EFFECTS OF DIFFERENT ROOTSTOCKS INMICROGRAFTING ON GROWING OFWASHINGTON NAVEL ORANGE PLANTSOBTAINED BY SHOOT TIP GRAFTINGG.Sertkaya .University of Mustafa Kemal, Faculty of AgricultureDepartment of Plant Protection, 31034-Hatay, Turkey

ABSTRACTIn this research, the efJects of different rootstocks in micrografting on rapid productionof virus-free Washington Navel orange (Citrus sinensis (L.) Osb.) plants obtained bythermotherapy (TT) and shoot tip grafting (STG) in vitro were studied. Shoot-tipgrafted plants on Troyer citrange (e. sinensis X Poncirus trifoliata) were cultured inliquid Murashige and Skoog mediumfor 6weeks and then micrografted onto Citrumelo(C. paradisi X P.trifoliota), Cleopatra mandarin (e. reshni Hart ex Tan), Macrophylla(C. macrophylla Wester.), Rough lemon (e. jambhiri Lush.), sour orange (C.aurantium L.), Troyer citrange (e. sinensis X P.trifoliata) and Volkamer lemon (C.Volkameriana Ten and Pas.). Microgafted plants were kept in greenhouse for 24months at a temperature 26±2°C and under 16/8 hours photoperiod (day/night). Thesuccess rates ofSTG and micrografting were 42.0% and 80.4%, respectively. Diametersof graft-union, stock and scion trunks, survival rates of the plants micrografted ontodifferent rootstock, and shoot lengtlı of scion were observed and measured. The shootlengths of the plants micrografted onto Citrumelo, Cleopaıra mandarin, Macrophylla,Rough lemon, sour orange, Troyer citrange, and Volkamer lemon were measured as122.6, 121.3, 148.3, 162.4, 158.7, 144.3, and 151.7 in cn'?, 12 months af termicrografting. Averages of graft-union-stock-scion trunk diameters of the plantsmicrografted onto Citrumelo, Cleopatra mandarin, Macrophylla, Rough lemon, sourorange Troyer citrange, and Volkamer lemon at 10 cm under and above the grafi-union werefound as 13.80-11.02-8.20,12.55-10.20-8.45, 15.5,5-11.10-8.75, 14.95-11.90-8.63, 15.58-10.50-8.42, 15.24-10.77-9.72 and 17.23-11.30-9.90 in mmrespectively, 24 months af ter micrografting. Many of plants had been reached to thesize of indexingfor main virus diseases in a short time (in 6-8months) by micrograftingmethod. The plants micrografted onto Rough lemon and sour orange produced thelongest shoot. As a result of the study, it was concfuded that so ur orange could besuggested to use as a rootstock in micrografting studies for rapid development ofplant obtained by TT + STG.

IntroductionA great number of viruses and other graft-transrnissible infectious agents were knownto affcct and endanger the citrus industryworldwide (Salibe, 1986; Roistacher, 1991).

Recently, a new virus disease has appearedin citrus plantations in Eastern Mcditerra-nean Region of Turkey (3). Because of theusing infected plant material to produce ofcitrus seedlings, some important virus dis-

Biotechnol. & Biotechnol. Eq. 19/2004/2 82

T)

eases of citrus have been presently spreadout to new areas in Turkey.Obtaining of pathegen-free citrus plants isone of the most important step in "CitrusVariety Improvement Program". Numerousmethods have been developed to re-co vervirus-free plants. Shoot-tip grafting (STG)in vitro, which was studied by Murashige etal (1972) and deseribed in details by Navar-ro et aL.(ı97S), is the most effective tech-nique for elimination of all major virus andvirus-Ilke pathogens, inc1uding those noteliminated by thermotherapy. Plants ob-tained by STG are true-to type and they donot have juvenile characters, Thus, theseplants could be used for budwood produc-tion after theyare indexed (12).In Turkey, virus-free citrus plants have beenrecovered successfully by using of standardprocedure of shoot-tip grafting in vitro andindexing of that plants about 12-14 monthsafter transplanting into special growing mix-ture (7, 17).Transplanting the seedlings obtained by stan-dard procedure of STG into soil mixtureneeds a long time (approximately ıo-ısmonths). In order to shorten the growingperiod of plants for indexing stage, somemodified procedures and rnethods, i.e. mi-crografting technique, have been developed(4,8). Unfortunately, Rough lemon iCı janı-bhiri Lash.), which is used as rootstock inthe micrografting studies, could be sensitiyeto Phytophtora, a soi lborne fungus that hasthe capability to kill seedlings with in a fewweeks, Ci 9). In this case, there is a high riskto loss the plants obtained by a difficult andlength procedures of STG in vitro,This study was conducted to determine themost suitable citrus varieties as rootstock inmicrografting technique and to short the pe-riod of the obtaining virus-free WashingtonNavel orange (C sinensis (L.) Osb.) seed-lings as budwood source.

Materials and MethodsAll the mature leaves of IS W. Nave1 or-ange plants grown in glasshouse were ex-cised by cutting and the seedlings were keptin a elimatical room at 32±2°C temperatureand under 8/16 hours photoperiod (daylnight) conditions for thermotherapy treat-ment for 3 months. The shoots from acti ve-ly growing branches on the plants were usedas' source of shoot-tips in STG. Af ter germi-nation period in Murashige and Skoog(1962) culture solution solidified with 1%Bacto agar at 26±I°C in continuous dark-ness for 2 weeks, Troyer citrange (C sinen-sis X P trifoliata) seedlings which are widelyused as rootstock for sweet orange in STGwere removed from the germination medi-um and decapitated leaving 2-4 cm at theepicotyl and 2-A cm of the root. Their coty-ledons and axilIary grafts were also removed.The excised shoot-tips of W. Navel orangecomposed of the apical meristem and 3 pri-mordia (O. ıs-o. ı8 mm in height) were seton the cortex surface in an inverted-T inci-sion (12, 13). The plantlets were cultured inliquid MS nutrient solution at a constant2rC and exposed 16 hours daily to ı000lux illumination for 4 weeks and than for 2additional weeks under a higher light irıten-sity (about SOOO lux). The plantlets, havingat least 2-3 expanded leaves (Fig.1-B), weremicrografted on different rootstocks approx-imately 6 weeks after STG (De Lange,i978). ı2 plantlets (6 weeks-old) obtainedby STG culture in vitro were micrograftedon each 7 different rootstock species (9-10morıths-old) on the date of 19.03.1997 (Ta-ble 1).All the rootstocks were grown in plastic potsfilled with S litre of sterilised mixture ofSO% peat and SO% tuff in controlled green-house at 2S±2°C temperature and under 8116 photoperiod (day/night) conditions. Themicrografted plants were alsa kept under the

83 BiotechnoJ. & BiotechnoJ. Eq. 19/2004/2

Fig.l-A. Shoot tip grafted plant; shoot tip of Washington Navel orange was placed on Troyer citrangerootstoek by inverse T-insieion.B. In vitro eulture of shoot tip grafted plants.C. Mierografting of Washington Navel orange plantlet on sour orange rootstoek.D. Binding of the mierografted plantlet with streeh parafilm.E. The development of bud union (Troyer eitrange part of rootsoek) and seion (Washington Navel

orange) on mierografted sour orange rootstoek.

same greenhouse conditions and fertilisedevery week by using fertigation system.The upper part of STG plantlets with the firstrootstock, Troyer citrange (in i0-1 S mmlength) were cut by an incision of inc1inedcut and placcd into a T-incision on secondrootstock (Fig.l-C). The micrografted seed-lings were bound with stretched parafilm(Figure I-D), and covered by a transparentplastic bag until the scions were well adapt-ed to the new conditions approximately 10days after micrografting.After the beginning of the shoot develop-ment on micrografted seedlings, the upper

Bioteehnol. & Biotechnol. Eq. 191200412

parts of rootstocks were cut at 2-3 cm abovegraft-union.In order to determine the effect of the dif-ferent rootstocks, width on width of graft-union, rootstock and scion stems of the mi-crografted plants had been measured by acalliper compass (in 1120 sensitivity) dur-ing the period of 24 months at 4 week-irıter-vals. Stock and scion diameters (mm) weremeasured at iO cm under and above the graft-union. One-way ana1ysis of variance wasconducted on all data and Duncan's multi-ple range test was used to separate the mearıs.In order to determine the effect of the root-

84

TABLE i

The citrus species used as rootstock in shoot tip grafting andmicrografting experiments for obtaining of virus-free WashingtonNavel orange seedlings as source of budwood in a short time

Techniques Rootstock SpeciesShoot tip grafting Troyer citrange (Citrus sine/ıs is X Poncirus ırifoliataıMicrografting Citrumelo CC paradisi X P. trifoliota)

Cleopaıra mandarin (C reshııi Hart cx Tan)Macrophylla CC macroplıylla Wester.)Raugh lernon (C jambhiri Lush.)Sour orange CC aurantium L.)Troyer ci tran ge CC sinensis X P. ırifoliataıVolkamer leman CC volkamerlana Ten and Pas.)

TABLE 2The numbers and the success rates of micrografting ondifferent rootstocks

Rootstock Speciesi

No. of Survival Plarıts Success Rate of iNo. of Grafted Plants Grafting (%)

Citrumelo 10/12 83.3Cleopatra mandarin 7/12 58.3Macrophylla 9/12 75.0

Rough lemon 11/12 90.1

Sour orange 11/12 90.1

Troyer citrange 11/12 83.3Volkamer leman 10/12 90. ı

stocks on Jength of shoots, the plants whichwere linearly grown as a sirıgle shoot hadbeen measured from graft-union to top ofthe flush by using a tape measure (in cm)periodically for 12 months at 4 week-inter-vals.

Results and DiscussionIn shoot tip studies, about 20 shoot tips hadbeen grafted on Troyer citrange seedIingsin one-hour period. Totally 228 plantletswere grafted by STG method. Shoot tipswere survived on Troyer citrange rootstocksbut shoot formation was not observed by10% of grafted plantlets in 4th weeks after

in viıro culture. So, the plantIets with shootdevelopment were accepted as successfullygrafted plant, The plantlets with 3-4 expand-ed leaves in vitro culture were used in mi-crografting on different rootstocks, 6 week safter STG. Shoot regeneration was observedin all plantlets micrografted on Rough lem-on seedhngs. The success rates of micrograft-ing on Rough lemon, sour orange, Volkamerlemon, Citrumelo, Troyer citrange, Macro-phylla and Cleopatra mandarin were obtainedby 90. ı%, 90.1 %, 90. ı%, 83.3%, 83.3%,75.0%, and 58.3% respectively (Talıle 2).Rough lemon had the largest rootstockgrowth, followed by Volkamer lemon, Mac-

85 Biotechnol. & Biotechnol. Eq. ı912004/2

rophylla, sour orange, Troyer citrange, Cleo-patra mandarin and Citrumelo, respective-Iy. Macrophylla, sour orange and Troyer ci-trange were not statistically different fromeach other in rootstock growth (Table 3).Volkamer lemon was placed in the first groupby the width of graft-union (Fig. I-E). Sourorange and Macrophylla were in the secondgroup, Troyer citrange, Rough lemon werein the third group, Citrumelo was in thefourth group and Cleopatra mandarin wasin the fifth group.Volkamer lemon rootstock was placed in thefirst group by the width of sciorı. Troyer ci-trange placed in the second group whileMacrophylla and Rough lemon in the thirdgroup, Cleopatra mandarin, Sour orange andCitrumelo were placed İn the fourth group.The average s of shoot lengths of W. Navelorange on different rootstocks at 6-monthintervals were shown in Fig. 2. Rough lem-on and sour orange had the longest shootlength İn cm followed by Volkamer lemon,Macrophylla, Troyer citrange, Citrumelo,and Cleopatra mandarin in ı62.4, ı58.7,IS 1.7, 148.3, 144.3, ı22.6, and ı21.3 respec-ti vel y, 12 months after micrografting.STG resulted in 42% of shoot formed plants.

The success rate of grafting was similar withother studies (l2, 13). The success rate ofmicrografting ranged from 58.3% (Troyercitrange) to 90.1 % (Rough lemon). De Lange(1978) reported 90% of success rate with mi-crografting on Rough lemon. Although manyof that rootstocks had been studied as root-stock in S'TGexperimenrs (4, 5, 12, 13,14),there was no study to cornpare the effects of

, these species on seedling development in mi-crografting studies.The graft -union + rootstock (Troyer ci trange)part of shoot tip grafted plantlets micrograft-ed on different rootstocks were exhibitedgreater tissue development than both in root-stock and scion parts of seedlings. Especial-ly in Volkamer lemon, a very clear overgrowwas observed at the graft-union in all mi-crografted plants. This situation might be dueto incompatibility or being of a few part ofTroyer citrange which was used as rootstockin STG, between scion and new rootstockspecies which were used in micrograftingexperiments (Figure ı-E). Similar1y, root-stocks with trifoliate orange parentage reg-ularly overgrowth thcir sciorıs was reportedby Ferguson et al. (6). L. Navarro et aL.(1975) reported that the success rate of shoot

TABLE 3Graft-union, rootstock and scion growth of micrograftedplantlets on different rootstocks

Diarneter of Diarneter of Diarneter ofRootstock Species Graft-uniorı Rootstock Scion

(rrırn)" (mm)* (mm)*

Citrumelo 13.80 c+ 9.90 c 8.20 cCleopatra mandarin 12.55 d 10.20 be 8.45 cMacrophylla 15.55 b 11.10 ab 8.75 beRough lernon 14.95 be 11. 90 a 8.63 beSour orange 15.58 b 11.05 ab 8.42 cTroyer citrarıge 15.24 bc 10.77 ab 9.72 bVolkamer lemon 17.23 a 11.30 b 9.98 a

Biotechnol. & Biotechnol. Eq. 19/2004/2 86

(

f(

(

tip grafting on Troyer citrange when lemonshoots weı~e used as source shoot tip waslower in the STG studies.The plants transferred to soilless mixtureafter STG culture are required 2 or 3 timeslonger growing period to reach a suitable sizefor indexing than micrografted ones (7, 13,18). Especially plants grafted on sour orangeand Rough lemon reached to optimum size(60-80 cm) for indexing within 6-8 months(Figure 2). Other rootstocks such as Macro-phylla and Troyer citrange reached to satis-factory size withirı 8- iO months. In Citrurne-lo, Macrophylla and Volkamer lemon root-stocks, the shoot growth from rootstocks wasat a high leveJ. These shoots slowed downthe growth of grafted plants (especially thepart of scion). Moreover, two of plants graft-ed on Rough lemon dried due to foot rot(gummosis) and root rot.Foot rot and root rot cansed by Phytophtoraspp. ereare major problems in nurseries.Some Rough lemon sources are highly sus-ceptible to infection while Cleopatra man-

darin and sour orange rootstocks are resis-tant or tolerant (2, 6, 19).This situation creates risk of losing healthyplant candidates obtained from STG andmicrografting which are detailed, difficultand time consuming methods. Sour orangeis widely used (95%) rootstock in citricul-ture in Turkey (1). The results of our studyshowed that, besides Rough lemon, sour or-ange was found to be alsa suitable for mi-crografting studies for rapid development ofplant after thermotherapy + STG in vitro.Reasons of this are as following;1) Root and root neck problem s are at a min-

imum level with sour orange rootstock.2) Sour orange is commercially propagated

in a large quantities in the controlledglasshouses and theyare reguired lesseramount of time and labour when com-pared with the other rootstocks.

3) Sour orange rootstock could be obtainedfrom nurseries growing rootstocks pcri-odically by the quantities and ages (8-10month) as theyare requested.

:l.19.03.1997* ~14.09.1997 017.03.1998

ıt 200 a** ab bec cE 150 d d2-..ı:::+-'ol 100cOJ

(5o 50..ı:::u)

O 1~)~~1&~1 ~,~~~:~~ fg~~t\\ ~~~~,o ro c ro ..ı::: c OJ '- OJ '-ın '- 'c ol o ol OJ ol OJro >. E cE ro ::J E c », c oo, "D ..ı::: o ~ 2 ro ro:::ı o c

o, o:: ~ .b ~ E.b OJ ro 2 o i- 'u (5 ~G G E '-o :::ı >ro o

~ Rootstocksu)

Fig. 2. Averages of shoot lengths of scion (Washington Navcl orange) micrografted on differerıt rootstocks(cm). *Shoot lengths in grafting stage were between 0.2-0.4 cm. **Means by the different letters withineach column indicate significant differerıces (Pd : 0.01, n:7).

87 Biotechnol. & Biotechnol. Eq. 19/2004/2

4) Other rootstocks require additicnal placetime, labour and cost for the propagation,seed obtaining, seed storage and seed pro-duction. Alsa, periodical seeding is re-quired to obtain rootstock at a suitableage with others.

5) Sour orange rootstock has advantages ofhigh success rate of micrografting and

.plant growth.Sour orange has been routinel Yused as root-

stock in our micrografting studies to rap-id growth virus-free citrus budwoodsources.

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8. Hong-Ji S., Jan- Yang C. (1984) Proc. InLSoc. Citriculture, 1, 332-334.9. Korkmaz S. (1997) Doğu Akdeniz Bölgesiturunçgillerinde vektörlerle taşman virüs benzeribir hastalığm yayılması, taşınması, duyarlı türve çeşitlerin belirlenmesi ve tanısı üzerindearaştırmalar (Phf) Thesis), Çukurova Üniv. FenBiL. Enst. 70.s.10. Murashige T., Skoog F. (1962) PhisioL.Plant, 15,473-497 .ll. Murashige T., Bitters W.P., Ragan T.S,Nauer E.M., Roistacher C. N., Holliday P. B.(1972) Hort. Scien. 7 (2),118-119.12. Navarro L. (1984) Proc. FAO/NORWAYSymp. on plant tissue culture, technologyandutilization, micropropagation of selected rootcrops,palms, citrus and omamental plants. PlantProduction and Protection Paper, 59, 113-154.ı3. N avarro L., Roistacher C. N., Murashige T.(1975) 1. Amer. Soc.Hort. Sci., 100 (5), 471-479.14. Roistacher C.N., Kitto S.L. (1977) PlantDisease Reporter, 61, 594-596.15. Salibe A.A. (1986) A programme for citrusimprovement and protection İn Turkey. Report tothe Government of Turkey. FAO, Rome, p. 66.16. Şaş G. (1991) Turunçgillerde GA, uygula-

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17. Tamer Ş. (1988) Virüs ve virüs benzerihastalık etmenlerinin Navel portakallarındanarındırılması. (Phl) Thesis), Çukurova Ün iv. FenBiL. Enst.), 97 S.

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i