Turk J Biol

27 (2003) 95-100
© TÜB‹TAK

In Vivo and in Vitro Micrografting of Pistachio,

Pistacia vera L. cv. “Siirt”

Ahmet ONAY*, Vedat P‹R‹NÇ, Filiz ADIYAMAN, Çi¤dem IfiIKALAN, Engin T‹LKAT, Davut BAfiARAN
Department of Biology, Faculty of Science and Literature, University Dicle, Diyarbak›r - TURKEY

Received: 08.10.2002

Abstract: In this study, the success of in vivo and in vitro micrografts of pistachio (Pistacia vera L. cv. "Siirt") materials are
presented. The only variable tested was age (1, 5, 10, and 30-year-old trees). Ten- to 12-day-old axenic seedlings germinated in
vitro or seedlings (3 to 5 months-old) grown in pots in vivo were used as rootstocks. Shoot tips collected from the four age classes
of mature trees of pistachio were the source of scions. Firm contact between the scion and rootstock was assured through the use
of parafilm tape at the graft junction for in vivo micrografts. The in vivo micrografting system provided good growth and
development for new axillary shoots. These plantlets were successfully transplanted and no problems were encountered with the
establishment of micrografted plants in soil. The recovery of microscions was slow, but the use of micrografts onto herbaceous
rootstocks proved a useful technique.

Key Words: Micrografting, Pistacia vera L., Vegetative propagation

Antepf›st›¤›n›n (Pistacia vera L. cv. "Siirt") ‹n Vitro ve ‹n Vivo Mikro Afl›lanmas›

Özet: Bu çal›flmada Antep f›st›¤›n›n (Pistacia vera L. cv. "Siirt") in vitro ve in vivo mikro afl›lanmas› araflt›r›ld›. Çal›flmada farkl› yafl
grubu a¤açlardan (1, 5, 10, 30 y›ll›k) al›nan mikroçeliklerin in vivo ve in vitro ortamda afl› tutma oranlar› rapor edildi. ‹n vitro mikro
afl›lamada, laboratuvarda sterilize edilen tohumlardan, Murashige ve Skoog (MS) besi ortamlar›nda çimlendirilen 10-12 günlük
fideler anaç olarak kullan›l›rken, in vivo mikro afl›lamada 3 ayl›k fideler anaç olarak kullan›ld›. ‹n vivo mikro afl›lamada, çelik ile anaç
aras›ndaki destek ve kaynaflmay› sa¤lamak için parafilm bant kullan›ld›. ‹n vivo mikro afl›l› fidelerde daha fazla sürgün olufltu¤u ve
daha iyi büyüme gözlendi¤i gibi tarla koflullar›na aktar›ld›ktan sonra geliflmelerine devam ettiler. Yumuflak dokulu genç anaçlarda
mikroçeliklerin (meristemlerin) geliflmesi de¤erlendirildi¤inde, Antep f›st›¤›n›n klonal ço¤alt›m› için faydal› bir teknik olabilece¤i
kan›s›nday›z.

Anahtar Sözcükler: Mikro afl›lama, Pistacia vera L., Vejetatif ço¤altma

Introduction solution to the problems of clonally propagating elite

Pistachio, a member of the Anacardiaceae family
which includes 11 species of the genus Pistacia, is a semi-
tropical nut tree (1). Being a natural outbreeder and wind
pollinating, the establishment of clonal orchards raised
from superior genotypes is desirable for crop
improvement. Since pistachio is difficult to propagate by
cuttings, clonal propagation is achieved by grafting buds
from elite clones onto heterozygous rootstocks. As the
species is dioecious, it is common to see male and female
scions grafted onto one rootstock. Incompatibility
between rootstock and scion frequently necessitates
intergrafting. Micrografting thus offers a possible
pistachio varieties.
Micrografting was developed in the 1980s (2,3) and
consists of the placement in aseptic conditions of a
miniaturised scion onto an in vitro or in vivo grown
rootstock. The results of in vitro micrografting and the
plant material deriving from it can be further cultivated in
tissue culture conditions, or acclimatised to outdoor
conditions. In addition to the benefits of traditional
grafting, micrografting shoot tips can be an efficient
means of regenerating plant material free of endogenous
contaminants (4) and with enhanced potential for true-
to-type cloning mature plants (5) the possibility of

* Correspondence author

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In Vivo and in Vitro Micrografting of Pistachio, Pistacia vera L. cv. “Siirt”
micrograft less differentiated shoot tip tissues may also
help in reducing compatibility problems between scion
and stock (3).
Micrografting has been applied in the improvement
and rejuvenation and/or reinvigoration of other tree
species (6-10). Pistachio has benefited little from the use
of this relatively new approach. The available information
on micrografting in this species concerned the first
attempt to rejuvenate mature materials by micrografting
in vitro mature scion shoot tips onto juvenile Pistacia vera
rootstocks (11). In this study, the very slow growth of
the grafted scion was observed and no elongation was
obtained. Pistachio has also been reported to have been
micrografted in vitro as well as in vivo (12), but no
rejuvenation was reported when elite mature trees were
used as scions. The benefits of applying in vivo and in
vitro micrografting to the pistachio are obvious, especially
when considering the need to improve the genetic quality
of the planting stock of this slow-growing species while
increasing crop potential. Application of in vitro
micrografting for the improvement of pistachio would
require the establishment of in vitro shoots derived from
mature trees as a source of scion, in vitro germinated
seedling rootstock and techniques of grafting these in
vitro and in vivo. Axillary shoot proliferation and
organogenesis from juvenile pistachio has been achieved
by many researchers (13-15). The establishment of in
vitro shoots from mature tree tissues was a slow process
(16) but juvenile tissues responded faster and axillary
shoots were proliferated (15). Possible ways forward are
to regenerate clones via in vivo and in vitro micrografting
induced in material explanted from mature and proven
elite clones.
The capacity of in vivo and in vitro micrografting
juvenile-mature P. vera trees was therefore investigated
and the results presented here for the clonal propagation
of pistachio from elite-mature trees represent the first
stage of a study of micrografting.
Materials and Methods
Establishment of the rootstock
In in vitro studies, freshly picked mature dry nuts
were used to raise in vitro seedlings that were used as
rootstocks. Mature kernels, from which the outer
pericarp and shells had been removed, were sterilised by
a 20% (v/v) sodium hypochlorite solution for 30 min. The
96
testas were then removed and the kernels washed three
times with sterile distilled water before being placed in
contact with the culture medium. After removal of the
seed coat and partial trimming of the cotyledons, the
embryos were cultured singly in Magenta GA7 vessels
(Chicago Corp.) containing 50 ml of MS (M-0404, Sigma
Ltd.) medium with Gamborg vitamins with 20 g/l sucrose
and 7 g/l agar. All cultures were incubated at a 25 °C/16
h photoperiod. Ten- to 14-day-old in vitro seedlings with
an open leaf were used after decapitation above the
cotyledons as rootstocks in micrografts. In in vivo studies,
rootstocks consisted of 3- to 5 month-old P. vera
seedlings grown under glasshouse conditions in a mixture
of soil and sand (1:1; v/v).
Scion source
Different age classes (1-, 5-, 10- and 30-year-old-
trees) of plant material scions were collected from
pistachio trees of the "Siirt" cultivar grown in an orchard
in Diyarbak›r province, south-east Turkey. Juvenile plant
material scions originated from 10-12 month-old
seedlings, container cultivated in the greenhouse close to
mature trees. This was adopted especially since the main
objective of the current investigation was to develop a
working system which could be used in rejuvenating
and/or invigorating mature pistachio material. Sample
collections were performed on the same date
simultaneously for the four age classes. In in vitro
micrografting, the 10-15 mm shoot tips were surface-
sterilised by a 30-min soak in 20% NaOCl. After 4-5
rinses in sterile distilled water, the shoot tips (5-10 mm)
were micrografted directly or they were cultured in
Magenta GA7 vessels containing 50 ml MS medium with
Gamborg vitamins with 200 mg/l casein hydrolysate, 1
mg/l BA and 30 g/l (w/v) sucrose. The regenerated shoots
from the forced shoot tips were micropropagated, and
subcultures were made every 3 weeks over a 1-year
period. The scion preparation procedures used were
similar to those described for in vitro micrografting (see
above) except that scions were 5-10 mm in length. Scions
from the four age classes were used either directly or
after BA (1 mg/l) treatment on the cut surfaces of scion
and stock. Grafting was performed in March 2002 in
Diyarbak›r.
In vitro micrografting method
Slivers of a stainless razor blade mounted on a handle
were used for cutting the plant material. Freshly picked
current year shoot tips and in vitro regenerated shoot tips
 A. ONAY, V. P‹R‹NÇ, F. ADIYAMAN, Ç. IfiIKALAN, E. T‹LKAT, D. BAfiARAN

were used as microscions. To prepare the initial scions of Table 1. In vivo and in vitro micrografting success rates.
the micrografts, the leaves from those portions were
Micrografting1
eliminated, and two wedge-shaped oblique cuts were
made in their basal parts. Explants were previously Age In vitro success rate In vivo success rate
surface sterilised by soaking in a 20% sodium classes
hypochlorite solution for 20 min and rinsed twice with Direct Regenerated shoots Direct Forced shoots
sterile water. After 4 weeks of culture, apical tips were
micropropagated. The rootstock was decapitated to 1 14/20 18/20 18/20 19/20
5 9/20 13/20 13/20 7/20
remove all leaves and a vertical slit was made on the
10 5/20 14/20 7/20 2/20
stump; the scion base, cut in a v-shape, was fitted to the 30 4/20 6/20 3/20 0/20
slit.
1
Twenty micrografts were performed for each treatment.
In vivo micrografting method
Freshly picked current year shoot tips were directly or
when the regenerated shoots were used in vitro, whereas
after a 1 mg/l BA treatment used as scions. To ensure a
proper matching of stem size between scions and using scions from 30-year-old trees most of the grafts
rootstocks, plant material scions for all tested age classes failed. The main visible effects of the four age classes:
were collected from the basal part of shoots arising from poor elongation of the scions was frequently observed
the lower branches of the trees. Regardless of age class, and a few graft unions showed axillary shoot
scions consisted of one apical tip, 5-10 mm in length. The development on the scions explanted from 10- or 30-
basal part of the tips trimmed to form a “V” 2-3 mm in year-old trees (Figure 1). At this stage, the decapitated
length was then inserted into the vertical slit made in the roots did not grow either, but instead became dark and
central part of the rootstocks. Parafilm tape was used to necrotic.
tie the scion to the stock. The grafted stock was then Throughout in vivo studies, the best results were also
placed under 50% shade with intermittent moisture over obtained with scions from 1-year-old trees, which gave a
3 to 4 weeks to avoid any desiccation damage until the 95% successful graft rate, when the forced 1-year-old
scion was successfully established.
Evaluation of in vivo and in vitro micrografting
success
The in vivo micrografting success rate was established
6 weeks after grafting by recording the number of scions
still alive out of the 20 grafts performed for the four age
classes. Elongating scions were also recorded and
measured for each sample 2 months after the date of
micrografting.

Results
Table 1 shows that the in vivo and in vitro
micrografting of pistachio was greatly influenced by the
age of the scion used. The success levels obtained
decreased with the age of the scions in both types of
micrografts. A good result was obtained with the scions
of 1-year-old trees, which gave a 70% successful graft
rate when the shoot tips were used directly as
microscions in in vitro studies. In addition, on average
Figure 1. Shoot tips of P. vera L. cv. "Siirt" micrografted onto P.
90% of scions coming from 1-year-old plant material vera L. seedling rootstocks grown in vitro after 4 weeks
were successfully grafted and developed into new shoots in culture.

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In Vivo and in Vitro Micrografting of Pistachio, Pistacia vera L. cv. “Siirt”

Figure 2. 30-year-old P. vera L. cv."Siirt" shoot tips micrografted onto P. vera L.

seedling rootstocks in vivo after 8 weeks under in vivo conditions.

Table 2. Comparative mean shoot length 2 months after in vivo and in vitro
micrografting.

Micrografting1

Age In vitro success rate In vivo success rate

classes
Direct Regenerated shoots Direct Forced shoots

1 7.78 ± 0.62 10.61 ± 0.60 10.38 ± 0.49 8.28 ± 0.77

5 6.66 ± 0.60 10.14 ± 0.61 10.62 ± 0.34 7.80 ± 0.86
10 5.14 ± 0.50 7.2 ± 0.65 10.28 ± 0.68 4.00 ± 1.00
30 4.00 ± 0.40 5.4 ± 0.44 7.33 ± 0.88 0.00 ± 0.00

1
Twenty micrografts were performed for each treatment.

shoot tips were used as microscions, whereas all of the
grafts failed when using scions from 30-year-old trees.
After 8 weeks of micrografting, most of the grafts
already had elongated shoots and new axillary shoots
(Figure 2). Of the initial 20 micrografts, 90%, 65%,
70% and 30% established successful graft unions when
the microsicons of the different age classes were grafted
directly onto 1-year-old rootstocks. This indicated
differences between the different age classes and
applications as regards their capacity for in vivo
micrografting.
The mean shoot length of in vivo and in vitro pistachio
micrografting varied according to the graft tested (Table
2). Although the growing scions from juvenile sources (1-
and 5-years old) were generally observed to elongate
faster than their homologues from the mature plants
(10- to 30-year old), there were differences in terms of
scion length between the four age classes 2 months after
micrografting. In general, in both in vivo and in vitro
micrografts the mean elongation of scions recorded was
similar over 8 weeks. Few axillary shoots developed on
scions explanted from 1- or 5-year-old trees. In in vitro

98
 A. ONAY, V. P‹R‹NÇ, F. ADIYAMAN, Ç. IfiIKALAN, E. T‹LKAT, D. BAfiARAN
micrografts, no problems were observed during union
formation and development under in vitro conditions, but
after ex vitro transplantation there was a less than 50%
survival rate on the micrograft for all age classes.
However, for in vivo micrografts, no problems were
observed after ex vitro transplantation.
Discussion
Our study indicates that although pistachios can be
grafted either in vivo or in vitro, the success rates are
highly dependent upon the age of the donor tree. In vivo
and in vitro micrografts usually fail through
incompatibility between stock and scion, oxidative
browning of cut surfaces, poor contact or poor
development of the root system (3,17).
The aim of this study was to find a micrografting
method provide suitable for producing rejuvenated elite
materials of P. vera shoots, which have been reported
to be recalcitrant as far as vegetative manipulation is
concerned (18-20). This fact has also been confirmed
by the results of studies on in vitro rooting (13,15,21).
A successful preliminary in vitro micrografting
technique was reported for P. vera cv. Mateur by
Abousalim and Mantell (12). High levels of graft union
were achieved when the regenerated shoots of 4-year-
old P. vera cv. Mateur were grafted onto in vitro raised
seedling rootstock. It was reported that poor
elongation of the scion was observed and no axillary
shoots developed on the scions (12). However, in this
study we have reported successful micrografts with
shoots derived from 10- to 30-year-old-trees using
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