Juglans regia L

Seria: Biologie
UNIVERSITATEA DIN CRAIOVA
Horticultură
UNIVERSITY OF CRAIOVA
Tehnologia prelucrării
produselor agricole
Ingineria mediului
Vol. XV ( XLXI ) - 2010
EMBRIOLOGIC AND CYTOLOGIC STUDIES ON FRUIT SETTING IN WALNUT
TREE (Juglans regia L.)
KEY WORDS: walnut, apomixis, fruit, structure
ABSTRACT
Cosmulescu Sina 1 , Simeanu C. 1 , Achim Gh. 2
Apomixis is an important characteristic of walnut. Fruit-setting through apomixis presents
theoretical and practical importance; the embryo is homozygous and it loyally transmits the
characters of mother plant. Research on apomictic formation of the embryo in walnut tree have been
carried out in several countries; the mechanism of apomixis in walnut has been reported as
adventitious embryony, apospory or diplospory. The paper aims at following-up the embryo
development throughout its different stages, cell structure and development in walnut (fruits obtained
by apomixis and by free pollination).
INTRODUCTION
Apomixis is an important feature of walnut, particularly in the northern cultivation
areas (Loiko, 1990). Research carried out in walnut on fruit-setting have shown that some
cultivars can generate fruits even without fecundation, through parthenocarpy or apomixis;
but it was found out that fruit-setting is feeble, almost zero in some cases, while apomictic
fruit-setting cannot be expected (Zhang et al., 2000;. Mu et al., 2001; Şan and Dumanoğlu,
2006; Guoliang et al., 2007, 2010). Rate of apomictic fruits differs from year to year and
depends on climate and variety (Asadian et al., 2005).
Possibility of fruit-setting without pollination in walnut, under usual conditions,
has raised special interest to researchers on flowering biology and seed formation. Sartorius
and Stösser (1997) made the investigations aimed to explain the development of apomictic
embryos by histological methods. Badalov (1989) also reported that apomictic embryos
have developed mainly from the egg cell with subsequent doubling of chromosome
numbers, leading to homozygosity. The mechanism of apomixis in walnut has been
reported as adventitious embryony (Valdiviesso, 1990), apospory (Terziiski and Stefanova,
1990), or diplospory (Sartorius and Stosser, 1991; Pintea, 2004). Ultrastructure of the
fleshy pericarp and seed coat cells was systematically investigated by using transmission
electron microscopy, and this study makes a significant contribution to our understanding
of ultracellular events in developing walnut fruit (Wu et al., 2009). In Romania there were
1 University of Craiova, Faculty of Horticulture, Al.I. Cuza street, no.13, 200585, Craiova
2 University of Craiova, SCDP Vâlcea, Calea lui Traian Street, no. 464, 240273, Rm. Vâlcea
* Corresponding author: sinacosmulescu@hotmail.com
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not found out any cultivars or hybrids to possess valuable characteristic of apomixis (Cociu
et al., 2003).
The aim of this study was to determine embryo development thougout different
stages, structure and development of cells in walnut (fruits obtained by apomixis and by
free pollination).
MATERIALS AND METHODS
Cultivars were used by including all types of dichogamy: protandry, protoginy,
homogamy. To obtain apomictic fruits, flowers isolation with pergamin paper pocket was
carried out before stigmata bifurcation; and pockets opening was carried out when stigmata
were fully dried (Cociu et al. 1989). For cross sections of apomictic fruit and normally
developed fruit, fruits were collected (Figure 1-3), then fixated in FAA fixator (80%
ethanol: 10% glacial acetic acid: 10% formol) and then kept in the refrigerator. Cross and
longitudinal sections were visualized by microscope and photographed.
Figure 1-3: Fruits obtained by free pollination and apomictic fruits
RESULTS AND DISCUSSIONS
From cross sections made in apomictic fruits and freely polinated fruits, the
folowing facts were found out:
In 'Jupâneşti' cultivar (Figure 4), the cross section made through endocarp and seed
-a fruit that is obtained by free pollination- showed that the unilayered skin lies at the
exterior, equipped with a thick cuticle. Epicarp is made of 4-5 layers of cells that have
colenchimatized walls. From place to place, at the limit between epicarp and mesocarp
there are isolated cells or groups of large tanniniferous cells. Mesocarp is well developed; it
is made of large spheroidal or ovoidal cells that contain numerous chloroplasts. In the
mesocarp there are many conducive wood-free fascicles that are disorderly arranged and
getting to the endocarp. Endocarp is made of small, parenchymatic cells that are
tangentially elongated. There is no sclerification process in cells; the seed has thin
tegument, made of 3-4 layers of small, parenchymatic cells that are slightly tangentially
elongated. For the rest, the seed is made of spheroidal or ovoidal cells that are slightly
larger than those in the tegument, but they do not have reserve substance at the interior.
These cells have small intercellular spaces.
In apomictic fruit (Figure 5), 'Jupâneşti' cultivar, epidermis is unilayered and
equipped with thick cuticle. Tanniniferous cells go inside the mesocarp. Epicarp is made of
2-3 layers of cells with slightly colenchimatized walls, and underneath there are numerous
tanniniferous cells, some of them large and radially elongated that are goign inside the
mesocarp. Mesocarp is made of spheroidal and ovoidal cells, with small spaces between
them, and numerous chloroplasts at the interior. In the mesocarp there are conducive woodfree
cells that are smaller than in pollinated form; and they are located somehow orderly on
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two circles; one is located under the epicarp, while the other is located at the exterior of
endocarp; and in other areas they are disorderly arranged. Endocarp is made of 2-3 layers of
cells with thin walls, slightly elongated tangentially. There is no sclerification process
observed in cell walls. Seminal tegument cannot be seen in the seed; the cells located
underneath the endocarp -that belong to the seed- do not contain any stored reserve
substance at the interior.
Figure 5: 'Jupâneşti'
cultivar (apomictic fruit),
cross section made through
endocarp and seed (40x10)
Figure 4: 'Jupâneşti'
cultivar (pollinated fruit),
cross section made through
endocarp and seed (40x10)
Figure 7: General view – in
'Jupâneşti' cultivar
(apomictic fruit) (4x10)
Figure 6: General viewepicarp,
mesocarp,
endocarp and seed in
'Jupâneşti' cultivar (4x10)
Figure 8: Tanniniferous
cells, in 'Jupâneşti' cultivar
(apomictic fruit) (40x10)
Figure 9: Epidermis,
epicarp, mesocarp in
'Fernet' cultivar (apomictic
fruit) (40x10)
General view (Figure 6) – epicarp, mesocarp, endocarp and seed in 'Jupâneşti'
cultivar; fruit is obtained by natural pollination; and general view (Figure 7) – epicarp,
mesocarp, endocarp and seed in 'Jupâneşti' cultivar, with apomictic fruit. In apomictic fruit
there are tanniniferous cells; they are radially elongated, of spherodial and ovoidal shape,
which go deeply into mesocarp (Figure 8).
In 'Fernet' cultivar, the cross section through epidermis, epicarp and part of
mesocarp through apomictic fruit (Figure 9), has uni-layered epidermis equipped with very
thin cuticle, and numerous pluricellular hairs that are elongated and club-shaped. The fruit
is during the period right after fecundation (black-coloured stigmata). During this stage of
development, the epicarp is poorly differentiated, made of 4-5 layers of cells with thin
walls; the cells are slightly elongated tangentially. Mesocarp is made of spheroidal and
ovoidal cells, without chloroplasts at the interior. Conducive fascicles are under
differentiation stage, and they are disorderly arranged. Endocarp is not differentiated and
one cannot see the difference between mesocarp, endocarp and seed. In some areas the
internal epidermis of the ovar can be observed.
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In conclusion, embriologic and cytologic studies are outlining the different stages
in the embryo’s development. Further precise studies are required to outline morphological
differences between apomictic and pollinated fruits.
ACKNOWLEDGEMENTS
This paper is carried out as a result of the research contract CNCSIS –
UEFISCSU, the project: PNII – IDEI code 430 /2008.
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