1937 1033

On the Fertilization of Nelumbo nucifera

ByIchiro Ohga

(With One Plate)

Early in 1904 York studied the American species, Nelumbo lutea

and described its fertilization, floral development and embryo forma

tion. While studying the Japanese species, N. nucifera we have had

an opportunity to investigate the pollination and fertilization of

this plant for the past few years. In the present paper some results

of the experimental and cytological observations carried out during

last summer will be presented.

Material was collected from a Chinese variety grown and

cultivated in the lotus field at Kisarazu, Tiba-prefecture. The ovaries

at various developmental stages, their lower half being removed, were

fixed at intervals of half an hour from 6 a.m. to 6 p.m., August 20 to

23, 1936, in either Bouin's fixative or Navashin's solution. The latter

gave better results than the former. The material was passed

through the alcohols as usual and imbedded in paraffin. Sections cut

from 10 to 15ƒÊ in thickness were stained with Heidenhain's iron

alum haematoxylin.

On the first day of flowering and on the morning of the second

day, the embryo-sac already comes to maturity, in which state an

egg and one fused or two as yet unfused polar nuclei are noticeable

in their fine spireme stages (Plate 39, Figs. 1-4). The egg is

spherical and near its central region one nucleus is located (Plate 39,

Fig. 4). The synergids are found almost always to have degenerated.

The polar nuclei are situated a short distance below the egg cell. In

some embryo-sacs the two polar nuclei are still a little apart; while

in other cases they are beginning to fuse. Most commonly these

nuclei are found in close contact with each other, though in some

cases they have already partially fused (Plate 39, Figs. 1-4).

On the early morning of the second flowering day, pollen grains

reach the stigma by the aid of small insects and there begin to

germinate. Only a few of the pollen tubes thus developed are able

to attain the ovary through a short style having a length of about

1mm. The pollen tube enters the embryo-sac directly through the

micropyle and penetrates five or six layers of the nucellus tissue, the

apex attacking the lateral side of the egg (Text-fig. 1).

It was proved in the present case of N. nucifera that the pollen

tube penetrates the style and reaches the embryo-sac during a period

1034 I. OHGA Cytologia, Fujii jub. vol.

between about 6-8 hours after pollination occurred at about 6 to 7 a.m. on the second day of flowering; and that the fertilization follows soon after. In Text-fig. 2 two densely stained male nuclei which became free from the pollen tube are found in the micropylar

portion of the embryo-sac. They are smaller than both egg nucleus and polar nuclei and retain their ellipsoidal form during fusion (Text-figs. 3, 4). One of the male nuclei fuses with the large polar-fusion nucleus (Text-fig. 4), while the other fuses with the egg nucleus at about the same time (Text-fig. 3).

The fusion of the three nuclei is not carried out in a uniform way in different

plants or even in the same plant under varying conditions. A male nucleus and two polars fuse simultaneously in

Text-fig. 1. Pollen tube attacking

the egg. e, egg nucleus; s,

sperm nucleus. •~1300.

Potamogeton lucens (Cook, 1908) and in Nelumbo lutea (York, 1904), while in Castalia odorata (Cook, 1902) and Nymphaea advena (Cook, 1902, Seaton, 1908) the second male nucleus is added to the polarfusion nucleus as in the present case of N. nucifera.

The primary endo

sperm nucleus under

goes division immediately after its formation (Plate 39, Figs. 5, 6). The axes of the first and second cleavage figures of the fertilized egg are nearly perpendicular to the transverse axis of the embryo-sac (Plate 39, Figs. 7, 8) resulting in a linear three-celled embryo, as described by Cook (1902) in Castalia and Nym

phaea.In concluding, the

pollination in N. nucifera occurs with the aid of small insects

about one or two hours after the opening of the flower which

Text-figs. 2-4. 2, micropylar portion of the embryo-sac,

a pollen tube entering. 3, fusion of female and male

nuclei. 4, fusion of a sperm nucleus with a large

polar-fusion nucleus (p). e, egg nucleus. s, sperm

nucleus. •~2000.

Cytologia, Fujii jub. vol., 1937 Plate 39

Ohga: On the Fertilization of Nelumbo nucifera

1937 On the fertilization of Nelumbo nucifera 1035

takes place usually at about 5 a.m. on the second day of flower

ing. Fertilization is accomplished within about 6 to 8 hours after

pollination and is followed immediately by division of the primary

endosperm nucleus. According to Schnarp's compilation (1929),

Phaseolus vulgaris and Secale cereale are good examples in showing

the shortest time intervals from pollination to fertilization; in the

former 8-9 hours (Weinstein, 1922) and in the latter 7 hours (Jost,

1907), both obtained under green house conditions. The present

case of N. nucifera seems to add one more example to these, which

example however has been obtained under natural conditions.

The writer wishes to thank Mr. Kazuo Suzuki of the Botanical

Institute, Faculty of Agriculture, Tokyo Imperial University, who so

gladly offered assistance in preparing materials and photomicro

graphs.

Botanical Institute, Faculty of

Agriculture, Tokyo Imp. University

Literatures Cited

Cook, M. T. 1902. Development of the embryo-sac and embryos of Castalia odorata

and Nymphaea advena. Bull. Torr. Bot. Club, 29: 211-220.

- 1908. The development of the embryo-sac and embryo of Potamogeton listens. ib.,

35: 209-218.

Jost, L. 1907. Uber die Selbststerilitat einiger Bldten. Bot. Zeit., 65: 77-116.

Schnarf, K. 1929. Embryologie der Angiospermen. Berlin.

Schurhoff, P. N. 1926. Die Zytologie der Blutenpfianzen. Stuttgart.

Seaton, S. 1908. The development of the embryo-sac of Nymphaea advena. Bull.

Torr. hot. Club, 35: 283-290.

Weinstein, A. I. 1926. Cytological studies on Phaseolus vulgaris. Amer. Journ. But.

13: 248-263.

York, H. H. 1904. The embryo-sac and embryo of Nelumbo. Ohio Naturalist, 4:

167-176.

Explanation of Plate 39

All photomicrographs are made at a magnification of 2100 diameters except Fig. 9

(•~ca. 210) and reduced 2/3 in reproduction.

Figs. 1-3. Successive stages of fusion of two polar nuclei.

Fig. 4. Micropylar end of a matured embryo-sac showing an egg (upper) and a

polar-fusion nucleus (lower).

Fig. 5. An egg and a primary endosperm nucleus after double fertilization ac

complished.

Fig. 6. Anaphase of the first mitosis of the endosperm nucleus.

Fig. 7. Telophase of the same. Two celled embryo formed.

Fig. 8. Partition wall forming between two daughter embryo-sac nuclei.

Fig. 9. Showing an embryo of two cell stage and two celled embryo-sac.