Genetic Transformation of Citrus

State of the art

Until recently, citrus species have been recalcitrant to transformation. Kobayashi and Uchimiya (1989) obtained transgenic callus from Trovita sweet orange by poly- ethylene glycol (PEG) treatment of cell sus- pensions with a plasmid containing the marker gene nptII, but regeneration of trans- genic plants from that callus was unsuccess- ful. Vardi et al. (1990) produced transgenic callus from Rough lemon (C. jambhiri Lush) by PEG treatment of protoplasts with a plas- mid containing the marker genes cat and nptII, and obtained several stably transgenic embryogenic lines, but only two of them regenerated whole plants. Hidaka et al. (1990) produced transformed callus of Washington navel and Trovita oranges by co-cultivation of embryogenic cell suspen- sion lines with Agrobacterium tumefaciens, but only one transgenic plantlet of Washington navel was regenerated. Moore et al. (1992) produced two transgenic plantlets of Carrizo citrange by co-cultiva- tion of internodal stem segments from in vitro grown seedlings with A. tumefaciens. More recently, this group reported a slight increase in transformation effi ciency using basically the same procedure and regener- ated two sour orange, nine lime and nine Carrizo citrange plants with the major coat protein gene of CTV (Gutié rrez et al., 1997). Hidaka and Omura (1993) obtained trans- formed Ponkan mandarin callus by electro- poration of protoplasts, but no plants were regenerated. Yao et al. (1996) reported trans- formation of Page tangelo embryogenic cells using particle bombardment, and produced 15 transgenic embryo lines, but they did not progress further. Kayim et al. (1996) bom- barded nucellar cells of lemon, but only b- glucuronidase (GUS) expression from those cells was reported.

The fi rst report of effi cient and reliable production of citrus transgenic plants was published by Kaneyoshi et al. in 1994. They obtained transformation effi ciency higher than 25% by co-cultivating etiolated epi-

cotyl segments of trifoliate orange with A. tumefaciens. This allowed them effi ciently to incorporate the human epidermal growth factor (hEGF) (Kobayashi et al., 1996) and the rolC gene from Agrobacterium rhizo- genes (Kaneyoshi and Kobayashi, 1999) into this species. The same procedure with slight modifi cations has been used by Bond and Roose (1998) to transform Washington navel orange, and by Luth and Moore (2000) to transform grapefruit. Pé rez- Molphe and Ochoa-Alejo (1998) have reported effi cient production of transgenic lime plants by co-cultivation of internodal stem segments from in vitro grown seedlings with A. rhizogenes. Yang et al. (2000) have used A. tumefaciens to trans- form epicotyl segments of Rio Red grape- fruit, and have reported production of transgenic plants containing an untranslat- able version of the major coat protein gene from CTV and the Galanthus nivalis agglu- tinin gene. Co-cultivation of epicotyl seg- ments with A. tumefaciens has been also used by Gentile et al. (1998) and LaMalfa et al. (2000) to regenerate transgenic plants of Tarocco orange and Troyer citrange with rol genes from A. rhizogenes and with the green fl uorescent protein gene (gfp) from the jellyfish Aequorea victoria, respec- tively, and by Koltunow et al. (2000) to pro- duce transgenic limes containing genes for decreased seed set. A different approach was followed by Fleming et al. (2000) who used the gfp gene as both a selectable and reporter marker to transform Itaborai sweet orange protoplasts with PEG, produce embryogenic callus from them and recover whole transgenic plants through somatic embryogenesis.

The recalcitrance of citrus to genetic transformation is mainly due to several fac- tors: ineffi ciency of bacterial vectors in the transformation of citrus cells, since citrus species are not natural hosts of Agrobacterium; diffi culties in regenerating shoots only from the transformed cells at the same time avoiding the recovery of escapes; and difficulties in rooting the transgenic shoots. Our group started to work in genetic transformation of citrus in

1993, and since then we have been able to develop effi cient and reliable procedures to produce transgenic plants from Carrizo cit- range (Peñ a et al., 1995a; Cervera et al., 1998a), using epicotyl segments from in vitro grown seedlings as source material, and from Pineapple sweet orange (Peñ a et al., 1995b; Cervera et al., 1998b), lime (Peñ a et al., 1997; Domí nguez et al., 2000), sour orange (Ghorbel et al., 2000), alemow, lemon and Cleopatra mandarin (Ghorbel et al., 2001a), using internodal stem segments from greenhouse-grown seedlings as source material. The use of the appropriate Agrobacterium strain super-transforming citrus as vector, the establishment of the appropriate infection and co-cultivation conditions and culture media, as well as adequate selection conditions and culture media, the use of source plant material in a good physiological state, the determination of the cells competent for transformation in citrus explants, the use of appropriate marker genes, and the rapid production of whole transgenic plants through grafting of regenerating transgenic shoots into vigor- ous rootstocks fi rst in vitro and later in the greenhouse have been crucial to enabling the regeneration of transgenic citrus plants at high effi ciencies. Furthermore, we have been able to transform mature material, recovering transgenic plants that fl owered and set fruits in 1–2 years after Agrobacterium infection (Cervera et al., 1998b). We are now incorporating trans- genes of potential agricultural interest into citrus species. These procedures and the recent developments of our projects are the subjects of this chapter.

Transformation from seedling explants

Four- to 12-month-old greenhouse-grown (18–27°C) sweet orange, sour orange, lime, alemow, lemon and mandarin seedlings are used as the source of tissue for transforma- tion. Stem pieces (20 cm in length) are stripped of their leaves and thorns, disin- fected for 10 min in a 2% (v/v) sodium hypochlorite solution and rinsed three

times with sterile distilled water. For Carrizo citrange, stored seeds originating from the same tree stock are peeled, remov- ing both seed coats, disinfected for 10 min in a 0.5% (v/v) sodium hypochlorite solu- tion containing 0.1% (v/v) Tween-20, and rinsed three times with sterile distilled water. Five-week-old germinating seedlings are used as the starting material for genetic transformation. These seedlings are grown in MS salt solution (Murashige and Skoog, 1962) plus 10 g/l agar, pH 5.7, at 26˚ C in darkness for the fi rst 2 weeks, and under a

16 h photoperiod and illumination of 45

mEm–2 s–1 for an additional 3 weeks.

Agrobacterium tumefaciens strain EHA105 (Hood et al., 1993) carrying a binary plasmid is used as the vector system for transformation. The T-DNA of the binary plasmid must contain, apart from the gene or genes of interest, a selectable marker gene, such as nptII, and a reporter marker gene, such as uidA. The binary plas- mid is introduced into Agrobacterium by electroporation. Bacteria are cultured overnight in an orbital shaker at 28˚ C and

200 r.p.m. in LB medium containing the appropriate antibiotics for the binary system to grow. Bacterial cells are pelleted at 3500 r.p.m. for 10 min, resuspended and diluted to approximately 4 ´ 107 cells/ml in liquid inoculation medium, which consists of MS salt solution, 0.2 mg/l thiamine hydrochloride, 1 mg/l pyridoxine hydro- chloride, 1 mg/l nicotinic acid and 3% (w/v) sucrose, pH 5.7.

Sweet orange, sour orange, lime, alemow, lemon and mandarin internodal stem segments, and citrange epicotyl seg- ments (~1 cm long) are cut transversely and incubated for 15 min in 10 cm diameter plates containing 15 ml of the bacterial sus- pension in inoculation medium by gentle shaking. The infected explants are blotted dry on sterile fi lter paper and placed hori- zontally on plates with CM medium for a 3 day co-cultivation period. CM medium con- sists of MS salts, 1 mg/l thiamine hydrochloride, 1 mg/l pyridoxine hydro- chloride, 1 mg/l nicotinic acid, 3% (w/v) sucrose, 2 mg/l indole-3-acetic acid, 1 mg/l

2-isopentenyladenine, 2 mg/l 2, 4- dichlorophenoxyacetic acid and 0.8% (w/v) agar, pH 5.7. Co-cultivation in a medium rich in auxins provides to the plant cells at the cut ends of the explants an appropriate treatment to shift them to a competent state for transformation, involving dedifferentia- tion, induction of cell division and callus proliferation (Peñ a et al., 1997; Cervera et al., 1998a).

After co-cultivation, the explants are blotted dry with sterile fi lter paper and transferred to SRM medium, which consists of MS salts, 0.2 mg/l thiamine hydrochlo- ride, 1 mg/l pyridoxine hydrochloride, 1 mg/l nicotinic acid, 3% (w/v) sucrose, 1% (w/v) agar, pH 5.7, plus 100 mg/l kanamycin for the selection of transgenic shoots and 250 mg/l vancomycin and 500 mg/l cefotaxime to control bacterial growth. This medium is supplemented with 3 mg/l benzylaminopurine (BAP) for sweet orange and citrange, 1 mg/l BAP for lime, lemon, alemow and mandarin, and 1 mg/l BAP plus 0.3 mg/l naphthaleneacetic acid (NAA) for sour orange. Cultures are main- tained in the dark for 4 weeks at 26˚ C and then are transferred to a 16 h photoperiod, with 45 mEm–2 s–1 illumination at 26˚ C. Culture of the explants in the dark favours callus formation and thus the progress of transformation events to regenerate trans- genic shoots and, at the same time, avoid regeneration of escape shoots that could be stimulated by the exposure of explants directly to the light (Peñ a et al., 1997; Cervera et al., 1998a). For sour orange, it can also be speculated that the combination of BAP and NAA in the SRM medium is more favourable than BAP alone in stimu- lating cell divisions and re-differentiation from the transgenic competent cells to undergo transformation events (Ghorbel et al., 2000).

Small pieces of the shoots emerging from the explants are assayed for histo- chemical GUS activity (Jefferson et al., 1987) and then apical portions are grafted in vitro onto Troyer citrange seedlings (Navarro et al., 1975; Navarro 1992; Peñ a et al., 1995a, b). Rootstock preparation is as

follows: Troyer citrange seeds are peeled, removing both seed coats, disinfected for 10 min in a 0.5% (v/v) sodium hypochlorite solution containing 0.1% (v/v) Tween-20 and rinsed three times with sterile water. The germination medium is MS salts with 10 g/l agar, pH 5.7. Seeds are sown individ- ually in tubes and grown in darkness at 27°C for 2 weeks. Troyer citrange seedlings are decapitated, leaving 1–1.5 cm of the epi- cotyls. The roots are shortened to 4–6 cm and the cotyledons and their axillary buds are removed. Then, the regenerated shoot apical ends are placed on the top cut sur- faces of the decapitated citrange epicotyls, in contact with the vascular ring, or, when larger than 0.4 cm, they are inserted into a lateral incision or in a vertical incision along the length of the epicotyl, starting at the point of decapitation (Navarro et al., 1975; Peñ a et al., 1995a, b, 1997). Grafted plants are cultured in a liquid medium composed of MS inorganic salts, 100 mg/l m-inositol, 0.2 mg/l thiamine-HCl, 1 mg/l pyridoxine-HCl, 1 mg/l nicotinic acid and 75 g/l sucrose, pH 5.7. The cultures are kept at 25°C, with a 16 h photoperiod and 45 mE m–2 s–1 illumination. Shoots of only 0.1 cm in length can be used to regenerate trans- genic plants following this protocol. A fre- quency of 100% successful grafts is usually obtained. Scions develop 2–4 expanded leaves 3–4 weeks after grafting. A new graft- ing of the in vitro growing plants on vigor- ous rootstocks in the greenhouse allows the rapid acclimatization and development of the plants.

Alternatively, for certain purposes, rooting of the transgenic shoots allow rapid analyses of the effect of the transgene of potential interest to be performed even in vitro when the new inserted trait could affect not only to the aerial part of the plant but also the roots (i.e. tolerance to salinity, plant size, etc.). Although development of whole plants is slower and less effi cient than performing in vitro grafting, rooting can be obtained by cutting 0.5–1 cm regen- erated shoots from the explants and trans- ferring them to SRM medium supplemented with 0.3 mg/l BAP for 7–10

days, and then to SRM medium supplement with 5 mg/l indolebutyric acid. At least for lime and lemon, roots develop within a 3–6 week period with 90–100% effi ciency.

Standard polymerase chain reaction (PCR) techniques are used to detect the presence of the transgene(s) in leaf samples from the regenerated putative transgenic plantlets. Southern blot analyses are per- formed to confi rm the stable integration of the transgene(s), and northern blot, western blot and enzyme-linked immunosorbent assay (ELISA) analyses allow the detection of their expression in the transgenic plants. Defi ning the transformation effi ciency as the frequency of whole transgenic regener- ated plants established in a greenhouse per Agrobacterium -inoculated explant, effi - ciencies of more than 40% for citrange, about 20% for sweet orange and lime, between 5 and 10% for alemow and sour orange, and less than 5% for lemon and mandarin are currently obtained in our lab- oratory.