Tarım Bilimleri Dergisi Journal of Agricultural Sciences

Tar. Bil. Der.

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www.agri.ankara.edu.tr/dergi www.agri.ankara.edu.tr/journal

Morphological and Anatomical Investigations on in Vitro Micrografts

of OHxF 333 / Pyrus elaeagrifolia Interstock / Rootstock Combination

TARIM BİLİMLERİ DERGİSİ — JOURNAL OF AGRICULTURAL SCIENCES 20 (2014) 269-279

in Pears
Hatice DUMANOĞLUa, Aysun ÇELİKa, H. Nurhan BÜYÜKKARTALb, Saeed DOUSTİa
a
Ankara University, Faculty of Agriculture, Department of Horticulture, 06110, Ankara, TURKEY
b
Ankara University, Faculty of Sciences, Department of Biology, 06100, Ankara, TURKEY

ARTICAL INFO
Research Article
Corresponding Author: Hatice DUMANOĞLU, E-mail: dmanoglu@agri.ankara.edu.tr, Tel: +90 (312) 596 12 99
Received: 03 December 2013, Received in Revised Form: 02 January 2014, Accepted: 13 January 2014

ABSTRACT

In this study, possibility of creating a specific interstock / rootstock combination to obtain a clonal, semi dwarf, composite
pear rootstock tolerant to various stresses by micrografting was investigated. In vitro shoots of ‘Old Home x Farmingdale
333’ (Pyrus communis L.) which is a clonal semi dwarf pear rootstock resistant to fireblight and pear decline was used
as interstock, and in vitro Pyrus elaeagrifolia Pallas seedlings known as tolerant to Fe-chlorosis, salinity and drought
stresses was used as rootstock. Cleft grafting was applied in micrografts. Grafted seedlings were cultured on Murashige
and Skoog basal medium with ½ strength of macronutrients for 8 weeks under white fluorescent light for 16 h day-1.
Cultures, except the control, received complete darkness either 1 or 2 weeks at the beginning of incubation. Graft take
success in the control treatment was significantly higher (97.9%) than darkness treatments of 1 or 2 weeks (90.5% and
82.5%, respectively). Ultrastructural observations with transmission electron microscope revealed that dividing cambial
initials reached to 2-3 layers, and new xylem and phloem elements distinctly differentiated in transverse sections of the
graft union 8 weeks after micrografting in the control and darkness treatments. The results indicated a successful graft
union formation.
Keywords: Micrografting; Graft union; Interstock; Darkness treatment

Armutlarda OHxF 333 / Pyrus elaeagrifolia Ara Anaç / Anaç

Kombinasyonunun in vitro Mikroaşılarında Morfolojik ve Anatomik
İncelemeler
ESER BİLGİSİ
Araştırma Makalesi
Sorumlu Yazar: Hatice DUMANOĞLU, E-posta: dmanoglu@agri.ankara.edu.tr, Tel: +90 (312) 596 12 99
Geliş Tarihi: 03 Aralık 2013, Düzeltmelerin Gelişi: 02 Ocak 2014, Kabul: 13 Ocak 2014
Morphological and Anatomical Investigations on in Vitro Micrografts of OHxF 333 / Pyrus elaeagrifolia Interstock..., Dumanoğlu et al

ÖZET

Bu çalışmada, mikroaşılama yoluyla çeşitli streslere tolerant, klonal, yarı bodur, birleşik bir armut anacı elde etmek
için spesifik bir ara anaç / anaç kombinasyonu oluşturmanın olabilirliği araştırılmıştır. Ateş yanıklığı ve armut göçüren
hastalıklarına dayanıklı, yarı-bodur armut klon anacı ‘Old Home x Farmingdale 333’ (Pyrus communis L.)’ün in
vitro sürgünleri ara anaç ve demir klorozu, tuzluluk ve kuraklık streslerine tolerant olarak bilinen Pyrus elaeagrifolia
Pallas’ın in vitro çöğürleri anaç olarak kullanılmıştır. Mikroaşılarda yarma aşı tekniği uygulanmıştır. Aşılanmış
çöğürler, makro element düzeyi ½ olan Murashige ve Skoog temel besin ortamı üzerinde, 8 hafta süreyle, 16 h gün-1
süreyle beyaz floresan ışık altında inkübe edilmiştir. Kontrol dışındaki kültürler, inkübasyonun başlangıcında, 1 ya da
2 hafta süreyle tamamen karanlık koşullara alınmıştır. Aşı başarısı kontrol uygulamasında (% 97.9), 1 ya da 2 hafta
karanlık uygulamalarından (sırasıyla, % 90.5 ve % 82.5) önemli düzeyde daha yüksek olmuştur. Mikroaşılamadan 8
hafta sonra aşı kaynaşma yerinden alınan enine kesitlerde, transmisyon elektron mikroskopu ile yapılan ultrastrüktürel
gözlemler, kontrolde ve karanlık uygulamalarında kambiyal inisyallerin bölünerek 2-3 sıraya ulaştığını ve yeni ksilem
ve floem elemanlarının belirgin biçimde farklılaştığını ortaya koymuştur. Bulgular başarılı bir aşı kaynaşmasının
meydana geldiğini göstermiştir.
Anahtar Kelimeler: Mikroaşılama; Aşı kaynaşması; Ara anaç; Karanlık uygulaması

© Ankara Üniversitesi Ziraat Fakültesi

1. Introduction propagation methods, and also for studying more

Native Mediterranean Pyrus species P. elaeagrifolia in depth the relationships between genetically
Pallas which has habitat ranging from Turkey to different tissues and cells (Monteuuis 2012). In
Southeastern Europe and Ukraine (Bell 1990), is a initial studies, the objective of micrografting was
potential pear rootstock in areas where lime, salt and elimination of some virus diseases in tree fruit
drought are limiting factors for growing many Pyrus species (Jonard 1986). Murashige et al (1972) and
species (Lombard & Westwood 1987; Matsumoto et Navarro et al (1975) were first to consider the use
al 2006). Scions of cultivars (Pyrus communis L.) of micrografting technique in Citrus species in
grafted on P. elaeagrifolia grow vigorously similar order to eliminate virus diseases. Micrografting
to pear seedlings causing a long juvenile period. This has been successfully applied in Citrus (Edriss &
problem could be overcome by using a dwarf or semi Burger 1984; Sharma et al 2008), Prunus (Barba
dwarf clonal Pyrus interstocks such as ‘OHxF 333’ et al 1995; Jarausch et al 1999; Conejero et al
on P. elaeagrifolia. It is known that interstocks may 2013), Malus (Huang & Millican 1980; Bisognin
reduce vegetative growth and enhance reproductive et al 2008) and Pyrus (Faggioli et al 1997)
growth of the tree. Such double-worked plants species to get plants free from virus or virus like
have two unions, one between the rootstock and organisms. Besides, micrografting has been used for
interstock and one between the interstock and the micropropagation, rejuvenation of mature tissues,
scion. The interstock piece is budded or grafted or determination of graft incompatibility and root to
bench grafted onto the rootstock prior to combine shoot communication, transport or cryopreservation
with the scion cultivar (Hartmann et al 2011). in Citrus (Obeidy & Smith 1991; Parthasarathy et
However, production of grafted-rootstock plants al 1997; Volk et al 2012), Prunus and Amygdalus
using conventional techniques takes time for at least (Ozzambak & Schmidt 1991; Ghorbel et al 1999;
more than one year considering the growth time of Amiri 2006; Yıldırım et al 2010; Isikalan et al
rootstock lines (Elivar & Dumanoğlu 1999). 2011), Malus (Lane et al 2003; Nunes et al 2005;
In vitro grafting is an original and skillful Dobranszki & Silva 2010), Pyrus (Musacchi et al
technique which deserves greater consideration 2004; Espen et al 2005; Hassanen 2013), Pistacia
for overcoming the limitations of other vegetative (Abousalim & Mantell 1992; Onay et al 2003; 2004;

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Can et al 2006; Ozden-Tokatlı 2010), Castanea and radical of the seed was in the medium. Five seeds
Corylus (Nas & Read 2003), Actinidia (Ke et al were placed in each petri dish (70 x 10 mm). The
1993), Olea (Toroncoso et al 1999), Morus (Ma et seeds were stratified in vitro at 4 °C in dark for 8
al 1996), Anacardium (Thimmappaiah et al 2002), weeks in order to break dormancy. Chilled seeds
Opuntia (Estrada-Luna et al 2002) and Carica were germinated in a growth chamber at 25 ± 1
(Nava et al 2011) species. Creation of rootstocks by °C under 16/8 h day-1 (light/dark) photoperiod
interstock/rootstock combination is possible by in (cool white fluorescent light, 35 µmol m-2 s-1)
vitro micrografting which is very fast, using in vitro for 10-14 days. MS basal medium (Murashige &
rooted young rootstock plantlets and in vitro grown Skoog 1962) with half-strength of macronutrients,
interstock scions. Thus, micrografting technique containing 3% (w v-1) sucrose and 1% (w v-1) Difco
offers new possibilities for mass production of Bacto agar was used in seed stratification and
grafted rootstocks which might be later grafted in germination studies. The pH was adjusted to 5.8
vitro or in vivo with the scion cultivar. Up to date, before adding agar and autoclaving at 121 °C for
we are unaware of micrografting studies which 20 min.
have been done for micropropagation of rootstocks
grafted with interstock. In vitro shoots obtained from shoot-tip culture
of ‘OHxF 333’ were used as scion (interstock).
The objective of this study was to develop a Shoot-tip cultures were established from actively
quick in vitro micrografting technique to create growing shoots of plants. Shoot tips approximately
a specific pear rootstock combination using semi
20 mm long were collected, washed in running
dwarf clonal interstock (‘Old Home x Farmingdale
tap water for 30 min, and sterilized by immersion
333’, (OHxF 333)) and the vigorous seedling
in a solution of sodium hypochlorite (1% active
rootstock tolerant to different stress conditions (P.
chlorine) for 20 min followed by 3 rinses with
elaeagrifolia), and to study graft union formation on
sterile distilled water for 5 min. Approximately
in vitro micrografted and complate darkness treated
10 mm long explants were cultured in glass tubes
plantlets, since complete darkness may prevent
(120 x 25 mm) containing 10 mL of medium under
internal auxin degradation, by morphological and
aseptic conditions. The shoots (>10 mm long) from
anatomical (Transmission Electron Microscope)
investigations. initial cultures were subcultured four times by four
week intervals in glass flasks (250 mL) containing
50 mL of medium.
2. Material and Methods
For initial culture and subcultures, MS basal
2.1. Rootstock and scion (interstock) sources for medium containing 3% (w v-1) sucrose and 0.7% (w
micrografting experiments v-1) agar was used. MS medium was supplemented
In vitro germinated seedlings of P. elaeagrifolia with 4.4 µM BA and 0.9 µM GA3 which were were
Pallas were used as the rootstock. The seeds were added to media before autoclaving at 121 °C for 20
processed before culturing in aseptic conditions min. The pH was adjusted to 5.8 before adding agar.
as follows; (1) the seeds were scarified in sulfuric All cultures were incubated at the same conditions
acid (Merck, 95–98% H2SO4) for 2.5 min followed as seed germination.
by rinsing in running water for 5 min, (2) the seeds
2.2. In vitro micrografting experiments
were dipped in 85% ethanol for 3 min, and then
sterilized in a solution of sodium hypochlorite Cleft grafting was applied in micrografts under
(2.5% active chlorine) for 30 min followed by 3 aseptic conditions. About 10-14 day-old seedlings
rinses for 5 min with sterile distilled water. Then, with horizontal cotyledonary leaves, without true
the seeds were placed on germination medium in leaves, with roots about 15-30 mm in length were
petri dishes by positioning vertically so that the used as rootsock in grafts (Figure 1A).

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Figure 1- Micrografting procedure of interstock ‘OHxF 333’ on Pyrus elaeagrifolia seedling under aseptic
conditions. An in vitro germinated P. elaeagrifolia seedling (A); forming a cleft at about 2-3 mm in length
on the top of hypocotyle of seedling which was cut just below the cotyledon leaves (B); an in vitro grown
seedling ready for micrografting (C); a scion prepared from an in vitro grown interstock ‘OHxF 333’ micro
shoots ready to be inserted into the cleft (D); an inserted scion into the cleft (E); a graft union wrapped
around with sterile aluminum foil (F); a micrografted plantlet taken out from in vitro conditions after 8
weeks of grafting (G)
Şekil 1- Pyrus elaeagrifolia çöğürleri üzerine ‘OHxF 333’ ara anacının, aseptik koşullarda mikroaşılama
işlemi. İn vitro koşullarda çimlendirilmiş P. elaeagrifolia çöğürleri (A); kotiledon yaprakların hemen altından
tepesi vurulmuş fidelerin hipokotilinin üst kısmında yaklaşık 2-3 mm uzunluğunda bir yarık oluşturulması (B);
mikroaşılama için hazır bir in vitro çöğür (C); in vitro koşullarda geliştirilmiş ara anaç ‘OHxF 333’den kalem
olarak hazırlanmış, yarık içerisine yerleştirilmeye hazır mikro sürgünler (D); yarık içerisine kalemin yerleştirilmesi
(E); aşı yerinin steril alüminyum folyo ile sarılması (F); aşılamadan 8 hafta sonra in vitro koşullardan çıkartılmış
bir mikroaşılı bitkicik (G)

The roots were cut to ~20 mm length. The tip of medium in glass tubes (120 x 25 mm) containing
hypocotyl was decapitated just below the cotyledons. 10 mL of half-strength MS basal medium without
The stem was cut longitudinally 2-3 mm deep growth regulators containing 3% (w v-1) sucrose and
(Figure 1B & C). The scion, prepared from in vitro 0.7% (w v-1) agar. The pH was adjusted to 5.8 before
shoots of ‘OHxF 333’ was about 10 mm long and adding agar and autoclaving.
contained the apical meristem with 2-3 leaves. The For the micrografting experiments, grafted
basal end of the scion was cut to form a wedge about seedlings received either complete darkness for 1 or
2-3 mm long (Figure 1D). The scion was inserted 2 weeks in a growth chamber or 16/8 h day-1 (light
into the cleft and the graft junction was wrapped / night) photoperiod (control) at 25 ± 1 °C. At the
with a sterile 5 x 20 mm piece of aluminum foil (12 end of the dark treatments, cultures were incubated
µ thick) which was folded 4 times (Figure 1E & F). under the photoperiod as the control received.
The basal end of grafted seedlings was placed in the The micrografting experiments were a completely
medium with the graft junction above the level of randomized design with four replications with

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25 micrografts (tubes) in each replication. The transferred to 100% propylene oxide and embedded
experiment was repeated at least twice to ensure the in Epon 812 (Luft 1961). Ten blocks were prepared
accuracy of results. for each treatment. Ultrathin sections were stained
with uranyl acetate (Stempak & Ward 1964) and
2.3. Morphological investigations of micrografted lead citrate (Sato 1967). Ultrastructural observations
plantlets were made under Jeol CXII Transmission Electron
Grafted plantlets were removed from in vitro Microscope at 80 Kv.
conditions 8 weeks after micrografting and the
aluminum band was removed (Figure 1G). Percent 3. Results and Discussion
graft take, graft junction rating on a scale of 1 to 3 (1
= weak, 2 = medium, 3 = good) by visual inspection, In this study, very high graft take success was obtained
shoot length and number of leaves on the shoots in the micrografts of ‘OHxF 333’ / P. elaeagrifolia
were recorded. Morphological data were subjected seedlings. Based on morphological observations, the
to analyses of variance (ANOVA) at P<0.05, 0.01 highest graft take and graft junction rating was 97.9%
or 0.001 using MINITAB statistical software and 2.70%, respectively (Table 1).
(MINITAB Inc., UK). Means were separated Hassanen (2013) found that the highest percentage
by Duncan’s multiple range test at P = 0.05. The of successful grafts was 83% in the Le-Cont pear /
percent data were transformed into angle values Pyrus betulaefolia combination. Nunes et al (2005)
prior to analyses. reported that in vitro micrografting techniques offer
the potential for effective propagation of high quality
2.4. Preparation of epon blocks and anatomic genetic material in a short time under controlled and
investigations aseptic conditions. Grafting techniques and initial
Tissue samples in size of 2 mm lenght were taken treatments applied prior to in vitro micrograftings
from the graft union 8 weeks after the micrografts are subjects of concern. Cleft grafting was used
were made. The samples were fixed in 3% in this study because it has been reported as a
glutaraldehyde buffered with 0.1 M phosphate (pH successful micrografting technique in different fruit
7.2) for 3 h and then fixed in 1% osmium tetraoxide species such as cherry, pistachio, olive (Ozzambak
(in 0.1 M Na-P buffer) for 3 h at room temperature. & Schmidt 1991; Abousalim & Mantell 1992;
Specimens were dehydrated in an ethanol series, Toroncoso et al 1999; Onay et al 2004).

Table 1- The effect of complete darkness treatments applied at the beginning of incubation, on graft
take success and shoot development in ‘OHxF 333’ / Pyrus elaeagrifolia seedling interstock/ rootstock
combinations in vitro (after 8 weeks of micro-cleft graftings)
Çizelge 1- İn vitro koşullarda inkübasyon başlangıcında uygulanan tamamen karanlık uygulamalarının ‘OHxF
333’ / Pyrus elaeagrifolia çöğürü ara anaç / anaç kombinasyonunda aşı başarısı ve sürgün gelişimi üzerine
etkileri (mikro-yarma aşılamadan 8 hafta sonra)
Graft take Graft junction rating Shoot length Number of
Treatments
(%) (1-3) (mm) leaves
Control 97.9a 2.70 ± 0.06a 13.9 ± 0.8a 6.8 ± 0.5a
Complete darkness for a week 90.5b 2.33 ± 0.13b 10.6 ± 0.4b 3.9 ± 0.0b
Complete darkness for two weeks 82.5b 2.09 ± 0.13b 10.9 ± 0.6b 4.5 ± 0.3b
P 0.001 0.011 0.011 0.000
Graft junction rating 1, weak; 2, medium; 3, good. Means in each column followed by the same letter were not significantly different
at P = 0.05, according to Duncan’s new multiple range test

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We applied complete dark treatment for 1 or 2 Transverse sections of a region near graft
weeks as an initial procedure with an expectation elements showed that parenchymatous cells forming
of increasing graft take success by promoting callus callus tissue were large and had larger intercellular
formation due to its role in internal auxin biosynthesis spaces. In some of these cells the cytoplasm was less
(Yin et al 2012). Endogenous growth regulators dense and contained vacuoles and a few organelles
have been proposed to play a key role in grafting, (Figure 2B). Some enlarged cells and occasionally
with special reference to auxin which is known to divided cambial initials were observed at the graft
be synthesized in shoot apices and degraded by union. Cambial cells were formed by couple of cell
light. Our results showed that although graft take layers (Figure 2C), and cytoplasm of some of the
success, graft junction rating, leaf number and shoot cells were dense (Figure 2D). Xylem elements and
development were satisfactory in all treatments, the phloem cells were formed by couple of cell layers
control treatment performed significantly better than and they were starting to differentiate. Tracheal
the dark treatments (Table 1). The control treatment elements which were in the process of differentiation
had the highest graft take among the treatments had reached their normal size and the lumen of the
(P = 0.001) which was followed by 1 week (90.5%) cell was dense with electrons (Figure 2E). Phloem
and 2 weeks (82.5%) darkness treatments although cells were found to be round, isodiametric or
the difference between last two was not statistically hexagonal in shape, thin walled and dispersed.
significant. The grade for level of graft junction
In one week complete darkness treatment,
was also high on grafts and the differences were
parenchymatous cells with rough walls forming the
significant. The control treatment had the highest
callus tissue between the graft elements were plenty
grade (2.70 ± 0.06) followed by 1 week (2.33 ±
and the cytoplasm stained dark (Figure 3A). At this
0.13) and 2 weeks of complete darkness (2.09
stage, the cambial relation was present between
± 0.13) treatments. (Table 1). The results are in
graft elements. Cambium cells were formed with
agreement with the report of Monteuuis (1996)
cells of different sizes and occasionally formed 7 or
that the application of dark treatment for 2 weeks
8 layers. These cells had thin walls, were rectangular
in in vitro micrografts did not improve the results
in shape, and formed radial rows. The cytoplasm
in Acacia mangium. On the other hand, Monteuuis
stained very dark in some cells. There were few
(1994) observed beneficial effects of 2 weeks to
newly formed xylem and phloem elements (Figure
3 weeks in darkness immediately after grafting in
3B).
vitro seedlings of Picea abies which resulted in
52.4% success as compared to 32.6% for the control. In two weeks of complete darkness treatment,
Similarly, 5 days of dark treatment shortened the callus tissue between the graft elements at the graft
time required for grafting and increased the graft union was dense in samples treated. The cells at the
union rate (90.6%) compared with the control union tissues were generally flattened and dispersed.
(80.7%) in the plug seedling of rose rootstock (Han However, parenchymatous cells producing the
et al 1998). callus tissue were formed by large cells with large
inter-cellular spaces. The cytoplasm in some cells
Ultrastructural observations with transmission
electron microscope in ‘OHxF 333’ / P. elaeagrifolia was dense. Cambium was formed of couple of cell
micrografts revealed the successful graft union rows and the cytoplasm in some cells was dense.
formation, and new xylem and phloem elements At this stage, the established cambial relation
differentiated in all of the treatments. In the control, between graft elements was very clear. New xylem
observation of callus tissue between graft elements and phloem elements were few in number and they
at the graft union revealed that the cells at the graft started to differentiate (Figure 4).
union were generally oval in shape and scattered Espen et al (2005) indicated that the incompatible
(Figure 2A). heterograft (Pyrus communis L. cv. ‘Bosc’ (B) /

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Figure 2- Transversal sections of the graft union in the control treatment after 8 weeks of micrografts.
Rs, rootstock (P. elaeagrifolia seedling); Sc, scion (OHxF 333 interstock); Xy, xylem; C, cambium; Pa,
parenchymatous cell. Semithin section showing parenchymatous cells at the graft union Bar= 20 µm (A);
electron micrograph showing detail of parenchymatous cell cytoplasm Bar= 3 µm (B); semithin section
showing cambial cells and newly differentiated xylem and phloem cells Bar= 50 µm (C); the cytoplasm of
the active cambial cells Bar= 3 µm (D); detail of the newly differentiated xylem cells Bar= 3 µm (E)
Şekil 2- Mikroaşılamadan 8 hafta sonra kontrol uygulamasında aşı kaynaşma yerinden alınan enine kesitler.
Rs, anaç (P. elaeagrifolia çöğürü); Sc, ara anaç (OHxF 333); Xy, ksilem; C, kambiyum; Pa, parankimatik
hücre. Aşı kaynaşma yerinde parankimatik hücreleri gösteren yarı ince kesit Bar= 20 µm (A); parankimatik
hücre sitoplazmasının ince yapısını gösteren elektron mikrografisi Bar= 3 µm (B); kambiyum hücreleri ve yeni
farklılaşmış ksilem ve floem hücrelerini gösteren yarıince kesit Bar= 50 µm (C); aktif kambiyum hücrelerinin
sitoplazması Bar= 3 µm (D); yeni farklılaşmış ksilem hücrelerinin ince yapısı Bar= 3 µm (E)

Cydonia oblonga Mill. East Malling clone C BH) and the compatible heterograft (BH / EMC).
(EMC)) showed a marked delay in internode Thus we believe that, ‘OHxF 333’ / P. elaeagrifolia
cohesion compared with the autografts of B / B, combination would form a compatible heterograft.
and Pyrus communis L. cv. ‘Butirra Hardy’ (BH /

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Figure 3- Transversal semithin sections of the graft union in 1 week complete darkness treatment after 8
weeks of micrografts. Rs, rootstock (P. elaeagrifolia seedling); Sc, scion (OHxF 333 interstock); Xy, xylem;
Ph, phloem; C, cambium; Pa, parenchymatous cell. Parenchymatous cells at the graft union Bar= 20 µm
(A), cambial cells, newly differentiated xylem and phloem cells Bar= 20 µm (B)
Şekil 3- Mikroaşılamadan 8 hafta sonra 1 hafta tamamen karanlık uygulamasında aşı kaynaşma yerinden alınan
enine kesitler. Rs, anaç (P. elaeagrifolia çöğürü); Sc, ara anaç (OHxF 333); Xy, ksilem; Ph, floem; C, kambiyum;
Pa, parankimatik hücre. Aşı kaynaşma yerinde parankimatik hücreler Bar= 20 µm (A), kambiyum hücreleri, yeni
farklılaşmış ksilem ve floem hücreleri Bar= 20µm (B)

Figure 4- Transversal sections of the graft union in 2 weeks complete darkness treatment after 8 weeks of
micrografts. Cambial ring and newly differentiated xylem and phloem cells Bar= 20 µm Rs, rootstock (P.
elaeagrifolia seedling); Sc, scion (OHxF 333 interstock); Xy, xylem; Ph, phloem; C, cambium
Şekil 4- Mikroaşılamadan 8 hafta sonra 2 hafta tamamen karanlık uygulamasında aşı kaynaşma yerinden
alınan enine kesitler. Kambiyum halkası ve yeni farklılaşmış ksilem ve floem hücreleri Bar= 20µm Rs, anaç (P.
elaeagrifolia çöğürü); Sc, ara anaç (OHxF 333); Xy, ksilem; Ph, floem; C, kambiyum

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Armutlarda OHxF 333 / Pyrus elaeagrifolia Ara Anaç / Anaç Kombinasyonunun in vitro Mikroaşılarında Morfolojik..., Dumanoğlu et al

4. Conclusions vera L. var. Siirt, on wild pistachio rootstocks. Journal

of Molecular Cell Biology 5: 25-31
In vitro micrografting technique was successfully
Conejero A, Romero C, Cunill M, Mestre M A, Martínez-
applied to create ‘OHxF 333’ / P. elaeagrifolia
Calvo J, Badenes M L & Llácer G (2013). In vitro
seedling combination. Thus, with mass shoot-tip grafting for safe Prunus budwood exchange.
micropropagation it is possible to shorten the Scientia Horticulturae 150: 365-370
required time for production of a pear rootstock
Dobranszki J & Silva J A T (2010). Micropropagation of
consisted of rootstock and interstock which is apple - A review. Biotechnology Advances 28: 462-
clonal, semi dwarf, tolerant to Fe-chlorosis, salinity 488
and drought stresses, and resistant to fireblight and
Edriss M H & Burger D W (1984). Micro-grafting shoot-
pear decline less than a year. This is the first report of tip culture of Citrus on three trifoliolate rootstocks.
in vitro micrografting on Pyrus elaeagrifolia Pallas Scientia Horticulturae 23: 255-259
wild pear, and micropropagation of rootstock plants Elivar D E & Dumanoğlu H (1999). Ayaş (Ankara)
which are micrografted with interstock in vitro. koşullarında elma, armut ve ayvada bir yaşlı fidan
Abbreviations and Symbols üretiminde ilkbahar sürgün ve sonbahar durgun göz
aşılarının karşılaştırılması. Tarım Bilimleri Dergisi
C cambium 5(2): 58-64
MS murashige & skoog Espen L, Cocucci M & Sacchi G A (2005). Differentiation
Pa parenchymatous and functional connection of vascular elements in
Ph phloem compatible and incompatible pear / quince internode
micrografts. Tree Physiology 25: 1419-1425
Rs rootstock (P. elaeagrifolia seedling)
Estrada-Luna A A, Lopez-Peralta C & Cardenas-Soriano
Sc scion (OHxF 333 interstock) E (2002). In vitro micrografting and the histology of
Xy xylem graft union formation of selected species of pricly
pear cactus (Opuntia spp.). Scientia Horticulturae 92:
317-327
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