African Journal of Biotechnology Vol. 11(30), pp. 7670-7675, 12 April, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.1192 ISSN 1684–5315 © 2012 Academic Journals Full Length Research Paper An investigation on mechanisms of blanked nut formation of hazelnut (Corylus heterophylla fisch) Jian-feng Liu, Yun-qing Cheng*, Kun Yan and Qiang Liu College of Life Sciences, Jilin Normal University, Siping 136000, China. Accepted 22 July, 2011 The occurrence of blank nuts is common in Corylus heterophylla Fisch orchards of China. This study was aimed to find the possible mechanisms involved in blank nuts formation in wild C. heterophylla Fisch species. The effects of pollination, defoliation and girdling on fruit production of C. heterophylla Fisch were studied from northern China. The effect of pollination on various aspects of the reproductive output of C. heterophylla Fisch was studied by performing hand pollination, open pollination and no pollination. Different pollination types significantly affected flower cluster set including no flower cluster set produced in no pollination treatment. However, pollination type had no direct effect on nut and kernel traits. Three defoliation treatments (control, 50 and 100% leaf removal) were applied at branch level on 10 trees. Six branches were used per treatment in each tree and half of these branches were girdled (a ring of bark and cambium was removed from the branch base). Leaf removal from ungirdled branches had little effect on pistillate flower cluster set, fruit cluster set and nuts per cluster. However, these variables decreased as the extent of 100% defoliation increased on girdled branches. Defoliation and girdling reduced nut and kernel weight which was the result of a reduction in the kernel weight rather than nut coat reduction. Control of the carbohydrate supply to the reproductive shoots by girdling and defoliation made no difference to nut number and size but the kernel percent and blank nut ratio were highly sensitive to carbohydrate availability. Resource importation not exportation by fruiting branches might be a mechanism to reduce blank nut in this species. Key words: Corylus heterophylla Fisch, pollination, defoliation, girdling, blank nut. INTRODUCTION Corylus heterophylla Fisch, the Asian Hazel is a species the number of female flower setting fruit. Thus, maxi- of hazel native to eastern Asia including northern China, mizing fruit set is an important measure to increase hazel eastern Mongolia, Korea, Japan and southeastern production. The occurrence of blank nuts is common in C. Siberia (Whitcher and Wen, 2001). Although the nuts of heterophylla Fisch orchards. Shell-kernel weight ratio is C. heterophylla Fisch have smaller and thicker shells the main determinate of quality and price of hazelnuts. characteristics; the important wild species in China was The most common defect “blank nuts” in Chinese cultivar cultivated commercially for some desirable and econo- have a significant effect on the shell-kernel weight ratio. mically important traits such as flavor, nonsuckering “Blank” means a filbert containing no kernel or a kernel growth habit, and tolerance to alkaline soil, and filling less than one-fifth capacity of the shell. Most exceptionally early maturation and cold hardiness. C. species of hazelnut are largely self-incompatible and a heterophylla Fisch is at present an expanding crop in number of studies have suggested that self-incom- China due to increased demand by the processing patibility was often associated with a higher frequency of industry. Fruit production of hazelnuts mainly depends on blanks (Erdogan and Mehlenbacher, 2001; Beyhan and Marangoz, 2007). One of the more intriguing aspects of the reproductive biology of hazelnuts is the temporal separation of pollination and fertilization. At the time of *Corresponding author. E-mail address: pollination, the ovary is not formed and grows only if the [email protected] Tel: +86-434-3294489. flower is pollinated. Liu et al. 7671 The formation of ovules begins in March and No pollination was made on individual branches or the entire trees fertilization occurs by the end of May or during the first depending on plant size. three weeks of June; two to three months after pollination Some unpollinated flowers were collected when styles were visible. Hand pollinated flowers were collected after hand pollination when the diameter of the nuts is 7 to 10 mm (Germain, for 24 h. When staminate catkins elongate and are about to shed 1994). Therefore, different environmental stressors often pollen, they were collected, placed on a sheet of paper in the occur at different stages between pollination and fertili- laboratory and allowed to dry overnight at room temperature (24°C). zation and lead to poor nut set and higher frequency of The following morning, the catkins were discarded and pollen was blanks (Solar and Stampar, 2001). Hazelnuts fill their collected from testers and stored in cotton stopper glass vials at fruits in the months of June, July and August. C. 0°C until used. Some female flowers were pollinated by hand when styles were visible outside the bud or were exerted beyond the red heterophylla Fisch has high fruit set and large amount of dot stage (>2 mm). The numbers of treated flowers and harvested photosynthates are needed during kernel filled stage nut clusters were counted and percent cluster set was calculated as while photosynthate sinks closest to the leaves tend to be the ratio of nut clusters to flowers pollinated. Pistillate flower the strongest, so insufficient photosynthates importing clusters were harvested 24 h after hand and no pollination, and into nuts in the filled stage can result in shriveled kernels styles were processed for fluorescence microscopy for pollen germination and tube growth as follows: for cytochemical assays (Kholupenko et al., 2003). Thus, some authors suggested with bright field and epifluorescence observations using a light that the limited resource translocation of carbohydrates microscope, the sampled material was fixed in formalin-aceto- from the photosynthetic pool was a possible cause of alcohol (FAA) for 48 h and then transferred to 70% ethanol for blank nuts. We investigated possible mechanisms storage. Fixed samples were then dehydrated in an ethanol series involved in the blank kernels formation in wild C. (50, 80, 95, 100, 100%: 12 h each) and transferred to an embedding solvent (xylene; Panreac Quimica SA, Montcada i heterophylla Fisch species. The following two hypotheses Reixac, Spain) through a xylene-ethanol series (30, 50, 80, 100, were set and tested: 100%: 12 h each) and finally saturated with paraffin (Paraplast Xtra; Sigma, St Louis, USA). Sections (10 µm thick) were cut with a H1: The self-incompatible characteristics in C. rotary microtome (Nahita 534; Auxilab SA, Beriain, Spain) and heterophylla Fisch have an effect on the cluster set of attached to adhesive-treated microscope slides (polysine slides; nuts but not the reason of blanked nut. Menzel GmbH & Co KG, Braunschweig, Germany). Samples were embedded in paraffin, sectioned at 10 mm in a rotary microtome H2: The lack of assimilate substances during and stained with hematoxylin or safranin-fast green (Odabas, development of fertilization fruits is the possible cause of 1976). A girdling treatment was applied to the fruit branch to inhibit blanks and shriveled kernels. the supply of assimilates and/or other substances to the fruit via phloem transport. The effect of this girdling on the occurrence of blanks on the tree was then investigated. The effect of defoliation and girdling on nut characteristics of C. MATERIALS AND METHODS heterophylla was studied after flowering finished. From 28 May 2008, we selected 10 trees for study. After measuring the number of The study was conducted at an area located in the Siping region leaves and the fruits on these twigs, 18 tagged shoots per tree were (Jilin, China, 43° 09′ 20″ N, 124° 30′ 16″ E) from March 2008 to selected for defoliation. Three defoliation treatments (control, 50 October 2009. The area has a slope around 5% with a south- and 100% leaf removal) were applied at branch level in 10 trees. western aspect. The adjacent vegetation is mainly natural Six branches were used per treatment in each tree and half of evergreen forest with some cleared farmland nearby. The C. these branches were girdled. A subset of reproductive shoots was heterophylla Fisch species are grown in the shrub growing form in girdled by removing a ring of bark and cambium approximately 1.0 China, thus the experimental unit was shrub growing system. cm wide from the base of the shoot and 5 mm in diameter. This Samples were collected from five systems (six plants per shrub procedure interrupts phloem transport but does not affect xylem system). All studied individuals were exposed to sunlight. Plant transport (Obeso, 1998). Other tagged shoots that were neither height ranged from approximately 1.2 to 2.3 m. Pistillate flower defoliated nor girdled, acted as controls. The presence or absence clusters (cymule) and fruit clusters of wild C. heterophylla Fisch of fruit developed from each flower was recorded so as to species were examined from 2008 to 2009. The effect of pollination determine the fruit set in late-May (the green fruit period just after on various aspects of the reproductive output of C. heterophylla flowering, hereafter called initial fruit set), in mid-July (the middle Fisch was studied by performing hand pollination, open pollination stages of seed maturation, hereafter called middle fruit set) and in and no pollination. Open-pollinated flowers were collected weekly mid-September (the final stages of seed maturation, hereafter during the flowering period. For artificial pollination, one to two called final fruit set). To examine the effect of assimilate limitation branches of each tester tree were emasculated by clipping catkins and pollination on fruit traits, the following variables were and were covered with Tyvek bags (1*0.5 m) in late March.