Indian Journal of Biotechnology Vol I. October 2002, pp 344-349

Factors Influencing GUS Expression in Cucumber (Cucumis sativus Linn.)

A Vasudevan ', A Ganapathi' *, N Selvara/ and G Vengadesan ' I Department of Biotechnology, Bharathidasan University, Tiruchirappalli 620024. India

2Department of Botany. Periyar E V R College (Autonomous), Tiruchirappalli 620023, India

Received 18 Decell/ber 200 I .. accepted 15 April 2002

The transformation efticiency of two strains of Agrobacteril/lIl tlllllejaciells, EHA 105 and LBA 4404 with three cucumber cultivars, viz. Endeavor, Green Long and Poinsett 76 have been investigated. Factors such as age of explants, co-cultivation period, efticiency of strains, acetosyringone treatment, use of Kanamycin and Basta as selection agents were assessed. GUS histochemical assay was performed to assess transformation efticiency. 5-day-old cotyledons and 3-day co-cultivation period for Endeavor and Green Long and 2-day co-cultivation period for Poinsett 76 were suitable for ellicient transformation. EHA 105 was more virulent and the transformation elliciency was two to three folds higher than LBA 4404. Among the three cultivars, Endeavor was found to be more responsive to transformation as evidenced by GUS expression followed by Green Long and Poinsett 76. Acetosyringone (20 iJ.M-50 iJ.M) enhanced the efticiency of transformation in all the three cultivars. Basta at 10 mgll was more efficient selection agent than Kanamycin at 100 mg/1. Shoot regeneration occurred within 6 weeks after co-cultivation.

Keywords: Cl/cl/lIlis .I'ativlIs, Agrobacterilllll, acetosyringone, Kanamycin, Basta

Introduction Cucumber (CuculIlis sativus Linn.) belonging to the

family Cllcurbitaceae is an important horticultural crop. It is mainly cultivated for its fruits, which are used for slicing, pickling and juice extraction. In India, both the fruits and seeds have long been used in the manufacture of traditional medicines, which act as skin conditioner, diuretic and body coolant. Except viral resistance (Chee & Slightom, 1991) and fungal resistance (Raharjo et al, 1996) other potentially useful agronomic traits have not been engineered into cucumber genome. Cucumber is a potential candidate for edible vaccine also (Sharma et al, 1999). Recently, the genes that regulate sex expression in cucumber have been cloned successfully (Kahana et ai, 1999). To accomplish these tasks, a reliable transformation and regeneration protocol is essential. So far, there have been only few reports on cucumber transformation (Chee, 1990a; Sarmento et al, 1992; Chee & Slightom, 1991; Raharjo et al, 1996; Nishibayashi et ai, 1996). In India, studies on cucumber ti ssue culture and transformation are very limited (Ganapathi & Perl-Treves, 2000). To optimize the process of gene transfer via Agrobacterium, three cucumber genotypes were selected and co-cultivated

* Author for correspondence: Tel: 091-0431-660386; Fax: 091- 0431- 660245, 660320 E- mail : ganap @bdu.crnel. in

separately with two bacterial strains namely, EHA 105 and LBA 4404.

Materials and Methods Plallt Material

Seeds of cucumber cultivars, namely, Poinsett 76 [Indo-American Hybrid Seeds (India) Pvt, Ltd, Bangalore, India] Endeavor and Green Long (Mahyco Seeds Co, Maharashtra, India) were procured and used for transformation experiments.

The sterilized seeds were kept on sterile moist cotton for 24 hrs . Then their seed coats were separated and aseptically removed without disturbing the cotyledons. The cotyledons were carefully dissected from the embryonic axis. The distal end of the cotyledon explants was cut or injured slightly and the cotyledonary pieces (5 mm) were vertically inoculated in such a way that the distal end touches the medium. MS medium containing BA (6-benzylamino purine) (l mg/I) was used for shoot regeneration. The cultures were kept at 25±2°C with a 16 hrs photoperiod with the light intensity of 80 Ilmol m-2

S-I under cool white fluorescent lamps.

Agrobacterium tumefaciens Strains and Co­cultivatioll

Disarmed A. tumefaciens strains, EHA 105 and LBA 4404 were used to study strain efficiency on

VASUDEVAN el ({f: GUS EX PRESSION IN CUCU MBER 345

transformat ion. Both the st rains contain a binary plasmid (pGA492G I) with IIptll (neomycin phophotransferase), and 35SGUS fu sion (The GUS with a plant intron), and bar (35S-phosphinothricin acetyltransferase). EHA 105 and LBA 4404 strains were kindly provided by Rafael Perl-Treves, Bar lIan University, Israe l. The presence of ch imeric gllsA gene with a plant inlron makes it a useful tool for studyi ng early events of transformation in plant ti ssues. The two bacterial strains were grown at 28°C on LB (Luria Broth) medium containing, casein hydrolysate (10 g/l ), yeas t extract (5 g/l ), sodi um chloride (5 g/l ), Kanamycin (50 mg/l) and Tetracyc line (10 mg/l ). Prior to infection, si ngle bacterial colony of each stra in was inoculated separately in 50 ml of LB medium containing 50 mg/l Kanamycin and 20 mg/l Tetracycline and incubated at 28°C on a shaker at 200 rpm for 24 hrs. The agrobacterial suspension ( I OD) was diluted ( I :50) in half strength MS liquid medium conU.lInlng Kanamycin (50mg/ l). One hour before the co­culti vation of ex plants, ace tosyringone ( 10-50 )..lM) was added. The prox imal end of the coty ledon explants was pri cked with a sterile needle to induce agro-infection. The explants (100-120 per treatment) were dipped in bacterial suspension fo r 10 min (each of two strai ns were evaluated In separate ex periments).

GUS Assay Ex plants/regenerants were assayed for the

express ion of gllsA gene foll owing the hi stochemical procedure described by l efferson et (11 (1987). Transient gllsA ex pression was measured immediately after co-cultivat ion (8 hrs). The percentage of GUS expression was calculated by the number of explants showi ng GUS positive divided by the number of ex plants subjected to co-culti vation and multiply by 100 (Cao et (11, 1998).

Each treatment consisted of at least 20 ex plants and each experiment was repeated thrice. A complete randomi zed des ign was used in all experiments and analys is of vari ance and mean separati ons were carried out using Duncan' s Multiple Range Test (DMRT). Significance was determined at 5% level (Gomez & Gomez, 1976).

Results and Discussion There have been a few reports in cucumber

transformation (Srivastava et aI, 1990; Chee, 1990a; Chee & Slightom, 199 1; Raharj o et aI, 1996;

Nishibayashi et (11, 1996). The transformation frequency in these studi es varied from 12 to 52%. Such a problem has largely been attributed to several fac tors, which have not been full y ana lyzed for cucumber gene transfer. Therefore, an effort was made to evaluate the two important strains of A. tllJllej'aciel1 s (EHA 105 and LBA 4404) on three commercial culti vars namely, Poinsett 76, Endeavor and Green Long.

Direct regeneration system was adopted so that the GUS expression could be scored within the meristematic region of the coty ledon, whi ch was highly organogenic . This system has already been employed successfully to recover transgeni c plants (Tabei et aI, 1998; Ganapathi & Perl-Treves, 2000) . In addi tion, direct regeneration system has been considered as an ideal one to get true to type regenerants (B urza & Plader, 1995; Plader er aI, 1998) .

Effect of Explant Age Oil GUS Expression Age of the explant is a critical factor, which

intluences transformation effici ency (DeBondt er (11 , 1994). 1-5 -day-old cotyledonary explants and three week-o ld you ng shoots that arose from coty ledonary explants were analyzed for GUS ex pression to assess the transformation efficiency. Five-day-old explants in all cultivars showed higher GUS ex press ion (Fig. I) with EHA 105 strain . The freq uency of GUS expression was more (75 %) in Endeavor followed by Green Long (62%) and Poinsett 76 (55 %). GUS ex press ion on the cotyledon exp lants of Endeavor and Green Long was signi ficant ly higher than Poinsett 76. Cao er al (1998) observed in blueberry that the age of the ex plant did not have sign ificant effect on the GUS

80 ,--------.--- .- ._--- --- .-- - ------- ,

70 .

10

o

IT] Endeavor Im Greenlong m Boinsett76

2 3 4 5 Explant age (Days)

Fig. I - Effect of age of the cotyledon ex plants on UGS expression in Cucumber culti vars co-clllli v::l tec\ wi th EHA 105

346 INDIAN J BIOTECHNOL, OCTOBER 2002

Fig, 2-Transformation of CucUll/is salivllS Linn. Cotyledon explants by AgrobacteriulIl IUlI1ejaciel/s. A, B & C, 5-day-old cotyledon expIants of Poinsell 76 after co-cultivation with AgrobacleriulII tllll/ejaciel/s strain EHA 105 showing strong GUS activity at the meri stematic region of the proximal end. Meri stematic zones were seen by GUS assay after 8 hrs of infection; D, E & F, 5-day-old cotyledon explants of Endeavor showing GUS positive buds after 3 days of co-cultivation with EHA 105; G & H, Developing shoot primordia (Poinsell 76) showing GUS activity after 3 weeks of culture on Kanamycin containing (100 mg/I) media; I, 5-day-old­cotyledon explants showing transformed (blue) and non-transformed (yellow) meri stematic regions after 2 days co-cultivated with LBA 4404; & J, Proliferating shoots from cotyledon explants (Poinsett 76) after 4 weeks of culture on Kanamycin (100 mg/I) containing medium.

V ASUDEV AN et at: GUS EXPRESSION IN CUCUMBER 347

expression. But the studies on cucumber transformation proved that age of the explant played vital role (Chee, 1990; Chee & Slightom, 1991; Raharjo et al, 1996; Nishibayashi et al, 1996).

Influence of Co-cultivation Period on GUS Expression

The initial transformation optimization experiments were performed to determine the effective co­cultivation time. GUS staining was often observed in other parts of the explants other than the meristematic zones and they were not considered as transformed regions. The intensity of GUS expression in the meristematic region had increased when the co­cultivation time was prolonged to fourth day.

Visible GUS expression was observed at the meristematic zones of the ex plants (Fig.2A-J) in three cultivars 8 hrs after transferring to selection medium containing Cefotaxime (300 mg/l) and Kanamycin (100 mg/I). This observation was in contrast to the findings of Cao et al (1998) in blueberry and Cervera et at (1998) in Citrange, where no GUS expression was observed immediately after co-cultivation. The GUS expression gradually increased after a day of co­cultivation, reaching to a maximum on the fifth day in all the three cultivars (Table 1). EHA 10S strain was more efficient than LBA 4404 in evoking better GUS

expression. Among the three cultivars, GUS expression was maximum in Endeavor followed by Green Long and Poinsett 76 (Table 2) . Prolonged co­cultivation period of more than three days has been successfully used for transformation of certain plants (DeBondt et ai, 1994; Cao et ai, 1998; Cervera et ai, 1998; Mourgues et ai, 1996). In earlier studies on cucumber transformation, the co-cultivation period varied from 2-S days for different explants (Chee, 1990b; Chee & Slightom, 1991 ; Raharjo et ai, 1996; Nishibayashi et ai, 1996). In the present study, co­cultivation period of 3 days for Endeavor and Green Long and 2 days for Poinsett 76 was found optimum for effective transformation .

Effect of Strains on GUS Expression Raharjo et al (1996) achieved 11-14%

transformation in Endeavor using EHA lOS. Nishibayashi et al (1996) used EHA 101 strain in the transformation of cucumber pure line 1021 with a frequency of SO%. Our studies are in conformity with the observations of Hood et al (1986, 1993) who reported strains EHA 101 and EHA lOS were more effective than strain LBA 4404 since both were derived from super virulent strain A281 whereas, strain LBA 4404 was derived from less virulent strain AchS (Hoekema et ai, 1983).

Table I-Effect of co-cultivation period on GUS expression in cotyledon explants derived from 1-5 day old seedlings of cucumber cultivars co-cultivated with EHA 105 and LBA 4404

Cultivar Percentage of GUS expression Days of co-cultivation

2 3 4 5 Endeavor 40a.3 1a 64a, 57a 76a, 70a 89a, 84a 98a,91a

Green Long 31ab, 22b 52ab,40b 62ab,49b 71b,68b 86b, 79ab

Poinsett 76 20c, 16bc 31c, 27bc 45c, 38bc 64bc,57bc 78bc,61c

Each value represents the treatment means of twenty ex plants and repeated three times. Values with the same letter within columns are not significantly different according to Duncan's Multiple Range Test (DMRT) at 5% level.

Table 2-Intensity of GUS expression in 5-day-old cotyledon ex plants of cucumber cultivars co-cultivated with EHA 105 and LBA 4404.

Cultivar Agrobacterium Intensity of GUS expression No. of shoots per explant Strains in cotyledon explants showing GUS

Endeavor EHA 105 +++++ 10

LBA 4404 ++ 4

Green Long EHA 105 ++++ 7

LBA 4404 + 2

Poinsett 76 EHA 105 +++ 5

LBA 4404 +

+++++ - very strong, ++++ - strong, +++ - moderate, ++ & + - weak

348 INDIAN J B10TECIINOL, OCTOBER 2002

Illfluellce of Acetosyrillgone 011 GUS Expression The effect of acetosyringone treatment during co­

cu lti vation of cucumber explants with EHA 105 and LBA 4404 strains has been studied. Acetosyringone is a phenolic compound produced during wounding of plant cell that induces the transcription of the viru lence genes of A. tumefaciell s.

The explants of three cu lti vars were co-cu ltivated using EHA 105 and LBA 4404 strains with different concentrations of acetosyringone (10-50 /lM). In control treatments (without acetosyringone), GUS expression was found at a low frequency. Further, the explanls, which were co-cu lt ivated without and with acetosyringone for two and three days were transferred to the shoot induction medium containi ng Kanamycin for two to three weeks . The frequency of GUS expression in the regenerated shoots that arose from the explants treated with acetosyringone had increased. Among the three culti vars, the freq uency of GUS expression in the regenerated shoots was maximum in Endeavor followed by Green Long and Poinsett 76. A concentration of 20 /lM acetosyringone was optimum for Poinsett 76, beyond which shoot recovery was not possible due to bacterial overgrowth in explant. Acetosyringone at 50 /lM concentration was optimum for Endeavor and Green Long (Table 3). In the present study, the acetosyringone treatment that was used during infection and a period of two to three day's co-cultivation were effective in enhancing the frequency of shoot regeneration in all three culti vars over control. Further, it was evident that irrespective of genotypes, the nature of the bacterial strain coupled with acetosyringone treatment was essential in cucumber transformation . Enhanced

transformation efficiency using acetosyringone in cucumber had been reported earlier (Nishibayashi et ai, 1996; Mohinuddin et ai, 2000).

Effect of Selection Agents (Kallamycin and Basta) To select the better selection medium for effective

transgenic recovery of cucumber, AgrobacferiulIl EHA 105 strain containing bar and nptl! genes within T- DNA border coding for PPT (Phosphinothricin) and Kanamycin resistance, respectively was used. Among the three culti vars, Endeavor exhibited better response. Explants were cultured in MS medium containi ng various concentrations of Kanamycin (0, 25,50,75, 100, 150 mg/I) . At 100 mg/I concentration of Kanamycin, shoot formation was completely inhibited in non-eo-cultivated explants. On the other hand, transformed shoots grew normally in 100 mg/I Kanamycin . GUS assay was performed in the regenerated shoots.

The explants were inoculated in MS medium containing various concentrations of Basta (0, 2, 4, 8, 10, 12 mg/I) . The frequency of shoot regeneration gradually decreased with the increase in Basta concentrations and at 10 mg/I, all the shoots stopped growing and became yellow showing symptoms induced by Basta. From these results, it was evident that 10 mg/I PPT concentration was optimum for transgenic recovery. Our results are in agreement with the observations of De Block et al (1987) in tobacco and Akama et al (1992) in Arabidopsis. The occurrence of a certai n degree of escapes in Kanamycin selection in this study suggests that Basta selection would be suitable more appropriately for cucumber transformation studies.

Table 3- Effect of acetosyringone on GUS expression in 5-day-old coty ledon explants of cucumber cult ivars co-cultivated with EHA 105 and LBA 4404

Acetosyringonc Percentage of GUS expression in explants treatment (flM) After 8 hrs End of co-culti vation

E GL P E (72 hrs) GL (72hrs) P (48 hrs) EHA LBA EHA LBA EHA LBA EHA LBA EHA LBA EHA LBA 105 4404 105 4404 105 4404 105 4404 105 4404 105 4404

10 3ab Ib 2b Ibc 5a 3a 17a 8a 14b 6b 12b 5bc

20 7ab 3b 5b 3bc ll a 6a 22ab lOb 20c 8bc 50a 44a

30 llb 5b 8ab 4bc 16a 7a 33b 17b 31bc 14bc 6 1a 33a

40 13b 6b Ilbc 6bc 19a 9a 52a 30a 44ab 23ab

50 30a 17a 24b 14b 21bc 12bc 65a 56a 58ab 52ab

E-Endeavor, GL-Green Long & P- Poinsett 76 Each value represents the treatment means of twenty explants and repeated three times. Values with the same letter within columns are not significantly different according to Duncan's Multiple Range Test (DMRT) at 50/0 level.

VASUDEVAN et a/: GUS EXPRESSION IN CUCUMBER 349

In conclusion, our experiments have clearly indicated that all the three cucumber cultivars are competent for Agrobacteriul1I mediated transformation with EHA 105 strain under defined physical conditions.

References Akama K et al. 1992. Efficient Agrobacterill/II - mediated

transformation o f Arabidopsis tlll/liallo using the bar ge ne as selectable marker. Plallt Cell Rep. 12, 7- 11.

Burza W & Plader S, 1995. Direct plant regeneratio n fro m leaf ex plant in Cucumber (Cllcllllli.\' sativlIS L.) is free o f stable gc: ne tic variation. Plallt Breed, 12, 34 1-345.

Cao X et al. 199X . GUS express ion in blueberry (Vaccillilllll spp.): Facto rs influencing Agrobacterilllll -mediated gene tran sfer effici ency. Plallt Cell Rep, 18, 266-270.

Cervera M et al. 1998. Agrobacterill/n medi ated transformation o f Citrange: Factors affecting transformation and regeneration. Plallt Cell Nep , 18, 27 1-278 .

Chee P P, 1990a. Transformation of Cllclllllis Sa/iIlIlS ti ssue by Agrobacrerilllll tUlllej(lciells and the regenerati on of transformed plants. Plallt Cell Rep. 9, 245-248.

Chee P P. 1990b. High frequ ency o f somati c e mbryogenesis and recovery of fertile cucumber plants. Hortic Sci, 25, 792-793.

Chee P P & Sl ightom J L, 199 1. Transfer and expression o f C ucumber Mosaic Virus Coat protein gene in the genome of CllclIlIli.\' satil'IIS. } AIIl Soc Hortic Sci, 116, 1098- 1102.

De Block M el al. 1987. Use of bar as a selec table marker gene and for the producti on of herbicide- resistance rice plants from protoplasts. EM BO 1. 6, 2513-2518.

DeBondt A el al. 1994. Agrobacterilllll mediated tran sformatio n of apple (MaillS x dOlllestim Borkh) : An assessment of factors affecting gene transfer e ffici ency during early transformation steps. Plant Cell Rep , 13,587-593.

Ganapathi A & Perl-Treves R, 2000. AgrobacteriUln-mediated transformatio n in CllcllInis sativus L. via direct organogenesis. Acta Hortic. 510, 405-407.

Gomez K A & Gomez K A, 1976. Statistical Procedures for Agricultural Research with Emphasis of Rice. International Rice Research Institute, Las Banos, Manila, Philippines.

Hoekema A et al. 1983. A binary vector strategy based on separation of vir- and T- region of the Agrobacterilllll tlllneJaciells Ti- plasmid. Natllre (Lolld), 303, 179- 180.

Hood E E et al. 1986. The hypovi rulenee of Agrobacterilllll IllllleJaciells A281 is encoded in the region o f pTi Bo 542 outside the T-DNA. } Bacteriol, 168, 1291- 1301.

Hood E E et al. 1993. New Agrobaclerilllll vectors for plant tran sformation. Tran.l'gellic Res, 2.208-2 18.

Jefferson R A & Kavanagh T A, 1987. GUS fusions: ~­Glucuronidase as a sensiti ve and versatile gene fu sion marker in hi gher plants. EMBO j , 6, 390 1-3907.

Kahana A et al. 1999. Expression of ACC ox idase genes differs among sex gcnotype and sex phases in cucumber. Plallt Mill Bioi, 41 , 517 -528.

Mohinuddin A K M et al. 2000. Influence of acetosyringon . on Agrobacterilllll · mediated transformation of cucum ber (CllclI/ni.l· sativlI.I' L. ). Plo III Tisslle CIIII , 10, 167- 173 .

Mourgues F et al. 1996. Efficient Agrobacterilllll- med iated transformati on and recovery of transgenic plants from pear (Pyms COllllllllni.l' L.). Plant Cell Rep, 16,245-249

Ni shibayashi S et al. 1996. Tran sformation of cucumber (CIIClllll is sativlIs L.) plants using Agrobacterilllll tllllle/clciens and regeneration from hypocoty l explants. Plwlt Cell Rep. IS . 809-814.

Plader W et al. 1998 . The re lationship between the regeneratio n syste m and genetic variability in the cucumber (ClIclllll is .lalivlIS L. ). ElIpliytica, 103,9- 15 .

Rah3lj o S H T et al. 1996. Transformation o f pickling cucumber with chitinase-e ncoding genes using Agrobacterilllll tlllllejclciens. Plant Cell Nep, IS, 591-596.

Sarmento G G el al. 1992. Factors influencing Agrobacterilllll tllll1e{aciells-mediated transformation. Plant Cell TisslIe Organ Cult, 31 , 185-193.

Sharma A K et al. 1999. Transgenic plants for the production of edible vaccines and antibodies for immunotherapy . ClIrr Sci. 77, 524-529.

Srivastava D K et ai, 1990. Genetic transform ation of foreign gene expression in cucumber ti ssues (CuclIlIlis sativlIs L. cv Kustovoi). Abstr IAPTC Congr, Amsterdam, 77.

Tabe i J et al. 1998. Transgenic cucumber plants harbouring a rice chitinase gene exhibit enhanced resistance to grey mold (Botyrytis cinerea). Plant Cell Rep, 17,159-164.