Histological study on the stages of pollination and

fertilization in the cul tivars of red seedless and

ghezel-ozum grapes

Mahdi Mohammadi

*, Mohammad-Reza Dadpoor

, Mahboobe Aliasgharpoor

Elham Mohajel-Kazemi

, Hamed Dolati-Baneh

and Jaber Panahandeh

Pomology, Department of Horticultural Science, Faculty of Agricultural Sciences, University of Tabriz,

Tabriz, Iran

Plant Sciences, Faculty of Life Sciences, University of Tabriz, Tabriz, Iran

Agricultural Research Centre at Seed and Plant Improvement, Kahriz, Urmia, Iran

Olericulture, Faculty of Agricultural Sciences, University of Tabriz, Tabriz, Iran

ABSTRACT

The Vitaceae is one of the most important plant families,which it includes the Vitis genus with all its economic

characteristics. In the meantime, the European seedless grapes for their high-quality fruitshave popularity. However,

in the breeding works, the progenies from the seedless cultivars have a low frequency. In Sultana cultivar (known as

Thompson seedless in the United States) which is the most important seedless cultivar, abortion of the embryo after

pollination and fertilization has been mentioned as a reason for being seedless. Investigating the structure of the

seed and determining the time of abortion can be important in terms of tissue culture, embryo rescue and biology.

In this research, the comparative study of the seed development of the red seedless grape with Ghezel-Ozum seeded

cultivars considered using the histological techniques. Samples were harvested from pre-pollination till seed matu-

ration every  ve days from the Research Station of Kahriz, Urmia and  xed in the FAA  xator. The  xed samples

were immersed in the paraf n and then were cut with a microtome. After staining with PAS-Hematoxilen, samples

were studied with the light microscope and photography. Observations showed that the growth and development of

zygote and endosperm were delayed and eventually stopped in the red seedless cultivar after the double fertilization.

KEY WORDS: ABORTION; DOUBLE FERTILIZATION; ENDOSPERM; POLLINATION; SEEDLESS GRAPES; SEEDED GRAPES

306

ARTICLE INFORMATION:

*Corresponding Author: MahdiMohamadi1982@yahoo.com

Received 27

Dec, 2016

Accepted after revision 2

March, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2017: 4.31 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

Biosci. Biotech. Res. Comm. Special Issue No 1:306-317 (2017)

Mahdi Mohammadi et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS HISTOLOGICAL STUDY ON THE STAGES OF POLLINATION AND FERTILIZATION 307

INTRODUCTION

The Vitaceae is one of the most important plant families,

which it includes the Vitis genus with all its economic

characteristics. Furthermore, this family has about 15

genera with 900 species (Zhang et al. 2015). The Vitis

genus has a special importance in horticulture. Itis

divided into two sub-genera of Euvitis and Muscadine.

The commercial cultivars are related to the  rst sub-

genus. Their  owers have 5 sepals, 5 petals, 5 stamens

linked to the petals and alternating with the disc-shaped

edges and the pistil with di-carpel ovary, in which each

ovary there are the two standing ovules (Ghahraman

1993; Ghanadha et al. 2004).

The different species of the sub-genus Euvitis (2n=38)

have very little differencesin terms of the chromosomal

structure, and their intercourse with each other is pos-

sible. Therefore, only the geographical boundaries and

ecological barriers have separated them from each other

and created the European, American and Asian varieties

(Ghahraman 1993; Ghanadha et al. 2004).

The European grapes due to having high-quality fruit

as the commercial varieties are popular, and it has been

found that polyphenolic compounds existing in those

which include the  avonoid compounds are useful in

preventing the heart diseases and cancer in human (Kalt

2001; Lepiniec et al. 2006). The American grapes are also

used as the stock due to resisting pests and diseases. The

varieties that are resistant to cold and frost can also be

found in the Asian grapes (Ghahraman 1993; Ghanadha

et al. 2004).

The origin of the European grapes is the area between

the Caspian Sea to the Black Sea and from there to the

Mediterranean area and Europe has expanded (Wen

2007). Although, grapevines are the dioecious plants,

but following selective breeding done by man that has

continued for many years, the male stocks have been

removed from the European grapes and now the female

stocks and self-pollinated hermaphrodite are cultivated.

Since being seedless is desirable characteristic in many

fruits for table utilizations, in the grape has also paid

special attention to this feature. However, the seed-

less varieties and their progenies have a low frequency.

Perhaps the most main factor is dif culties in making

hybrid and producing seed in these varieties (Bharathy

et al. 2005; Liu et al. 2003; Ramming et al. 1991; Sharma

et al. 1996; Yang et al. 2007).

In Sultana cultivar which is the most important seed-

less cultivar, being seedless is the result of digesting the

embryo after pollination and fertilization. Ebadi et al.

(2010) demonstrated that the high frequency of abnor-

mal ovules and single fertilization can be considered as

two other reasons of seedlessness. The remainders of

undeveloped ovule inside fruitcan be seen until its har-

vest stage. However, due to the small size of these imma-

ture seeds, it does not feel when eating fruit. Although

this feature is very favorable in terms of production,

but in terms of eugenic objectivesand obtaining the

reproductive progenies would be problematic (Bharathy

et al. 2005; Farsi &Bagheri 2004; Farsi &ZolAli 2006; Liu

et al. 2003; Wakana et al. 2002; Yang et al. 2007).

Knowing how to digest the ovule and its morphologi-

cal disorders and the appearance time of this phenom-

enon is very important and necessary. Perhaps only by

this way, the separating of embryo and timely rescue it

can be performed (Bharathy et al. 2005; Liu et al. 2003;

Yang et al. 2007).

Therefore, the comparative study of the development

of the ovule, the formation of embryo sac and seed in the

seedless and seeded cultivars of grapes not only explain

the structural and morphological differences between

them, but it can also use to identify and utilize the

appropriate methods to prevent the hollowness appear-

ance of the seed (in breeding purposes) (Pratt 1971).

More processes of the development of ovule and the

appearance of embryo occur at the microscopic level

thereforethe use of appropriate histological methods for

the structural studying of seedless in the grapes will be

inevitable. However, preparing the microscopic samples

of the perennial trees has speci c problems which the

hardness of tissuesas well as tannins and other phenolic

compounds in them can be noted, and their combina-

tion with chemical materials used in the  xator prevents

the optimal  xation of the tissues. Therefore, cellular

and histological studies on tree species much less than

herbaceous plants, and vines are not also an exception.

In the annual and biennial plants such problems are less

common (Ruzin 1999).

The purpose of this study was a documentation of his-

tological information in vitis geneus with a comparative

study of the seed development of the red seedless grape

with Ghezel-Ozum seeded cultivar to exact determine

the time of embryo abortion. This study can be useful in

studies and practices of tissue culture and embryo rescue.

Because, to know an appropriate time of exiting seeds can

be more successful in tissue culture and embryo rescue.

MATERIALS AND METHODS

PLANT MATERIAL AND SAMPLING

Samples ( ower buds,  owers and fruits during sam-

pling) were collected from Seed and Plant Improvement

Research Institute, Kahriz, Urmia, Iranin Jun to August

2007. Samples were collected from 10 days before and

40 days after the loss of cap (with a 7-day intervals)

(one in orescence per branch, and 10 branches per plant

and 20 plants in the total of the experiment for each

Mahdi Mohammadi et al.

308 HISTOLOGICAL STUDY ON THE STAGES OF POLLINATION AND FERTILIZATION BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Table 1. The dates of sampling of the Ghezel-Ozum cultivar

Date Developmentstage Features

2008/05/28 The stage of pollination The peak stage of anthesis

The length of ovule: 1-1.5 mm

2008/06/02 5 days after pollination The length of ovule: 2-3 mm

The length of fruit: 4-6 mm

2008/06/07 10 days after pollination The length of seed: 2-4 mm

The length of fruit: 8-10 mm

2008/06/12 15 days after pollination The length of seed: 5-6 mm

The length of fruit: 10-14 mm

2008/06/17 20 days after pollination The length of seed: 6-7 mm

The length of fruit: 14-16 mm

2008/06/24 27 days after pollination The length of seed: 6-8 mm

The length of fruit: 14-18 mm

2008/06/27 30 days after pollination The length of seed: 6-8 mm

The length of fruit: 18-20 mm

2008/07/03 36 days after pollination The length of seed: 6-8 mm

The length of fruit: 18-20 mm

2008/07/07 40 days after pollination The length of seed: 6-8 mm

The length of fruit: 18-20 mm

2008/07/13 46 days after pollination The length of seed: 6-8 mm

The length of fruit: 18-20 mm

2008/07/18 51 days after pollination The length of seed: 6-8 mm

The length of fruit: 18-20 mm

2008/10/30 5 months after pollination The length of seed: 6-8 mm

The length of fruit: 18-22 mm

The stage offull maturity of

embryo and a ripe fruit

variety), and immediately  xed in the FAA  xator (for-

maldehyde- acetic acid-alcohol) and transferred to the

laboratory. The local varieties that were used for experi-

ments, including the red seedless cultivar (containing

stenospermocarp seed) and Ghezel-Ozum seeded cultivar

(containing the actual seed) were selected for compari-

son with each other.

Samples were harvested from the clusters that were in

the similar phonological stage, and were marked for next

sampling. Samples were harvested in the early morn-

ing to avoid shrinking and losing the water. The 8-year

plants were used and cultivated by using the method of

Top Wire Cordon (2×4 Meter).

Samples were kept in the refrigerator during the

experiment period. Then, based on the length (in mm) of

pistil (or ovarian), and fruit were dividedin the early and

later stages, respectively. These divisions in the early

stages with the graph paper, and at later stages with the

caliper under the simple (loop) microscope were done In

the maturity stages, seeds were separated from the fruits

and their length were also measured.

Separating under the loop was done using forceps and

sharp-pointed needles, and cap and stamens were pre-

cisely separated, and immediately placed in the  xator

materials. Seeds were also separated from the fruit in the

same method. At this stage, it has paid attention to the

time of separating not to damage the samples. Further-

more, in the mature seed, the embryo was removed from

the seed and examined under a microscope. Sampling

of the Ghezel-Ozum and red seedless cultivars were per-

formed in accordance with Tables 1 and 2.

PREPARING THE SAMPLES AND

HISTOLOGICAL ASSAY

The samples were immersed in the FAA  xator (formal-

dehyde 37%, 5 ml; ethanol 50%, 90 ml; and 5 ml of

glacial acetic acid) for 12-24 hrs. After suf cient wash-

ing with running water and dehydrating with increasing

levels of ethanol, the samples were clari ed with xylene

and saturated with paraf n. The samples after molding

in paraf n, with the rotary handle microtome (R Jung

Heidelberg) were cut at the thickness of 8 to 10 μm. The

slices after removing paraf n and water were stained

with Hematoxylin and PAS-Hematoxylin (Jenson 1962).

The staining of Light Green, and Sudan Black and Red

were used for studying proteins and lipids, respectively

(Gahan 1984). The microscopic investigatingand photo-

graphing of samples were done with the light micro-

scope (Nickon, E200-LED, USA).

Mahdi Mohammadi et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS HISTOLOGICAL STUDY ON THE STAGES OF POLLINATION AND FERTILIZATION 309

RESULTS

THE CHANGES OF PRE- AND POST-POLLINATION

STAGES IN THE RED SEEDLESS CULTIVAR

In the  owers of Red Seedless cultivar in the anthesis (stage

4; stages 1-3 are not shown here) based on sampling date

and the morphological studies with the light microscopy,

the pollen shad germinated and pollination had occurred.

It should be noted that the  owers in grapes are on the

clustered in orescences and at one in orescence,  ow-

ers can be in several different phonological stages, and in

examinations should be considered this issue.

Three days after pollination (stage 5), the fertiliza-

tion occurred in all the  owers, and micropyle was been

closed, which was due to growing upand becoming mas-

sivethe internal walls.At this stage, the presence of pol-

len tube into the embryo sac was signi cant (Fig.1).

The microscopic sections prepared from  owers pol-

linated at the stages of 10 and 15 days after pollination

showed that the double fertilization in this cultivar was

successfully occurred; so that the  rst division of the

zygote was observed in 15 days after pollination. The

apical and basal cells were formed in high and low den-

sities, respectively and the basal cells were observed in

its  rst division (Fig.2).

In the stage of 8 (20 days after pollination), a number

of the free nuclei within the embryo sac were observed

which have been resulted from the division of synergids

(Fig.2). Whereas at this stage the growth of the zygote

had been stopped and the cell had been degenerating.

The formation of the brown sediments around the cell

that was degenerating was signi cant (Fig.3). The sig-

ni cant point at this stage was distinctively the thick-

ening of the transverse walls of calot cells which had

likely been started from the previous stage (the tangen-

tial walls). Furthermore, sometimes the wall thickening

was observed in the radial walls (Fig.3).

In addition, other notable phenomenon was the sepa-

ration of the inner integumentfrom the outer integument

which was started from this stage and at the following

stages, this space was increased (Fig.4).

In the stage of 9 (30 days after pollination) in the

red seedless cultivar, the nucellus were strongly pressed

and the inner integument completely kept away from

the outer integumentand an empty space was created

between them (Fig. 4b).

In the stage of 10 (40 days after pollination) in the red

seedless cultivar, the shrinkage of nucellus was contin-

ued and nucellus in the form of crumpled on one side of

the seed was observed. The stages of degeneration were

along with the shrinkage and becoming small of the

seed. Thickening in the transverse walls of the calot cells

were also continued at this stage. In the outer integu-

ment, the needle-shaped crystals of calcium oxalate

were observed which large groups of them were formed

within the idioblast (Fig. 5).

THE CHANGES OF PRE- AND POST- POLLINATION

STAGES IN THE GHEZEL-OZUM CULTIVAR

Five days after pollination in the Ghezel-Ozum cultivar,

zygoteand synergids were observed in the embryo sac

(Fig.6).

Table 2. The dates of sampling of the red seedless cultivar

Date Developmentstage Features

2008/06/13 Flower bud

2008/06/14 The beginning of anthesis

2008/06/16 30 to 50 percent anthesis

2008/06/18 The time of pollination The peak stage of anthesis

(80 percent  owers in anthesis)

The length of ovule: 1-1.2mm

2008/06/21 5 days after pollination The length of seed: 1.2-1.5 mm

The length of fruit: 2-4 mm

2008/06/27 10 days after pollination The length of seed: 1.5-2 mm

The length of fruit: 4-6 mm

2008/07/03 15 days after pollination The length of seed: 2-2.5 mm

The length of fruit: 6-10 mm

2008/07/08 20 days after pollination The length of seed: 2.5-3 mm

The length of fruit: 10-10 mm

2008/07/15 30 days after pollination The length of seed: 2.5 mm

The length of fruit: 12-14 mm

2008/07/28 40 days after pollination The length of seed: 2-2.3 mm

The length of fruit: 12-14 mm

310 HISTOLOGICAL STUDY ON THE STAGES OF POLLINATION AND FERTILIZATION BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Mahdi Mohammadi et al.

FIGURE 1. The longitudinal section of the seed

of the red seedless cultivar in 3-5 days after

pollination (stage 5), stained with the PAS-

Hematoxilen method, a) Ovule-synergids and

polar nuclei within the embryo sac were distin-

guished. b) Fig.¬a with further magni cation,

the entering of the pollen tube to synergids

was distinguished. c: calot, es: embryo sac, nu:

nucellus, pt: pollen tube.

Ten days after pollination, a number of the free nuclei

were observed within the embryo sac. The free nuclei,

after the double fertilization, were resulted from the divi-

sions of the synergids which would form the endosperm.

The zygote as well as the synergids was observed in the

form of the shrunk mass by the side of that (Fig.7). In the

outer epidermis of the inner integument, the tangential

divisions were seen with the increasing of the lengthin

the radial direction. In the inner epidermis of the outer

integument were also added on the reddish brown com-

pounds.

In Ghezel-Ozum cultivar, the formation of the globu-

lar embryo was observed in simultaneous with the stage

of 8 (20 days after pollination) (Fig.8). In addition, in the

Ghezel-Ozum cultivar, endosperm was formed with cell

wall and also both internal and external integuments grew.

In the  nal stages, the entire seed with the mature

embryo and the cell endosperm was observed. The

FIGURE 2. To occur the double fertilization

process in the red seedless cultivar, stained with

the PAS-Hematoxilen method. a) The zygote

has fertilized and its  rst division has done. b)

Endosperm nuclei within the embryo sac are

identi ed. en: endosperm nuclei, es: embryo sac

embryo in this cultivar had thoroughly evolved and

consisted of an axial section, which the apical meristems

of the root and stem formed at the two ends of that, and

storages-accumulated cotyledons were on the both sides

of the apical meristem of stem (Fig.9).

In the mature stage of the Ghezel-Ozumseed, the pro-

tein compounds within the endosperm tissue were thor-

oughly observed which were stained with Light Green

(Fig.10). Furthermore, the needle-shaped crystals of cal-

cium oxalate in the form of the groups of idioblast were

also observed in the  nal stages in this cultivar.

DISCUSSION

One of the important cultivars of grapes is red Sultana

or red seedless which is one variety of stenospermocarps

and consumed freshly and for producing sultana. So far,

few studies, histologically, was performed on this culti-

var. Stott (1936) was the  rst to report stenospermocarpy

and he applied this word for the immature seeds. He

Mahdi Mohammadi et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS HISTOLOGICAL STUDY ON THE STAGES OF POLLINATION AND FERTILIZATION 311

FIGURE 3. Longitudinal section of red seed-

less cultivar in 20 days after pollination,

stained with the method of Hematoxilen .a) The

degenerated pre-embryonic cells were shown

at the micropylar pole. B) Fig.a. With further

magni cation. Brown sediments are observed

around the purple mass. c: calot, de: degener-

ated embryos, mi: micropyle

FIGURE 4. The longitudinal section of red seed-

less cultivar in 20-30 days after pollination

(stages 8 and 9), stained with the method of PAS-

Hematoxilen. a) The initiation of separating the

inner integument from the outer integument is

shown. Photographed with dark  eld microscope,

20 days after pollination (stage 8). b) Separating

the inner integument from the outer integument,

30 days after pollination (stage 9). c,e) Fig. a

with further magni cation.d, f) Fig. b with fur-

ther magni cation.The nucellus has degenerated.

es: embryo sac.

stated that the growth of embryo in Thompson Seedless

varieties continues to globular embryo stage (according

to the Pommer et al. 1995).Thereafter, it has been more

considered the breeding aspects of grapevines, which

the most important subjectswere embryo rescue with the

aim of disease resistance, improving fruit quality and

enhancing the performance in the stenospermocarp cul-

tivars. In these studies, genotype, culture medium, sam-

ple age and harvest time have been introduced as impor-

tant factors for successin embryo rescue, however, any

exact research has not been performed on the subject of

ovule and immature seeds of stenospermocarp (Sharma

1996; Yang et al. 2007).

Studies showed that pollination at the stenosper-

mocarp cultivars is just a factor to develop fruit, and fol-

lowing growth and development of endosperm and the

embryo have dif cult (Hanania et al. 2007; Pratt 1971).

Pratt (1971) noted that the outer integument of ovule at

the stenospermocarp cultivars is without sclerenchyma

cells and ovules or aborted seeds remain just as a small

object into cubes. The development of ovule may be

normal, such as seeded cultivars that have nucellus, one

or two integuments and the cord, and or is nearly nor-

mal. Pratt (1971) noted that pollen is usually fertile and

self-pollination occurs in these cultivars. It was found

that the being seedless characteristic is heritable (Bou-

quet and Danglot 1996; Pratt 1971). Liu et al. (2003)

demonstrated that the main reason of stenospermocarp

in Sultana seedless grapeis unknownup to now.

GERMINATION OF POLLEN

It was found that pollen affects the growth and size of

seed, because, the half of the embryo genes and one

third of endosperm genes are sourced from paternal

parent (Ebadi & Dehghani 2002). Pratt (1971) said that

stenospermocarpc ultivars have usually alive pollen. In

our surveys on the red seedless grape, viability and ger-

mination of pollen on the stigma surface were con rmed

by  uorescence microscope, however, because of style

tissue thickness, the observation of its penetration from

stigma surface to style was impossible. In sectioning to

view in bright  eld light microscope, the pollen tube and

its transition place in the micropylar region and calot

were observed which shows the pollen tube penetrates

into the ovule and then embryo sac.

Mahdi Mohammadi et al.

312 HISTOLOGICAL STUDY ON THE STAGES OF POLLINATION AND FERTILIZATION BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 5. The longitudinal section

of red seedless cultivar in 40 days

after pollination (stage 10), Stained

with the method of Hematoxilen.

a,b) Parenchymal cells containing

a mass of calcium oxalate crystals

(Idioblast).

FIGURE 6. The longitudinal section of

Ghezal-Ozum seedless cultivarin pol-

lination and fertilization stage (stage

5), stained with the method of PAS-

Hematoxilen. a) Longitudinal section

of ovule at the Ghezel-Ozum cultivar.

b) Embryo sac in the micropylar pole

with further magni cation has been

determined. c) Zygote cells and ovule

nuclei have been identi ed. e: embryo,

es: embryo sac, pn: polar nuclei, mi:

micropyle

FERTILIZATION AND ZYGOTE FORMATION

In studies, it has been found that the pollen tube reaches

the micropyle within 12 hrs and fertilization take place

24 hrs after pollination (Pratt, 1971). However, some lit-

eratures demonstrated that it occurs 2 to 3 days after

pollination (Batigiana 2006). In an effort to breed seed-

less varieties by Sahijram and Kanamadi (2003), oocytes

were histologically investigated 24 hrs after pollina-

tion, and the formation of zygotes were observed in 4

days after pollination at all studied crosses. Endosperm

mother cell was also formed in all of them, and it means

that double fertilization was successful (Sahijram &

Kanamadi 2003). These  ndings correspond with our

observations. The studying sample appeared that ferti-

lization occurred in 3 to 5 days after pollination (due to

a clustering of in orescence and intervals for sampling).

In the studied microscopy sections at this stage, the pres-

ence of pollen tubes inside the synergids con rms this.

Also, fertilization at Ghezel-Ozum cultivar took place in

3 to 5 days after pollination.

POST-FERTILIZATION

Endosperm formation

In our surveys, after fertilization, free nuclei were

formed from divisions of the synergids nucellus in the

embryo sac. However, 30 to 40 days after pollination,

it was found that the endosperm cells were gradually

degenerated at this stage; a type of shrinkage was also

created in nuclei.

In some literatures were noted that the growth of

embryo can be stopped and aborted at the immature

stage which are mentioned different reasons for this. For

example, it can refer to the lack of proper nutrition of

embryo by the endosperm (Bharathy et al., 2005; Liu et

al., 2003; Ramming et al., 1991; Sharma et al.,1996; Yang

et al., 2007). It was also distinguished that the death of

the embryo can be due to toxin production by endosperm

and incompatibility of embryo and endosperm (Bhar-

athy et al. 2005; Yang et al. 2007). In red seedless culti-

var, it seems that one of the reasons for stopping growth

and development of zygote is the stopping the growth

and development of the endosperm; however also the

delay in the growth and development can be another

Mahdi Mohammadi et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS HISTOLOGICAL STUDY ON THE STAGES OF POLLINATION AND FERTILIZATION 313

FIGURE 7. The longitudinal section

of the seed of Ghezel-Ozum cultivar

in the 10 days after pollination

(stage 6), stained with the method

of PAS-Hematoxilen. a) Zygote and

primary endosperm nuclei are shown

at the micropylar pole. B) Fig.a.with

further magni cation, zygote and

endosperm nuclei which are dividing

have been identi ed. c) Zygote with

further magni cation was shown.

ic: inner integument (coat), mi:

micropyle, ne: nuclear endosperm, z:

zygote, c:calot

cause. Pachno et al. (2014) suggested that, in apomictic

dandelions, the persistent synergids may play a role in

the nutrition of the developing embryo.

In Ghezel-Ozum seeded cultivar, endosperm tissue is

formed and fully developed after double fertilization.

Ten days after pollination, the release endosperm nuclei

were observed in large numbers, which their growth has

continued in the following stages in 15 to 20 days after

pollination and eventually was fully shaped into cell. In

our investigations, proteins, polysaccharides, oil com-

pounds and star-shaped crystals of calcium oxalate were

observed in the mature endosperm.

Endosperm in the seeded cultivars of grape has irreg-

ular shape and is mainly composed of thick-wall cells

(Pratt 1971). It was found that the endosperm can con-

tinue to grow even without fertilization (Chaudhury et

al. 1998; Raghavan 2006). Endosperm effect on embryo

morphogenesis is probably due to physical pressures and

in the production of somatic embryos can be observed

abnormal mode due to imbalances in pressure (Farsi

&Bagheri 2004; Farsi&ZolAli2006).

GROWTH OF INTEGUMENT AND CLOSING

THE MICROPYLE

Batigiana (2006) demonstrated that a series of severe

divisions after fertilization occurs in the funicle, navel,

chalaza and integuments.Also, in the seedless cultivar,

after fertilization, the growth of cells of inner integu-

ment was observed and therefore micropyle was closed.

Then any division was not observed at the integument

cells, while in Ghezel-Ozum seeded cultivar, the growth

of outer integument was impressive. Carraro et al. (1979)

reported when micropyle remains open it is due to the

disruption of pollination, and increases the embryo

abortion and stenospermic in grapes. In our observa-

tions, micropyle opened before fertilization, and after

fertilization due to the growth of inner integument cells

had been closed. Haughnand Chaudhury (2005) report-

edthat there is a relationship between the growth of

endosperm and integument which is related to genetic

control. It means that with growth and development of

the endosperm, integument growth continues. Striem et

al. (1992) showed that seed integument and endosperm

formation and development in the stenospermocarp

grapes are independent of each other, and therefore, seed

integument will be emerging without endosperm and

embryo. In our study, independent of the integument

formation from endosperm and embryo was rejected;

because in this sample, growing integument of embryo,

i.e. the seed, was stopped from 20 days after pollination

and even earlier when also stops growth of endosperm.

In our studies, it was found that the outer integu-

ment at micropylar pole was formed from two to three

cell layers and in chalazal pole was added to the num-

ber of layers and it is 6-7 cell layers.In the red seed-

less cultivar, this integument has color compounds that

are likely polyphenols or anthocyanins. Vessels at the

chalazal pole of outer integument were completely vis-

ible.The Color of outer integument was different with

inner integument and has transparent cells that contain

clearcontents.The inner integument by two to three lay-

ers has the compressed, stretched and  attened cells

astangential. The inner integument surface was covered

by the cuticle layer which was determined after staining

with black Sudan. These observations correspond with

the  ndings of Pratt (1971).

In the following stages of the red seedless cultivar,

we saw the separation of the inner integument from the

outer integument. The beginning of this phenomenon

was observed 20 days after pollination. Furthermore,

nuclei were gradually suffering from degeneration and

Mahdi Mohammadi et al.

314 HISTOLOGICAL STUDY ON THE STAGES OF POLLINATION AND FERTILIZATION BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 8. Axial sections of the seed of Ghezel-Ozum cultivar in

20 days after pollination (stage 8), stained with method of Hema-

toxilen. Photographed with dark and bright  eld microscope.a)

Globular embryo is shown at the micropylar pole. Photographed

with a bright  eld microscope.b) Globular embryo is shown at

the micropylar pole. Photographed with a dark  eld microscope.

c) Fig. a is shown with further magni cation. d) Fig.b. Is shown

with further magni cation. ge: globular embryo, ic: inner integu-

ment (coat), mi: micropyle.

FIGURE 9. Longitudinal sections of mature embryo of the

Ghezel-Ozum cultivar, stained with the method of Hematoxilen.

a) Longitudinal section from isolated embryo, meristem of roots,

shoots and cotyledons has been speci ed. b) Longitudinal section

from embryo inside the seed, meristem of shoots and cotyledons

has been speci ed. c) Root meristem. d) Shoot meristem. c: coty-

ledons, rm: root meristem, sm: shoot meristem.

Mahdi Mohammadi et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS HISTOLOGICAL STUDY ON THE STAGES OF POLLINATION AND FERTILIZATION 315

FIGURE 10. Longitudinal sections of mature seed of the Ghezel-Ozum cultivar, the different

parts of the embryo and endosperm components are shown, stained with the method of

PAS_Hematoxilen. a) Embryo is shown inside the endosperm tissue. b) Endosperm tissue

that stained with the method of PAS- Light. In this  gure, polysaccharide walls and intra-

cellular storage are shown green.c) Endosperm tissue that stained with the method of Light

green. In this  gure, protein components are shown. d,e) Endosperm tissue that stained

with the method of black and red Sudan, plasma membrane of cells with Sudan Black, is

stained black. c: cotyledon, e: endosperm, rm: root meristem, sm: shoot meristem.

shrinkage. These  ndings are corresponded with studies

of Vallania et al. (1987) in terms of shrinkage of nucleo-

lus and separating integument.

FINAL STAGES

With further investigations it was found that the zygote

divided 7 or 15 days after pollination. In fact, the zygote

has done  st division and then stopped.The maximum

size of seed is created at the end of the  rst stage and

then its growth stops and just continuing the growth

and development of the embryo, endosperm and berries

would be observed. In our observations and investiga-

tions, also seed size was completed 20 days after polli-

nation, but then, growth of embryo had stopped.

In the red seedless cultivar, following the investiga-

tions, on 20 to 30 days after pollination, brown sediments

around the zygote which is dividing, were observed that

seems be the compressed polyphenols and tannins and

may be effective in the death of embryo.This compact

tannin role as an agent for dormancy and preventing

earlier germination of the seed was documented which

are in the seed coat (Debeaujon et al. 2000). Further-

more, nucleolus had been suffering from shrinkage in

this stage. At the  nal stages, needle-shaped crystals

of calcium oxalate were observed at the outer wall that

large groups of them formed within idioblast, which

their roles are for regulation of calcium, plant protec-

tion, detoxi cation of heavy metals, ion balance,  rm-

ness and so on (Vincent et al. 2005).Also, Rosianski et

al. (2016) reported that the pollinated fruit of  g had a

larger diameter and weight and improved  rmness com-

pared to the parthenocarpic fruit. These groups of cal-

cium oxalate were also observed in the seeded cultivar.

Mahdi Mohammadi et al.

316 HISTOLOGICAL STUDY ON THE STAGES OF POLLINATION AND FERTILIZATION BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

The calcium oxalate crystals were also observed within

the seedin the  nal stages. Pratt (1971) noted that this

case can be observed in samples with abnormal ovules

that oxalate crystals are replaced by nucellar cells. These

abnormal ovules consist inovules with nucleolus and

embryo sac with defection at growth and development,

abnormal curvature of ovule in the ovary, necrosis of

part of chalaza and so on.

CONCLUSION

The  ndings showed that growth and development of

zygote and endosperm were delayed and  nally stopped

in red seedless cultivar after double fertilization. It means

that  rst zygote was divided in 15-20 days after pollina-

tion when apical and basal cells had been formed, but

after ward their divisions were ceased. Also in 20 days

after pollination the series of brown sediments were

observed around the zygote. Mean while, in Ghezel-

Ozum cultivar, the formation of globular embryo and

endosperm were observed in 20 days after pollination.

In red seedless cultivar, endosperm cell was divided as

nuclear that release nuclei were observed in the embryo

sac and then divisions were stopped. Moreover, in the

transverse walls of calot cells, thickening was seen quite

clear. At the nuclei in the  nal stages, shrinkage and

degeneration were observed. Also, internal integument

was separated from outer integument.

ACKNOWLEDGMENT

Authors like to thank Agricultural Research Centre at

Seed and Plant Improvement, Kahriz, Uremia, Iran for

facilities that give in conducting this project.

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