Analysis of Flowering and Nectar Characteristics of Major Four Chestnut Cultivars (Castanea spp.)

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1 Original Article Journal of Apiculture 32(3) : 237~246 (2017) DOI: /apiculture Analysis of Flowering and Nectar Characteristics of Major Four Chestnut Cultivars (Castanea spp.) Sea Hyun Kim, Andosung Lee, Hae Yun Kwon, Uk Lee and Moon Sup Kim* Division of Special Purpose Tree, National Institute of Forest Science, Suwon, Korea (Received 29 June 2017; Revised 29 August 2017; Accepted 30 August 2017) Abstract In order to provide a basic data of Castanea spp. as a honey plant, flowering time and nectar characteristics were investigated using Korean prevailing chestnut cultivars: Daebo, Okkwang, Riheiguri and Tsukuba planted in clonal archive in Hwaseong city. There were differences among four cultivars in the beginning of flowering and the end of the flowering. In 2015, female flower bloomed approximately eight days earlier than the male flower. Also, there were differences among four cultivars in nectar volume per catkin as the values were 54.3µl and 6.7µl in Okkwang and Daebo, respectively. On the contrary, in the sugar content, the values were 15.6µg/µl and 69.3µg/µl in Okkwang and Daebo, respectively. Therefore, it is possible to assume the sugar content per catkin using the total nectar per catkin and the total amount of free sugars. Based on the formula, we estimated the values: Tsukuba was µg; Okkwang was 777.5µg; Riheiguri was 708.9µg; and Daebo was 486.4µg. Moreover, we investigated the amino acid content. The result was that proline, glutamic acid, asparagine, and alanine were major amino acids in four cultivars. Also, C. spp. showed different values in many aspects such as catkin morphology, nectar characteristics, sugar content, and amino acid content. The sugar content per catkin which determines a honey yield considered, it is believed that Tsukuba is highly valued as a honey tree. It would be needed to further study related to the nectar characteristics of female flower to exactly estimate the total nectar volume per tree and the total honey yield per tree. Key words: Amino acid contents, Chestnut, Cultivar, Nectar secretion, Sugar contents (Fagaceae) (Castanea), 10. (C. crenata), (C. mollissima), (C. dentata), (C. Sativa) (, 2001). *Corresponding author. honeytree@korea.kr 237

2 238 ( ), (Dryocosmus kuriphilus),, 1968.,, (, 2001). 55,592,,,,,,,, (, 2016).,,. (, 2003;, 2011)., (, 1963;, 1974;, 1977;, 1993)., C. sativa kynurenic acid, kynurenic acid (Turski et al., 2016)., 1-phenylethanol 2- aminoacetophenone compound, cis-cinnamyl alcohol p- hydroxyacetophenone (Alissandrakis et al., 2011). C. crenata, (, 2007), 6 (, 2009). tyrosinase (, 2010)., (, nectar). (nectary) 80%, (sugar) 20%,,,,,. 20%., (Nicolson et al., 2007) Beutler(1953), (1984) Micro-capillary tube. (, 2014a, 2014b;, 2015).,,, 4,.

3 239 Table 1. The names, pedigrees and origins of four major chestnut cultivars (Castanea spp.) used in this study Cultivar name Korean English Pedigree Origin Daebo C. crenata (C. crenata C. mollissima) hybrid Korea Okkwang C. crenata Korea Riheiguri C. crenata C. mollissima Japan Tsukuba C. crenata hybrid (Ganne Hayadama) Japan A: Shape of tree B: Female flower C: Male flower (Catkin) Fig. 1. Pictures of a chestnut tree including male and female flowers. 8m, 30cm 4 (,,, ) 3. 12, 60~80%. Table 1. (Fig. 1), 4 (Table 2)., (catkin) 4 20 (NA S). 3, (Microfuge 16, Beckman Coulter, USA) 2,000 rpm µl microliter syringe(hamilton, USA) (GMK-703T, Giwonhitech, Korea)., HPLC 0.45µm membrane filter(whatman, UK), 80% 10 Eppendorf vial ( 70 C).

4 240 Table 2. The criteria based on the flowering stages of female and male flowers Gender Flowering stages Criteria Flowering beginning When a main pistil projects out and styles grow almost 4mm. Female flower Full bloom beginning When a size of side pistil is larger than 4 mm and a plant flowers approximately 30% among a whole flower bud of plant. Full bloom end When a plant flowers approximately 70%. Flowering end When the color of a stigma turns black, styles are browning and staminate flower abscise. Male flower Flowering beginning When anthers emerge from staminate flower and some of them flower. Full bloom beginning When a plant flowers approximately 30% among a whole flower bud of plant. Full bloom end When a plant flowers approximately 70%. Flowering end When the color of staminate flowers turns brown, staminate flower abscise. Table 3. Flowering period of female and male flowers from the major four chestnut cultivars in 2015 Cultivar Female flower Male flower FB z FBB FBE FE FB FBB FBE FE Daebo 6/3 6/8 6/12 6/16 6/13 6/15 6/17 6/22 Okkwang 6/3 6/7 6/13 6/17 6/10 6/13 6/15 6/21 Riheiguri 6/3 6/6 6/11 6/15 6/8 6/10 6/12 6/17 Tsukuba 5/31 6/2 6/8 6/14 6/11 6/13 6/15 6/21 z : Abbreviations flower characteristics; FB: Flowering beginning, FBB: Full bloom beginning, FBE: Full bloom end, FE: Flowering end. HPLC(Dionex ultimate 3000, Dionex, USA). 3, 0.5 ml/min, 80 C. Ri-101 detector (Shodex, Japan), Aminex 87P column (Bio-rad, USA)., Sucrose, Glucose, Fructose, Galactose(Sigma, USA). O-phthalaldehyd(OPA)-Fluorenylmethyl chloroformate (FMOC). Borate buffer, OPA/Mercaptopropionic acid(mpa), FMOC, HPLC(1200 series, Aglient, USA). 10mM Na 2 HPO 4, 10mM Na 2 B 4 O 7 10H 2 0 A (ph8.2) Water: Acetonitrile:Methanol=10:45:45 B A :B 100:0 (v/v,%) 26~28 55:45, 28~30.5 0:100, :0. 1.5ml/min. 1ml 40 C Inno column C18 (Innopiatech, Korea)., 338nm, OPA 450nm, 340nm, FMOC 305nm, 266nm. SPSS program(statistical Package for Social Science, ver. 12.), Spearman, ANOVA,

5 241 Duncan. (FB; Flowering beginning), (FBB; Full bloom beginning), (FBE; Full bloom end), (FE; Flowering end) 4 (, 2011) 4 Table , , 8. 9, 6 8,,,. (FBE) (FE). 4 14, (, 2003;, 2011). (2003), 27, (2011) 10,.,, µl, µl (, 2014a), Silene 3 (S. alba, S. dioica, S. nutans) (Witt et al., 1999)., Cucurbita pepo, Euterpe precatoria.., 90%, 10% (Table 4). Duncan cm, mm, Catkin Table 4. The morphological characteristics of catkins and their nectar from the major four chestnut cultivas Cultivar Length of catkin (cm) Diameter of catkin (mm) Daebo a z b a b a Okkwang ab a ab a d Riheiguri b b b b b Tsukuba b a b ab c Mean z : Duncan s multiple range test (Significant at p=0.05). Number of flower per catkin (ea) Total nectar per catkin (µl) Soluble solid content (%Brix)

6 242 Table 5. Correlation coefficients by Spearman between catkin and nectar characteristics from the major four chestnut cultivars Charcteristics Length of catkin Diameter of catkin Total nectar per catkin Soluble solid content Diameter of catkin n.s. Total nectar per catkin n.s ** Soluble solid content n.s ** ** Number of flower per catkin ** n.s. n.s. n.s. **, n.s.: Significant at p=0.01 and non-significant, respectively. Table 6. Total amount and composition of sugars in floral nectar of staminate flower from major four chestnut cultivars Cultivar Sucrose (µg/µl) Glucose (µg/µl) Fructose (µg/µl) Galactose (µg/µl) Total amount of free sugars (µg/µl) Sugar content per catkin Y (µg/catkin) Daebo a Z Okkwang b Riheiguri a Tsukuba b Mean z : Duncan s multiple range test (Significant at p=0.05). Y : Sugar content per catkin (µg/catkin) = Total nectar per catkin Total amount of free sugars. Fig. 2. Ratio of sugar composition in nectar from the major jour chestnut cultivas µl µl., 57.1%Brix 18.2%Brix.,.,. Spearman Table 5.,

7 243 Table 7. The composition of amino acids in nectar of male flowers from the major four chestnut cultivas Amino acid (%) Cultivar name Aspartic acid c z b bc a p=0.00 Glutamic acid c c b a p=0.00 Asparagine a a a b p=0.02 Serine b ab ab a p=0.03 Glutamine Histidine Glycine Threonine * bc a c ab p=0.01 Arginine Alanine a b a a p=0.01 GABA d a b c p=0.00 Tyrosine Valine * Phenylalanine * a d b c p=0.00 Isoleucine * ab a ab b p=0.02 Leucine * Lysine * Proline Methionine * Tryptophan * z : Duncan s multiple range test (Significant at p=0.05). * : Essential amino acid. Daebo Okkwang Riheiguri Tsukuba Mean ANOVA p value. (r=0.950, p=0.01),., (r=0.844, p=0.01). (r= 0.725, p=0.01). (2014a) (r= 0.746, p=0.01). HPLC Table 6., (Pacini et al., 2003). HPLC, 4. 4,.,, µg, 777.5µg, 708.9µg, 486.4µg., 4..

8 244 Fig. 2 HPLC. 4 Hexose (glocose, fructose, galactose) Sucrose, Hexose fructose 71.4%. Sucrose/Hexose ratio 0.12, hexose-dominant (Baker and Baker, 1982). Kim et al.(2012) fructose 44.3~77.9%, 0.22(hexose-rich) 0.72(sucrose -rich)., (2014a) fructose 12.1% 19.6%, sucrose 71.4%, 52.8%, 2.5, 1.2(sucrose-dominant),. HPLC 4 Table 7. proline, glutamic acid, asparagine, alanine. proline, asparagine, alanine, glutamic acid, glutamine, 69.9%. proline, asparagine, GABA(Gamma aminobutyric aicd), serine, glutamic acid 68.5%. proline, asparagine, alanine, glutamic acid, GABA 64.4%, proline, glutamic acid, serine, aspartic acid, alanine 62.6%. proline, asparagine,, proline glutamic acid. proline 28.1%. proline proline (Hrassnigg et al., 2003)., 4 tryptophan, methionine., aspartic acid, glutamic acid, asparagine, serine, threonine, alanine, GABA, phenylalanine, isoleucine 9.,, alanine, GABA. GABA phenylalanine (Petanidou et al., 2006). GABA 7.4%, phenylalanine 1.7%. phenylalanine, 1.0%, 67.2% (, 2014a; Kim et al., 2015;, 2015).. 4 (,,, )., cm, 2.7mm.,

9 µl, 6.7µl. 16.4µg µl, 69.3µg µl., µg, 777.5µg, 708.9µg, 486.4µg. fructose 71.4% hexose-dominant., proline, glutamic acid, asparagine, alanine. 4,,,,.,. (No ).,,,,,,,,,,,,,, pp.16.,,,, ,. 92(4): ,,,. 2014a.,. 103(1): ,,,,. 2014b.. 29(4): ,,,,, ,. 30(4): : ,,,,,,,, (2): ,,,,, pp pp. 282., ,. 29: , : (2): ,,, (1):17-21., : ,,,,,,, (1): Alissandrakis, E., Tarantilis, P.A., Pappas, C., Harizanis, P.C. and Polissiou, M Investigation of organic extractives from unifloral chestnut (Castanea sativa L.) and eucalyptus (Eucalyptus globulus Labill.) honeys and flowers to identification of botanical marker compounds. Food Science and Technology 44: Baker, H.G. and Baker, I Chemical constituents of nectar in relation to pollination mechanism and phylogeny. In: M.H. Nitechi (Ed.), University of Chicago Press. Biochemical Aspects of Evolutionary Biology Beutler, R Nectar. Bee World. 34: , , Hrassnigg, N., Leongard, B. and Crailsheim, K Free amino acids in the hemolymph of honey bee queens (Apis mellifera L.). Amino Acids 24: Kim, M.S., S.H. Kim, J. Han and J.S. Kim Analysis of secretion quantity and sugar composition of nectar from Tilia amurensis Rupr. Journal of Apiculture 27(1): Kim, M.S., S.H. Kim, J.H. Song, H.Y. Kwon, H. Kim and Y.S. Choi Honeybee visiting and floral nectar characteristics of Styrax japonicus Sieb. & Zucc. Journal of apiculture 30(1): Nicolson, S.W., Nepi, M. and Pacini, E Nectar production and presentation. Nectaries and Nectar pp Pacini, E., Nepi, M. and J.L. Vesprini Nectar biodiversity: a short review. Plant Systematics and Evolution 238: 7-

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