern . Hum. Welfare Environ. 2, (Ed. MIYAMOTO, J.) Pergamon Press, pp. 145-150, Oxford 1983.
Received February 25, 1992; revised/orm accepted May 29, 1992 Authors' address: Dr. T. KIYOSUE, Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki-ken, 305, Japan .
23*
BPP 188 (1992) 5
347