Kim, C. S.

Lee, C. H.

Lee, G. P.

Park, H. S.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 44 (4), 195–196 (2005)Research NoteIn vitro grafting of grape withphylloxera resistant rootstockcultivarsC. S. KIM1), C. H. LEE1), H. S. Park2) and G. P. LEE2)1) Division of Bioindustrial Science, College of Life and Environ-mental Science, Korea University, Seoul, Korea2) Department of Applied Plant Science, Chung-Ang University,Ansung, KoreaK e y   w o r d s :  micrografting, grape, scion, Phylloxeraresistance rootstock.Introduction: Grape phylloxera (Daktulosphairavitifoliae Fitch) feed on the sap of grape roots, causingdamage and often death of vines (MAKEE et al. 2004). Incase of phylloxera infestation current solutions include rip-ping out infested vines, fumigation of soils with methyl bro-mide, and replantation with tolerant rootstocks (MOTOSUGIet al. 2002).Micrografting has been successfully used in a widerange of horticultural plants including grape, as an effectivemethod for the acquisition of clones which are free of soil-borne pathogens, including viruses and virus-like diseases.This technique is also used to detect graft compatibilityduring early stages. Some important success has been re-ported for fruit trees, including citrus (OIYAMA 1992), cherry(OZZAMBAK and SCHMIDT 1991), apple (RICHARDSON et al. 1996)and grapes (MARTINO 1992).The objective of the present study was to develop anefficient and robust micrografting system for the protectionof grapes from phylloxera, using Millardet et de Grasset01-14, Kober 5 BB, Couderc 3309 C and Rupestris du Lotrootstocks and, for mass production, favorable table grapesas scions.Material and Methods:  P l a n t   m a t e r i a l   a n d   e x-p l a n t   p r e p a r a t i o n :  Grape cultivars and rootstockswere sampled from the field at the National HorticulturalResearch Institute in Suwon, Republic of Korea, from lateJune until the first week of August in 1999 and 2003. Thecultivars which were used to produce experimental scionsincluded Kyoho, Campbell Early, Schuyler, and Tamnara.Millardet et de Grasset 101-14, Kober 5 BB, Couderc 3309 Cand Rupestris du Lot were used as rootstocks. Shoot tipswere sterilized with 70 % ethanol for 1 min; stirred for 12 minin 15 % sodium hypochloride, and rinsed 4 times in sterilewater. The sterilized shoot tips were cut into pieces (30 mmin length), which were cultured (6-7 per jar).R o o t s t o c k   a n d   s c i o n   m u l t i p l i c a t i o n :For shoot multiplication we used MS basal medium(MURASHIGE and SKOOG 1962) supplemented with plantCorrespondence to: Dr. G. P. LEE, Department of Applied PlantScience, Chung-Ang University, Ansung-city 139-774, Korea. Fax:+82-31-676-6714. E-mail: gplee@cau.ac.krgrowth regulator (0.3-0.5 mg·l-1 benzyladenine, 3 % sucrose,and 0.8 % Gum Agar, Sigma). The pH of the media was ad-justed with NaOH to 5.7 prior to autoclaving.M i c r o g r a f t i n g   m e d i u m :  For micrografting weused half-strength MS medium which was free of growthregulators, but supplemented with 4 % sucrose and 0.8 %Gum Agar, Sigma). The pH of this medium was also adjustedto 5.7 prior to autoclaving (121 °C, 15 min).M e t h o d   o f   g r a f t i n g :  3-week-old shoots wereused as scions after the final subculture in vitro. Each scionconsisted of a 5-10 mm long herbaceous stem with an axil-lary bud. Ten-week-old stems (length: 15-20 mm) were usedfor rootstocks. Cleft-grafting was applied as a micrograftingmethod, thereafter the cultures were incubated in darknessat 27± 1°C for three d, then transferred to light (40 W cool-white fluorescent bulbs, average photosynthetic photon fluxdensity: 100 µmol·s-1·m-2) in a growth room at 27 ± 1°C witha forced cooling fan system.Results and Discussion: The success of micrograftingprimarily depends on the grafting technique. In this studycleft grafting proved to be the most successful and efficientmethod for in vitro micrografting (Figure A, B and C). Graft-ing success was determined by callus formation and root-ing. Calluses tended to be formed at the junction of a graftunion, arising from the living cells of both, scion and root-stock (Figure D, E, F, and G). MOORE and WALKER (1983)observed that adjacent callus masses tend to graft success-fully in the absence of vascular differentiation. However,callus formation was determined by independent of graftingsuccess, although callus growth constitutes a key factor inthe development of the graft union; e.g. it physically joinsthe scion to the rootstock in the pear cactus (Opuntia)(JEFFREE and YEOMAN 1983).Grafted shoots began to grow approximately 10 d aftergrafting. When the scion produced about 6 new leaves, theplants were transferred into small pots which contained anartificial soil mixture (vermiculite: peat moss = 5 : 1, v/v). Thepots were enclosed in polyethylene bags and after 3-7 d thebags were gradually opened. After 4 weeks, the plants weretransferred to large pots and moved to a greenhouse (FigureH and I).After grafting cvs Campbell Early and Kyoho to differ-ent rootstocks we observed that, Kyoho exhibited a highergraft compatibility rate than Campbell Early (Table). Whensoftwood graftings were compared with hardwood graftings,the former proved to be superior with regard to successfulunions. In Korea, it has been reported that Kyoho, graftedto SO 4 is arable, as defined by evaluations of soluble sol-ids, coloring, berry shattering and yield. Campbell Earlygrafted to each of the tested rootstocks had a significantlyhigher shoot vigor compared to non-grafted seedlings. Therootstock Couderc 3309 was successful in rooting, callusformation, and growth with all scion types (Table). Amongthe rootstocks or scions, we found that grafting compatibili-ties ranged between 11 % (Tam-nara/Rupestris du Lot) and100 % (Schuyler/Couderc 3309).In Korea, most grapevines were field-grafted withphylloxera-resistant rootstocks, although the production ofrootstocks and scions in the field is an extremely time-con-196 C. S. KIM et al.T a b l eEffects of micrografting of cvs Kyoho, Campbell Early, Tamnara, and Schuyler to various rootstock cvsScions Rootstocks Scion Rate of Rate of Rate oflength (cm) rooting (%) callus formation (%) micrografting (%)Kyoho Millardet et de Grasset 101-14  9.56 a*) 0.80 a 0.63 a 19 bCouderc 3309 7.17 b 0.83 a 0.83 a 72 aRupestris du Lot 12.60 a  0.69 ab 0.72 a  61 abKober 5 BB 5.26 b 0.27 b 0.44 a  27 abCampbell Millardet et de Grasset 101-14 14.00 a 0.50 a 0.61 a 38 aEarly Couderc 3309 11.36 a 0.66 a 0.66 a 66 aRupestris du Lot 12.43 a 0.83 a 0.83 a 83 aKober 5 BB 10.74 a 0.55 a 0.66 a 55 aTamnara Millardet et de Grasset 101-14  7.64 b 0.75 a 0.83 a 75 aCouderc 3309 12.44 a 1.00 a 0.91 a 91 aRupestris du Lot  4.70 b 0.11 b 0.00 b 11 bKober 5 BB  5.03 b 0.22 b  0.55 ab 22 bSchuyler Millardet et de Grasset 101-14 11.50 a 0.83 a 0.83 a 50 bCouderc 3309 10.68 a 1.00 a 1.00 a 100 aRupestris du Lot  9.03 a 0.55 a 0.66 a 22 cKober 5 BB  7.56 a 0.61 a 0.72 a  38 bc*) Mean separation in columns within scions by Duncan’s multiple range test at P < 0.05. The mean values are from10 replications (jars) with 3 subsamples (graftings) per jar.Figure: In vitro grafting of grape to Phylloxera resistant rootstocks(Schuyler/Couderc 3309). (A) no leaf, (B) one leaf, and (C) twoleaves, (D) with callus, (E) no callus, (F) and (G) graftings after6 weeks, (H) and (I) graftings after 8 weeks.suming process. In this study, we propose mass productionof phylloxera-tolerant grapevines via micrografting, involv-ing in vitro culturing for the production of virus-free scionsand rootstocks. Moreover, micro-grafting can also be suc-cessfully applied to predict more rapidly the grafting com-patibility between newly developed scion and rootstockcultivars.This study was supported by Ministry of Education andHuman Resources Development in Korea (2005) to Dr. C. H. LEEand partially supported by Specific Research-promoting JointProjects, RDA, Republic of Korea in 2005 (20050301-033-279-079-02-00) to Dr. G. P. LEE.JEFFREE, C. E.; YEOMANM, M. M.; 1983: Development of intercellularconnection between opposing cells in a graft union. New Phytol.93, 491-509.MAKEE, H.; CHARBAJI, T.; AYYOUBI, Z.; IDRIS, I.; 2004: Evaluating resist-ance of some rootstocks to grape phylloxera with in vitro andexcised root testing systems. In Vitro Cell. Dev. Bio-Plant. 40,225-229.MARTINO, L.; 1992: In vitro micrografting of grapevine. Petria 2,17-25.MOORE, R.; WALKER, D. B.; 1983: Studies of vegetative compatibility-in-compatibility in higher plants. VI. Grafting of Sedum andSolanum callus tissue in vitro. Protoplasma 115, 114-121.MOTOSUGI, H.; NARUO, T.; KOMAZAGI, S.; YAMADA, M.; 2002: Resistanceof autotetraploids of grapevine rootstock cultivars to phylloxera(Daktulosphaira vitifoliae FITCH). Vitis 41, 103-106.MURASHIGE, T.; SKOOG, F.; 1962: A revised medium for rapid growth andbioassays with tobacco tissue cultures. Physiol. Plant. 15,437-497.OIYAMA, I.; 1992: Studies on polyploidy breeding in citrus with specialreference to the production of tetraploid breeding. Bull. FruitTree Res. Stn. 3, 68.OZZAMBAK, E.; SCHIMDT, H.; 1991: In vitro and in vivo micrografting ofcherry (Prunus avium L.) Gartenbauwissenschaft 56, 221-223.RICHARDSON, F. V. M.; SAOIR, S. M. A.; HARVEY, B. M. R.; 1996: A study ofthe graft union in vitro micrografted apple. Plant Growth Regul.20, 17-23.Received March 18, 2005

In vitro grafting of grape with phylloxera resistant rootstock cultivars

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In vitro grafting of grape with phylloxera resistant rootstock cultivars

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