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(American Journal of Botany. 2002;89:766-776.)
© 2002 Botanical Society of America, Inc.

Physiology and Development

Induction of vivipary in Arabidopsis by silique culture: implications for seed dormancy and germination1

Val Raghavan

Department of Plant Biology, The Ohio State University, 1735 Neil Avenue, Columbus, Ohio 43210 USA

Received for publication September 27, 2001. Accepted for publication December 14, 2001.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Culture of excised fruits (siliques) of different ages of Arabidopsis thaliana in a solidified mineral salt medium supplemented with vitamins, myo-inositol, and 3% sucrose induces vivipary. Whereas early stage and immature embryos complete their full development before germinating viviparously in seeds enclosed in the silique, mature green embryos enclosed in green ovules germinate without further growth in culture. Vivipary is not observed in cultured siliques enclosing brown ovules with yellowish mature embryos inside. Suggestive of a role for abscisic acid in preventing vivipary on the mother plant, addition of the hormone to the culture medium is found to inhibit vivipary in cultured siliques. Although dried green ovules enclosing mature embryos require a cold treatment for germination, undried ovules of the same age do not germinate even after a cold treatment. This indicates that mature embryos enclosed in green ovules that germinate viviparously are cold resistant and have not become dormant at the time of culture of siliques. The circumvention by silique culture of a cold treatment and light exposure normally required for germination of isolated seeds of A. thaliana provides new possibilities to study the molecular biology of vivipary and seed germination in this model plant.

Key Words: Arabidopsis thaliana • Brassicaceae • embryogenesis • seed germination • silique culture • vivipary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
In the vast majority of flowering plants, embryogenesis is terminated with the development of the seed enclosed within the fruit. Following drying and desiccation of the seed, the embryo enters a period of quiescence or dormancy. Whereas quiescent seeds germinate when provided with the appropriate conditions necessary for resumption of growth of the enclosed embryo, dormant seeds germinate only when some additional hormonal, environmental, metabolic, or physical conditions are met. For example, dormancy of freshly harvested seeds of Arabidopsis thaliana is broken by a short chilling treatment of imbibed seeds at 2°C for 4 d or by a prolonged dry storage for 4–8 wk, both followed by light exposure or by gibberellic acid (GA) application in the dark (Karssen and Laçka, 1985 ). At least three genes (ABSCISIC ACID INSENSITIVE [ABI], FUSCA3 [FUS3], and LEAFY COTYLEDON1 [LEC1]) are known to control the initiation and maintenance of dormancy in A. thaliana seeds by suppressing the development of germination characteristics in mature embryos (Keith et al., 1994 ; Meinke et al., 1994 ; Nambara et al., 1995 ). The ABI gene originally isolated on the basis of the ability of seeds to germinate in the presence of exogenous abscisic acid (ABA) now includes five different genes (ABI1–ABI5); mutations in these genes map to different loci and mutant seeds display different degrees of dormancy and sensitivity to ABA (Koornneef, Reuling, and Karssen, 1984 ; Finkelstein, 1994 ). In contrast to genes that are apparently essential for dormancy induction and germination delay, the COMATOSE (CTS) gene has been shown to promote seed germination, as a mutation in this gene severely impairs the germination potential of embryos (Russell et al., 2000 ).

Vivipary or germination of immature seeds within the fruit still attached to the mother plant has been described as an infrequent occurrence in cultivated plants such as tomato and maize and as a natural way of life in mangroves and trees and shrubs of estuarine habitats. Characteristically, embryos of viviparous plants skip the usual period of quiescence or dormancy typical of those of normal plants and display an uninterrupted transition from embryo development to germination (Sussex, 1975 ). In maize, where the genetics of vivipary has been extensively investigated, at least nine recessive mutant alleles are known to control vivipary. Genetic and physiological studies have shown that besides causing premature germination, mutations in some viviparous genes interfere with other facets of grain maturation, especially ABA synthesis (McCarty, 1995 ). The involvement of a gene in the induction of vivipary in A. thaliana has been established by the isolation and characterization of ABA-deficient (aba) mutants, whose seeds and fruits (siliques) have lower levels of endogenous ABA than those of the wild type. The relationship between a low level of ABA in the seed and vivipary was strengthened by the demonstration that whereas mature seeds of mutant lines germinate in siliques attached to plants held in an atmosphere of high humidity, with little or no evidence of arrested embryo growth, immature seeds germinate in siliques incubated on wet filter paper (Karssen et al., 1983 ). On the other hand, some abi mutants (abi1, abi2, and abi3) had increased or similar levels of ABA in their fruits and seeds as the wild type. Mutant seeds of the monogenic lines were generally nondormant and germinated in high numbers in continuous light, whereas siliques of aba1/abi3 double mutants incubated on wet filter paper showed vivipary (Koornneef, Reuling, and Karssen, 1984 ; Koornneef et al., 1989 ). These observations have raised some questions about the precise role of ABA in the induction of dormancy and seed development in A. thaliana.

During the course of investigations on the relationship between embryo growth and seed dormancy in A. thaliana, it was found that seed germination can be easily and reproducibly induced by the simple expedient of culturing immature siliques in a defined medium. Whereas early division phase embryos in cultured siliques germinate after completing their full development, mature embryos germinate without further growth. Although the fruit is not attached to the mother plant, germination of seeds maturing in cultured siliques is considered as vivipary, because it occurs within the fruit while seeds are connected to the maternal tissues by the funiculus (Goebel, 1905 ; Raz, Bergervoet, and Koornneef, 2001 ). Vivipary contrasts with precocious germination commonly observed when immature embryos excised from ovules bypass the later stages of embryogenesis and germinate in culture (Dieterich, 1924 ). As embryos become both morphologically and physiologically fully mature before they germinate in cultured siliques, the phenomenon described here is closer to vivipary than to premature germination described in detached siliques of single mutants and recombinants among lec1, lec2/fus3, and aba1/abi3 mutants of A. thaliana, cut open and placed on agar media. Moreover, premature germination occurs after ovules sever vascular connection with maternal tissues and before embryos become mature (Raz, Bergervoet, and Koornneef, 2001 ).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plant materials
Most of the experiments described here were carried out using seeds of a Columbia ecotype of Arabidopsis thaliana (L.) Heynh. obtained from the Arabidopsis Biological Resource Center (ABRC), The Ohio State University. Seeds of other ecotypes and mutants used in this work were also supplied by ABRC, whereas those of the transformed Columbia ecotype carrying the cycAt::CDBGUS construct were kindly provided by Dr. John L. Celenza, Boston University, Boston, Massachusetts, USA. Seeds were routinely sown in 11.5-cm (4-inch) pots containing sterilized soil–peat moss–vermiculite mixture and exposed to 4°C for 4 d to overcome dormancy and ensure uniform germination. Pots were immediately transferred to a growth chamber under a photoperiod of 18 h at 25°C; germinated seedlings were maintained with regular nutrient feeding under this condition.

Tissue culture and cytology
For culture, siliques of different ages enclosing ovules with single-terminal cell (following the first division of the zygote producing a small terminal cell and an elongate basal cell)/two-terminal cells (following the first longitudinal division of the terminal cell and one or two transverse divisions of the basal cell) stage embryos to mature embryos were harvested from the primary inflorescence axes of plants. They were surface-sterilized for 10 min in 12% chlorine bleach and washed three times in sterile water. After excising the basal part of the pedicel damaged by chlorine bleach treatment, siliques were transferred to the surface of 10-mL Murashige-Skoog mineral salt medium supplemented with vitamins, myo-inositol, and 3% sucrose and solidified with 0.8% agar, contained in 30-mL (1-ounce) screw-capped French square bottles. In early experiments, stages of embryo development in ovules enclosed in siliques were determined arbitrarily by comparing lengths of cultured siliques to embryo stages in siliques of the same lengths collected from another plant. In later experiments, embryo developmental stages were determined more accurately by fixing a 1–2 mm piece excised from the stigmatic end of cultured siliques ("cut siliques") in acetic alcohol (glacial acetic acid : 70% ethanol, 1 : 3). The fixed materials were examined later by clearing in Hoyer's solution (7.5 g gum Arabic, 5 mL glycerol, 100 g chloral hydrate, and 30 mL water) (Vernon and Meinke, 1994 ). Siliques enclosing ovules with single-terminal cell/two-terminal cells stage embryos were obtained by tagging flowers immediately after anthesis and collecting them 24 h later for culture. Siliques were planted horizontally on the medium or vertically with the pedicel inserted in the medium. Cultures were maintained in incubators at 25°C in complete darkness or in continuous light provided by two fluorescent tubes (7.0 µEinstein/µmol). Observations on the first appearance of the radicle or plumule through the pericarp were made beginning 8 d after culture and at 2-d intervals thereafter up to 40 d. In each experiment, at least ten siliques containing ovules with single-terminal cell/two terminal cells stage embryos as well as globular, heart-shaped, torpedo-shaped, bent-cotyledon, and mature embryos as defined by previous investigators (Mansfield and Briarty, 1991 ; Jürgens and Mayer, 1994 ) were cultured under the conditions described above. Because the number of cultures that can be conveniently handled in one experiment is limited, some quantitative data presented here are based on pooled results from several experiments. Whole-mount observations of embryo development were routinely made at 1–2 d intervals by clearing ovules and seeds from cultured whole siliques or cut siliques in Hoyer's solution. For histological studies, ovules collected from dark-cultured whole siliques enclosing single-terminal cell/two terminal cells stage embryos were fixed in acetic alcohol at 24 h-intervals up to 10 d, dehydrated in ethanol, n-propanol, and n-butanol series and embedded in glycol methacrylate following standard procedures (Feder and O'Brien, 1968 ). Sections cut at 7 µm thickness on a rotary microtome equipped with a steel knife were double-stained in periodic acid-Schiff and toluidine blue and mounted in Euparal (O'Brien and McCully, 1981 ).

ß-Glucuronidase (GUS) staining and detection
GUS activity was detected histochemically using 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide (X-Gluc; Sigma, St. Louis, Missouri, USA) as a substrate, according to the method of Donnelly et al. (1999) . Ovules dissected from siliques were placed in 90% acetone on ice for 15 min, followed by X-Gluc buffer (750 µg/mL X-Gluc, 100 mol/L sodium phosphate pH 7.0, 1.5 mmol/L potassium ferricyanide, 1.5 mmol/L potassium ferrocyanide, 10 mmol/L EDTA, and 0.1% Nonidet-P40) under vacuum for 16–20 h at laboratory temperature (25°C). Ovules were subsequently rinsed in water and cleared in Hoyer's solution for observation of embryos.

Germination assays
For germination experiments, seeds or dried ovules were sown on the surface of 25-mL 0.8% agar-distilled water medium contained in 10 cm diameter petri dishes. Following treatment in the dark for 4 d at 4°C in the refrigerator or at 25°C in the incubator, samples were exposed to continuous fluorescent light (7.0 µEinstein/µmol) at 25°C for 4 d before germination counts were made. Emergence of the radicle outside the seed coats as seen in the dissecting microscope was used as the criterion for germination. The ABA (mixed isomers; Sigma, St. Louis, Missouri, USA) was cold-sterilized through a Millipore filter before use in silique culture and in germination experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Growth and germination of embryos in cultured siliques
Whole siliques and cut siliques of A. thaliana enclosing ovules of various embryo developmental stages remain green for 7–10 d when cultured horizontally in the dark on a solidified mineral salt medium supplemented with vitamins, myo-inositol, and 3% sucrose; subsequently the pericarp begins to turn yellow or brown. Periodic dissection of whole siliques and cut siliques confirmed that ovules remained attached to the placenta through the funiculus for several days after the pericarp turned yellow. Appearance of the radicle or plumule outside the pericarp as the first sign of vivipary occurs as early as 8 d after culture in siliques enclosing bent-cotyledon and older embryos, whereas in siliques enclosing earlier stage embryos, vivipary is progressively delayed (Fig. 1). Microscopic examination of siliques dissected at the first sign of vivipary showed that embryos were in various stages of germination with partially elongated radicle and plumule (Fig. 2). Irrespective of the stage of embryo development, there was no synchrony in vivipary in cultured siliques. On average, cut siliques cultured in the dark at the bent-cotyledon and mature embryo stages show vivipary in 15–20 d (50% vivipary = 19–20 d), whereas those cultured at the torpedo-shaped, heart-shaped, and globular embryo stages become viviparous in 18, 24, and 25 d (50% vivipary = 20–29 d), respectively. Siliques cultured in the dark at the single-terminal/two-terminal cells stage of embryos became viviparous in about 27 d (50% vivipary = 29 d); however, only a few ovules produced mature embryos and seedlings from these siliques. This is attributed to the failure of fertilization, as many shrunken or shriveled ovules were usually observed along with germinating seeds in siliques dissected at the end of the experiment. However, other possible explanations such as nutritional deficiency in the medium and absence of maternal signals have not been ruled out to account for this observation. About 1% of flower buds cultured 1 d before anthesis produced small viviparous siliques, whereas the rest of the buds did not open and became brown or, if opened, did not produce siliques. Assuming that pollination and fertilization had occurred in culture, this is considered as evidence that vivipary can be induced even in siliques maturing in culture beginning at the single-celled zygote stage.



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  		Fig. 1. Time course of vivipary in dark-cultured whole siliques enclosing single-terminal cell/two-terminal cells stage embryos and dark-cultured cut siliques enclosing embryos of other developmental stages of A. thaliana. Both whole siliques and cut siliques were planted horizontally on the medium. Data points indicate the number of siliques (expressed as additive percentages of the total number of uncontaminated cultures) showing the first signs of vivipary at days indicated, beginning 8 d after culture. Figures in parentheses against each embryo developmental stage refer to the total number of cultures counted. Siliques collected 1 d later from flowers tagged on the day of opening have been established to contain ovules with single-terminal cell/two-terminal cells stage embryos. Other embryo developmental stages were determined by examining ovules from cut pieces of siliques

 


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  		Figs. 2–4. Vivipary in cultured whole siliques and cut siliques of A. thaliana. 2. Part of the population of ovules from a cut silique enclosing bent-cotyledon stage embryos cultured horizontally in the dark and examined on the day of appearance of the first sign of vivipary outside the pericarp (16 d after culture). Embryos are at different stages of germination. Scale bar = 200 µm. 3. A silique cultured vertically in the dark for 18 d showing the outgrowth of the plumule (arrow). 4. A silique cultured vertically in the dark for 23 d showing the emergence of the radicle from two germinating embryos (arrows) and of the plumule (arrowheads) from additional embryos. Siliques shown in Figs. 3 and 4 contained embryos at about the torpedo-shaped stage of development. Scale bars for Figs. 3, 4 = 1 mm

 
In siliques cultured vertically in the dark with the pedicel inserted in the medium, vivipary became evident by the radicle or the plumule of one or more embryos piercing the pericarp and appearing outside (Figs. 3, 4). Appearance of the first seedling outside was followed within 4–8 d by the germination of embryos and production of seedlings from most of the enclosed ovules, leading to the separation of the pericarp halves. When the radicle was the first embryonic organ to appear outside the pericarp in vertically cultured siliques, it invariably showed positive gravitropism and continued to grow toward the agar medium. In vertically cultured siliques, it was also not uncommon to see both radicle and plumule of germinating embryos growing in the air due to the failure of the latter to establish contact with the culture medium. Positive gravitropic growth of the radicle into the medium and negative gravitropic growth of the plumule were, however, the norm in horizontally cultured siliques (Figs. 5, 6). Both vertical and horizontal planting of siliques and light and dark conditions favored vivipary in cultured whole siliques and cut siliques of all ages, although continuous light and horizontal planting favored earlier germination than complete darkness and vertical planting (data not shown). Vivipary was also induced in whole siliques and cut siliques cultured in a liquid medium of the same composition as the solid medium. Embryos completed their development and germinated while ovules were still enclosed within the silique and seedlings subsequently shed from the silique formed a "float" culture. Although the culture medium was chosen arbitrarily, siliques were found to display vivipary when they were cultured horizontally in the light in a distilled water-agar solidifed medium without any mineral salts, vitamins, myo-inositol and sucrose. Using siliques enclosing heart-shaped and torpedo-shaped embryos, it was also possible to induce vivipary in each segment of a silique cut transversely into two, four, or six pieces and cultured in the same or different bottles containing the regular medium (Fig. 7).



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  		Figs. 5–7. Vivipary in horizontally cultured siliques and silique pieces of A. thaliana. 5. A culture bottle showing negative gravitropic growth of the plumule of viviparously germinating embryos from a silique planted horizontally in the dark; photographed 25 d after culture. Scale bar = 5 mm. 6. A silique cultured horizontally in the dark for 25 d and in the light for 1 d, showing the emergence of numerous seedlings. Arrowheads point to the two separated halves of the silique. Scale bar = 1 mm. 7. Vivipary in individual segments of a silique cut into six pieces and cultured in the same bottle in the dark for 28 d. Numbers i to vi indicate cut pieces of the silique in sequence beginning from the pedicel end. Scale bar = 1 mm. Siliques shown in Figs. 5–7 enclosed ovules containing embryos at about the torpedo-shaped stage of development

 
Examination of whole mounts and sectioned materials revealed that single-terminal cell/two terminal cells stage embryos enclosed in ovules of cultured whole siliques began to undergo morphogenesis within 1–2 d and completed the embryo developmental program as they progressed through the quadrant, octant, globular, heart-shaped, torpedo-shaped, bent-cotyledon, and mature stages within 8–10 d (Figs. 8–16). Division sequences of these early stage embryos in cultured siliques were identical to those described during embryogenesis on the intact plant (Mansfield and Briarty, 1991 ; Jürgens and Mayer, 1994 ). Following several days of physiological maturation when no further morphological growth occurred, embryos entered the germinative mode, resulting in the elongation of the radicle or plumule through the pericarp. Globular, heart-shaped, torpedo-shaped, and bent-cotyledon stage embryos contained in ovules of cultured cut siliques showed a pattern of morphogenesis similar to that of one-terminal cell/two-terminal cells stage embryos as they completed the remaining stages of embryogenesis before germination. Mature embryos of cultured cut siliques did not grow further before germinating viviparously. These observations show that embryos developing in ovules of cultured siliques do not skip any stages of development preparatory to vivipary.



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  		 Figs. 8–16. Embryogenesis in siliques of A. thaliana enclosing single-terminal cell/two-terminal cells stage embryo, cultured horizontally in the dark. 8. Section of the ovule showing a single-terminal cell embryo at the time of culture. Arrow points to the large basal cell that forms the suspensor. 9. Section of the ovule showing an embryo with two terminal cells at the time of culture. Arrow points to the basal cell that has probably divided once. 10. Section of the ovule showing a quadrant stage embryo 1 d after culture. Arrow points to the three-celled suspensor. 11. Section of the ovule showing an early globular embryo, at the stage of protoderm (p) differentiation, 4 d after culture. h, hypophysis of the suspensor. 12. Section of the ovule showing a late globular embryo, 5 d after culture. p, protoderm. Scale bars for Figs. 8–12 = 10 µm. 13. Section of the ovule showing a heart-shaped embryo 6 d after culture. 14. Section of the ovule showing a torpedo-shaped embryo 6 d after culture. Scale bars for Figs. 13, 14 = 20 µm. 15. Section of the ovule showing a bent-cotyledon shaped embryo 8 d after culture. Scale bar = 40 µm. 16. Section of the ovule showing a mature embryo 11 d after culture. c, cotyledons; r, root apex; s, shoot apex. Scale bar = 50 µm

 
Embryos of all ages maturing in cultured siliques undergo several days of physiological maturation before they germinate viviparously and thus require more time for germination than embryos of seeds maturing on the plant. It is considered unlikely that embryos lapse into dormancy during physiological maturation, because they germinate in cultured siliques without a cold treatment and light exposure normally required to break dormancy and potentiate germination of isolated seeds of the ecotype used in this work. It has not been established here whether mature seeds isolated from cultured siliques behave like seeds maturing on planta with regard to their germination requirements.

To determine whether embryos maturing in cultured siliques had ceased mitotic activity and entered the growth phase, cell division profiles of embryos of transgenic A. thaliana plants harboring Arabidopsis thaliana Cyclin1 gene including cyclin destruction box (CDB) fused in frame with GUS reporter gene construct (cycAt::CDBGUS) were assayed. The fusion of CDB to the GUS gene in this construct leads to the degradation of CDBGUS protein at the end of mitosis, thus restricting the reporter gene expression to those cells passing through the mitotic cycle. Dense, blue-staining GUS protein was observed in virtually all cells of normally developing globular to heart-shaped embryos of the transgenic plant. There was a decrease in the number of cells with GUS activity in the torpedo-shaped embryo, although a disproportionately large number of stained cells were concentrated in the cotyledons. Confirming the observations of Raz, Bergervoet, and Koornneef (2001) , GUS-expressing cells decreased further in the bent-cotyledon stage embryos and completely disappeared in mature embryos enclosed in green and brown ovules. A similar pattern of distribution of GUS-expressing cells was observed in globular embryos maturing in cultured siliques, with a complete absence of GUS-reacting cells as embryos were phased from the bent-cotyledon stage into the mature stage (Figs. 17–21). These results show that as in embryos maturing normally on the plant, physiological maturation of embryos in cultured siliques is entirely due to cell elongation.



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  		Figs. 17–21. Histochemical localization of ß-glucuronidase activity in embryos of cleared ovules from cultured cut-siliques of transgenic A. thaliana carrying the cycAt::CDBGUS reporter gene construct. In all photographs, the focus is on the embryo, as seen through the cleared ovule. 17. A globular embryo in an ovule at the time of culture. Arrow indicates the point of origin of the suspensor. 18. A heart-shaped embryo, 4 d after culture. c, incipient cotyledons. Scale bars for Figs. 17, 18 = 10 µm. 19. A torpedo-shaped embryo, 5 d after culture. Scale bar = 20 µm. 20. A bent-cotyledon stage embryo, 9 d after culture. 21. A mature embryo, 11 d after culture. c, cotyledons; r, root apex. Scale bars for Figs. 20, 21 = 40 µm

 
In small experiments, vivipary was also induced in cultured whole siliques of A. thaliana ecotypes Landsberg erecta and Wassilewskija, and aba1, abi1, abi2, and abi3 mutants derived from Landsberg erecta. Comparison of embryo developmental stages in siliques of the same lengths as those cultured showed that cultured siliques of the wild type and mutants enclosed torpedo-shaped and bent-cotyledon stage embryos. Vivipary was observed in cultured siliques of mutants before or after the pericarp turned yellow, and occurred earlier than in cultures of their wild-type counterparts (data not shown).

Effects of ABA on vivipary
Results of previous studies, also confirmed in the present investigation, have shown that 10 µmol/L ABA causes nearly 90% inhibition of germination of seeds of A. thaliana and that germination is completely inhibited by 30 µmol/L ABA (Koornneef et al., 1982 ; Koornneef, Reuling, and Karssen, 1984 ). In view of the extreme sensitivity of seed germination to ABA, it was of interest to determine the effect of the hormone on vivipary in cultured siliques. For this purpose, cut siliques enclosing globular embryos were cultured horizontally in the dark in media supplemented with 1.0–400 µmol/L ABA. Although the first sign of vivipary was delayed by about 7 d in siliques cultured in 1.0 µmol/L ABA, percentages of siliques showing vivipary at the end of a 45-d experimental period remained nearly as high as in the basal medium. Higher concentrations of ABA (10 and 50 µmol/L) progressively inhibited vivipary in cultured siliques and no vivipary was observed in siliques cultured in a medium containing 100 µmol/L ABA (Table 1). Dissection of nonviviparous siliques cultured in a medium containing 100 µmol/L ABA at the end of the experiment showed that in some ovules embryos had developed to the mature stage, whereas in others the radicle had elongated outside the ovule, but further growth in the viviparous pathway was inhibited (Fig. 22). In both viviparous and nonviviparous siliques, abnormal embryos consisting of a globular mass of cells, unequal cotyledons, and laterally expanding cotyledons were also frequently observed. Dissection of siliques cultured even in media containing 200–400 µmol/L ABA at the end of the experiment revealed the presence of heart-shaped to mature embryos. Taken together, these results indicate that although embryogenic development is insensitive to ABA, to the extent that germination involves growth of the radicle and/or plumule, the hormone is an effective inhibitor of normal and viviparous germination of seeds.


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  		Table 1. Effect of different concentrations of ABA on vivipary in cut siliques cultured at the globular embryo stage. Data are pooled from four experiments. Final counts of cultured cut siliques showing vivipary were made 45 d after culture. Numbers in parenthesis indicate the total number of uncontaminated cultures in each case

 


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  		Figs. 22–26. Whole mounts of germinated embryos of A. thaliana. 22. Part of an ovule from a cut-silique enclosing globular embryos, cultured horizontally in the dark in a medium containing 100 µmol/L ABA and examined 45 d after culture; note the outgrowth of the radicle (r). Scale bar = 50 µm. 23. A seedling formed from a dried ovule enclosing mature green embryo, cold-treated for 4 d and photographed 4 d after exposure to continuous light. Arrow points to the remains of the ovule still attached to the seedling. 24. Plumular part of another seedling formed from a dried ovule enclosing mature green embryo, treated as above, showing leaf primordia (arrow). 25. A seedling formed from a dried ovule enclosing mature green embryo, treated for 4 d at 25°C and photographed 4 d after exposure to continuous light. 26. Germination of another dried ovule enclosing mature green embryo, treated for 4 d at 25°C and photographed 4 d after exposure to continuous light; only the radicle (arrowhead) has grown out of the ovule. Arrow points to an ungerminated ovule. Figure 26 scale bar = 200 µm applies to Figs. 23–26

 
Vivipary in cultured siliques in relation to embryo dormancy
In the above experiments, cultured siliques at the mature embryo stage that showed vivipary were green in color enclosing green ovules. In this investigation, an embryo was considered mature when it remained green or turned yellow and almost filled the ovule, and tips of cotyledons had reached the level of the radicular meristem. To determine whether mature embryos had entered a period dormancy at the time of culture of siliques, the first ten siliques formed on the main inflorescence axis of plants were tagged, and as the first two siliques formed on each inflorescence axis began to turn to yellow, all the tagged siliques were collected. The length of each silique in the series from each plant was measured for identification purposes. Siliques were subsequently sterilized and each silique was cut transversely at two-thirds its length toward the pedicel end into a large piece and a small piece ("silique pieces"); the small piece was cultured horizontally in the basal medium in the dark to monitor vivipary. After determining the stage of embryo development, ovules dissected from the large piece were dried on the laboratory bench (25°C) for 4 d and sown on the surface of agar-distilled water medium contained in two sets of petri dishes for germination tests. The results showed that cultured silique pieces enclosing green ovules with mature green embryos or bent-cotyledon stage embryos, invariably became viviparous, while virtually no vivipary was observed in silique pieces enclosing brown ovules with mature yellowish embryos (Table 2). Because mature seeds are characterized by the presence of brown testa and yellowish embryo (Koornneef and Karssen, 1994 ), this indicates that embryos of mature seeds are not prone to viviparous germination by silique culture. (More mature siliques than those used in these experiments break open during handling and sterilization preparatory to culture, and so it was not possible to establish that siliques enclosing fully mature seeds would not show vivipary in culture.)


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  		Table 2. Vivipary in cultured silique pieces and germination of dried ovules. Data are pooled from five experiments using siliques from 2–3 tagged plants in each experiment. Siliques from each plant are grouped in column 1 according to their age (Group I, oldest; Group X, youngest). Final counts of cultures showing vivpary were made 40 d after culture of silique pieces and are given as percentages of the total number of uncontaminated cultures (in parentheses). For germination experiments, ovules from each silique piece were dried and sown on the surface of agar-distilled water medium contained in two sets of petri dishes. One set of petri dishes was kept at 4°C and the other at 25°C, both in the dark for 4 d, followed by continuous light at 25°C for 4 d. Percentages of germination determined at the end of the light treatment are given, followed, in parentheses, by the total number of dried ovules counted

 
With regard to their requirement for germination and extent of growth of the radicle, hypocotyl, and plumule, three types of dried ovules could be identified in the above experiment. Compared to the high percentages of germination of dried brown ovules enclosing yellowish mature embryos (Groups I and II) and slightly brown and green ovules enclosing green mature embryos (Groups III and IV) subjected to a cold treatment (4°C), only small percentages of these ovules germinated in the absence of the cold treatment (25°C). In addition, germinating cold-treated dried ovules produced long roots and hypocotyls and robust plumules consisting of fully open cotyledons with or without visible leaf primordia (Figs. 23, 24), compared to the short roots and hypocotyls, and plumules with closed or partially open cotyledons formed from ovules kept at 25°C (Fig. 25). A few seeds in the latter group also germinated producing just only the radicle (Fig. 26). Although cold-treated dried green ovules enclosing green mature embryos (Groups V–VII) germinated in large numbers, seedlings formed had shorter roots and hypocotyls than those produced by cold-treated ovules of Groups I–IV and were somewhat closer in appearance to the small number of seedlings formed from corresponding ovules kept at 25°C. No appreciable germination was observed in ovules of Groups VIII–X enclosing bent-cotyledon stage embryos, irrespective of whether they were cold treated or not. The few seedlings that were formed from ovules in these groups had short roots and hypocotyls and unexpanded cotyledons. It was noted that upon drying, ovules of Groups III–X had become fully brown with yellowish embryos. The fact that siliques in Groups III–VI all show vivipary, but dried ovules of these same groups require a chilling treatment for germination suggests that dormancy is a secondary effect of biochemical changes in the embryo and testa induced by drying.

The above results also suggest that mature embryos enclosed within green ovules that germinate viviparously are probably prone to become dormant at the time of culture of siliques and that dormancy begins to take effect as the embryo is phased from the bent-cotyledon stage into the mature stage (Group VII); if this were not the case, dried ovules of Groups I–VII would have germinated in large numbers without a cold treatment. To test this hypothesis, ovules dissected from staged siliques were sown immediately on the surface of agar-distilled water medium contained in two sets of petri dishes and exposed to 4°C or 25°C for 4 d, followed by 4 d continuous light at 25°C as in the previous experiment. Germination counts made at the end of the light treatment (Table 3) showed that only the cold-treated brown ovules with yellowish mature-stage embryos (Group I) germinated fully to produce robust seedlings, whereas no germination occurred in the other groups of cold-treated ovules. However, ovules phasing into the mature embryo stage from the bent-cotyledon embryo stage (Groups IV–VI) germinated in high numbers to produce seedlings with short roots and hypocotyls, and plumules with unexpanded cotyledons even without the cold treatment, although, not unexpectedly, brown ovules containing yellowish mature-stage embryos and green to brown ovules containing green mature-stage embryos (Groups I–III) did not germinate appreciably under this condition. Ovules enclosing bent-cotyledon stage embryos (Groups VII–X) did not also germinate irrespective of whether they were cold-treated or not. Examination of nongerminating cold-treated ovules at the end of the experiment showed that whereas the ovular tissues had become brown, embryos had turned white and did not grow beyond the stage at culture. These observations reflect the fact ovules which enclose mature-stage green embryos (Groups II–VI) are cold-resistant/intolerant and do not become dormant until they are desiccated; on the other hand, those of Groups VII–X, which enclose bent-cotyledon stage embryos, are cold-intolerant.


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  		Table 3. Germination of undried ovules from siliques of different ages. Siliques from three tagged plants are grouped in column 1 according to their age (Group I, oldest; Group X, youngest). Ovules were collected from siliques, and, after determining the embryo developmental stages, they were immediately transferred to the surface of agar-distilled water medium contained in two sets of petri dishes. One set of petri dishes was kept at 4°C and the other at 25°C, both in the dark for 4 d, followed by continuous light at 25°C for 4 d. Percentages of germination determined at the end of the light treatment are given, followed, in parentheses, by the total number of ovules counted

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
There are reports of germination of seeds in which the embryo completes its development in cultured ovaries, but ovary or fruit culture as a means to induce vivipary has received little attention (Guha and Johri, 1966 ; Johri and Sehgal, 1966 ). Moreover, germination described in these reports appears to have occurred after seeds maturing in cultured ovaries or fruits are shed into the medium. The present work has clearly shown that A. thaliana embryos that complete their development in culture germinate viviparously in the silique without lapsing into a dormant phase which is normally overcome by cold treatment and light exposure of seeds. Although vivipary has been observed in some monogenic and digenic lines among the aba, abi, fus, and lec mutants of A. thaliana by keeping plants under high humidity or by incubating excised siliques under humid conditions (Karssen et al., 1983 ; Koornneef, Reuling, and Karssen, 1984 ; Koornneef et al., 1989 ; Raz, Bergervoet, and Koornneef, 2001 ), vivipary in attached or detached siliques of wild-type plants has not been reported before. To the extent that excision of the silique and its culture results in a loss of the late embryogenesis pathway leading to dormancy, silique culture may be said to have the same effect as mutations in the genes that induce dormancy and prevent germination of the embryo. Whether yellowing or senescence of the cultured silique directly or indirectly contributes to vivipary has not been determined.

In the development of the seed of Gossypium hirsutum (cotton), continued embryo growth has been attributed to the supply of regulatory factors by the mother plant through the funiculus, whereas ABA synthesized subsequently in the ovular tissues apparently prevents vivipary and precocious germination of the embryo (Ihle and Dure, 1972 ). In analogy with cotton, constituents of the culture medium might be considered to substitute for the regulatory factors from the mother plant in promoting embryo growth in ovules of cultured siliques of A. thaliana. Inhibition of vivipary in cultured siliques by ABA might indicate that ovular tissues are unable to synthesize this hormone in sufficient quantity to prevent vivipary; however, reciprocal crosses between aba mutants and wild-type A. thaliana have shown that seed dormancy is due to the production of ABA by the embryo and not by the maternal tissues (Karssen et al., 1983 ). Based on the germination of isolated seeds and vivipary in excised siliques of double mutants between lec/fus and aba/abi, Raz, Bergervoet, and Koornneef (2001) have implicated two sequential processes in the development of seeds of A. thaliana, one regulated by FUS3 and LEC1 genes promoting embryo growth arrest foreshadowing seed maturation and the other by ABA, inducing dormancy. According to this model, double mutations affecting both developmental processes produce viviparous phenotypes. Both FUS3 and LEC1 genes have been cloned and are known to encode transcription factors; especially LEC1 gene product has been identified to control the activation of several target genes during embryogenesis such as those affecting suspensor formation, cotyledon identity, desiccation tolerance, and initiation of embryogenic program in vegetative cells (Lotan et al., 1998 ; Luerßen et al., 1998 ). If transcription factors encoded by FUS3 and LEC1 genes are involved in preventing mitotic activity in mature embryos as a prelude to dormancy, as implied in this model, results of the present study indicate that they are synthesized during embryogenesis in culture drawing upon the constituents of the medium and act during a narrow developmental window. The fact that vivipary in cultured siliques is inhibited by ABA without any effect on the growth of embryos is consistent with the model of Raz, Bergervoet, and Koornneef (2001) implicating an ABA-mediated step in the control of vivipary, but data on the changes in levels of endogenous ABA in cultured siliques are necessary to confirm this view.

It has generally been assumed that mature embryos of the vast majority of plants lapse into quiescence or dormancy following drying and desiccation of the seed. However, conditions during seed development and the duration of storage affect dormancy of seeds of A. thaliana, and consequently, requirements for their release from dormancy are varied (Derkx and Karssen, 1993 ; Koornneef and Karssen, 1994 ). The high percentages of germination of dried ovules enclosing late-stage embryos triggered by a cold treatment reveal that embryos are potentially capable of becoming dormant when they are phased into the mature stage from the bent-cotyledon stage. According to Kermode and Bewley (1985a) , germination of embryos of prematurely desiccated seeds of Ricinus communis (castor bean) is confined to a brief period before they naturally desiccate and pass into quiescence or dormancy. At the biochemical level, premature drying of castor bean seeds suppresses developmental protein synthesis in the embryo and endosperm, but induces the synthesis of germination proteins and their corresponding mRNAs in these tissues (Kermode and Bewley, 1985b, 1986 ; Kermode, Pramanik, and Bewley, 1989 ). However, virtually nothing is known about the biochemical signals that induce dormancy in developing seeds of A. thaliana, although desiccation of the embryo followed by chlorophyll breakdown and development of testa pigments are likely candidates. As undried ovules harboring various mature-stage embryos (Groups II–VI, Table 3) do not germinate in response to the cold treatment, it is reasonable to conclude that embryos of these stages in cultured siliques have not lapsed into dormancy. Although the morphological appearance of the embryo has been used as a criterion to classify its state of maturity, this does not necessarily indicate the attainment of physiological maturity.

Because dormancy of seeds of various plants can be broken by a chilling treatment, storage at dry temperature, light, or GA administered singly, the requirements for a chilling treatment or prolonged dry storage followed by exposure to light or GA to induce germination of seeds of A. thaliana can be considered unusual (Bewley and Black, 1994 ). Whereas phytochrome involvement has been clearly established in the photocontrol of germination of seeds of A. thaliana (Shinomura et al., 1994 ), the mechanism by which the chilling treatment overcomes seed dormancy is not known. Interestingly, inhibition of germination of seeds of A. thaliana cold treated in the presence of 5 µmol/L of ABA leads to the accumulation of transcripts and proteins of the ABI5 gene, but no such regulatory molecules are detected in the absence of the hormone (Lopez-Molina, Mongrand, and Chua, 2001 ). The ease of germination of seeds maturing in cultured siliques without a cold treatment and light exposure normally required for germination of isolated seeds opens up an interesting new methodology to study the molecular biology of vivipary and seed germination in A. thaliana, especially the origin, perception, and transduction of signals involved in the induction and breakage of dormancy; it also facilitates large-scale screening of seedling mutants and mutants defective in embryogenesis.


   FOOTNOTES
 
1 The author thanks the Arabidopsis Biological Resource Center (ABRC), The Ohio State University, for supply of wild-type and mutant seeds used in this investigation; Dr. John L. Celenza, Boston University, Boston, Massachusetts for supply of seeds of the transgenic plant; Ms. Luz Rivero of ABRC for access to growth chamber facilities and for help and advice in growing plants; and Ms. Jill Williams and Dr. Jochen Schwuchow, both of this Department for help in the preparation of Fig. 1 . This work was supported by an allocation from the Department of Plant Biology, The Ohio State University. Back


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 ABSTRACT
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 DISCUSSION
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