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Improvement of Direct Somatic Embryogenesis in Rice by Selecting the Optimal Developmental Stage of Explant and Applying Desiccation Treatment

Improvement of Direct Somatic Embryogenesis in Rice by Selecting the Optimal Developmental Stage... NII-Electronic Library Service Plant Prod. Sci. 3 (2) : 114-123 (2000) Improvement of Direct Somatic Embryogenesis in Rice by Selecting the Optimal Developmental Stage of Explant and Applying Desiccation Treatment Totik Sri Mariani*, Hiroshi Miyake and Yoji Takeoka (Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan) Abstract : By selecting the optimal developmental stage of zygotic embryos used as explants and applying desiccation treatment, we improved direct somatic embryogenesis in rice scutellum from two cultivars, Nipponbare and Sasanishi­ ki. Zygotic embryos isolated 14-17, 21, 28-30 and 35-40 dafter anthesis (DAA) from Nipponbare and 14-17, 18-21, 28-30 and 40-42 DAA from Sasanishiki were cultured on the embryo induction medium (ElM) . Then they were transferred to embryo maturation medium (EMM) and germinated on the embryo germination medium (EGM). Only immature zygotic embryo isolated 14-17 DAA from Nipponbare and Sasanishiki could develop somatic embryos that germinated. Explants from embryos at other developmental stages could develop somatic embryos only until the elongating or scutellar stage. They enlarged and formed callus without further development. The ElM and EMM consisted of N6 macronutrients, B5 micronutrients, and B5 vitamins, supplemented with 0.1 g L - casein hydrolysate, 1 1 1.5 g L - proline and 1 g L - MES buffer. EGM consisted of MS macro- and micronutrients and MS vitamins without 1 1 1 organic supplement. In addition, 2 mg L- 2,4-D was added to ElM, 1 mg L - 2,4-D to EMM and 0.01 mg L- zeatin to EGM. Developmental processes of somatic embryos derived from the explants were observed by scanning electron microscopy. Desiccation treatment of maturing somatic embryo was proved to produce fully mature somatic embryos capable of germinating vigorously. Key words : Desiccation treatment, Developmental stage, Immature zygotic embryo, Mature zygotic embryo, Nipponbare, Sasanishiki, Scanning electron microscopy, Somatic embryo. Somatic embryogenesis is an amazing process because pattern will be achieved through the optimalization of the bipolar structure possessing shoot and root resem­ media and culture protocols for each individual stage of bling zygotic embryo is produced from somatic cells embryo development (Merkle et al., 1995). through an orderly series of characteristic morphological The developmental stage of the zygotic embryo used stages (Emons, 1994). Particularly, direct somatic em­ as an explant is critical in direct somatic embryogenesis. bryogenesis is advantageous for plant propagation Geneve and Kester ( 1990) reported that immature zygotic embryos of Cercts canadencis with an initial fresh because there is no intervening callus stage, and, there­ weight between 4 and 12 mg exhibited competence to fore somaclonal variation can be reduced (Williams and form somatic embryos. No somatic embryos could be Maheswaran, 1986). induced from the mature zygotic embryos used as ex­ Jones and Rost ( 1989) reported direct somatic em­ bryogenesis from rice scutellum using American plants. Merkle et al. ( 1998) also reported that more cultivars. We previously examined the morphogenic immature seeds gave a higher frequency of somatic process of direct somatic embryogenesis in rice cv. embryogenesis than mature seeds in sweetgum. Nippon bare by scanning electron microscopy (SEM). Desiccation tolerance is one of the most fundamental This process commences with the initiation of proembryo properties of seeds. It is acquired late in seed develop­ ment and is considered necessary for completion of the leading to embryogenic nodule formation. Subsequently, plant's life cycle, as an adaptive strategy to enable seed the embryogenic nodule underwent proliferation and survival during storage or environmental stress, and to mass propagation (enlargement), histodifferentiation (determination of polarity) , maturation and germination ensure better dissemination of the species (Leprince et (Mariani et al., 1998). However, germination was spon­ al., 1993). The natural development of a zygotic embryo taneous and the germinating somatic embryos showed was close to the optimal condition and therefore the abnormal structures. somatic embryo should follow the same developmental The more closely the pattern of somatic embryo gene program as the zygotic embryo in the seed (Bewley and Black, 1994). Desiccation of the somatic embryo serves expression matches that of the zygotic embryo, the greater the chance of obtaining efficient regeneration two purposes. First, in some species, desiccation breaks systems. Such normalization of the gene expression the dormancy of the somatic embryo. Secondly, desicca- Received 27 January 1999. Accepted 8 November 1999. Corresponding author: H. Miyake 81-52-789-4064) "'Present address. Department of Biology, Bandung Institute of Technology, Bandung 40132, Indonesia. Mariani et al. --Improvement of Rice Somatic Embryogenesis Table 1. The composition of media for producing rice somatic embryos. Basal medium Carbon source Medium------ Vitamin Hormone Gelling agent Macro Micro Sucrose Sorbitol 1 3 1 ElM N6 > B5 > B5 2mgL- 2,4-D 1% 2% 0.25%Gelrite EMM N6 B5 B5 lmgL- 2,4-D 2% 4% 0.25%Gelrite 2 1 EGM MS > MS MS O.OlmgL- zeatin 1% 2% 0.4% Gelrite ElM and EMM were supplemented with 0.1 g L -l casein hydrolysate, 1.5 g L -l proline and 1 g L -t MES (2-(n-morpholino) ethanesulfonic acid). I) Chu et al. (1975) 2) Murashige and Skoog (1962) 3) Gamborg et al. (1968) tion promotes the accumulation of nutrients, such as two layers of sterilized filter papers in petri dish for 24 h storage protein, for use by the embryo during germina­ and then transferred onto EGM. The temperature of tion (Finer, 1994). culture room was 25·c. Culture on ElM and EMM was To our knowledge, there have been no reports on carried out in the dark and that on EGM under 16-h 2 1 direct somatic embryogenesis in the zygotic embryo iso­ light (20 ,umoles m- s- with a fluorescent lamp) and lated at different developmental stages and the effect of 8-h dark regime. These experiments were replicated 2 desiccation on the development of somatic embryos in times. rice. The objective of this study was to improve the direct Globular somatic embryo formation was observed somatic embryogenesis in rice by selecting the develop­ after a 2-wk culture on ElM. Elongate and scutellar mental stage of zygotic embryo optimal for use as an stages of somatic embryos were observed after a 2-wk explant and applying desiccation treatment. We utilized culture on EMM. Coleoptilar stage and germinated Nipponbare and Sasanishiki cultivars because they are somatic embryos were observed after a 2- and 3-wk the typical Japanese varieties that are widely used in culture on EGM, respectively. The percentage of ex­ agriculture and research work. plants producing somatic embryos at each develop­ mental stage was calculated per dish. The germination Materials and Methods rate (GR) was scored per dish as follows: 1. Materials GR= Number of explants with germinated somatic embryo Zygotic embryos isolated from rice ( Oryza sativa L.) Total number of explants X 100(%) cv. Nipponbare 14-17, 21, 28-30, 35-40 dafter anthesis (DAA) and Sasanishiki 14-17, 18-21, 28-30, 40-42 DAA Germination in the present study means the emer­ were used as explants. The seeds were sown in 0.02 m gence of both radicle and shoot. Routine observation was pots in early May 1997, and cultivated in a phytotron at done using a stereo microscope. 30/23·c (day/night). The plants commenced heading at Significance of the treatment effects on the percentage the end of July 1997. The seeds were refrigerated for 2 of explants with somatic embryos and the germination wk to 1 mon before starting experiments. rate was determined using analysis of variance. One-way analysis was used if there was an interaction between the 2. Methods factors. Percentage data were subjected to arc sine trans­ (1) Culture of zygotic embryo formation prior to statistical analysis. The rice seeds of both cultivars at various develop­ mental stages were sterilized using 1% N a-hypochlorite (2) Scanning electron microscopy (SEM) for 15 min and washed three times with sterilized distilled The morphogenic development of somatic embryos water. The sterilized seeds were placed onto the embryo was examined by SEM. In each experiment, additional induction medium (ElM) and cultured for 1 wk. Table petri dishes of cultures were prepared for SEM. The 1 shows the composition of media. The N6 basal medium procedures of SEM were described in detail previously was used for its high concentration of nitrogen compared (Mariani et al., 1998). toMS basal medium according to Wernicke et al. (1981) Results and Tsukahara et al. ( 1996). Then, the embryos isolated from the seeds ( explants hereafter) were cultured on 1. Effects of explant stage and cultivar ElM in a plastic petri dish for 2 wk. Four petri dishes Somatic embryos formed and developed on the scutel­ with 10 explants each were used for each treatment. lum of the explant (zygotic embryo). The ability to form Thereafter, all the explants were transferred onto the somatic embryos varied with the physiological conditions embryo maturation medium (EMM) , cultured for 3 wk of the explant and cultivar used. Fig. 1 shows the forma­ and germinated on the embryo germination medium tion of somatic embryos from each explant. Only the (EGM) for 2 to 3 wk. For desiccation treatment, the immature explants 14-17 DAA from both Nipponbare explants cultured on EMM for 3 wk were desiccated on and Sasanishiki produced somatic embryos capable of NII-Electronic Library Service NII-Electronic Library Service Plant Production Science Vol. 3, 2000 D globular [] elongate ~ scutellar • coleoptilar • gennination 14-17 DAA 18-21 DAA 28-30 DAA 40-42 DAA 14-17 DAA 21 DAA 28-30 DAA 35-40 DAA Nippon bare Sasanishiki Fig. 1. Effect of the developmental stage (days after anthesis, DAA) of the explants (zygotic embryos) on the percentage of the explants with somatic embryos at various stages. Bars represent standard errors (n=8). The formation of somatic embryo was significantly influenced by the developmental stage of explant at P = 0.001 or 0.01 except for the formation of globular embryo in cv. Nipponbare. germination. The explants of Sasanishiki obtained 14-17 development of somatic embryos from an early globular DAA produced globular somatic embryos on 91% of the stage to the stage at which the embryo is capable of explants, but elongate stage embryos only 68% of the germination. They developed from the scutellum surface explants. The percentage of the explants that produced of the explants obtained 14-17 DAA from Nipponbare scutellar and coleoptilar embryos was 40 and 39%, (Figs. 2a-c) and Sasanishiki (Figs. 2d-f). Fig. 2a shows respectively. After all, the percentage of explants with an early globular somatic embryo. The globular embryos germinated somatic embryos was 32%. The formation of developed into elongate somatic embryos as shown in somatic embryos in the explants of Nippon bare obtained Fig. 2b. Fig. 2c shows the formation of a notch on the 14-17 DAA was similar to that of Sasanishiki obtained scutellum of somatic embryo, which shows the develop­ 14-17 DAA, although the percentage of the explants with ment of the elongate somatic embryo to early scutellar stage. The scutellar notch is a marker of the differentia­ somatic embryos at each developmental stage was higher in Sasanishiki. tion of the shoot pole and the root pole. Fig. 2d shows the Explants of Sasanishiki obtained 18-21, 28-30 DAA late scutellar stage and Fig. 2e shows the somatic embryo and 40-42 DAA developed somatic embryos up to scutel­ 6 days after transfer to EGM. The scutellar notch lar stage. Explants of Nippon bare obtained 21 and 28-30 became deeper and wider and the early coleoptile devel­ DAA developed somatic embryos that reached the oped from the inner region of the notch. The somatic scutellar stage, whereas the explants obtained 35-40 embryo that had been desiccated for 2 h germinated DAA developed somatic embryos that reached only the completely 2 wk after transfer to EGM, as shown in Fig. elongate stage. Thereafter, the somatic embryos at the 2f. The coleoptile, first leaf and root were observed. The coleoptile and root grew from the ventral side of the elongate and scutellar stages in both cultivars could not develop further. Based on one-way analysis of variance, scutellum. This pattern of germination closely resembled somatic embryo formation was significantly influenced that of the zygotic embryo as shown in Figs. 6a and b. by the developmental stage of the explant at P = 0.001 or Fig. 3a shows the scutellum surface of the explant of 0.01 except for globular embryo formation in Nippon bare Sasanishiki 40-42 DAA and cultured for 1 wk on ElM. Many globular somatic embryos (GSE) are observed at which was not significant. There was a strong interaction between the developmental stage of the explant and this stage. After 3-wk culture on EMM, a few elongate· cultivar (P = 0.001). Therefore, the developmental stage and scutellar-stage somatic embryos developed (Fig. 3b). of the explant influenced somatic embryo formation However, most of the globular- and elongate-stage somatic embryos enlarged and formed undifferentiated somewhat differently in the two cultivars. Scanning electron micrographs show morphogenic structures (callus). Mariani et al. --Improvement of Rice Somatic Embryogenesis NII-Electronic Library Service 118 Plant Production Science Vol. 3, 2000 NII-Electronic Library Service Mariani et al. --Improvement of Rice Somatic Embryogenesis 119 NII-Electronic Library Service Plant Production Science Vol. 3, 2000 embryos affected the germination rate and vigor of Fig. 4 shows the scutellum surface of the explant of Nipponbare obtained 35-40 DAA and cultured for 3 wk germinated somatic embryo. As a result, the somatic on EMM (Fig.4). The enlarged globular somatic embryo embryo subjected to desiccation developed plants more appeared to have formed a callus through the disordered vigorously compared to that without desiccation treat­ growth, and this character was peculiar to Nipponbare. ment. Figs. 6a and b show the zygotic embryo 2 wk after germination. The coleoptile and root grew from the Fig. 5 shows a transverse section of the scutellar-stage somatic embryo formed in the explant of Sasanishiki ventral side of the scutellum. Figs. 7a and b show the obtained 14-17 DAA and cultured for 2 wk on EMM. somatic embryos cultured for 2 wk on EGM, after The vascular bundle of scutellum is visible in the upper desiccation and without desiccation treatment, respec­ region. Protoderm developed to the epidermal layer of tively. The desiccated somatic embryos germinated and scutellar somatic embryo. developed to plantlets, which was characterized by the formation of coleoptile and root from the ventral side of 2. Effect of desiccation treatment the scutellum (Fig. 7a). The pattern of germination of Desiccation treatment applied to the maturing somatic somatic embryos after desiccation treatment closely NII-Electronic Library Service Mariani et al. --Improvement of Rice Somatic Embryogenesis 121 cious germination. resembled that of the zygotic embryos. The coleoptile Fig. 8 shows the effect of desiccation treatment on the and root developed after desiccation treatment were percentage of explants that produced germinating more vigorous than those developed without the desicca­ tion treatment (Fig. 7b). In the plantlets developed somatic embryos. The percentage was 63% after desicca­ without desiccation treatment, the coleoptile and the tion treatment and 30% without desiccation treatment in scutellum were swollen and the root and the first leaf Sasanishiki, and 53% after desiccation and 24% without were scanty. Probably, the somatic embryos that ger­ desiccation in Nipponbare. Using two-way analysis of minated without desiccation treatment underwent preco- variance, effect of desiccation and varietal difference are significant at P = 0.00 1. 70 (ill] Desiccated Discussion D Not desiccated Only immature zygotic embryos isolated 14-17 DAA produced the somatic embryos capable of germination in both Nipponbare and Sasanishiki. Williams and Ma­ heswaran ( 1986) described that direct somatic em­ bryogenesis may occur only at certain developmental stages, and in particular cell types. Cells in the maturing scutellum of rice may be heterogeneous in their speciali­ zation and only limited cells may be capable of forming somatic embryos, while the cells in the immature scutel­ lum are meristematic and easily form germinable somatic embryos. There may be varietal difference in the development of somatic embryos from the explants at different develop­ mental stages. Jones and Rost (1989) observed sponta­ Sasanishiki Nippon bare neous germination of somatic embryos from mature rice Fig. 8. Effect of desiccation treatment on the germination rate of scutellum of American cultivars, although they did not somatic embryos. Bars represent standard errors (n = 8). describe the germination rate. In the present experiment Effect of desiccation and varietal difference are significant at P=O.OOI. with cv. Nipponbare and Sasanishiki, development of Explanation of figures Figs. 2a-f. Morphogenic development of somatic embryo on the scutellum surface of the explant (immature zygotic embryo) obtained 14-17 DAA from Nipponbare (Figs. 2a-c) and Sasanishiki (Figs. 2d-f) (Bars = 0.1 mm). Fig. 2a shows the early globular somatic embryo (eGSM) after 1-wk culture on the embryo induction medium (ElM). Fig. 2b shows the late globular somatic embryo (lGSE) and elongate somatic embryo (ElSE) after 2-wk culture on ElM. Fig. 2c shows the early scutellar somatic embryo (eSSE) after 2-wk culture on embryo maturation medium (EMM). The scutelar notch (SN) is the marker of the somatic embryo polarity. Fig. 2d shows the late scutellar somatic embryo (lSSE) after 3-wk culture on EMM. Fig. 2e shows the early coleoptilar somatic embryo (eCpSE) 6 days after the transfer to EGM. The early coleoptile (eCp) grew from the scutellar notch (SN). Fig. 2f shows the germinated somatic embryo (gSE) cultured for 3 wk on EGM after desiccating for 24 h. Cp, coleoptile ; fL, first leaf; R, root ; rSc, rudimentary scutellum. Figs. 3a and b. The scutellum surface of the explant (mature zygotic embryo) obtained 40-42 DAA from Sasanishiki and cultured for 1 wk on the ElM (a) and then for 3 wk on EMM (b) (Bars 1 mm). Fig. 3a shows the early globular somatic embryos (eGSE), and Fig. 3b shows the globular somatic embryo (GSE), elongate somatic embryo (ElSE), early scutellar somatic embryo (eSSE) with its scutellar notch (SN), enlarged globular somatic embryo (enGSE) and callus (Ca). Fig. 4. The enlargement of callus (Ca) on the scutellum surface of explant (mature zygotic embryo) obtained 35-40 DAA from Nipponbare and cultured for 3 wk on EMM. Enlarged and disordered growth of globular and elongate somatic embryos are observed (Bar 0.5 mm). Fig. 5. The transverse section of the scutellar somatic embryo (SSE) in the explant obtained from Sasanishiki 14-17 DAA and cultured for 2 wk on EMM. The vascular bundle of scutellum (Sc VB) is observed in the upper region. The well-developed protoderm (Pr) is visible as the epidermis layer of scutellar somatic embryo (SSE) (Bar = 0.1 mm). Figs. 6a and b. The zygotic embryo of Sasanishiki 2 wk after germination. The coleoptile (Cp) with the first leaf (fL) is observed, as well as root grown from the ventral scutellum (X 12). Fig. 6a shows the dorsal side of the scutellum, but Fig. 6b shows the ventral side. Figs. 7a and b. The somatic embryo of Sasanishiki after a 2-wk culture on EGM, preceded by desiccation treatment (7a) and without desiccation (7b) (X 48). First leaf (fL), coleoptile (Cp) and root (R) developed from somatic embryo subjected to desiccation treatment look healthy, while the first leaf and root are scanty and coleoptile is swollen in the somatic embryo without desiccation. Scutellum (Sc) is swollen in the embryo without desiccation treatment, but rudimentary (rSc) in the embryo after desiccation treatment. NII-Electronic Library Service 122 Plant Production Science Vol. 3, 2000 somatic embryos from mature scutellum was limited and Georgia, for sharing the knowledge in the discussion of the somatic embryos sometimes changed to callus, which this paper. was particularly the case in Nipponbare. References Desiccation treatment of maturing rice somatic Bewley, J.D. and Black, M. 1994. Seeds: Physiology of Develop· embryo (scutellar stage) improved the germination rate ment and Germination. Second edition. Plenum Press, New of the somatic embryos and the vigor of germinated York. 1-445. plantlet. Precocious germination was observed in the Chu, C.C., Wang, C.C., Sun, C.S., Hsu, C., Yin, K.C., Chu, C.Y. somatic embryos without desiccation whereas normal and Bi, F.Y. 1975. Establishment of an efficient medium for germination occurred in those after desiccation treat­ anther culture of rice through comparative experiments of the ment. It has been reported in many species that desicca­ nitrogen sources. Sci. Sin. 18: 659-668. tion treatment of somatic embryos enhances their subse­ Dronne, S., Label, P. and Lelu, M.·A. 1997. Desiccation decreases quent germination and growth and gives a better syn­ abscisic acid content in hybrid larch (Larix x Leptoeuropaea) chronization of root and shoot growth (Merkle et al., somatic embryos. Physiol. Plant. 99 : 433-438. 1995). Abscisic acid (ABA) is accumulated in both Emons, A.M.C. 1994. Somatic embryogenesis: cell biological zygotic and somatic embryos during their development aspects. Acta Bot. Neerl. 43: 1-14. (Faure et al., 1998). ABA is necessary for the develop­ Faure, 0., Dewitte, W., Nougarede, A. and Onckelen, H.V. 1998. ment of both embryos but is inhibitory for the germina­ Precociously germinating somatic embryos of Vitis vinifera tion of embryos. In normal seed development, the ABA have lower ABA and IAA levels than their germinating content decreases during the desiccation process of the zygotic counterparts. Physiol. Plant. 102: 591-595. Find, ].I. 1997. Changes in endogenous ABA levels in developing seed, and accordingly germination occurs after rehydra­ somatic embryos of Norway spruce (Picea abies (L.) Karst.) in tion without the inhibitory effects of ABA in the zygotic relation to maturation medium, desiccation and germination. embryo. In the same way, desiccation treatment of Plant Sci. 128 : 7 5-83. somatic embryos leads to the destruction of endogenous Finer, J.J. 1994. Plant regeneration via embryogenic suspension ABA, and enhances the germination of somatic embryos cultures. In R.A. Dixon and R.A. Gonzales eds., Plant Cell (Dronne et al., 1997; Find, 1997; Samoylov et al., 1998). Cultures. A Practical Approach. Second edition. IRL Press, Merkle et al. (1995) stated that desiccation is neces­ Oxford Univ. Press, Oxford. 99-125. sary to induce the final maturation of the somatic Gamborg, O.L., Miller, R.A. and Ojima, K. 1968. Nutrient embryo and that the embryo must reach desiccation requirements of suspension cultures of soybean root cells. Exp. tolerance (i.e., physiological maturity) before imposing Cell Res., 50: 151-158 desiccation on it. Presumably, the physiological acquisi­ Geneve, R.L. and Kester, S.T. 1990. The initiation of somatic tion of desiccation tolerance as in the zygotic embryo embryos and adventitious roots from developing zygotic promotes the final maturation process of somatic embryo explants of Ceris canadensis L. cultured in vitro. Plant embryo, which leads to the normal germination. In fact, Cell Tissue Organ Cult. 22 : 71-76. Samoylov et al. (1998) reported that the somatic em­ Jones, T.J. and Rost, T.L. 1989. The developmental anatomy and bryos of soybean at the cotyledon stage were ready for ultrastructure of somatic embryo from rice ( Oryza sativa L.) desiccation within 4 wk in the maturation medium. scutellum epithelial cells. Bot. Gaz. 150:41-49. Leprince, 0., Hendry, G.A.F. and McKersie, B.D. 1993. The Then, the desiccated somatic embryos germinated and mechanism of desiccation tolerance in developing seeds. Seed produced roots and shoots with leaves after germination Sci. Res. 3 : 231-246. on embryo germination medium. In the present experi­ Mariani, T.S., Miyake, H. and Takeoka, Y. 1998. Changes in ment, the somatic embryo of rice at the scutellar stage surface structure during direct somatic embryogenesis in rice seems to be ready for desiccation treatment which scutellum observed by scanning electron microscopy. Plant supported the final maturation, and the desiccation Prod. Sci. 1 : 223-231. decreased the ABA content and increased the germina­ Merkle, S.A., Parrott, W.A. and Flinn, B.S. 1995. Morphogenic tion rate of the somatic embryo. aspects of somatic embryogenesis. In T.A. Thorpe ed., In Vitro In conclusion, we found that immature zygotic embryo Embryogenesis in Plants. Kluwer Academic Publishers, Dor­ of rice cv. Nipponbare and Sasanishiki, isolated 14-17 drecht. 155-203. DAA is capable of producing somatic embryos that Merkle, S.A., Neu, K.A., Battle, P.J. and Bailey, R.L. 1998. germinated. Desiccation treatment proved to prevent Somatic embryogenesis and plantlet regeneration from im· precocious germination, and improved the vigor of mature and mature tissues of sweetgum (Liquidambar styraci­ somatic embryo and increased the germination rate. flua). Plant Sci. 112 : 169-178. Murashige, T. and Skoog, F. 1962. A revised medium for rapid Acknowledgment growth and bioassays with tobacco tissue cultures. Plant Physiol. 15: 473-497. This study was financially supported by a Grant-in­ Samoylov, V.W., Tucker, D.M., Thibaud.Nissen, F. and Parrott, Aid for Scientific Research from the Japanese Ministry W.A. 1998. A liquid-medium·based protocol for rapid regen­ of Education, Science, Culture and Sports (Monbusho). eration from embryogenic soybean cultures. Plant Cell Rep. The authors would like to thank Dr. Wayne Parrott, 18:49-54. Department of Crop & Soil Sciences, University of NII-Electronic Library Service Mariani et al. --Improvement of Rice Somatic Embryogenesis Tsukahara, M., Hirosawa, T. and Kishine, S. 1996. Efficient plant Z. Pflanzenphysiol. 103:361-365. regeneration from cell suspension cultures of rice ( Oryza sativa Williams, E.G. and Maheswaran, G. 1986. Somatic embryogenesis: L.). J. Plant Physiol. 149: 157-162. Factors influencing Coordinated behavior of cells as an em­ Wernicke, W., Bretteli, R., Wakizuka, T. and Potrykus, I. 1981. bryogenic group. Ann. Bot. 57: 443-462. 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Improvement of Direct Somatic Embryogenesis in Rice by Selecting the Optimal Developmental Stage of Explant and Applying Desiccation Treatment

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Abstract

NII-Electronic Library Service Plant Prod. Sci. 3 (2) : 114-123 (2000) Improvement of Direct Somatic Embryogenesis in Rice by Selecting the Optimal Developmental Stage of Explant and Applying Desiccation Treatment Totik Sri Mariani*, Hiroshi Miyake and Yoji Takeoka (Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan) Abstract : By selecting the optimal developmental stage of zygotic embryos used as explants and applying desiccation treatment, we improved direct somatic embryogenesis in rice scutellum from two cultivars, Nipponbare and Sasanishi­ ki. Zygotic embryos isolated 14-17, 21, 28-30 and 35-40 dafter anthesis (DAA) from Nipponbare and 14-17, 18-21, 28-30 and 40-42 DAA from Sasanishiki were cultured on the embryo induction medium (ElM) . Then they were transferred to embryo maturation medium (EMM) and germinated on the embryo germination medium (EGM). Only immature zygotic embryo isolated 14-17 DAA from Nipponbare and Sasanishiki could develop somatic embryos that germinated. Explants from embryos at other developmental stages could develop somatic embryos only until the elongating or scutellar stage. They enlarged and formed callus without further development. The ElM and EMM consisted of N6 macronutrients, B5 micronutrients, and B5 vitamins, supplemented with 0.1 g L - casein hydrolysate, 1 1 1.5 g L - proline and 1 g L - MES buffer. EGM consisted of MS macro- and micronutrients and MS vitamins without 1 1 1 organic supplement. In addition, 2 mg L- 2,4-D was added to ElM, 1 mg L - 2,4-D to EMM and 0.01 mg L- zeatin to EGM. Developmental processes of somatic embryos derived from the explants were observed by scanning electron microscopy. Desiccation treatment of maturing somatic embryo was proved to produce fully mature somatic embryos capable of germinating vigorously. Key words : Desiccation treatment, Developmental stage, Immature zygotic embryo, Mature zygotic embryo, Nipponbare, Sasanishiki, Scanning electron microscopy, Somatic embryo. Somatic embryogenesis is an amazing process because pattern will be achieved through the optimalization of the bipolar structure possessing shoot and root resem­ media and culture protocols for each individual stage of bling zygotic embryo is produced from somatic cells embryo development (Merkle et al., 1995). through an orderly series of characteristic morphological The developmental stage of the zygotic embryo used stages (Emons, 1994). Particularly, direct somatic em­ as an explant is critical in direct somatic embryogenesis. bryogenesis is advantageous for plant propagation Geneve and Kester ( 1990) reported that immature zygotic embryos of Cercts canadencis with an initial fresh because there is no intervening callus stage, and, there­ weight between 4 and 12 mg exhibited competence to fore somaclonal variation can be reduced (Williams and form somatic embryos. No somatic embryos could be Maheswaran, 1986). induced from the mature zygotic embryos used as ex­ Jones and Rost ( 1989) reported direct somatic em­ bryogenesis from rice scutellum using American plants. Merkle et al. ( 1998) also reported that more cultivars. We previously examined the morphogenic immature seeds gave a higher frequency of somatic process of direct somatic embryogenesis in rice cv. embryogenesis than mature seeds in sweetgum. Nippon bare by scanning electron microscopy (SEM). Desiccation tolerance is one of the most fundamental This process commences with the initiation of proembryo properties of seeds. It is acquired late in seed develop­ ment and is considered necessary for completion of the leading to embryogenic nodule formation. Subsequently, plant's life cycle, as an adaptive strategy to enable seed the embryogenic nodule underwent proliferation and survival during storage or environmental stress, and to mass propagation (enlargement), histodifferentiation (determination of polarity) , maturation and germination ensure better dissemination of the species (Leprince et (Mariani et al., 1998). However, germination was spon­ al., 1993). The natural development of a zygotic embryo taneous and the germinating somatic embryos showed was close to the optimal condition and therefore the abnormal structures. somatic embryo should follow the same developmental The more closely the pattern of somatic embryo gene program as the zygotic embryo in the seed (Bewley and Black, 1994). Desiccation of the somatic embryo serves expression matches that of the zygotic embryo, the greater the chance of obtaining efficient regeneration two purposes. First, in some species, desiccation breaks systems. Such normalization of the gene expression the dormancy of the somatic embryo. Secondly, desicca- Received 27 January 1999. Accepted 8 November 1999. Corresponding author: H. Miyake 81-52-789-4064) "'Present address. Department of Biology, Bandung Institute of Technology, Bandung 40132, Indonesia. Mariani et al. --Improvement of Rice Somatic Embryogenesis Table 1. The composition of media for producing rice somatic embryos. Basal medium Carbon source Medium------ Vitamin Hormone Gelling agent Macro Micro Sucrose Sorbitol 1 3 1 ElM N6 > B5 > B5 2mgL- 2,4-D 1% 2% 0.25%Gelrite EMM N6 B5 B5 lmgL- 2,4-D 2% 4% 0.25%Gelrite 2 1 EGM MS > MS MS O.OlmgL- zeatin 1% 2% 0.4% Gelrite ElM and EMM were supplemented with 0.1 g L -l casein hydrolysate, 1.5 g L -l proline and 1 g L -t MES (2-(n-morpholino) ethanesulfonic acid). I) Chu et al. (1975) 2) Murashige and Skoog (1962) 3) Gamborg et al. (1968) tion promotes the accumulation of nutrients, such as two layers of sterilized filter papers in petri dish for 24 h storage protein, for use by the embryo during germina­ and then transferred onto EGM. The temperature of tion (Finer, 1994). culture room was 25·c. Culture on ElM and EMM was To our knowledge, there have been no reports on carried out in the dark and that on EGM under 16-h 2 1 direct somatic embryogenesis in the zygotic embryo iso­ light (20 ,umoles m- s- with a fluorescent lamp) and lated at different developmental stages and the effect of 8-h dark regime. These experiments were replicated 2 desiccation on the development of somatic embryos in times. rice. The objective of this study was to improve the direct Globular somatic embryo formation was observed somatic embryogenesis in rice by selecting the develop­ after a 2-wk culture on ElM. Elongate and scutellar mental stage of zygotic embryo optimal for use as an stages of somatic embryos were observed after a 2-wk explant and applying desiccation treatment. We utilized culture on EMM. Coleoptilar stage and germinated Nipponbare and Sasanishiki cultivars because they are somatic embryos were observed after a 2- and 3-wk the typical Japanese varieties that are widely used in culture on EGM, respectively. The percentage of ex­ agriculture and research work. plants producing somatic embryos at each develop­ mental stage was calculated per dish. The germination Materials and Methods rate (GR) was scored per dish as follows: 1. Materials GR= Number of explants with germinated somatic embryo Zygotic embryos isolated from rice ( Oryza sativa L.) Total number of explants X 100(%) cv. Nipponbare 14-17, 21, 28-30, 35-40 dafter anthesis (DAA) and Sasanishiki 14-17, 18-21, 28-30, 40-42 DAA Germination in the present study means the emer­ were used as explants. The seeds were sown in 0.02 m gence of both radicle and shoot. Routine observation was pots in early May 1997, and cultivated in a phytotron at done using a stereo microscope. 30/23·c (day/night). The plants commenced heading at Significance of the treatment effects on the percentage the end of July 1997. The seeds were refrigerated for 2 of explants with somatic embryos and the germination wk to 1 mon before starting experiments. rate was determined using analysis of variance. One-way analysis was used if there was an interaction between the 2. Methods factors. Percentage data were subjected to arc sine trans­ (1) Culture of zygotic embryo formation prior to statistical analysis. The rice seeds of both cultivars at various develop­ mental stages were sterilized using 1% N a-hypochlorite (2) Scanning electron microscopy (SEM) for 15 min and washed three times with sterilized distilled The morphogenic development of somatic embryos water. The sterilized seeds were placed onto the embryo was examined by SEM. In each experiment, additional induction medium (ElM) and cultured for 1 wk. Table petri dishes of cultures were prepared for SEM. The 1 shows the composition of media. The N6 basal medium procedures of SEM were described in detail previously was used for its high concentration of nitrogen compared (Mariani et al., 1998). toMS basal medium according to Wernicke et al. (1981) Results and Tsukahara et al. ( 1996). Then, the embryos isolated from the seeds ( explants hereafter) were cultured on 1. Effects of explant stage and cultivar ElM in a plastic petri dish for 2 wk. Four petri dishes Somatic embryos formed and developed on the scutel­ with 10 explants each were used for each treatment. lum of the explant (zygotic embryo). The ability to form Thereafter, all the explants were transferred onto the somatic embryos varied with the physiological conditions embryo maturation medium (EMM) , cultured for 3 wk of the explant and cultivar used. Fig. 1 shows the forma­ and germinated on the embryo germination medium tion of somatic embryos from each explant. Only the (EGM) for 2 to 3 wk. For desiccation treatment, the immature explants 14-17 DAA from both Nipponbare explants cultured on EMM for 3 wk were desiccated on and Sasanishiki produced somatic embryos capable of NII-Electronic Library Service NII-Electronic Library Service Plant Production Science Vol. 3, 2000 D globular [] elongate ~ scutellar • coleoptilar • gennination 14-17 DAA 18-21 DAA 28-30 DAA 40-42 DAA 14-17 DAA 21 DAA 28-30 DAA 35-40 DAA Nippon bare Sasanishiki Fig. 1. Effect of the developmental stage (days after anthesis, DAA) of the explants (zygotic embryos) on the percentage of the explants with somatic embryos at various stages. Bars represent standard errors (n=8). The formation of somatic embryo was significantly influenced by the developmental stage of explant at P = 0.001 or 0.01 except for the formation of globular embryo in cv. Nipponbare. germination. The explants of Sasanishiki obtained 14-17 development of somatic embryos from an early globular DAA produced globular somatic embryos on 91% of the stage to the stage at which the embryo is capable of explants, but elongate stage embryos only 68% of the germination. They developed from the scutellum surface explants. The percentage of the explants that produced of the explants obtained 14-17 DAA from Nipponbare scutellar and coleoptilar embryos was 40 and 39%, (Figs. 2a-c) and Sasanishiki (Figs. 2d-f). Fig. 2a shows respectively. After all, the percentage of explants with an early globular somatic embryo. The globular embryos germinated somatic embryos was 32%. The formation of developed into elongate somatic embryos as shown in somatic embryos in the explants of Nippon bare obtained Fig. 2b. Fig. 2c shows the formation of a notch on the 14-17 DAA was similar to that of Sasanishiki obtained scutellum of somatic embryo, which shows the develop­ 14-17 DAA, although the percentage of the explants with ment of the elongate somatic embryo to early scutellar stage. The scutellar notch is a marker of the differentia­ somatic embryos at each developmental stage was higher in Sasanishiki. tion of the shoot pole and the root pole. Fig. 2d shows the Explants of Sasanishiki obtained 18-21, 28-30 DAA late scutellar stage and Fig. 2e shows the somatic embryo and 40-42 DAA developed somatic embryos up to scutel­ 6 days after transfer to EGM. The scutellar notch lar stage. Explants of Nippon bare obtained 21 and 28-30 became deeper and wider and the early coleoptile devel­ DAA developed somatic embryos that reached the oped from the inner region of the notch. The somatic scutellar stage, whereas the explants obtained 35-40 embryo that had been desiccated for 2 h germinated DAA developed somatic embryos that reached only the completely 2 wk after transfer to EGM, as shown in Fig. elongate stage. Thereafter, the somatic embryos at the 2f. The coleoptile, first leaf and root were observed. The coleoptile and root grew from the ventral side of the elongate and scutellar stages in both cultivars could not develop further. Based on one-way analysis of variance, scutellum. This pattern of germination closely resembled somatic embryo formation was significantly influenced that of the zygotic embryo as shown in Figs. 6a and b. by the developmental stage of the explant at P = 0.001 or Fig. 3a shows the scutellum surface of the explant of 0.01 except for globular embryo formation in Nippon bare Sasanishiki 40-42 DAA and cultured for 1 wk on ElM. Many globular somatic embryos (GSE) are observed at which was not significant. There was a strong interaction between the developmental stage of the explant and this stage. After 3-wk culture on EMM, a few elongate· cultivar (P = 0.001). Therefore, the developmental stage and scutellar-stage somatic embryos developed (Fig. 3b). of the explant influenced somatic embryo formation However, most of the globular- and elongate-stage somatic embryos enlarged and formed undifferentiated somewhat differently in the two cultivars. Scanning electron micrographs show morphogenic structures (callus). Mariani et al. --Improvement of Rice Somatic Embryogenesis NII-Electronic Library Service 118 Plant Production Science Vol. 3, 2000 NII-Electronic Library Service Mariani et al. --Improvement of Rice Somatic Embryogenesis 119 NII-Electronic Library Service Plant Production Science Vol. 3, 2000 embryos affected the germination rate and vigor of Fig. 4 shows the scutellum surface of the explant of Nipponbare obtained 35-40 DAA and cultured for 3 wk germinated somatic embryo. As a result, the somatic on EMM (Fig.4). The enlarged globular somatic embryo embryo subjected to desiccation developed plants more appeared to have formed a callus through the disordered vigorously compared to that without desiccation treat­ growth, and this character was peculiar to Nipponbare. ment. Figs. 6a and b show the zygotic embryo 2 wk after germination. The coleoptile and root grew from the Fig. 5 shows a transverse section of the scutellar-stage somatic embryo formed in the explant of Sasanishiki ventral side of the scutellum. Figs. 7a and b show the obtained 14-17 DAA and cultured for 2 wk on EMM. somatic embryos cultured for 2 wk on EGM, after The vascular bundle of scutellum is visible in the upper desiccation and without desiccation treatment, respec­ region. Protoderm developed to the epidermal layer of tively. The desiccated somatic embryos germinated and scutellar somatic embryo. developed to plantlets, which was characterized by the formation of coleoptile and root from the ventral side of 2. Effect of desiccation treatment the scutellum (Fig. 7a). The pattern of germination of Desiccation treatment applied to the maturing somatic somatic embryos after desiccation treatment closely NII-Electronic Library Service Mariani et al. --Improvement of Rice Somatic Embryogenesis 121 cious germination. resembled that of the zygotic embryos. The coleoptile Fig. 8 shows the effect of desiccation treatment on the and root developed after desiccation treatment were percentage of explants that produced germinating more vigorous than those developed without the desicca­ tion treatment (Fig. 7b). In the plantlets developed somatic embryos. The percentage was 63% after desicca­ without desiccation treatment, the coleoptile and the tion treatment and 30% without desiccation treatment in scutellum were swollen and the root and the first leaf Sasanishiki, and 53% after desiccation and 24% without were scanty. Probably, the somatic embryos that ger­ desiccation in Nipponbare. Using two-way analysis of minated without desiccation treatment underwent preco- variance, effect of desiccation and varietal difference are significant at P = 0.00 1. 70 (ill] Desiccated Discussion D Not desiccated Only immature zygotic embryos isolated 14-17 DAA produced the somatic embryos capable of germination in both Nipponbare and Sasanishiki. Williams and Ma­ heswaran ( 1986) described that direct somatic em­ bryogenesis may occur only at certain developmental stages, and in particular cell types. Cells in the maturing scutellum of rice may be heterogeneous in their speciali­ zation and only limited cells may be capable of forming somatic embryos, while the cells in the immature scutel­ lum are meristematic and easily form germinable somatic embryos. There may be varietal difference in the development of somatic embryos from the explants at different develop­ mental stages. Jones and Rost (1989) observed sponta­ Sasanishiki Nippon bare neous germination of somatic embryos from mature rice Fig. 8. Effect of desiccation treatment on the germination rate of scutellum of American cultivars, although they did not somatic embryos. Bars represent standard errors (n = 8). describe the germination rate. In the present experiment Effect of desiccation and varietal difference are significant at P=O.OOI. with cv. Nipponbare and Sasanishiki, development of Explanation of figures Figs. 2a-f. Morphogenic development of somatic embryo on the scutellum surface of the explant (immature zygotic embryo) obtained 14-17 DAA from Nipponbare (Figs. 2a-c) and Sasanishiki (Figs. 2d-f) (Bars = 0.1 mm). Fig. 2a shows the early globular somatic embryo (eGSM) after 1-wk culture on the embryo induction medium (ElM). Fig. 2b shows the late globular somatic embryo (lGSE) and elongate somatic embryo (ElSE) after 2-wk culture on ElM. Fig. 2c shows the early scutellar somatic embryo (eSSE) after 2-wk culture on embryo maturation medium (EMM). The scutelar notch (SN) is the marker of the somatic embryo polarity. Fig. 2d shows the late scutellar somatic embryo (lSSE) after 3-wk culture on EMM. Fig. 2e shows the early coleoptilar somatic embryo (eCpSE) 6 days after the transfer to EGM. The early coleoptile (eCp) grew from the scutellar notch (SN). Fig. 2f shows the germinated somatic embryo (gSE) cultured for 3 wk on EGM after desiccating for 24 h. Cp, coleoptile ; fL, first leaf; R, root ; rSc, rudimentary scutellum. Figs. 3a and b. The scutellum surface of the explant (mature zygotic embryo) obtained 40-42 DAA from Sasanishiki and cultured for 1 wk on the ElM (a) and then for 3 wk on EMM (b) (Bars 1 mm). Fig. 3a shows the early globular somatic embryos (eGSE), and Fig. 3b shows the globular somatic embryo (GSE), elongate somatic embryo (ElSE), early scutellar somatic embryo (eSSE) with its scutellar notch (SN), enlarged globular somatic embryo (enGSE) and callus (Ca). Fig. 4. The enlargement of callus (Ca) on the scutellum surface of explant (mature zygotic embryo) obtained 35-40 DAA from Nipponbare and cultured for 3 wk on EMM. Enlarged and disordered growth of globular and elongate somatic embryos are observed (Bar 0.5 mm). Fig. 5. The transverse section of the scutellar somatic embryo (SSE) in the explant obtained from Sasanishiki 14-17 DAA and cultured for 2 wk on EMM. The vascular bundle of scutellum (Sc VB) is observed in the upper region. The well-developed protoderm (Pr) is visible as the epidermis layer of scutellar somatic embryo (SSE) (Bar = 0.1 mm). Figs. 6a and b. The zygotic embryo of Sasanishiki 2 wk after germination. The coleoptile (Cp) with the first leaf (fL) is observed, as well as root grown from the ventral scutellum (X 12). Fig. 6a shows the dorsal side of the scutellum, but Fig. 6b shows the ventral side. Figs. 7a and b. The somatic embryo of Sasanishiki after a 2-wk culture on EGM, preceded by desiccation treatment (7a) and without desiccation (7b) (X 48). First leaf (fL), coleoptile (Cp) and root (R) developed from somatic embryo subjected to desiccation treatment look healthy, while the first leaf and root are scanty and coleoptile is swollen in the somatic embryo without desiccation. Scutellum (Sc) is swollen in the embryo without desiccation treatment, but rudimentary (rSc) in the embryo after desiccation treatment. NII-Electronic Library Service 122 Plant Production Science Vol. 3, 2000 somatic embryos from mature scutellum was limited and Georgia, for sharing the knowledge in the discussion of the somatic embryos sometimes changed to callus, which this paper. was particularly the case in Nipponbare. References Desiccation treatment of maturing rice somatic Bewley, J.D. and Black, M. 1994. Seeds: Physiology of Develop· embryo (scutellar stage) improved the germination rate ment and Germination. Second edition. Plenum Press, New of the somatic embryos and the vigor of germinated York. 1-445. plantlet. Precocious germination was observed in the Chu, C.C., Wang, C.C., Sun, C.S., Hsu, C., Yin, K.C., Chu, C.Y. somatic embryos without desiccation whereas normal and Bi, F.Y. 1975. Establishment of an efficient medium for germination occurred in those after desiccation treat­ anther culture of rice through comparative experiments of the ment. It has been reported in many species that desicca­ nitrogen sources. Sci. Sin. 18: 659-668. tion treatment of somatic embryos enhances their subse­ Dronne, S., Label, P. and Lelu, M.·A. 1997. Desiccation decreases quent germination and growth and gives a better syn­ abscisic acid content in hybrid larch (Larix x Leptoeuropaea) chronization of root and shoot growth (Merkle et al., somatic embryos. Physiol. Plant. 99 : 433-438. 1995). Abscisic acid (ABA) is accumulated in both Emons, A.M.C. 1994. Somatic embryogenesis: cell biological zygotic and somatic embryos during their development aspects. Acta Bot. Neerl. 43: 1-14. (Faure et al., 1998). ABA is necessary for the develop­ Faure, 0., Dewitte, W., Nougarede, A. and Onckelen, H.V. 1998. ment of both embryos but is inhibitory for the germina­ Precociously germinating somatic embryos of Vitis vinifera tion of embryos. In normal seed development, the ABA have lower ABA and IAA levels than their germinating content decreases during the desiccation process of the zygotic counterparts. Physiol. Plant. 102: 591-595. Find, ].I. 1997. Changes in endogenous ABA levels in developing seed, and accordingly germination occurs after rehydra­ somatic embryos of Norway spruce (Picea abies (L.) Karst.) in tion without the inhibitory effects of ABA in the zygotic relation to maturation medium, desiccation and germination. embryo. In the same way, desiccation treatment of Plant Sci. 128 : 7 5-83. somatic embryos leads to the destruction of endogenous Finer, J.J. 1994. Plant regeneration via embryogenic suspension ABA, and enhances the germination of somatic embryos cultures. In R.A. Dixon and R.A. Gonzales eds., Plant Cell (Dronne et al., 1997; Find, 1997; Samoylov et al., 1998). Cultures. A Practical Approach. Second edition. IRL Press, Merkle et al. (1995) stated that desiccation is neces­ Oxford Univ. Press, Oxford. 99-125. sary to induce the final maturation of the somatic Gamborg, O.L., Miller, R.A. and Ojima, K. 1968. Nutrient embryo and that the embryo must reach desiccation requirements of suspension cultures of soybean root cells. Exp. tolerance (i.e., physiological maturity) before imposing Cell Res., 50: 151-158 desiccation on it. Presumably, the physiological acquisi­ Geneve, R.L. and Kester, S.T. 1990. The initiation of somatic tion of desiccation tolerance as in the zygotic embryo embryos and adventitious roots from developing zygotic promotes the final maturation process of somatic embryo explants of Ceris canadensis L. cultured in vitro. Plant embryo, which leads to the normal germination. In fact, Cell Tissue Organ Cult. 22 : 71-76. Samoylov et al. (1998) reported that the somatic em­ Jones, T.J. and Rost, T.L. 1989. The developmental anatomy and bryos of soybean at the cotyledon stage were ready for ultrastructure of somatic embryo from rice ( Oryza sativa L.) desiccation within 4 wk in the maturation medium. scutellum epithelial cells. Bot. Gaz. 150:41-49. Leprince, 0., Hendry, G.A.F. and McKersie, B.D. 1993. The Then, the desiccated somatic embryos germinated and mechanism of desiccation tolerance in developing seeds. Seed produced roots and shoots with leaves after germination Sci. Res. 3 : 231-246. on embryo germination medium. In the present experi­ Mariani, T.S., Miyake, H. and Takeoka, Y. 1998. Changes in ment, the somatic embryo of rice at the scutellar stage surface structure during direct somatic embryogenesis in rice seems to be ready for desiccation treatment which scutellum observed by scanning electron microscopy. Plant supported the final maturation, and the desiccation Prod. Sci. 1 : 223-231. decreased the ABA content and increased the germina­ Merkle, S.A., Parrott, W.A. and Flinn, B.S. 1995. Morphogenic tion rate of the somatic embryo. aspects of somatic embryogenesis. In T.A. Thorpe ed., In Vitro In conclusion, we found that immature zygotic embryo Embryogenesis in Plants. Kluwer Academic Publishers, Dor­ of rice cv. Nipponbare and Sasanishiki, isolated 14-17 drecht. 155-203. DAA is capable of producing somatic embryos that Merkle, S.A., Neu, K.A., Battle, P.J. and Bailey, R.L. 1998. germinated. Desiccation treatment proved to prevent Somatic embryogenesis and plantlet regeneration from im· precocious germination, and improved the vigor of mature and mature tissues of sweetgum (Liquidambar styraci­ somatic embryo and increased the germination rate. flua). Plant Sci. 112 : 169-178. Murashige, T. and Skoog, F. 1962. A revised medium for rapid Acknowledgment growth and bioassays with tobacco tissue cultures. Plant Physiol. 15: 473-497. This study was financially supported by a Grant-in­ Samoylov, V.W., Tucker, D.M., Thibaud.Nissen, F. and Parrott, Aid for Scientific Research from the Japanese Ministry W.A. 1998. A liquid-medium·based protocol for rapid regen­ of Education, Science, Culture and Sports (Monbusho). eration from embryogenic soybean cultures. Plant Cell Rep. The authors would like to thank Dr. Wayne Parrott, 18:49-54. Department of Crop & Soil Sciences, University of NII-Electronic Library Service Mariani et al. --Improvement of Rice Somatic Embryogenesis Tsukahara, M., Hirosawa, T. and Kishine, S. 1996. Efficient plant Z. Pflanzenphysiol. 103:361-365. regeneration from cell suspension cultures of rice ( Oryza sativa Williams, E.G. and Maheswaran, G. 1986. Somatic embryogenesis: L.). J. Plant Physiol. 149: 157-162. Factors influencing Coordinated behavior of cells as an em­ Wernicke, W., Bretteli, R., Wakizuka, T. and Potrykus, I. 1981. bryogenic group. Ann. Bot. 57: 443-462. Adventitious embryoid and root formation from Rice leaves. NII-Electronic Library Service

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Plant Production ScienceTaylor & Francis

Published: Jan 1, 2000

Keywords: Desiccation treatment; Developmental stage; Immature zygotic embryo; Mature zygotic embryo; Nipponbare; Sasanishiki; Scanning electron microscopy; Somatic embryo

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