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Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele Vieţii Vol. 25 issue 2, 2015, pp.65-71

© 2015 Vasile Goldis University Press (www.studiauniversitatis.ro)

Correspondence: * Constantin Craciun, Ph. D., Babes-Bolyai University Cluj-Napoca, Electron Microscopy Center, 5-7 Clinicilor Street, Cluj-Napoca 400006, Romania, Phone: +40(0)262722, e-mail: [email protected] Article published: May 2015

ANATOMICAL, MORPHOLOGICAL AND CYTOLOGICAL

COMPARATIVE STUDY OF LEAVES AND COTYLEDONS FROM

FORESTRY SPECIES

II. COMPARISON BETWEEN THE MORPHO-ANATOMICAL AND

CYTOLOGICAL STRUCTURES OF COTYLEDONS AND LEAVES OF

ROBINIA PSEUDOACACIA L. Liviu BURESCU

1, Dorina CACHIŢA

2, Constantin CRACIUN

3)*

1University of Oradea, Faculty of Sciences;

2„V. Goldiş” University from Arad, Faculty of Sciences, Engeniering and Informatics;

3„Babeş-Bolyai” University from Cluj-Napoca, Electron Microscopy Center;

ABSTRACT: The role of cotyledons in seed physiology depends on the species it is experimented with. The black locust (Robinia pseudoacacia L) has an embryo with two epigeal cotyledons that, after the plant rises, turn green and their metabolism switches from a heterotrophic regime to an autotrophic photosynthetic process. The cotyledons supply the embryo with water, nutrients and energy that helps the plant to break the seed coat and the hypocotyl together with the cotyledons to raise above the ground. The life span of the cotyledons is approximately 40 days depending on the environmental conditions. After the black locust plantlets emerge, the epycotyl rises between the two cotyledons and produce odd pinnate leaves. They have elliptical-shaped folioles about 2.5 cm in lenght. The folioles are thinner than the cotyledons. At about 14 days after the onset of germination, the cotyledons have an average lenght of 1 cm and a thickness of about 3 - 4 mm. The foliar lamina is crossed by a main visible midvein that is well defined on their lower side. This morpho-anatomical formation is missing in cotyledons. The secondary veins are rare and thin. The folioles epidermis has stomata and the inferior one has live unicellular hairs. Between the two epidermises the foliole laminas have an assimilation mesophyll made of 5 – 7 layers of parenchyma cells. Here is also located the hypoderm whose cells contain secondary metabolites that are stained in blue with „Epoxy tissue stain”. The cotyledons have an elongated shape. The outline of the transversal sections is plano convex, the upper epidermis being planar and the lower epidermis is slightly convex. The cotyledon veins are scarce and are very fine. The cotyledons have a very well ordered tissue structure.

The upper and lower epidermis has rectangle shaped cells and both have only pre-stomata. Between the two epidermises there are 16 -20 assimilating parenchyma layers, the one right below the epidermis is of palisade type.

From a cytological point of view, the assimilating mesophyll of the cotyledons is poor in chloroplasts but both the foliary and cotyledonary mesophyll have in their vacuolar content secondary metabolites which toghether with the staining reagents, aggregate leading to corpuscular formations or electron-dense vacuolar structures.

Keywords: Robinia pseudoacacia L; leaf, cotyledons; morpho-anatomical structure.

INTRODUCTION:

The study of the cotyledons structure was generally

ignored by plant morphoanatomists because these

characteristic organs of embryos and plantlets

respectively although they play an important role in the

first weeks of life, they have a short but intense life.

In the plant kingdom, there are a diversity of

cotyledons depending on the plant species.

Taxonomically there are multicotyledonous (such as

gymnosperms), dicotyledonous (dicots) (for example

rosaceae, leguminosae, etc), monocotyledonous

(monocots) (for example gramineae) and plants that

have embryos with rudimentary cotyledons (such as

orchids and some parasitic plants).

Generally, in seed physiology, the cotyledons play

an important role in triggering the germination and in

embryo and plantlet growth. The role of the cotyledon

depends on the seed structure, if the cotyledon has

stored food reserves or if is in the endosperm

surrounding the cotyledons (for example at

gymnosperms or castor oil plant) or - at monocot plants

– the only cotyledon of the embryo is interposed

between the endosperm and the embryo.

At many dicot species, the two cotyledons of the

embryo are placed at the interface between the

hypocotyl and epicotyl.

Depending on the species, the cotyledons are

epigeal – when the hypocotyl raises above the ground-

or hypogeal when the cotyledons remain below ground.

Furthermore, there are species such as the beech or

cucumber at which the cotyledons – that are epigeal –

raise above ground turn green and became

photosynthetic. At other species such as beans, the

cotyledons will shrivel and fall shortly after raising

above the ground. Of the dicot plants, the black locust

was choosen to examine the morpho anatomical

structure of the cotyledons and to compare it with the

one the leaves of this species

Burescu L., Cachiţă D., Crăciun C.

Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele Vieţii Vol. 25, issue 1, 2015, pp. 65-71


66

At the black locust (Robinia pseudoacacia L), the

cotyledons are epigeal (Fig. 1 B), plano convex (with

the planary part orientated upwards and with the curved

part located below); they are green and about 3 - 4 mm

thick. The physiological role of the black locust

cotyledons is different from that of the albuminous

seeds because the storage parenchyma is lacking the

endosperm.

At few dicot plants – for example castor oil plant –

the cotyledons are foliaceous and are surrounded by an

endosperm that provides nutrients to the embryo. Most

of the dicot species lack endosperm and the stored

reserved substances are in the cotyledons.

After the black locust plant raises above the ground,

the cotyledons turn green and begin photosynthesis.

Thus, as in other species, the black locust cotyledons

have a complex role as long as the true leaves have not

yet emerged, situation hat involves a transformation of

their morphoanatomy. The black locust leaves are odd

pinnate (Fig. 1 A) and a foliole is more than double

compared with the cotyledon and as observed has a

different structural makeup than that of the folioles.

Fig. 1 A şi B. Morphological aspect of a odd pennate,

young black locust (Robinia pseudoacacia L) leaf (A);(B) black locust plantlet photographed on the 14

th

day of seeds germination in a plastic container on filter paper moistened with tap water (abbreviations: c (rst) – root-stem transition region; cot – cotyledons; ep – epicotyl; f – foliole; re – embryonary root; h – hypocotyl; b – budlet; R - rachis

In plant biotechnology, the explants taken from the

embryos (Cachiţă et al., 2004) and the plantlets

respectively, are valorised either as phyto – innocules

with high regenerative capacity or as experimental

models to examine their reaction as a function of

vitroculture conditions. Interesting are the

micrografting researches with the purpose of grafting

the top of the vitrocultivated plantlets when an apical

part of the hypocotyl with the pertaining cotyledons as

well as the epicotyl, formation that is used as a graft,

that is implanted in a parent stock (according to

Turnbull et al., 2002) (Fig. 2).

This operation is performed „in vitro” and there is a

good chance to obtain from the graft virus free plants

including the woody plant species at which the disease

free plants regeneration – by meristem culture

techniques – is extremely difficult.

Fig. 2 A – D. Micrografting technique of a plantlet

performed under aseptic conditions (according to Turnbull et al., 2002 –modified image) A – plantlet from

a sterile environment ( the arrow indicates the place where the plantlet was sectioned for detaching the graft); B „a” – trimming of the graft basal area and “b” splitting of the parent stock to introduce the graft; C –

placement of the graft on a parent stock ( with the apex removed and with an “infections free” certificate) D –

grafting zone (gz) can be consolidated with an autoclavable silicone sleeve (ss) (abbreviations: g – graft; ct – cotyledons; s – split; h – hypocotyls; b – budlet; pa – parent stock; Re – embryonary root; B „a” – graft; B „b” – parent stock; D „a” – fixation of the grafting area with a sterile silicone sleeve; D „b” – grafted area after the removal of the silicone sleeve)

The advantage of cotyledon-type phyto-innocules

in not only in the youthfulness of the tissues that make

up this type of explant but also in the fact that it’s cells

have nutrient reserves necessary to start and sustain the

growth processes of the embryo to which the cotyledon

pertains. This is very important mainly in the case of

vitrocultivated forestry species that can be hard to

propagate using the meristem culture technique

(Enescu et al., 1994).

In the literature (Stănescu, 1979), there are no

studies regarding the resemblances or differences

between the anatomy of the black locust cotyledons an

leaves. Therefore, the knowledge of these aspects is

important for explant prelevation in vitroculture

techniques.

MATERIALS AND METHODS: Black locust seeds were placed for germination in

plastic containers (colorless and transparent) on a filter

paper moistened with tap water, at room temperature.

The germination was carried out for 14 days at 23ºC,

the plantlets being exposed to a day/night cycle specific

for June, period in which the experiments were

performed. In the 14th

day of germination, the

cotyledons were taken from the plantlets (Fig. 1 B)

whereas the black locust folioles were take from

nature.

Anatomical, morphological and cytological comparative study of leaves and cotyledons from forestry species II. Comparison between the the morpho-anatomical and cytological structures

of cotyledons and leaves of black locust (Robinia pseudoacacia l)

Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele Vieţii Vol. 25, issue 1, 2015, pp. 65-71 © 2015 Vasile Goldis University Press (www.studiauniversitatis.ro)

67

The whole plant material was transversally

sectioned (Fig. 3) followed by fixation and processing

of the sections according to optical and transmission

electron microscopy (TEM) techniques.

The microscopic examinations were performed on

tissues that have been fixed prior to their use to obtain

semithin or ultrathin sections, using an Leica UC6

ultramicrotome.

The sections for light microscope examination were

around 500 nm (0.5 (µm) thick while the ultrathin

sections that can be examined using transmission

electron microscopy should have a thickness of about 40

- 70 nm.

Fig. 3. The procedure for obtaining tissular explants from black locust adult leaves or cotyledons (where: a and b – the way of sectioning of the folioles,

transversally along the midvein; lf – foliole lamina; mv – midvein; c – cotyledon; sec – the way of operating transversal sections through the cotyledons.

The preparation process for microscopy studies is

presented below. The tissue are first pre-fixed in 2.7%

glutaraldehyde solution prepared in 0.1 M, phosphate

buffer solution, pH 7.4, opperation followed by 4

succesive washings, 30 minutes each, with the same

buffer. The tissues are then post-fixed using a 1-2%

osmium tetroxide solution made in 0.1 M, pH 7.4,

phosphate buffer, then washed 2-3 times with the same

buffer. The samples are then dehydrated by placing them

in baths with increased acetone concentration. (50% to

100%). After that, the samples are passed through 2-3

baths containing propylene oxide solution. The tissue

samples thus prepared were placed in baths of Epon 812

(epoxy resin) mixed with anhydrous acetone with

increased resin concentration, the final bath containing

only the Epon resin. The samples impregnated with Epon

were embedded in gelatin capsules and kept at 50 - 60°C,

for 48 - 72 hours for polymerization of the resin which

will become hard and transparent. The blocks obtained

had modeled under a stereo microscope using a new

răzor blade to obtain a small „pyramid trunk" at one

end. For ultrathin sections, the top sides of the pyramid

trunk shoud be around 0.1 -0.2 mm. The sections were

obtained using a Leica UC 6 ultramicrotome equipped

with a Diatone diamond knife. The semi-thin sections

were stained with Epoxy tissue stain, a special epoxy

stain.

The sections for electron microscopy were contrasted

in the first stage with uranyl acetate solution, followed by

contrasting with lead citrate solution, technique which is

currently used in electron microscopy laboratories,

around the world (Cachiţă şi Crăciun, 1991; Hayat,

2000).

The semi-thin sections were examined by us using an

Olympus BX 51 light microscope equipped with a CCD

camera and the ultrathin sections were examined using

a JEM-JEOL TEM 1010 electron microscope. Selected

pictures were recorded using a MEGA WIEW III

camera.

The experiment described in this article was aimed to

highlight the resemblances and differences between the

leaves and cotyledons of black locust because these

organs (especially when young) can be a source of

explants in plant biotechnology.

RESULTS AND DISCUSSION: In the physiology of the plant seed and embryo

(Mayer and Poljakoff–Mayber, 1975), the cotyledons

have an important role, mainly regarding the supply of

the tissues and cells with water and nutrients.

Altohught the active life span of the cotyledons is

short, the metabolic processes at cellular level play an

essential role in supplying the embryo with water and

also energy that is produced following the metabolising

of nutrients from the storage tissues. For the embryo

this energy is necessary for the growing processes and

cellular proliferation and also for generating the force

necessary to break the seed coat and to sustain the

growth of the plantlet hipocotyl and epycotyl and their

raising above the ground.

As it results from figure 4A, the foliole of a young

black locust leaf is planary with a main vein (midvein)

along the central area.




68

Fig. 4 A - B – The structure of the foliole a young black locust (Robinia pseudoacacia L.) leaf, images observed in the optical microscope of the transversal sections through the central area of the lamina (A – ob. 40x) and in the median area of the half lamina limb (B and C – ob. 100x) (abbreviations: dv – vacuolar deposit; eps – upper epidermis; epi –

lower epidermis; h – hair; ma - assimilating mesophyll; mv – midvein; sv – secondary vein; x/pb – xylem/phloem bundle in the midvein; st – stoma).

This midvein is bulgy and prominent on the lower

side of the folioles. The two halves of the foliar lamina

are thin and from a hystological and cytological point

of view between the upper and lower epidermis are

about 6-8 layers of parenchyma cells out of wich about

5 represent the assimilating mesophyll.

Fig. 5 A – F. Hystological structure of the black locust (Robinia pseudoacacia L.) cotyledon. Optical microscopy images

of the transversal sections through such organ, taken from a 14 days (from the placing the seeds to germinate in transparent, colorless plastic containers exposed to laboratory ambient light) old plantlet (abbreviations: cit – cytoplasm; cl – chloroplasts; csst - substomatal cavity; dv – vacuolar deposit; epi – lower epidermis; eps – upper epidermis; pap – palisadic assimilating parenchyma; st – stomata; V – vacuole; where; A – ob. 40x; B – ob. 100x; C – ob. 20x; D – ob.

40x).

The epidermis cells have different shapes. The

stomata are rare and on the upper side of the epidermis

there are live unicellular hairs. Both under the upper

and lower epidermis and also on the upper epidermis,

the presence of a hypodermis tissues can be

distinguished, that have in their vacuolar content

compounds that with the reagent used for the

processing of the semithine sections turned dark blue.

This is probably due to the presence in the cells

vacuolar content of leuchoanthocyanins because the

folioles of black locust leaves are colored only in

green. The assimilating foliar mesophyll has rectangle-

shaped outline, arranged regularly similar with the

palisade parenchyma from the leaves that have such

structure.

From place to place, in the transversal sections, fine

veins can be distinguished with a xylem-phloem bundle

consisting only of a few cells specific for such tissue.

The cotyledons of black locust plantlets are green.

Their hysto-anatomical structure, in 14 days old

plantlets (germinated in laboratory under natural light)

is diffrent from that of the leaves. The cotyledons are

elongated and can reach up to 10 mm in lenght and a

with of about 5 – 7 mm. Their shape is lenticular,

plano-convex, the upper side being planary and the

lower side being slightly convex (Fig. 5 A – E).




69

It is interesting that in the cotyledons, after the

processing of the sections, there are no more cells with

vacuoles having their content colored in blue and the

epidermis cells (upper and lower epidermis) are very

ordered and lack hairs. The epidermises have many

stomata that have a substomatal cavity or, some

stomata are not fully developed and were considered

pre-stomata.

From the hystoanatomical conformation of black

locust cotyledons, the hypodermis is missing; instead

under the epidermis, all around, is a palisade

assimilation parenchyma very well organized made of

3 – 4 layers of elongated successive parenchyma cells

(Fig. 5 A – E)

That have small chloroplasts and big vacuoles.

The cotyledonary mesophyll has approximately 14

– 16 layers of well ordered assimilating parenchyma,

the first 3 – 4 layers from the immediate vicinity of the

epidermises being of palisate type. The gaps and

intercellular spaces are not very large (Fig. 5 E – F).

From place to place, in the foliar mesophyll, small

xylem-phloem veins can be distiguished, some of them

being placed towards the cotyledonary apex (Fig. 5 A) .

Regarding the ultrastructural aspects that can be

distinguished in the lamina of the folioles (Fig. 6 A –

F), it can be stated that during the processing of the

ultrathine preparations, for examination, the vacuolar

content of many cells have suffered a corpusculation

process, in the vacuoles appearing spherical formations

of different sizes that are electron-dense and confirm

the presence in the vacuole of either anthocyanins or

phospholipids. These formations were named vacuolar

deposits. In the cells cytoplasm, normally structured

chloroplasts can be seen (Fig. 6 A – D) and also some

large nuclei in which a very well represented nucleolus

can be observed (Fig. 6 F).

Fig. 6 A –F. Cytological aspects observed in the epidermis and foliar mesophyll of young black locust (Robinia pseudoacacia L.) from the ultrathin transversal sections examined with the transmission electron microscope (A and B – upper epidermis and the first two mesophyll cells layers; C – F – lower epidermis and the neighboring mesophyll cells layers; D – F – details of the aspects preserved in the central foliar mesophyll) (abbreviations: cl – chloroplasts; cit –

cytoplasm; dv – vacuolar deposits; eps – upper epidermis; epi – lower epidermis; h – hair; cw- cellular wall; N- nucleus; n – nucleolus; V – vacuole).

The epidermis of the folioles, on the lower side has

short unicellular live hairs whereas is lacking hairs.

Also, the epidermis of the folioles has a large number

of well structured stomata whereas the one of the

cotyledons has some undifferentiated stomata (pre-

stomata). Besides, depending on the environmental

conditions after about 3 – 4 weeks from the onset of the

germination, the cotyledons start to turn yellow and

finally fall whereas the leaves folioles remain active

until autumn.




70

Fig. 7 A – D. Transmision electron micrographs of the

transversal sections through the cotyledons of black locust (Robinia pseudoacacia L.) plantlets. The plantlets were 14 days old from the placement of the seeds to germinate in a colorless, transparent plastic container on a filter paper moistened with tap water and exposed

to the laboratory ambient light; A – the aspect of the

epidermis and first layer of the assimilating parenchyma; B – D – details regarding the structure of

the cells that make up the assimilating parenchyma or cotyledonary mesophyll (abbreviations: cit – citoplasm; dv – electron dense vacuolar deposits; eps – epidermis; G – gap; N – nucleus; cw – cellular wall; V – vacuole).

On a ultrastructural level (Fig. 7 şi 8), the

transversal sections through cotyledons have pointed

the presence in the cells of some cytoplasmic masses,

well represented, but poor in chloroplasts having

instead a relatively large nucleus and nucleolus (Fig. 8

E – F). Such structure suggests a rich metabolic

activity.

In the ultrathin sections performed in the central

area of the cotyledons, in the cotyledonary mesophyll,

the existence of some cell with vacuoles rich in

electron-dense deposits can be distinguished; further

away from this central area, the mesophyll cells close

to the epidermis have in their vacuoles numerous small

corpuscular deposits.

Fig. 8. A – F. Various cytological aspects regarding the structure of the cotyledons of 14 days old black locust (Robinia pseudoacacia L.) plantlets (14 days from the placing of seeds to germinate in colorless, transparent plastic containers, exposed to natural light), images obtained with the transmission electron microscope (abbreviations: cit – cytoplasm; cl – chloroplast; dv – vacuolar deposits; N – nucleus; n – nucleolus; cw- cellular wall; V – vacuole).




71

ACKNOWLEDGEMENTS: This work was supported by the strategic

grant POSDRU/CPP107/DMI1.5/S/80272, Project

“Doctoral and Post-doctoral programs of excellence

for highly qualified human resources training for

research in the field of Life sciences, Environment and

Earth Science” cofinanced by the European Social

Found within the Sectorial Operational Program

Human Resources Development 2007 –

2013. University from Oradea.

CONCLUSIONS:

The folioles of the black locust (Robinia

pseudoacacia L.) leaves are different from the plantlets

cotyledons. This difference comes from both an

morphoanatomical and physiological point of view as

well as ultrastructural.

Because the black locust is a dicot species with

epigeal cotyledons, as the cotyledons raise above the

ground, they turn green and apparently for a short

period of time (about 40 days from placing the seeds to

germinate) they photosynthesize. As the cells of the

cotyledonary mesophyll exhaust their stored reserve

substances, they move from a heterotophic to a

autotrophic nutrition and the energy resulted from the

catabolic metabolic processes serves for the growth of

the embryo.

The folioles of the black locust leaves are odd

pennate have an elipsoidal shape are flat, thin and

about 1.5 – 2.5 cm in lenght. Centrally, across them

thay have a prominent midvein on the back of the

folioles.

The cotyledons of the black locust embryos are

about 10 cm in lenght, plano-convex with a planar

upper side and a convex lower side.

From an anatomical point of view, the folioles –

between the upper and lower epidermis – have a foliar

mesophyll composed of 4 – 5 layers of assimilating

parenchyma with a palisade type structure. The

cotyledonary mesophyll has 16 – 26 layers of cells. In

cotyledons, the cells in the immediate vicinity of the

epidermises have a classical palisade tissue structure,

highly ordered with small intercellular spaces.

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