WFL Publisher

Science and Technology

Meri-Rastilantie 3 B, FI-00980 Helsinki, Finland e-mail: info@world-food.net

Journal of Food, Agriculture & Environment Vol.8 (2): 490-493. 2010

www.world-food.net

Graft union formation of spur apple varieties grafted on different rootstocks

Mehmet Polat 1*, Ouz Dolgun 2, Adnan Yildirim 3, M. Atilla Akin 1 and Zeliha Gkbayrak
1

Suleyman Demirel University, Faculty of Horticulture Dept. of Agriculture, Isparta, Turkey. 2 Adnan Menderes University, Sultanhisar Vocational College, Sultanhisar, Aydin, Turkey. 3 Suleyman Demirel University, Sutculer Vocational College, Sutculer, Isparta, Turkey. 4 Canakkale Onsekiz Mart University, Faculty of Horticulture Dept. of Agriculture, Canakkale, Turkey. * e-mail: ozmpolat@yahoo.com
Received 19 January 2010, accepted 6 April 2010.

Abstract
The aim of this research was to observe histological developments during the early stages of graft formation between some spur apple varieties and apple rootstocks in order to assess compatibility/incompatibility related to different growing rate of the plants used. As rootstocks, one year-old clonally propagated A2, B118, M111 (vigorous) and M7, M16, Pj80 (semi-dwarfing) were used. Scions were taken from one-year-old shoots of Scarlet Spur, Red Spur and Redchief spur apple varieties. Rootstocks were planted in the field in winter 2007 and whip-grafted in April 2008. An evident incompatibility was not observed in the combinations. Sufficient callus proliferation was visible in all the combinations. Successful cambial and vascular connection was apparent in the 90-day sections. In the 120th and following sections, it was observed that callus usually filled all the gaps between the rootstock and the scion. Xylem and phloem strands successively bridged the union. However, especially Pj80/Redchief and M16/Red Spur should be followed up in subsequent years in the field conditions because of the excessive callus formation between the stock and the scion. Key words: Apple, grafting, graft union, compatibility.

Introduction Histological developments between rootstock and scion during graft formation can provide some explanations in relation to compatibility/incompatibility. The development of a compatible graft is comprised of some major events such as cohesion of the stock and scion, proliferation of the callus cells, restoration of cambium and vascular continuity 1-3. A good histological union between rootstock and scion is necessary for the combination to be successful 4. Different histological developments and tissue organizations due to growing features of the stock and scion may influence graft union 5 and if the plants grow at different rates, grafting may fail as a result of different anatomical features and abnormal histological developments 2, 6, 7. Spur apple varieties are very fertile, therefore these varieties are preferred by apple growers in many countries. These varieties have short shoots and weak growth. On the other hand, there are many apple rootstocks that have different growing rate, ranging from vigorous to weak. According to Sitarek 8, many authors agree that the rootstock determines the success of grafting. Some of the rootstocks produce a very good junction with top varieties, but with some others, junction is poor and effectiveness of budding is very low. According to Soumelidou et al. 2, abnormal morphological features in graft unions of 1 to 2 year-old apple trees have been reported to be associated with varying degrees of dwarfism imposed by different rootstock. Therefore, before using a new dwarf rootstock, histological investigations on graft union provide us with very important information on compatibility or incompatibility.

The aim of this research was to observe the histological developments during the early stages of graft formation between some spur apple varieties and apple rootstocks in order to assess compatibility/incompatibility related to different growing rate of the plants used. Materials and Methods As rootstocks, one year-old and clonally propagated A2, B118, M111 (vigorous) and M7, M16, Pj80 (semi-dwarfing) were used. Scions were taken from one-year-old shoots of Scarlet Spur, Red Spur and Redchief spur apple varieties (Malus communis L.). Rootstocks were planted in the field in the winter 2007 and whipgrafted in April, 2008. Grafts were wrapped with artificial raffia followed by parafilm strips. Hundred plants were grafted for each combination. Grafting practices were carried out in the Experimental Orchard of the Suleyman Demirel University, Isparta, Turkey. First graft samples were taken in the second and fourth weeks, and afterwards every 30th day until 12 months passed after grafting. Samples of 3-5 graft unions from each rootstock/scion combinations were fixed in ethanol (70%). Samples were sectioned in 20-30 m thickness using a rotary microtome. Sections were colored and examined with Olympus mic-D digital microscope with coloring feature. During graft union formation five developmental stages were examined: 1) positions of necrotic layers, 2) proliferation of callus cells, 3) formation of callus bridge at the graft interface, 4) cambial continuity and 5) formation of vascular tissues. Graft samples were investigated in the laboratory facilities at the Adnan Menderes University, Aydn, Turkey.
Journal of Food, Agriculture & Environment, Vol.8 (2), April 2010

490

Results and Discussion The first 15 day samples were not cut as a complete section due to the poor connection of callus tissues. Therefore, they were taken by hand using a razor blade. In these sections of graft, combinations were not observed. Sufficient callus production and visual observations were not enough to describe histological developments. In 30-day sections, sufficient callus proliferation was visible in all the combinations. Callus which was produced by bark parenchyma and cut end cambium of scion and stock had started to spread into the gaps between the scion and the rootstock. Necrotic layer was clear in the outer sides and interfaces of the grafts. There was not a cambial and vascular differentiation. An intensive cellular activity in 60-day sections was observed. Increasing callus formation was exhibited in all the combinations. Callus formation was progressed filling the gaps between the graft members. An excessive callus formation was especially seen in Pj80/Redchief and M16/Red Spur combinations. Necrotic layer was visible in outer bark zone but necrotic layer placed in parenchymatic cortex zone had started to divide and being absorbed by the callus formation. Cambial continuity and vascular bridging were unclear in all the combinations. Successful cambial and vascular connection was apparent in 90day sections. Cambial continuity was clear. New vascular elements, xylem and phloem, were seen across the graft interface (Fig. 1). In Pj80/Redchief and M16/Red Spur combinations, cambium was crinkle because of the excessive callus formation. Necrotic layers were slightly visible along the cut surfaces. In 120th and following sections, it was observed that callus usually filled all the gaps between the rootstock and the scion. Xylem and phloem strands successively bridged the union. The graft lines were obliterated between the xylem and the bark. Necrotic layers were seen only in the outer bark regions. An abnormal histological development between stocks and scions was not observed. Abnormal histological developments related dwarfing effects and weak growth can cause incompatibility in grafting. Fruit trees are produced mainly by grafting. Some histological events or abnormalities that occurred in grafting process give us some introductory information on grafting which will be compatible or incompatible. Developmental abnormalities in graft union arise either during the formation of grafting as a response to contact between stock and scion, or during subsequent growth of the composite trees 2. Therefore, histological investigation on grafting is very important. In many histological investigations on graft formation it is underlined that first observable stage is the formation and position of the necrotic layer between stock and scion. During the grafting, as a result of cutting, some cells of scion and rootstock die and in the course of time these cells transform to dark points or mass as a result of oxidizing of cell contents. It is also notified that callus is produced from cambial cut end and cortical parenchyma of graft members 9, 10. In woody or dicotyledonous plants, grafting includes the formation of a necrotic layer and its subsequent reduction or elimination, callus growth, cohesion of stock and scion, and the differentiation of graft bridging vascular tissue and cambium, but these are independent events. While necrotic layer formation and cohesion are common in all grafts, one or more of the processes of callus formation, necrotic layer removal and vascular differentiation can be absent 11. In the present study, all processes of grafting
Journal of Food, Agriculture & Environment, Vol.8 (2), April 2010

were observed sequentially. Necrotic layers which formed as a result of cutting were seen along the cut surfaces in all the grafts, but in the course of time, necrotic layer was broken into pieces and then absorbed by the newly formed callus, especially in the cortex regions of the grafts. There was limited callus production along the xylem contact interface of graft members. Necrotic layer was kept as light dark strands or single points. It is seen that the removal of necrotic layer depended on cell division where callus was profuse. These results were in agreement with previous studies 1, 2, 9-12. In most grafts, callus production by the scion and stock is accepted as the first step of grafting. Thus, in addition to rupturing the necrotic layer that separates the stock and scion and providing cells at the graft interface through which vascular tissue subsequently differentiates 9, 13, 14 , the proliferation and interdigitation of callus cells also contribute significantly to the increase in tensile strength of grafts 1. On the other hand, cellular recognition of callus cells produced from stock and scion has an important role. Molecular alterations, deposition of some biochemical and toxins relating cells can lead to incompatibility, but this situation is generally seen in the genetically unrelated or different taxonomic group plants which have different growing features 9, 15. In this study, a good callus junction between two graft partners was obtained. In some combinations such as Pj80/ Redchief and M16/Red Spur, due to excessive callus formation, newly formed cambium was wavy. This may be result of different callus production related to growing rates. Differentiation of new vascular cambium and its continuity has also an important role in formation of the graft union. Some callus cells which received IAA (indole acetic acid) signal in newly formed callus or close to the cut ends of cambia of stock and scion transform to cambium cells 5, 16-18. After cambial continuity, generally, new cambium starts to produce vascular elements. In some case, callus cells can be directly differentiated into vascular elements in the callus 19, 20. In this study, callus formation, cambial continuity and vascular elements formation were seen in a form of sequential events. In 90-day and the following sections, vascular elements derived from new cambium were clearly visible. A direct transformation from callus cells to vascular elements was not observed. Conclusions In this study, some spur apple varieties were grafted on apple rootstocks with different growing rates and graft union was histologically investigated. An evidence related incompatibility was not observed, but the combinations, especially Pj80/Redchief and M16/Red Spur should be followed up in subsequent years in the field conditions because of the excessive callus formation between the stock and the scion. This may be a signal of the incompatibility. References
1

Moore, R. 1983. Studies of vegetative compatibility - incompatibility in higher plants. IV. The development of tensile strength in a compatible and an incompatible graft. Amer. J. Bot. 70(2):226-231. 2 Soumelidou, K., Battey, N. H., John, P. and Barnett, J. R. 1994. The anatomy of the developing bud union and its relationship to dwarfing apple. Annals of Botany 74:605-611. 3 Hartmann, H. T., Davies, D. E. and Geneve, F. T. 1997. Plant Propagation: Principles and Practices. 6th edn. Prentice Hall, Englewood Cliffs, NJ.

491

Errea, P., Garay, J. and Marin, A. 2001. Early detection of graft incompatibility in apricot (Prunus armeniaca L.) using in vitro techniques. Physiologia Plantarum 112:135-141. 5 Atkinson, C. J., Else, M. A., Taylor, L. and Dover, C. J. 2003. Root and stem hydraulic conductivity as determinants of growth potential in grafted trees of apple (Malus pumila Mill.). Journal of Experimental Botany 54(385):1221-1229. 6 Simons, R. K. and Chu, M. C. 1984. Tissue development within the graft union as related to dwarfism in apple. Acta Horticulturae 146:203210. 7 Simons, R. K. 1986. Graft-union characteristics as related to dwarfing in apple (Malus domestica Borkh.). Acta Horticulturae 160:57-66. 8 Sitarek, M. 2006. Incompatibility problems in sweet cherry trees on dwarfing rootstocks. Latvian Journal of Agronomy 9:140-145. 9 Moore, R. 1984. A model for graft compatibility-incompatibility in higher plants. Amer. J. Bot. 71(5):752-758. 10 Dolgun, O., Tekintas, F. E. and Ertan, E. 2008. A histological investigation on graft formation of some nectarine cultivars grafted on pixy rootstock. World Journal of Agricultural Sciences 4(5):565-568. 11 Stoddart, F. L. and McCully, M. E. 1980. Effects of stock and scion organs on the formation of the graft union in Coleus: A histological study. Botanical Gazetta 141(4):401-412. 12 Tekintas, F. E. and Dolgun, O. 1996. An investigation on compatibility in some peach and nectarine cultivars grafted on almond seedlings. Yuzuncu Yl Univ. Journal of Agric. Faculty 6(1):51-54. 13 Moore, R. and Walker, B. 1981a. Studies of vegetative compatibilityincompatibility in higher plants. I. A structural study of a compatible autograft in Sedum telephoides (Crassulaceae). Amer. J. Bot. 68:820830. 14 Moore, R. and Walker, B. 1981b. Studies of vegetative compatibilityincompatibility in higher plants.II. A structural study of an incompatible heterograft between Sedum telephoides (Crassulaceae) and Solanum pennellii (Solanacaceae). Amer.J.Bot. 68:831-942. 15 Jeffree, C. E. and Yeoman, M. M. 1983. Development of intercellular connections between opposing cells in a graft union. New Phytol. 93:491-509. 16 Parkinson, M. and Yeoman, M. M. 1982. Graft formation in cultured, explanted internodes. New Phytol. 91:711-719. 17 Aloni, R. 1987. Vascular differantiation of tissues. Annual Review of Plant Physiology 38:89-92. 18 Savidge, R. R. 1988. Auxin and ethylene regulation of diameter growth in trees. Tree Physiology 4:221-226. 19 Haris, J. M. and Barnett, J. R. 1975. Differantiated callus nodules in resin pockets of Pinus ponderosa Laws. New Zealand Journal of Forestry Science 5:226-229. 20 Weatherhead, J. and Barnett, J. R. 1986. Development and structure of unusual xylem elements during graft union formation in Picea sitchensis. Annals of Botany 57:593-598.

492

Journal of Food, Agriculture & Environment, Vol.8 (2), April 2010

A2 / Scarlet Spur

A2 / Red Spur

A2 / Redchief

B118/ Scarlet Spur

B118 / Red Spur

B118 / Redchief

M111 / Scarlet Spur

M111 / Red Spur

M111 / Redchief

M7 / Scarlet Spur

M7 / Red Spur

M7 / Redchief

M16 / Scarlet Spur

M16 / Red Spur

M16 / Redchief

Pj80 / Scarlet Spur

Pj80 / Red Spur

Pj80 / Redchief

Figure 1. Cross sections of 90 day samples of different apple stock/scion combinations.

C callus, NP new phloem, NX new xylem, NL necrotic layers, NC new cambium.

Journal of Food, Agriculture & Environment, Vol.8 (2), April 2010

493