TISSUE CULTURE RESULTS OF ALL WILD PISTACHIO SPECIES AND
SOME CULTIVARS IN IRAN
B.Sh. Behboodi
Department of Biology, Faculty of Science
University of Tehran
Iran
Keywords: Tissue culture, pistachio, wild species, cultivars, Pistacia vera, P. atlantica, P.
khinjuk
Abstract
Pistachio wild forests are distributed throughout most parts of Iran. Our country is
the first producer and exporter of pistachio nuts in the world. It is estimated that there are
more than 55 million pistachio trees in Iran. The pistachio is dioecious and for this reason
it has acquired large genetic diversity. Pistachio plants cannot be readily propagated from
cuttings taken from mature trees and are currently propagated by grafting. Therefore,
pistachio tissue culture, which produces calluses and shoots and finally suitable plants, is
proposed as a new and advanced method. This research has been conducted on: Pistacia
khinjuk, P. atlantica subsp. mutica, kurdica and cabulica, and P. vera from Sarakhs and
cultivars 'Akbary', 'Kalleghochi' and 'Fandoghi'. MS medium and different concentrations
of auxins: IBA, NAA, 2,4-D, and cytokinins: BAP, Kin were used. Explants were selected
directly form apical and nodal buds of mature plants and also from cotyledons, shoots and
apical buds of seedlings. The resulting calluses and shoots were studied comparatively.
The best results of callogenesis were obtained using NAA and BAP. BAP and IBA
resulted in maximum growth and shoot formation. Among the wild and cultivated
pistachio studied in our experiments, P. vera from Sarakhs showed maximum stem and
apical growth.
1. Introduction
There is no doubt that the native land of the domestic pistachio is the eastern part
of Iran and that the first pistachio trees were cultivated by Iranians. Today nearly 55
million domestic pistachio trees are grown in Iran. One of the reasons behind the great
genetic diversity in the pistachio is its dioecious nature. Pistachio cultivars cannot be
propagated readily by simply cutting a mature plant, and today, grafting has become a
popular method. This is why tissue culture has been proposed as a new and more
advanced method. Through culture, callus, shoot and finally the suitable plant can be
obtained. Wild pistachio forests are scattered throughout the vast country of Iran.
Unfortunately, they have undergone disastrous periods and no research has been
conducted on tissue culture. This study attempts to examine the effect of different culture
media and a variety of growth hormones on the samples of all wild trees and some
cultivars of domestic trees, and thus aims to identify the best method for creating callus
organogenesis for each species.
2. Materials and methods
2.1. Plant material
Pistacia khinjuk, P. vera and P. atlantica wild species, and subspecies: mutica,
kurdica, and cabulica were studied; as well as 'Fandoghi', 'Akbari' and 'Kalleghotchi'
cultivars. The useful organs included were: embryo-stem apical meristem, stem, leaf and
cotyledon, which were obtained from both old plants and seedlings.
2.2. Media and hormones
Proc. 3rd IS on Pistachios and Almonds
Eds. I. Battle, I. Hormaza & M.T. Espiau
Acta Hort. 591, ISHS 2002
399
MS, DKW, WP and LS were used as basic media for this study. Different
concentrations of auxin and cytokinin including IAA, IBA, 2,4-D, BAP and Kin were
tested on the above-mentioned samples.
2.3. Sterilization and culture
Mercury chloride at 1% was used for 15 minutes in order to sterilize the tissue
sections. This compound not only eliminated contamination but also reduced the
oxidation of phenolic compounds. Because of the oxidation of phenolic compounds in the
culture, new samples were taken every 7 days. Initially, the samples were left in the dark
for 30 days, then transferred and left 16 hours under light conditions, left where they were
then in the dark at 25o for 8 hours.
3. Results and discussion
3.1. Apical meristem culture
Generally, the apical meristem of mature pistachio hardly grows, and regenerates
due to its high degree of contamination and oxidative phenol compounds. The apical
meristem of the 'Akbari' cultivar grew better in the MS medium which had no hormone
supplement. The 'Kalleghoghi' cultivar started producing callus in the MS medium
without hormone supplement. With increasing NAA hormone in this medium the amount
of callogenesis increased and reached a maximum of 0.5 mg/l of NAA. In the Sarakhs
seedling meristem in the MS medium containing 0.1 mg/l of IBA and 2 mg/l BAP, first
the callus and then the vegetative buds were formed. This result was also obtained in the
culture medium containing 0.5 mg/l of NAA and 2 mg/l of BAP. However, the use of the
NAA hormone increased necrosis at the stem apex. These results agree with those of
Barghchi and Alderson (1985) and Yong and Lodders (1993). The apical meristem of the
wild subspecies mutica and kurdica were grown in the LS medium containing 4 mg/l of
BAP and the leaves were grown in the culture medium. These results agree with Barghchi
and Aldergon (1983 and 1985), the difference being that they worked with domestic
cultivars. The mutica meristem was grown in the LS culture containing 2.5 mg/l IBA and
the subspecies kurdica was grown in the LS culture containing 1.5mg/l IBA. However,
unlike Al Barazi et al. (1982) and Barghchi and Alderson (1983) who claimed that the
best growth results were obtained in a medium containing 2.5 mg/l IBA for rhizogenesis
of domestic cultivars, we did not observe root growth in these two subspecies in these
media. In the DKW culture medium, seedling apical meristems of mutica and khinjuk
grew directly and phenol oxidation compounds formed with some delay.
3.2. Stem culture
Sarakhs seedlings in MS culture containing 0.5 mg/l NAA and 2 mg/l BAP
proceeded with callogenesis and budding. This result does not confirm the records of
Yong and Ludders (1993) who claimed that P. vera gave the best results with 1 mg/l of
BAP and 5 mg/l of NAA. The genetic difference between Sarakhs P. vera and the
domestic P. vera is probably the cause of this difference. Stem explants of mutica in the
LS medium containing 4 mg/l BAP showed the maximum growth. With decreasing BAP
this growth was decreased and with the increase in BAP concentration callogenesis began.
This agrees with the results of Barghchi and Alderson (1983 and 1985). For this
subspecies the stem section in the LS culture without hormone showed the best
callogenesis. After adding 2,4-D to the medium, callogenesis was not observed. The
results from the kurdica subspecies stem culture showed that the best callogenesis occurs
in the LS culture containing 6 mg/l of BAP, but calluses go through necrosis over time.
Martinelli (1989) claimed that high concentrations of BAP at the beginning of culture
increased cellular division. In this study a decrease in growth was observed followed by
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necrosis.
3.3. Cotyledon culture
Cotyledons from the cabulica and muticu subspecies showed maximum
callogenesis in hormone-free LS culture medium. Increasing 2,4-D only increased the
cotyledon volume. If 0.5 mg/l BAP is added to the subspecies khinjuk, a callus is
produced and after some time organogenesis starts and buds and roots are formed. In
'Fandoghi' cultivar the best callogenesis is obtained from the LS medium containing 0.5
mg/l 2,4-D and 0.5 mg/l Kin.
3.4. Leaf culture
The NAA hormone caused callogenesis and rhizogenesis in all the wild cultivars
and species, however IBA hormone failed to cause rhizogenesis. These results contradict
those by Barghchi (1983) who claimed that IBA was the best hormone for rhizogenesis
induction. There are other researchers who claimed that using NAA in 1 mg/l
concentrations is useful for rhizogenesis (Bustamante et al., 1982). In our experiments in
the LS medium the best and fastest rhizogenesis results were obtained when the WAA
hormone was applied at 2 mg/l concentrations. The Sarakhs leaves cultured in the LS
medium containing 0.1 mg/l of NAA and 6 mg/l BAP first formed calluses and then grew
into leaves. We observed that in an LS medium containing 1 mg/l NAA and 4 mg/l BAP
formation of calluses eventually lead to root formation. There is no mention of
rhizogenesis of the Sarakhs species in former papers.
4. Conclusion
With regard to the wide scope of this research and the heterogeneity of past
reports, we can conclude that genetic diversity in the cultivars and species found in
different geographical areas leads to diverse results in pistachio tissue culture. Our results
disproved reports concerning the callogenesis effects of 2,4-D hormone, in the P.
atlantica species. According to Bailis (1977) it seems that this hormone can act as an
inhibitor in the G1 or G2 cellular division phases. Increase of NAA in pistachio causes an
increase in the oxidative phenolic compounds. On the contrary, increasing BAP decreases
these compounds and lowers callus necrosis. Another point is that increase in callogenesis
is incoherent with rhizogenesis increases. At times when the medium or hormone
compounds are not favourable for the culturing, explants turn brown at the beginning of
the culture. Probably this tissue is the result of the aggregation of phenolic compounds in
cells and a factor leading to growth decrease and early senescence in cells that are put
under stress. Probably just like Rijkenberg and Bulton (1977) mentioned for the citrus
plants, this is the result of intercellular metabolic changes that have not yet been
completely identified for Pistacia tissues. This is a major practical problem in Pistachio
tissue cultures.
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Fig. 1. Leaf explant of Sarakhs in LS medium containing 6 mg/l BAP.
Fig. 2. Leaf explant of 'Fandoghi' in LS medium containing 2.5 mg/l IBA and 6 mg/l BAP.
Fig. 3. Leaf explant of 'Akbari' in LS medium containing 0.1 mg/l NAA and 4 mg/l BAP.
Fig. 4. Leaf explant of subsp. mutica in LS medium containing (0.5 mg/l) NAA and 4
mg/l. BAP.
Fig. 5. Leaf explant of subsp. mutica in LS medium containing 2 mg/l BAP after 2
months.
Fig. 6. Callogenesis in leaf explant of subsp. mutica in LS medium containing 2 mg/l
NAA and 6 mg/l BAP, 2 months after culture.
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Fig. 7. Apical meristem of Sarakhs seedling in MS medium containing 0.1mg/l IBA and
2 mg/l BAP.
Fig. 8. Apical meristem of subsp. mutica in LS medium containing 4mg/l BAP.
Fig. 9. Callogenesis and organogenesis in stem explant of Sarakhs in MS medium
containing 0.5 mg/l NAA and 2mg/l BAP.
Fig. 10. Apical meristem of kurdica in LS medium containing 4 mg/l BAP.
Fig. 11. Cotyledon explant of subsp. mutica in LS medium without hormone.
Fig. 12. Cotyledon explant of kurdica in LS medium containing 0.5 mg/l IBA.
Fig. 13. Cotyledon of 'Fandoghi' in LS medium containing 1.5 mg/l 2,4-D and 0.1 mg/l
kin.
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