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TECHNICAL BULLETIN 139 PRODUCTION OF HEALTHY LAPITHOS LEMON PLANTS BY SHOOT-TIP GRAFTING IN VITRO Maria Ioannou, Anastasia Kyriakou and N. Ioannou -7 APR rULTURAL RESEARCH i ",-, INS11TUTE AGRICULTURAL RESEARCH INSTITUTE MINISTRY OF AGRICULTURE AND NATURAL RESOURCES CYPRUS NICOSIA NOVEMBER 1991
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TECHNICAL BULLETIN 139

PRODUCTION OF HEALTHY LAPITHOS LEMON PLANTS

BY SHOOT-TIP GRAFTING IN VITRO

Maria Ioannou, Anastasia Kyriakou and N. Ioannou

-7 APR rULTURAL RESEARCH i

",-, INS11TUTE

AGRICULTURAL RESEARCH INSTITUTE

MINISTRY OF AGRICULTURE AND NATURAL RESOURCES

CYPRUSNICOSIA

NOVEMBER 1991

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ISSN 0070·2315

All responsibility for the infonnation in this publication remains with the author(s). The use of trade names does not imply endorsement of or discrimination against any product by the Agricultural Research Institute.

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PRODUCTION OF HEALTHY LAPITHOS LEMON PLANTS BY SHOOT-TIP GRAFfING IN VITRO

Maria Ioannou, Anastasia Kyriakou and N. Ioannou

SUMMARY

The technique of shoot-tip grafting in vitro was used from 1988 to 1990 to recover the local lemon variety Lapithos free of exocortis and other virus and virus-like diseases. About 2,000 seedlings of three different rootstock cultivars were micrografted using scions from field or greenhouse-grown lemon plants. Among rootstocks citrumelo gave the highest grafting success (41 %), followed by sour orange (37%) and rough lemon (29%). Shoot tips from the field gave slightly higher grafting success (37%) than those from the greenhouse (32%), but the availability and quality of the former were season-dependent. For plant estab­lishment in vivo about 170 micrografted plantlets were regrafted on potted sour orange see­dlings, with 73% success. Indexing tests on 14-regrafted plants showed that 12 were free of exocortis (even though they originated from severely infected mother trees) as well as from tristeza, psorosis and infectious variegation. These plarrts are presently being propagated in order to develop a source of healthy budwood for Lapithos lemon.

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H EQyaoLa au'ti] £yWE O'tllv nEQLo()o 1988-90 !1£ oxono va ILEAE'tll8EL II ()Uva'to'tll'ta EqJUQlLOyi]<; 'tou ILEQLo'twlLa'tLX01J EW30ALaOILQU yLa anaUayi] 'tll<; V'toma<; AEILQVLa<; Aani]80u ano 'toY E!;WXOQ'tll xm aUa naQ6ILQLa na80yova. H 1L£80&o<; EqJaQIL60'tl]XE OE 2,000 onoQoqJU'ta an6 'tQLa ()LaqJOQE'tLXa uJtQxEL!1£va XQlJOLlLQnQLwvrcc; E!J.~6A.La AElLQvLac; ano avEJt'tuY/l£va Mv(){?U i] an6 vEaQa qJu'ta 8EQlLQxllnLou. Ta tl'llA.6'tE{?U noooo'ta EJtL'tUXLa<; £()WOE 'to unoxEL!1£vo citrumelo (41%) axoAou80u!1£vo ano 'tllV XL'tQo!J.llALa (37%) xm 'to rough lemon (29%). Ta E~oALa aVEJt'tuYIL£Vll<; AElLOvLa<; £()())(Jav x6.Jtw<; xaAu'tEQa ano'tEA£olLU'ta (37%) ano 'ta E~oALa 8EQILQXllnLou (32%), naQOuoLaoav 0ILW<; £V'tova 'to !1£LOV£x'tlllLa 'tll<; EJt0XLaxi]<; E!;aQ'tlloll<;. IIEQLnou 170 qJu'ta AElLovLa<; Aani]80u nou naQi]x8lloav o'to EQyaO'ti]QLO !1£ !J.EQLO'twlLU'tLXO EIL~ALaolL6 !;avaE~OALaOLl]XaV OLO 8EQlLQxi]mo navw OE avEJt'tuYIL£va onoQ0qJu'ta XL'tQolLllALa<; ILE noooo'to EJtL'tUXLa<; 73%. ~E 14 ano 'ta qJu'ta aULa £yWE LOAOYLXO<; £AEYX0<; nou £()ELl;E nw<; 12 i]oav anaUaY/l£va ano E!;WXOQ'tll (naQU 'tllV nQo£A.EOOll 'tou<; ano nQoo~~AlllL£vaILll'tQLxa MV()Qa) xa8w<; EJtLOll<; xm ano 'tL<; LWOEL<; 'tQLO't£~a, tIJOOQ<OOll xm ILOAUOILa'tLXi] nQLXLA.6XQ<OOll. Ta qJma aU'ta ea ano'tEA£oOUV 'tOY aQXLXO nUQi]va yLa 'tll ()ll!J.LOUQyLa ILll'tQLXi]<; qJU'tdac; 'tllC; AEILQVLac; Aani]eou yLa naQaywyi] UYLOUC; nOAAanAamaO'tLXou UALXOU.

INTRODUCTION developed a new in vitro technique known as shoot-tip grafting (STG), which consisted

Attempts to use shoot-tip culture in vitro of grafting a shoot apex from an infected for the production of pathogen-free citrus plant onto a disease-free seedling rootstock stocks have not been successful (Kartha, under aseptic conditions. This technique 1986). Thermotherapy has been satisfactory proved effective in recovering citrus plants in providing budwood free of certain viruses, free of several virus and virus-like patho­but has proved ineffective for the exclusion gens, including CEVd (Roistacher et ai., of citrus exocortis viroid (CEVd) and certain 1976). other pathogens (Calavan et aI., 1972). Ci­ In Cyprus, a recent study showed that the trus plants produced through nucellar em­ local lemon variety Lapithos is totally in­bryogenesis are free of most virus and virus­ fected by CEVd (Kyriakou, 1991). Since La­like pathogens, including those not eliminat­ pithos lemon is a very valuable variety for ed by thermotherapy, but unfortunately such Cyprus, the present work was carried out to plants exhibit juvenile characters which per­ investigate the possible use of STG in vitro sist for many years (Navarro et aI., 1975). for the recovery of this variety free of exo­

To overcome the above problems, Mura­ cortis and other virus and virus-like patho­shige et ai. (1972) and Navarro et ai. (1975) gens.

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MATERIALS AND METHODS

Shoot-tip grafting in vitro The fIrst step in the STG procedure was

the aseptic production of rootstock seedlings in vitro. The rootstock cultivars sour orange (Citrus aurantifolium L.), citrumelo (c. par­adisi Macf. x Poncirus trifoliata (L.) Raft) and rough lemon (C. jambhiri Lush) were tested. Seeds were peeled, disinfested and germinated in 25x150 mm culture tubes con­taining a simple nutrient medium composed of the salt base of Murashige and Skoog (1962) (Table 1) plus 2% sucrose and 0.7% agar. Seedlings were ready for grafting after incubation for 2 to 3 weeks at 25-30 °C in the dark.

Mature, bearing trees of Lapithos lemon, grown in commercial groves at Peristerona, Meneou, Paphos, Polis and Pyrgos, were se­lected as mother trees on the basis of their health state (determined by indexing), pro­ductivity, and trueness to type. Shoot tips were either obtained directly from the field­grown trees or from young potted plants es­tablished in the glasshouse from budwood taken from the selected trees in the field. Glasshouse plants were forced to produce new shoots by light pruning and defoliation. Such shoots were harvested when 2 to 4 cm long and were always used for scion prepara­tion the same day they were collected. In contrast, shoots from the fIeld were stored in a refrigerator and used for up to 6 days from collection.

Unless otherwise stated, in vitro grafting was carried out using the method of Navarro et al. (1975). A 2 to 3-week-old rootstock seedling was decapitated, leaving 1 to 1.5 cm of the epicotyl, and the cotyledons and their axilary buds were removed. An inverted T­incision was then made on the epicotyl through the cortex to the cambial region. For scion preparation a disinfested lemon shoot tip was placed under the stereoscope and all the foliar appendages near the shoot apex were removed. The shoot apex (about 0.2 mm long) consisting of the apical meristem plus 2 to 4 leaf primordia was then excised (using a no. 11 surgical blade) and inserted into the incision made on the rootstock see­dling.

Another method, compared with the pro­cedure described above during 1988/89 , in­

volved a triangular cut through the cortex at the point of decapitation of the epicotyl. The corresponding triangular piece of cortex tis­sue was removed and the scion was inserted into the cut, in contact with the cambium (Rizqui, personal communication). In either case the procedure was carried out aseptical­ly in a laminar flow cabinet taking all neces­sary precautions to minimize possible me­chanical transmission of exocortis viroid (Roistacher et aI., 1969; Kyriakou, 1991).

Micrografted plantlets were cultured in 25x150 mm culture tubes containing 25 ml of a liquid medium consisting of the salt base of Murashige and Skoog (1962), vita­mins, myoinositol and sucrose (Table 1). One plantlet was placed in each tube and supported with a Heller fIlter paper plat­fonn, as described by Navarro et al. (1975). Cultures were kept in a biological incubator at 25-30 °C under low-intensity artificial il­lumination (1500-2000 lux), with a 16-h photoperiod. Any adventitious shoots arising from the rootstock were removed periodical­ly following strict aseptic procedures.

Table 1. Nutrient medium used for the culture of grafted plants in vitro (pH 5.7)

Ingredient Concentration (mg)

MS salt base* 4303.5 Sucrose 74000.0 Myoinositol 100.0 Thiamine.HCI 0.2 Pyridoxine.HCI 1.0 Nicotinic acid 1.0

*Murashige and Skoog salt base (mg/l): NH4N03. 1650; KNGJ, 1900; CaClz.2HzO, 493.8; MgS04.7HzO, 370.6; KHZP04, 170; FeS04.7HzO, 27.8; Naz-EDTA. 37.3; H3BOJ. 6.2; MnS04.4HzO, 8.6; KI, 0.83; NazMo04.2HzO, 0.25; CuS04.5HzO. 0.025; CoClz.6HzO, 0.025.

Plant establishment in vivo and indexing for virus and virus-like pathogens

Successfully cultured plantlets with at least one leaf developed were established in vivo by regrafting onto 10 mm thick sour orange seedlings grown in a glasshouse (De Lange, 1978). The lemon scion along with a small chip from the in vitro roostock was in­serted into a T-incision made on the potted sour orange seedling and wrapped with PVC

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grafting tape. To prevent wilting, the scion was enclosed in a polyethylene bag for five days and then exposed progressively over a period of 15 days.

Regrafted plants were kept for about two months in a growth chamber (20-28 °C) be­fore moved back to the glasshouse and treat­ed as normal plants. Regrafted plants were indexed for virus and virus-like pathogens within 6 to 9 months from their establish­ment in the glasshouse. Each plant was test­ed at least twice by graft inoculation on the following indicators: a) Etrog citron, selec­tion Arizona 861 for CEVd, b) Mexican lime for citrus tristeza virus (CTV), c) Madam Vi­nus and Pineapple sweet orange for different forms of psorosis and d) Eureka lemon for infectious variegation. Mature Lapithos lem­on trees used as scion sources were also in­dexed at least once using the same set of in­dicator plants.

RESULTS AND DISCUSSION

Evaluation of micrografting procedures Of the two micrografting procedures test­

ed during 1988/89 only the method of invert­ed T-incision gave promising results, Le. 57 successful grafts out of 513 attempted (11 % success). In contrast, the level of success ob­tained with the triangular cut method was only about 1.5% (two out of 137). Refine­ment of technical details and manual dexteri­ty gained with practice proved also key fac­tors for improving STG success. Successful grafts with the inverted T-incision method

LJ

100 ~ greenhouse shoot tips

90 c:J field

~ 80 "* shoot

~ 01

70

60 J

U; 50 V'l

~ 40 v ~ 30

~ 20 r­10 ~

Jan Feb

increased from 11 % in 1988/89 to about 33% in 1990.

Influence of scion source and season on shoot-tip grafting success

In 1990, STG tests were carried out throughout the year (January to November) in an effort to assess the seasonal quality of shoot-tip scions from greenhouse and field­grown source trees. They involved 1429 see­dlings of which 656 were grafted with green­house material and 773 with field material, giving an overall frequency of successful grafts of 32% and 37%, respectively. Al­though scion material from the field gave higher grafting success, its availability and quality were season-dependent. In winter, there was no suitable grafting material avail­able, while in early spring (March) and late fall (November) only limited quantities of poor-quality material were available, as evi­denced by low levels of STG success which hardly exceeded 5% (Fig. 1). From April to October, however, field-grown source trees usually produced an abundance of high­quality shoot tips, especially in July-August, during which grafting success reached 90%.

Contrary to field material, greenhouse shoot tips were available the year round and resulted in grafting success with considera­bly lower seasonal variability (Fig. 1). Simi­lar results were obtained in other Mediterra­nean countries (Navarro et al., 1975, 1988). Other advantages of greenhouse source-

shoot tips -tips not available

c-r-

r­ .-­r-

r I Mar Apr May Jun Jut Aug Sep Oct Nov

Figure 1. Influence of season on micrografting success of Lapithos lemon shoot-tips from greenhouse and field-grown source plants.

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plants are the convenience in collection of shoots and the possibility of their immediate use.

Air temperatures in the glasshouse used in the present study varied from 14 to 37 °C, which cannot be considered ideal for the de­velopment of abundant and high-quality shoot tips. In Spain, a 60 to 70% grafting success was obtained by placing defoliated greenhouse source plants in a growth room at a constant temperature of 32 °C for 10 to 15 days (Navarro et al., 1988). Therefore, it may be possible to further improve STG suc­cess through an effective temperature control system.

Evaluation of rootstock cuItivars In vitro tests to evaluate the three root­

stock cultivars involved, 758 STG attempts were made during February-July 1990. Best results were obtained on citrumelo with an average success of 41%, followed by sour orange (37%) and rough lemon (29%) (Table 2). Citrumelo had the advantage of a trifoli­ate marker, which facilitated identification of successful grafts. Adventitious shoots arising from this rootstock could be readily detected and removed, thus minimizing their possible adverse influence on scion growth. Lower grafting success on rough lemon seedlings could be largely attributed to their slender epicotyls, which made grafting in vitro diffi­cult. Despite its rather poor performance in the present study, rough lemon was superior (27% success) to Troyer citrange (5% suc­cess) when used as rootstock for Ricote lem­on (Navarro et al., 1975).

Table 2. Effect of rootstock cultivar on micrografting success of Lapithos lemon shoot-tips

Rootstock No. of seedlings % successful cultivar grafted grafts*

Sour orange 289 37 Citrumelo 232 41 Rough lemon 237 29

* Differences between rough lemon and the other two rootstocks are significant (P=0.05) based on x2 test.

Regrafting and plant establishment in vivo Results of regrafting tests with 170 Lapi­

thos lemon plants produced by STG in vitro

on three different rootstocks are summarized in Table 3. About 90% (152/170) of the re­grafting attempts were successful, as evi­denced by records taken at the end of the 2­month incubation period under controlled temperature and light conditions. After trans­fer to normal greenhouse conditions, howev­er, some of the initially established grafts failed to develop, thus reducing success to about 73%. Regrafting success was similar for all three in vitro rootstocks (Table 3).

Table 3. In vivo establishment of shoot-tip grafted Lapithos lemon plantlets by regrafting onto potted sour orange seedlings

Number of plantlets Rootstock used for regrafted in viyo shoot-tip grafting in vitro Total Successful* Developed+

Sour orange Citrumelo

67 76

59 69

47 56

Rough lemon 27 24 21 Total 170 152 124

*Successful establishment on sour orange seedlings following a 2-month incubation at 2D-28°C.

+Full development on sour orange seedlings after transfer to normal greenhouse conditions.

Elimination of virus and virus-like patho­gens

Indexing tests showed that all mother lemon trees were infected with severe exo­cortis isolates; none was infected with either tristeza, psorosis or infectious variegation. Likewise, tests with 14 micrografted Lapi­thos lemon plants, which were established in vivo by regrafting on sour orange seedlings, showed that all were free of the above virus­es. Twelve plants were also found free of ex­ocortis viroid even though they originated from exocortis-infected mother trees. Inter­estingly, isolates of exocortis from the two infected plants were characterized as "mod­erate" and "mild", respectively, despite the fact that isolates from their mother trees were "severe". According to Duran et al. (1988) the exocortis disease is caused by a complex of viroids which in citron can be separated by STG, resulting in variable symptoms (Juarez et aI., 1990). The present results suggest that STG may also separate these viroids in lemon.

The recovery of a small number of

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plants, evidently free of exocortis and major virus diseases, is considered very important from the practical point of view. These plants comprise a valuable nuclear stock which is presently being propagated with a view to develop a source of healthy bud­wood for Lapithos lemon. This variety, undoubtedly very important for Cyprus, combines high productivity, superior horti­cultural characters and satisfactory tolerance to mal secco (Phoma tracheiphila), the most dreadful disease of lemon in the Mediterra­nean region. Sour orange, the main rootstock used in Cyprus, is highly tolerant to exocor­tis. Thus, despite its widespread incidence (Kyriakou, 1991), exocortis does not consti­tute at present a major citrus problem. How­ever, sour orange is susceptible to tristeza, a potentially catastrophic virus disease which was recently detected in Cyprus (Kyriakou and Polykarpou, 1989). To alleviate this pro­blem, other rootstocks, tolerant to tristeza, may have to be used in the future. These in­clude the trifoliates (Poncirus trifoliata L. Raft.) and the citranges (Poncirus x Citrus sinensis), which are highly susceptible to ex­ocortis. Consequently, it is very important to ensure that such rootstocks are grafted with exocortis-free budwood produced by STG in vitro.

AC~OLEDGEMENTS

The authors wish to thank Mr. S. Chris­toforou and Mr. D. Polykarpou for technical assistance.

REFERENCES

Calavan, E.C., C.N. Roistacher, and E.M. Nauer. 1972. Thermotherapy of citrus for inactiva­tion of certain viruses. Plant Disease Re­porter 56: 976-980.

De Lange, J.H. 1978. Shoot-tip grafting: A motified procedure. The Citrus and Subtropical Fruit Journal 533:13-15.

Duran-Vila, N., J.A. Pina, J.F. Ballester, J. Juarez, C.N. Roistacher, R. Rivera-Bustamante, and J.S. Semancik. 1988. The citrus exo­cortis disease: A complex of viroid-RNAs. Proceedings of the International Organiza­tion ofCitrus Virologists 10: 152-164.

Juarez, J., M.1. Molins, L. Navarro, and N. Duran­Vila 1990. Separation of citrus viroids by shoot-tip grafting in vitro. Plant Pathology 39: 472-476.

Kartha, K.K. 1986. Production and indexing of dis­ease-free plants. lIn Plant Tissue Culture and its Agricultural Applications (Withers, L.A., and P.G. Alderson, eds), pp. 219­238. University Press, Cambridge.

Kyriakou, A., and D. Polykarpou. 1989. Detection of citrus tristeza virus in Cyprus by indexing. FAO Plant Protection Bulletin 37: 131­132.

Kyriakou, A. 1991. Incidence of citrus exocortis vir­oid in Cyprus and its transmission by me­chanical means under Cyprus conditions. Plant Pathology (in press).

Murashige, T., and F. Skoog. 1962. A revised medi­um for rapid growth and bioassays with to­bacco tissue cultures. Physiologia Plantar­um 15:473-497.

Murashige, T., W.P. Bitters, E.M. Rangan, E.M. Nauer, C.N. Roistacher, and P.B. Holliday. 1972. A technique of shoot apex grafting and its utilization towards recovering vi­rus-free citrus clones. HortScience 7: 118­119.

Navarro, L., C.N. Roistacher, and T. Murashige. 1975. Improvement of shoot-tip grafting in vitro for virus-free citrus. Journal of the American Society for Horticultural Science 100:471-479.

Navarro, L., C.N. Roistacher, and T. Murashige. 1976. Effect of size and source of shoot tips on Psorosis-A and exocortis content of navel orange plants obtained by shoot-tip grafting in vitro. Proceedings of the Inter­national Organization of Citrus Virologists 7: 194-197.

Navarro, L., J. Juarez, LA. Pina, J.F. Ballester, and J.M. Arrequi. 1988. The citrus variety im­provement program in Spain after eleven years. Proceedings of the International Or­ganization of Citrus Virologists 10:400­406.

Roistacher, C.N., E.C. Calavan, and R.C. Blue. 1969. Citrus exocortis virus: Chemical inactiva­tion on tools, tolerance to heat and separa­tion of isolates. Plant Disease Reporter 53: 333-336.

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