Salih Parlak

Introduction

Mastic tree(Pistacia lentiscus var.chia)grows only on the coast of the island of Chios and the C?es?me Peninsula,Turkey,and has ecologically specific requirements.The C?es?me Peninsula is now on the verge of distinction,with only 250–300 trees remaining.C?es?me and its coasts are known to be home to mastic;it was taken to Chios later grafted.Mastic from P.lentiscus var.chia was a commercial commodity in 3000–4000 B.C.proven by shipwreck recoveries(Perikos 1993).

The therapeutic nature of mastic has also been known since the early ages,and the resin obtained from mastic is a natural antioxidant;therefore,it is used in cosmetics and pharmaceuticals,the food and spirit industry,and the health field.

It is traditionally used to treat problems such as abdominal pain,indigestion,gastric and duodenal ulcers,stomach problems,diabetes,cancer and tumours and to make oral antiseptic to treat dental diseases,ointments for burns and wounds,and vitamins(Al Said et al.1986;Perikos 1993;Dabosa et al.2010;Aissi et al.2016).It is also used to manufacture high quality painting dyes,lacquer for musical instruments,cosmetics and perfumes(Baytop 1968;Duke 1985;Yazgan et al.1986;Fu et al.1998;Assimopoulou et al.2005;Triantafyllou et al.2007;Balan et al.2007;Dabosa et al.2010;Glampedakia and Dutschk 2014).

Since female mastic trees do not yield high quality mastic,clonally propagated male trees are used to establish plantations and produce commercial mastic(Chenopoulos 1961).Clonal propagation is through cutting or grafting conventionally or tissue culture at laboratory,and each method has its own advantages and disadvantages.For example,some researchers reported that cuttings from Pistacia species had a very low rooting rate(Al Barazi and Schwabe 1982).The reason for rooting problems is associated with the fact that physical conditions cannot be optimized during the propagation of Pistacia species through cuttings(M?s?rl? et al.2002).Cuttings from 1-year shoots of the mastic trees yielded 76.6%rooting success(I˙sfendiyaroglu 1999,2000;Tas?k?n and I˙nal 2005)however;this success rate could not be extended to mass production in nurseries and greenhouses.

Propagation with tissue culture has failed due to the oxidation of phenolic compounds in the plant(Martinelli 1988;M?s?rl? et al.2002;Tas?k?n and I˙nal 2005).The classic immersion is not appropriate for mass seedling production,but other immersion methods have not been tried.

Nor has grafting,another clonal propagation method,on P.lentiscus in the field yet been successful(Whitehause 1957;Acar 1989;I˙sfendiyaroglu and Karak?r 1999).

Soil and climate conditions are the main factors that render the habitat and growing conditions of mastic special.Chios and C?es?me Peninsula provide a natural shelter against north winds,have a lot of sunlight during the day but cool winds at night,and their temperature rarely drops to minus degrees during the winter.These constraining climate conditions limit the distribution of mastic.

Mastic tree,a symbol of immortality known and used since the early ages,is unfortunately on the verge of disappearing due to the environmental pressure of many factors such as tourism and construction activities,new agricultural areas,new summer houses and loss of the old production culture.Clonal propagation is critical to protect the species in its natural distribution environment,revitalize its production potential and produce.

Male trees for high quality mastic,in commercial plantations thus,studies in this field are vital.The present study is the first one aimed at growing seedlings in a nursery after grafting mastic on species of Pistacia.

Materials and methods

Materials

Pistacia lentiscus and P.atlantica rootstocks(13–25 mm diameter)and scions originating from C?es?me were used in the study.

Preparation of grafting nursery

Before the rootstocks were planted,black polyethylene film was placed on the bed to prevent weed growth.Grafts,were shaded from direct sunlight by burlap that had a shade effect of 70%.A HOBO U-10 Model digital recording device was used to record the ambient humidity and temperature at every 20 min.Typical cultural maintenance such as shoot control,daily irrigation and weed control were practiced.Graft union formation and bud burst percentages were observed.Insecticides were used to kill aphids found on mastic buds that had started sprouting.

Origin and storage of scions

The scions used for grafting were taken from trees on the C?es?me Peninsula were used for mastic production.They not different from genetically those on Chios(Acar 1989).Scions were cut and transported using standard procedures(Childers 1995;Hartmann et al.1997).They were cleaned with tap water and soaked in sodium hypochlorite(1/10 in water)for 5 min and then rinsed with tap water.After that,all scions were soaked three times in fresh water for 5 min for thorough rinsing.Since the papers used for wrapping the scions had to be damp rather than wet,the scions were finally kept in a fungicide solution for 5 min and wrapped in damp paper,each holding 20 scions.Each bunch of scion was placed in two layers of polyethylene bags,which were then tightly tied.Since scions could be stored in refrigerators at 2–4 °C for 2 months(ozcan 1988;Garner 2007;Hartmann 1981),they were stored in the refrigerator until use.Because the lower buds of shoots in evergreen species that do not burst bud can be used for grafting(Hartmann et al.1997),those scions stored in refrigerator were used in T buds and chip buddings until 1 May,after which buds from the previous year that did not take were used.

Grafting methods

Since the physiology of trees changes during the year,the rate of graft union formation also varies(Hartmann et al.1997).Therefore,two Pistacia species were used as rootstocks to determine the best grafting time;20 grafts were placed in two replications every 15 days from 15 February to 15 October.T budding,cleft grafting and chip budding(Fig.1)were tested to determine the best method.

Scions were taken through cleft grafting from 15 February to 15 April.Since the rootstocks should have begun cambial activity and scions should not during the grafting(Hartmann et al.1997),scions that had been stored in the refrigerator were used.After grafting,they were wrapped with plastic graft tape,and the graft junctions and tips of scions were covered with grafting wax.A sapdrawer branch was retained on the rootstock to allow photosynthesis and prevent drying of the rootstock.

Fig.1 Grafting methods used in the study.a Split grafting;b chip budding;c T-budding

Chip budding,in which the bark is removed with a piece of wood and inserted into a cut made in the same size on the rootstock,is one of the most appropriate methods for grafting forest trees because it enables grafting over a longer period and does not require bark that‘slips’;thus,it can be performed in spring and fall when weather conditions are suitable for grafting(Hartmann et al.1997).Further,it can be easily performed when bark grafting is not suitable(Garner 2007;C?aglar and Kas?ka 1996).Chip budding can be done from 15 February to 15 October.

In T-budding,the bark of the rootstock is cut in a T shape without damaging the cambium,then raised and stripped carefully with a budding knife.After the bud is stripped properly,it is placed under the bark that was raised,and tied.This grafting method should be applied when the cambium is active in both the rootstock and scion.For successful T-budding,the bark needs to ‘slip’,that is,easily strip from the rootstock thus,indicating cambial activity.If the bark does not slip,grafting will most probably fail(Southwick 1979).In this respect,timing is very important for T budding.Buds can be grafted to rootstocks with a diameter of 6–26 mm.Since a plant can potentially be obtained from every bud eye,this method is more economical than scions(Hartmann et al.1997).In our study,T-budding could not be performed after 1 June,because the rootstocks of P.lentiscus and P.atlantica do not slip after that date.

Because grafting is appropriate at a height of 10–20 cm from the ground(Hartmann 1981),buds were grafted to the flat and smooth part of the rootstock at a height of 10–15 cm from the root collar to ensure easy handling and healthy growth of buds that took.

Statistical analyses

We used a two-factor randomized block design with grafting methods combined over the date of grafting and an analysis of variance(one way ANOVA)to access signif icant(P＜0.05)differences bud burst or not.The Duncan test was applied to create homogenous groups.SPSS(15.0)software package was used for all statistical analyses.Bud burst percentages for T budding were very low;therefore,they were not included in the assessments.Nor were the times that there was no union included in the analyses.

Results and discussion

Humidity and temperature are important for a successful scion fusion.The lowest temperature was 14.4°C(in February);the highest was 27.9°C(in July).The lowest relative humidity was 57.1%in May while the highest was 75.4%in November.Temperature and relative humidity were within the ideal range(Hartmann et al.1997)for successful callus graft(Table 1).

The highest union was achieved for grafts started on 15 February.The grafts in 15 February and 15 March were more successful than other times.Chip budding started in summer failed.The highest bud burst percentage was 55%in both rootstocks(Table 2).

The contact and union surface between the scion or bud and the rootstock differs depending on the type of graft.A callus is formed on nearly all the cut surface of the rootstock,while only a small portion of the cut surface of the scion forms a callus in grafts;therefore,the function of the rootstock is more important for a successful union(Hartmann et al.1997).The scions grafted on strong rootstocks are more robust compared to those grafted on weak rootstocks(Hartmann et al.1997;Garner 2007).To achieve a good union,the cuts on the rootstock and scion should be identical and preferably the cambium layer on both sides of the bud or scion should overlap with thecambium layer of the rootstock.If the cambium of each part overlaps well with a large contact surface,the success of graft union also increases,otherwise,only a partial union will form,and the graft will not be robust(Hartmann et al.1997).

Table 1 Mean temperature(temp.)and relative humidity(RH)during month of grafting

Accordingly,grafts on the strong rootstock P.atlantica developed better than those on P.lentiscus,a weak rootstock.The bud burst percentage for chip buddings made on P.atlantica was better than on P.lentiscus,because the surface roughness of P.lentiscus prevents the necessary overlap for a good graft.

Good grafting technique requires a long,proper cut through the surface of the rootstock and scion;if the wood tissue of the rootstock is hard,cutting and grafting are challenging.For example the branches and bark of a wild olive cultivar are hard,and the slow growth of the tree results in short internodes;a graft union barely forms(C?ec?en 1965).It has therefore been argued that a flat surface to place the bud eye is difficult to find.Any spaces between the rootstock and scion will prevent full overlap and thus prevent graft union(Anonymus 1992).

During the grafting procedure,the wood tissue of P.lentiscus was found to be harder than that of P.atlantica.On the other hand,when the bark is removed from P.lentiscus,there is little smooth surface for grafting,the surface tissue is rough and has a wart-like structure.Therefore,it is very difficult to fully overlap the bud eye toensure graft union.Since the internodes on the rootstock of P.atlantica are wide,budding is easier.Bud burst percentages for scions and chip budding were better on P.atlantica than on P.lentiscus.

Table 2 Grafting date and methods with resulting percentage bud burst on each rootstock

The analysis of variance on scions revealed no statistically significant difference in the bud burst percentage between the rootstocks and in the date×rootstock interaction.There was,however,a significant difference among the grafting dates(P＜0.01).There was a significant difference between date×rootstock interactions with respect to bud burst percentages(P＞0.05)and between the rootstocks(P＜0.001).

The highest percentage of bud burst in grafts on the rootstock of P.atlantica was obtained with grafts made on 15 February and 15 March,followed by grafts made on 1 March.The highest bud burst percentage for grafts on rootstock of P.lentiscus was obtained with grafts made on 15 February and 15 March.

An easy ‘slip’on rootstock and scions increases the success of grafting(C?ec?en 1965);therefore,graft union was not achieved in summer when the plants cambial activity slowed.A cambial activity indicated by wood slipping is important for the success of grafting,thus when cell division and cambial growth slow in summer and fall the bark is difficult to remove and the success of grafting decreases(Hartmann et al.1997).The highest percentage bud burst was obtained with grafts made on 15 February.For bud burst in general,the highest percentage was obtained in(15 February–15 March)when cambial activity was higher.The highest percentage bud burst on scions based on date was obtained for grafts made on 15 February,1 March and 15 March(Table 3).

The best grafting period for P.atlantica when used as the rootstock for grafting P.vera was reported to be between late May and late July(Kuru et al.1986).However,none of our grafting efforts for mastic were successful during that period.Formation of cambial union between the graft and scion depends on environmental conditions.Although graft union formation varies depending on the time of year,it usually takes place at 12.8–32 °C with an optimum at 26.5–29.5 °C.The frequency of callus formation increases at 4–32 °C and in a humid environment;however,the callus starts dying at temperatures over 40°C(Hartmann et al.1997;Janick 1979).

Temperature and air humidity influence cell division;therefore,it is very important for the success of grafting.The ideal ambient temperature for grafting is considered to be 15–30 °C,while lower or higher temperatures decreases callus formation(Y?lmaz 1970;Lewis and Alexander 2008).Because bark formation in trees is associated with environmental conditions and bark is formed through easy separation of thin-walled cambium cells that are activelydividing(Hartmann et al.1997),rootstocks do not slip in summer and fall and grafts made in those periods failed.

Table 3 Analysis of variance on bud burst percentages and Duncan test

Fig.2 Photographs taken at 3 months after grafting of a chip budding and b T-budding

Table 4 Recommendations for grafting methods for eachrootstocks based on date

Grafting of the rootstocks of pistachio when vegetative growth starts has been very successful,and success is lower later when the weather is warmer(Nikpeyma 1990).So unsurprisingly,grafting on both rootstocks in early spring was more successful in our study.Although the success of dormant budding in grafts of P.vera in fall was successful(Tekin et al.2001),chip budding for mastic in summer and fall was not.

As a general rule,graft tapes are untied 15–20 days after budding(Kuru et al.1986);however,removing them within 2.5–3 months is not a problem(C?aglar and Kas?ka 1996).In fact,when tapes were untied early,those mastic buds dried after a while.Therefore,although the buds burst and started sprouting,graft tapes should not be untied,and buds should be allowed to grow for 2.5–3 months(Fig.2).

Incompatibility refers to the lack of successful union of two different plants that are grafted(Hartmann et al.1997).No signs of incompatibility between the rootstock and scion or between the rootstock and bud were observed after the graft union formed and sprouting began.In the long run,however,since P.lentiscus is a rootstock with poor growth(Tekin et al.2001),it may not be able to support P.lentiscus var.chia,that forms a wider diameter,and it may break in the wind or from its own weight,which might happen when the diameter of the bud exceeds that of the rootstock.

Conclusion

We demonstrated that mastic tree can be propagated clonally through grafting and determined the best grafting method to use for the time of year.The best result was obtained using scions for both rootstocks.The highest bud burst percentages resulted using the rootstock of P.atlantica and chip budding,while the rootstock was not effective in scions.The success of T budding was low at all grafting dates.

Between 15 February and 15 March are the best grafting dates for chip budding and scion grafting on rootstock of both P.atlantica and P.lentiscus.Chip budding was not successful after 15 March.Recommendations for grafting and dates are summarized in Table 4.

Signs of incompatibility were not observed in the graftings or buddings;however,buds grafted on rootstocks of P.lentiscus and sprouted developed slower than those on P.atlantica.Since P.atlantica has strong growth characteristics,it is an appropriate rootstock for graft propagating mastic trees as long as signs of incompatibility are not observed in the long term.

Only two Pistacia species were used as rootstocks in the study,other Pistacia species should also be tested as rootstocks.Using more controlled conditions should also increase the success of grafting.

AcknowledgementsI thank the Aegean Forestry Research Institute and I˙zmir Regional Directorate of Forestry for their support of the study.

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