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a Department of Horticulture, University of Hawaii, 3190 Maile Way #102, Honolulu, Hawaii 96822–2279; and b Department of Botany, University of Hawaii, 3190 Maile Way #101, Honolulu, Hawaii 96822–2279
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND
METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: anatomy • Anthurium • Araceae • embryogenesis • histochemistry • morphology • ovule/embryo culture
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND
METHODSRESULTSDISCUSSIONREFERENCES |
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Most Anthurium cultivars are asexually propagated clones derived from hybrids. Some taxa fail to produce viable seeds due to incompatibilities (Sheffer and Kamemoto, 1976). Ovule and embryo culture can be used to overcome some of these problems (Raghavan, 1986). However, seed maturation is protracted and can take from 6 to 12 mo. Consequently, it is difficult to predict when embryo development stops and when intervention should occur. Correlative knowledge of the in vivo stages of embryogeny and the ability of ovules and embryos to mature in vitro would be very important for this genus. Somatic embryogenesis has the potential for rapid, mass propagation of clones and has recently been reported for Anthurium (Geier, 1982; Kuehnle, Chen, and Sugii, 1992). It is important to compare key events in somatic embryogenesis with zygotic embryogenesis to determine whether similar developmental events occur in somatic embryos and to assess their quality (Redenbaugh et al., 1986; Roberts et al., 1990; Flinn et al., 1991). Presently, there are insufficient data on zygotic embryogenesis of Anthurium to make such comparisons.
The goals of this study were to document the major anatomical events in embryo and endosperm development following controlled pollination of Anthurium andraeanum flowers, to use histochemical tests to document major changes in physiology during embryogeny, to use the above to compose a temporal framework for zygotic embryogenesis, and to correlate embryo maturation with the ability to produce plants from ovule or embryo cultures.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND
METHODSRESULTSDISCUSSIONREFERENCES |
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Microtechnique
Carpels or
ovules were removed from the flower and fixed in 2%
glutaraldehyde and 2.5% paraformaldehyde in 0.05 mol/L sodium
cacodylate buffer, pH 7.0 (Karnovsky, 1965). Specimens were washed three
times with buffer and dehydrated in ethanol (10% increments every
15 min). After two changes of 100% alcohol, specimens were
infiltrated with 3 mL of fresh alcohol and 1 mL of Historesin (Leica
Inc., Deerfield, Illinois) infiltration solution. Additional
infiltration solution (1-mL aliquots) was added every other day. After
1 wk, the Historesin-alcohol mixture was replaced by 100%
Historesin infiltration solution. This was changed every other day
until the tissue appeared translucent. Inverted Beem capsules with a
small hole in the pointed end were used for embedding so that explants
would lie flat against the lid for proper orientation. Addition of
resin through the small hole and placement of the sample under vacuum
aided polymerization by minimizing atmospheric contact with the
specimen.
Blocks containing specimens were cured at 60°C for at least 2 d prior to sectioning. This ensured a harder block, which decreased the amount of chatter. Glass knives were used for sectioning at 5 µm on a Sorval Porter-Blum MT2-B Ultra-Microtome. Sections were floated on distilled water and heated on a slide warmer at 40°C. Sections were stained with Periodic Acid Schiff (PAS) for carbohydrates and counterstained with naphthol (aniline)-blue-black for proteins (Feder and O'Brien, 1968). Results were based on observations of several pistils from flowers in the middle portion of the spadix at approximately the same maturity.
Ovule/embryo
culture
Flowers were obtained from a spadix at eight intervals
(every 2 wk from 8 to 24 wk postpollination). Tepals and stamens were
removed with the aid of a dissection microscope. The isolated pistils
were surface sterilized by two sequential 30-min soaks in 0.525%
sodium hypochlorite and 0.262% sodium hyphochlorite with Tween
20 (1 drop/100 mL) and rinsed three times with distilled sterile water.
Ovules were excised from pistils for the first three intervals
(8–12 wk postpollination). Both ovules and embryos were excised
from pistils for the last five intervals (14–24 wk
postpollination). Ten explants were plated per petri plate, with
20–23 explants per time interval.
A modified Kunisaki (1980) medium was used and consisted of half-strength MS (Murashige and Skoog, 1962) macronutrients, full-strength micronutrients, 2% sucrose, 100 mg/L myo-inositol, 25 mg/LNaFeEDTA, MS vitamins modified to include 0.4 mg/L thiamine-HCl, 2% (w/v) sucrose, and 15% (v/v) immature coconut water, pH 5.7. This was solidified with 0.25% (w/v) Gelrite. Following sterilization at 121°C (103.42 kPa for 15 min), the medium was dispensed into 100-mm petri plates (20 mL per plate). Each plate of embryos or ovules was placed in complete darkness or under continuous fluorescent light at 23°C.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND
METHODSRESULTSDISCUSSIONREFERENCES |
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Week 4
The zygote, located
at the micropylar end of the ovule, was indicated by a large, densely
stained nucleus with two prominent nucleoli (Fig.
7). The nucellus was composed of a single layer of cells
between the zygote and micropyle. Single synergid cells were often
observed between the zygote and nucellus, the other synergid having been
disrupted by the pollen tube during fertilization (Fig. 7). A section of a different sample at 4
wk postpollination shows the embryo had undergone oblique divisions to
become a proembryo (Fig. 8). The
inner integument of the ovule, which gives rise to the inner seed coat
or tegmen, was two layers thick. The outer integument, which becomes
the outer seed coat or testa, was also two layers thick (Fig. 8). Yellow deposits were predominantly
found in the inner integuments and are probably tannins, known to be
present in the seed coat.
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Week
6
The proembryo exhibited further oblique and transverse
divisions (Fig. 9) by the highly
vacuolate cells. Cell boundaries of the endosperm stained more intensely
and the yellow deposits were more prominent in the inner integument. At
this point in development most of the nucellus has been
degraded.
Week 8
Further cell divisions formed
a terminal region that comprised the embryo proper and suspensor
(Fig. 10). The cells of the endosperm
were more developed at the chalazal end of the ovule (Fig. 11). There was a strong PAS staining
reaction in this region of the endosperm and funiculus, suggesting
increased cell wall development and the onset of starch accumulation.
Thus, food reserves appear to be imported from the sporophyte through
the funiculus, which also stains strongly for carbohydrates and
proteins, to the developing endosperm and embryo. Cells in the
micropylar region of the ovule also stained heavily with naphthol
blue-black.
Week 10
The terminal region
increased in size and can now be called the embryo proper (Fig. 12). The cytoplasm of its cells stained
intensely with naphthol blue black. The suspensor was enlarged.
Suspensor cells distal to the micropyle were more vacuolated than those
closer to the embryo proper.
Week 12/week
14
Distinction between the embryo proper and the suspensor was
much greater after 12–14 wk and the suspensor was clearly
two-ranked (Fig. 13). The embryo
proper had elongated and appeared elliptical. Furthermore, it was
asymmetrically flattened along one side due to the localized development
of a lateral meristematic region composed of small, densely cytoplasmic
cells. These embryos closely resembled Luzula fosteri zygotic
embryos at the stage of shoot apex and cotyledon formation
(Souèges, 1923, cited by Raghavan, 1976, fig. 2.15), and A.
andraeanum somatic embryos with lateral notch formation (Kuehnle et al., 1992). The
cotyledon comprises the more vacuolate cells surrounding the lateral
notch, with the latter seen in later weeks to produce the shoot and root
apices (see weeks 16–20). Thus, cotyledon formation by A.
andraeanum embryos occurs by 12–14 wk (Fig. 13).
A distinct protoderm was also apparent (Fig. 13). Endosperm cells contained more cytoplasm except for those proximal to the embryo. These were devoid of cytoplasmic contents and their walls stained positively for carbohydrates with PAS, most likely indicating a transfer of nutrients to the developing embryo. This documents the stage at which the embryo starts to use the endosperm for nutrition. The outer integument contained large idioblasts with calcium oxalate crystals (raphides). Cells of the inner integument had yellow, tannin-like deposits (Fig. 14).
Week 16 through week 20
Embryo
maturation was extremely rapid at 14–16 wk postpollination, with
embryos well developed and ~2 mm long by week 16 (Fig. 15). Embryos at this stage of development
contained shoot and root apical meristems and a continuous procambium
(Figs. 16–18). Storage
products present in the embryo and endosperm included calcium oxalate,
proteins, and starch. The cotyledon was well developed and enveloped
the shoot apex.
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Week
20
Root and shoot meristems were well formed and leaf primordia
were present (Fig. 16). Procambial
connections between the shoot, root, and cotyledon were evident
(Figs. 17, 18). The two-ranked
suspensor was still present at the base of the embryo. Suspensor cells
were densely cytoplasmic and well formed, suggesting vitality. However,
they contained many small vacuoles and were devoid of amyloplasts. They
also lacked protein bodies, except where the suspensor joined the embryo
(Fig. 19). The seed continued to
store products in the cotyledon and endosperm (Fig. 20). Cells of the cotyledon were rich in
starch, as indicated by the pink stain, and the endosperm predominantly
contained protein, as indicated by the blue stain. Calcium oxalate
crystals were present in the cotyledon as well as at the base of the
embryo. Raphides in the Anthurium embryo appear as one or two
sets of parallel crystal arrays (Fig.
21).
Week 22
The seed has continued
to accumulate proteins and starch in the endosperm and cotyledon. Leaf
primordia showed further development and were located within the
protective sheath of the cotyledon, which encompassed the entire shoot
meristem. Shoot and root vascular connections were evident. A
suspensor, which was present in all previously viewed specimens, could
not be detected.
Week 24
The embryo was fully
mature and averaged 4 mm in length (Fig.
6). Leaf primordia were larger and tracheary elements were
present (Fig. 22). The cotyledon
contained starch grains with some protein bodies, while the endosperm
contained a mixture of starch and proteins in seemingly equal amounts.
The cells of the endosperm and cotyledon contained reduced amounts of
starch grains and proteins due to their probable utilization by the
embryo.
Ovule culture
The overall
frequency of morphogenic responses by ovules cultured in light or
darkness was the same, and 100% of the explants responded by week
20 (Table 1). Normal embryo
germination (Fig. 23) occurred in the
dark for ovules of all ages, with the frequency of germination being low
for young explants, until week 14. Germination was comparable to that
of mature seeds and led to the formation of whole plants. Frequency of
germination increased rapidly for older explants, from 44.4 to
100% between weeks 14 and 20. Light promoted callus formation in
addition to normal embryo ontogeny for young ovules aged 8–12
weeks postpollination. Callus was evident after prolonged culture (10
wk) and was firm and white. It appeared to arise from the outermost
layer of the ovule or from within the ovule, possibly from the embryo.
In all cases, the callus did not differentiate into plantlets or other
organized structures. Older ovules (from week 14 on) germinated
normally in the light, except for two (10%) at week 16 that
formed two shoots per ovule. These extra shoots appeared to have formed
without an intervening callus phase.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND
METHODSRESULTSDISCUSSIONREFERENCES |
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At 14 wk postpollination, the embryo had a clearly defined protoderm and shoot apex. This type of ontogeny in which the shoot apex is formed lateral to the cotyledon is characteristic of monocotyledonous embryos (Fahn, 1990). The formation of the lateral meristem by the embryos was correlated with major changes in physiology and morphogenic competence. At this time the embryo had begun to derive nutrition from the endosperm. Furthermore, there was a significant increase in the ability of cultured ovules to germinate.
Development of the embryo from 14 to 16 wk postpollination was extrememly rapid. Samples harvested at 15 wk postpollination closely resembled samples harvested at 16 wk, and an intermediate step between the two stages could not be obtained.
Starting at 16 wk postpollination, the emphasis in seed development was the accumulation of storage products in both the cotyledon and endosperm. Storage in the cotyledon was predominantly in the form of starch, while storage in the endosperm consisted of both starch and protein. After week 16, the embryo may be considered fully mature and capable of germination. It is at this stage that the cotyledons of the A. andraeanum embryos cultured under light were highly morphogenic and able to form somatic embryo-like structures. This is analogous to the highly regenerative scutellum from immature embryos of grasses such as maize and sorghum (Lorz et al., 1986). All embryos and ovules were capable of germination in vitro after week 20.
Protein and starch accumulation were greatest at week 20. In subsequent stages the cells within the cotyledon and endosperm appear more vacuolated. At week 22 the suspensor was no longer present and may have broken down after the embryo reached maturity or it may have been crushed against the seed coat after the embryo reached full maturity. Morphologically at this stage the spadices begin to distort and the berries start to swell, and by week 24 some of the berries are physically separated from the axis of the spadices. Histochemical staining of the cells of the cotyledon and endosperm indicates change from starch and protein accumulation to utilization, implicating the function of the suspensor in nutrient absorption.
In the fully matured Anthurium seed, the endosperm persists, unlike most other hermaphroditic genera in the Araceae (Grayum, 1991). The embryo was completely green while enclosed in the seed; and foliage leaves are fully formed in the shoot apex. Histochemical staining of the endosperm and embryo with PAS and naphthol-blue-black indicated mostly protein with some starch in the former, with starch predominant in the latter. Staining of fresh freehand sections with Sudan III did not show any significant amounts of lipids in the endosperm or embryo (T. Matsumoto, unpublished data). While Campbell (1905) reported rare occurrence of calcium oxalate in the endosperm of A. scandens, we did not observe any calcium oxalate in the endosperm of A. andraeanum sections. Calcium oxalate crystals were present in the form of raphides in the cotyledon of the embryo as well as the inner integument of the ovule.
This investigation provides a time frame for embryogenesis and a general overview with emphasis on key structures in embryogenesis. A combination of this study with the procedure for the fixation of mucilage (Matsumoto, Kuehnle, and Webb, 1995), which allows unobstructed observations of early stages of embryogenesis within the carpel locule, should provide the basis for further embryological work for taxonomic purposes.
From our observations, ovules or embryos at least 14–16 wk after pollination are highly suitable for ovule or embryo culture in vitro. Explants grown under light formed one to few plants. Explants grown under complete darkness produced multiple shoots or somatic embryos. This may be used as an embryo rescue method for important crosses with embryo and endosperm incompatibility. Somatic embryogenesis of Anthurium has been reported in spadix fragments (Geier, 1982) and from in vitro grown lamina (Kuehnle, Chen, and Sugii, 1992). Similarly to other species, histological sections reveal that somatic embryos closely resemble their zygotic embryo counterparts (Geier, 1982; Matsumoto, Webb, and Kuehnle, 1996). Finally, this paper documents zygotic embryos as an additional explant material for generating adventitious embryoids.
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND
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Campbell, D. H.1905Studies on Araceae. III. Annals of Botany 19:329 –349.
Croat, T. B.1990A comparison of aroid classification systems. Aroideana 13:44 –63.
———.1992Species diversity of Araceae in Colombia: a preliminary survey. Annals of the Missouri Botanical Garden79 :17 –28.
Engler, A.1905Pothoideae. In A. Engler, (ed). Das Pflanzenreich 21 :1 –330.
Fahn, A.1990Plant anatomy, 4th ed. Pergamon Press, New York, NY. 476–479.
Feder, N., and T. P. O'Brien.1968Plant microtechnique: some principles and new methods. American Journal of Botany 55 :123 –142.
Flinn, B. S., D. R. Roberts, D. T. Webb, and B. S. Sutton.1991Storage protein changes during zygotic embryogenesis in interior spruce. Tree Physiology 8:71 –81.
Geier, T.1982Morphogenesis and plant regeneration from spadix fragments of Anthurium scherzerianum cultivated in vitro. In A. Fujiwara [ed.], Proceeding of the Fifth International Congress Plant Tissue and Cell Culture, 137–138. Japanese Association for Plant Tissue Culture, Japan.
Grayum, M. H.1991Systematic embryology of the Araceae. Botanical Review 57:167 –203
Johansen, D. A.1950Plant embryology: embryogeny of the spermatophyta. Chronica Botanica Company, Waltham, MA.
Karnovsky, M. J.1965A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. Journal of Cell Biology 27 :137A .
Kuehnle, A. R., F. C. Chen, and N. C. Sugii.1992Somatic embryogenesis and plant regeneration in Anthurium andraeanum hybrids. Plant Cell Reports 11:438 –442.
Kunisaki, J. T.1980In vitro propagation of Anthurium andreanum Lind. HortScience15 :508 –509
Lorz, H., P. Brown, E. Goebel, B. Junker, R. Luhrs, A. Stolarz, and J. Zimny.1986In vitro manipulation of cereal crops. In J. Smeal [ed.], Somaclonal variation and crop improvement, 117–126. Martinus Nijhoff Publishers, Dordrecht, The Netherlands.
Matsumoto, T. K., A. R. Kuehnle, and D. T. Webb.1995Improvements for observing Anthurium (Araceae) floral structures by light microscopy. Biotechnic and Histochemistry 70 :205 –210.
———, D. T. Webb, and A. R. Kuehnle.1996Histology and origin of somatic embryo derived from Anthurium andraeanum Linden ex. André lamina. Journal of the American Society for Horticultural Science 121:404 –407.
Murashige, T., and F. Skoog.1962A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum 15:473 –497
Raghavan, V.1976Experimental embryogenesis in vascular plants. Academic Press, New York, NY.
———.1986Embryogenesis in angiosperms: a developmental and experimental study. Cambridge Press, New York, NY.
Redenbaugh, K., B. D. Paasch, J. W. Nichol, M. E. Kossler, P. R. Viss, and K. A. Walker.1986Somatic seeds: encapsulation of asexual plant embryos. Bio/technology4 :787 –791.
Roberts, D. R., B. S. Flinn, D. T. Webb, F. B. Webster, and B. S. Sutton.1990Abscisic acid and indole-3-butyric acid regulation of maturation and accumulation of storage proteins in somatic embryos of interior spruce. Physiologia Plantarum 78:355 –360.
Sheffer, R. D., and H. Kamemoto.1976Cross compatibility in the genus Anthurium. Journal of the American Society for Horticultural Science. 101: 709–713.
Yeung, E. C. and D. W. Meinke.1993Embryogenesis in angiosperms: development of the suspensor. Plant Cell. 5: 1371–1381.
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