Indian Journal of Experimental Biology Vol. 40, March 2002, pp. 329-333

In vitro organogenesis and genetic transformation in popular Cucumis sativus L. through Agrobacterium tumefaciens

E V Soniya & M R Das

Rajiv Gandhi Centre for Biotechnology, Jagathy, Thiruvananthapuram 695 014, India rgcbt@sancharneLin soniyacv@yahoo.com

Received 9 January 2QOO; revised 20 August 2001

The effect of growth regulators and culture conditions on the morphogenetic response of cotyledonary leaf discs was studied in popular cucumber variety (Cucumis sativus cv. Sheetal). Organogenesis was induced directly without any inter­vening callus phase on Murashige and Skoog medium supplemented with different concentrations of benzyladenine and in­dole propionic acid: Best results (93%)were obtained in the presence of the 4mgIL benzyladenine and 1 mgIL IPA. The elon­gated shoots were rooted in basal medium with I mgIL indole butyric acid, hardened and transferred to the field conditions. Genetic transformation system has been established for Cucumis sativus cv. Sheetal, plants by infecting cotyledonary ex­plants with Agrobacterium tumefaciens strain LBA4404 carrying binary plasmid pBI I 2 1 , which contains scorable marker, (3-glucuronidase and selectable marker nptll under the CaMV 35S promoter. Infection was most effective when ex plants were infected with Agrobacterium for 1 5 min and co-cultivated for 2 days in the co-cultivation medium. Shoots were regen­erated directly from cotyledonary leaf explants in the presence of kanamycin (50J.1g/ml) and analysed. Southern blot analysis confirmed that transformation had occurred. This method will allow genetic improvement of this crop by the introduction of agronomically important genes.

Cucumber (Cucumis sativus L) is an important vege­table crop that is cultivated extensively in America, Asian and European countries. This species has a nar­row genetic base and severe crossing barriers hamper the introduction of desired traits from related species 1 • Genetic improvement of Cucumis has been acheived by conventional plant breeding methods, but recent advances in genetic transformation techniques have opened new avenues for crop improvement. For the use of genetic manipulation techniques, efficient and reproducible regeneration procedures are needed. Even though, there are reports for regeneration in Cu­cumii·8, several factors like genotype, source tissue and composition of the medium, have been shown to influence regeneration frequency of plant tissue and hence would influence transformation efficiency. It was proven that a genotype could influence various stages of in vitro culture and plant regeneration abil­ity. Genetic engineering of Cucumis sativus has al­ready been reported9. 12, however, transformation fre­quencies are greatly influenced by the cultivar and the procedure usedl3. A suitable tissue culture protocol for regenerating shoots from tissue explants that can be transformed by Agrobacterium tumefaciens is a pre requisite for application of genetic transformation 14. The main objective of our study was to find an effi-

cient, quick and reliable protocol for direct plant re­generation in Cucumis sativus cv. Sheetal using coty­ledonary ex plants followed by transfomation of the cultivar with Agrobacterium carrying the binary vec­tor pBI 1 2 1 which is very much beneficial for the fu­ture improvement of the plant.

Materials and Methods Plant materials and Agrobacterium strain­

Seeds of Cucumis sativus L. cv. Sheetal used in this study were obtained from Agricultural college Vel­layani, Thiruvananthapuram. These were surface dis­infected with 70% alcohol for 2 min followed by 0. 1 % (w/v) aqueous mercuric chloride solution for 8 min and rinsed several times with sterile distilled wa­ter. The sterilized seeds were inoculated aseptically in culture tubes (25 x 1 50 mm) containing basal Mura­shige and Skoog mediuml5. The media were adjusted to pH 5.8 prior to the addition of 0.8%w/v agar and autoclaved at 1 2 1 °C for 1 5 min. The cultures were maintained at 26°± 2°C in the culture room with 16hr photoperiod with 3000 lux light intensity provided by cool white florescent tubes.

The disarmed Agrobacterium tumefaciens strain LBA440416 harboring binary plasmid pBI 12 1 17 was used as vector system for transformation studies. The

330 INDIAN J EXP BIOL, MARCH 2002

pBI121 plasmid contains nos/nptIl gene for selection on kanamycin containing medium and a gene CaMV3SS, GUS chimeric gene as the scorable marker. The binary vector was mobilized into Agro­bacterium by the freeze thaw method 1 8. The bacteria were grown at 28°C in YEP medium ( 1 % yeast, 1 %peptone and O.S% NaCl) containing 100 Ilg/ml kanamycin. Cotyledonary leaf explants of 6-8mm in length excised from 2-S day old seedling were placed on MS medium supplemented with different concen­trations and combinations of BA (0.8-6mg/L), IPA ( 1 -2mg/L), picloram ( 1 -4 mg/L) , NAA (0.8-2 mg/L) and kinetin (0.8-4mg/L) for organogenesis.

Co-cultivation and transformation - Bacterial col­ony was inoculated into SOml of liquid medium with 100)lg/ml kanamycin and incubated at 28°C on a shaker at 120rpm for overnight ( 1 6- 1 8h) and used in the late log phase (OD600=0.6). Bacterial culture was centrifuged at 3 ,SOO rpm for 10 min, the pellet was resuspended in SOml liquid half MS medium .The pre culture of cotyledonary leaf explants for 2 days on regeneration medium prior to Agrobacterium infec­tion was needed. The sensitivity of uninoculated Cucumis explants to kanamycin for effective selection of putatively transgenic plants was tested using differ­ent concentrations of kanamycin (0,2S,SO and I OOllg/ml) , added to the regeneration medium. At SOllg/ml and above the explants turned brown and did not show further growth. Hence SOllg/ml was used as selection pressure.

The pre cultured cotyledonary explants were in­fected by immersing them in the Agrobacterium sus­pension under gentle agitation for 10-60 min. After infection, they were placed on sterile filter paper and then placed horizontally on petriplates containing MS medium with appropriate hormones for regeneration (co-cultivation medium). Ten to 20 explants were used for each treatment, with three to five replica­tions. All explants were co-cultivated for a period of two days in darkness at 2so±2°C. Control experiments were also carried out without co-cultivation with Agrobacterium. Two days after co-cultivation ex­plants were transferred to regeneration medium with SOllg/ml kanamycin and 300J.!g/ml cefatoxime for selection. Sub culturing was done routinely to fresh medium at two weeks' intervals. Individual shoots (about 1 cm) were transferred to elongation medium and the putative transformants were selected.

DNA isolation and Southern blotting -Genomic DNA was isolated from fresh leaf tissue from in vitro

grown putative transgenics and control plants as de­scribed by Rogers and Benedictl 9. DNA from three GUS positive plants as well as 'from the control plants and the bacteria were digested with the restriction en­donuclease EcoR I separated by electrophoresis through a 0.8% agarose gel and transferred to Hybond N+ membrane (Amersham). The probe (4.Skb) was prepared from the vector by digesting with restriction enzymes Hindlll and EcoRl , labeled with a 32p [dCTP] using random primer kit (Prime-a-Gene La­beling system) from Promega. Southern blotting and hybridization were performed according to the stan­dard Sambrook et al. ,20 protocol. After hybridization, washi-ng was carried out under high stringency condi­tion (6S%, O. IX SSC, O.S% SDS).

Results and Discussion The most determining factor for regeneration com­

petence of Cucumber cotyledons was their develop­mental stage. Competence of cotyledon cells for re­generation is restricted to initial 2-S days of the seed­ling development. According to Colij-Hooymans et al} a sudden increase in ploidy level occurs, result­ing in cotyledonary cells with higher DNA contents which have an adverse effect on the regeneration ca­pacity of Cucumber cotyledons.

Direct shoot regeneration from cotyledonary leaf explant occurred within 28 days of culture initiation. Greenish rounded structures appeared on the cut end of the explants (Fig. lA) within two weeks of culture · on MS medium augmented with varying concentra­tions of BA (0.8-6 mg/L) and IPA (0.8-4 mg/L). The increase in regeneration frequency was obtained with increased concentrations of BA and was maximum at 4 mg/L BA and 1 mg/L IPA (Table 1 , Fig. I BC). Ad­dition of hormones like NAA and picloram at differ­ent concentrations produced callus along with BA. Similarly kinetin instead of BA also did not produce any shoots. Cotyledonary explants cultured on basal medium in the absence of any exogenous growth regulators never differentiated to produce shoot buds. According to Wehner and Locy21 , the Cucumber l ines and varieties differ from one another in their plant regeneration using hypocotyls and cotyledons. In Cu­cumis sativus it was reported that there exists a defi­nite preference in certain genotypes for the type of growth regulator used, for instance, for Cucumis sati­vus 'Borszczagowski' the combination of 2ip/2,4-D was most effective4, while for cv. 'Skierniewicki' NAA was best. Shoots elongated to about 4cm length were transferred to basal medium with Img/L IBA for

...

SONIY A & DAS: ORGANOGENESIS & GENETIC TRANSFORMATION IN CUCUMIS 331

rooting. The rooted plantlets, after hardening were transferred to field conditions where they showed 80-85% survival. In vitro regeneration of adventitious shoot is an essential component for most methods of genetic transformation I S , 22,23. Therefore a protocol to maximize the regeneration of adventitious shoots must be developed before attempting biological trans­formation using Agrobacterium as a vector24.

Cotyledonary leaf explants co-cultivated with Agrobacterium tumefaciens (Fig. ID) produced direct shoot buds on selection medium (50J.,lg/ml), after three weeks (Fig. 1 EF). Further increase in kanamycin con­centration drastically decreased the shoot bud forma­tion, although resulted in no escapes. Compared to non transformed controls, efficiency of direct shoot regeneration was markedly decreased by co-cultivation

Table 1 -Morphogenetic response of cotyledonary leaf explants cultured on Murashige and Skoog medium

Hormones (mgIL) Total no .of No. {)f explants No. of shoots Rate of survival explants showing formed (%)

BA IPA inoculated regeneration (Mean± SE) (% of regeneration)

0.8 42 50 35 (70%) 3+0.2 1 80. 1

2 42 I 3 44 2 4 1 3 4 (84%) 8±0.62 83.4 3 I 48 42 (88%) 14±0.5 1 8 1 .3 4 I 46 43 (93%) 22±0.3 1 85. 1 5 I 44 32 (72%) 9±0. 1 2 8 1 .2 2 2 46

Fig. 1 ...:....-Direct regeneration from cotyledonary leaf ex plants of both normal and putative transformants. (A) Initiation of shoot buds di­rectly from cotyledonary leaf explant. (B) Direct shoot buds from cotyledonary leaf explant. (C) Elongated shoot buds. (D) Co cultivated cotyledonary leaf explants in selection medium. (E) Initiation of direct shoot buds from the explant. (F) Elongated shoot buds. (G) Sepa­rated and subcultured shoot buds for elongation. Both white and green shoots (putative transformants) can be seen.

332 INDIAN J EXP BIOL, MARCH 2002

Table 2-Agrobacterium tumefaciens mediated transformation experiments in Cucumis sativus L.

No. of ex plants No. of shoots No. of 'gus' positive No. of plants Transformation plated regenerated plants survived frequency

70 1 2 2 1 63 1 0 2 1 67 1 4 3 1 75 20 4 1 73 9 1 1 76 1 1 2 2 55 1 0 2 1 69 1 3 2 1 65 12 2 1 75 9 I 2 74 1 1 2 1 72 8 1 1 72 10 2 I 75 14 2 1 70 8 I 1 74 9 1 1

1 1 25 1 80 30 1 8 2.6%

Transformation frequency (%) = Number of GUS positive plants x 100/ number of infected explants

C 2 J +n .' P M K b

• ._., 4 . :; 0

---.. ( ) ()X ---.. O X ;

Fig. 2- Southern blot analysis of transgenic plantlets - Lane C: negative control using non-transformed Cucumber DNA; 1 -3: Independent transgenic individuals; +ve: plasmid DNA; P: probe; M: lambda DNA digested with EcoR I and HindIII as molecular weight markers. Arrow head indicates the gus positive bands.

with Agrobacterium. According to Terresa et al.25, the cause of this reduction in regeneration efficiency may be related to hypersensitive response of explants to Agrobacterium tumefaciens infection. Some of the shoots were found to be whitish in colour, which will not develop further (Fig. 1 G). The green shoots were considered to be the resistant and putative transfor­mants. The duration of both inoculation and co­cultivation with bacteria for optimum transient GUS expression was 1 5 min and 2 days, respectively. Ear­lier report of transformation of cucumber showed suc-

cess from hypocotyl as well as cotyledonary leaf callus in the presence of acetosyringonel l - 1 3. But in the present study, we have used direct regeneration system with­out acetosyringone for the transformation studies. Be­cause of the potential genetic variability associated with callus regeneration, direct regeneration is of very much importance in the transformation studies. Southern blot analysis of the genomic DNA from three independent transgenic plants confirmed the integration of GUS gene sequences into the plant ge­nomes. With exception of control, the plants tested had sequences that hybridized to a 32p labeled DNA fragment of the GUS gene. Fig. 2 reveals the strong hybridization signal of 32p labeled probe hybridized with nuclear DNA of transgenic plants and plasmid DNA. Transformation frequency was calculated from number of GUS positive plants and the total number of explants used in the experiment (Table 2). The cal­culated transformation efficiency was 2.6% from the present study.

These results demonstrate that we have success­fully developed transformation protocol for Cucumis sativus cv. Sheetal using Agrobacterium tumefaciens from cotyledonary leaf explants which ailows the ge­netic improvement of this species by recombinant DNA technology. The transformation system we have established should facilitate the use of this species for studies on gene manipulation and expression and be available for introducing useful genes that will modify agronomic traits which otherwise could not be intro-

� I

.�.

r

SONIY A & DAS: ORGANOGENESIS & GENETIC TRANSFORMATION IN CUCUMIS 333

duced with traditional breeding methods. More . de­tailed studies of the transformation efficiency are now in progress using protocol established in the present study.

Acknowledgement The authors are thankful to Dr. N. S. Banerjee and

Mr. P. Manoj for their help throughout this work.

References I Den Nijs A P M & Custers J B M, Introducing resistance into

the Cucumber by interspecific hybridization, In: D M Bates (Ed),The biology and chemistry of the Cucurbitaceae (Cor­nell University Press, ltaca, New York) 1990,382.

2 Ziv M & Gadasi G, Enhanced embryogenesis and plant re­generation from Cucumber (Cucumis sativus L.) callus by activated charcoal in solid/ liquid double layer cultures, Plant Sci, 47 ( 1986) 1 1 5.

3 Malepzy S, Cucumber (Cucumis sativus L), in Biotechnology in Agriculture and Forestry, Vol 6.Crops II, edited by YPS Bajaj (Springer-Verlag Berlin Heidelberg) 1988, 277.

4 Sang-Go K, Joeng-Rahn C, Heijon Cheol C & Kwang­Woong L,Callus growth and plant regeneration in diverse cultivars of Cucumber (Cucumis sativus L), Plant Cell Tissue Organ Cult, 12 ( 1 988) 67.

5 Gambley R L & Dodd W A, An in vitro technique for the production of de novo multiple shoots in cotyledon ex plants of Cucumber (Cucumis sativus L), Plant Cell Tissue Organ Cult, 20 ( 1 990) 1 77.

6 Gambley R L & Dodd W A, The influence of cotyledons in axillary and adventitious shoot production from cotyledonary nodes of Cucumis sativus L (Cucumber), } Exp Bot, 42 ( 1 99 1 ) 1 13 1 .

7 Colijin-Hooymans C M , Hakkert J C, Jansen J & Custers J B M, Competence for regeneration of Cucumber cotyledon is restricted to specific developmental stages, Plant Cell Tissue Organ Cult, 39 ( 1 994) 2 1 7.

8 Burza W & Malepszy S, Direct plant regeneration from leaf explants in Cucumber (Cucumis sativus L) is free of stable genetic variation, Plant Breeding, 1 14 ( 1 995) 341 .

9 Trulson A J, Simpson R B & Shahin E A , Transformation of Cucumber plants with Agrobacterium rhizogenes, Theor Appl Genet, 73( 1986) 1 1 .

10 Chee P P, Transformation of Cucumis sativus L. tissue by Agrobacterium tumefaciens and regeneration of transformed plants, Plant Cell Rep, 9( 1990) 245.

I I Sarmento G G. Alpert K, Tang F A & Punja I K, Plant Cell Tissue Organ Cult, 3 1 ( 1 992) 1 85.

12 Nishibayashi S, Kaneko H & Hayakawa T, Transformation of Cucumber (Cucumis sativus L.) plants using Agrobacte­rium tumefaciens and regeneration from hypocotyls explants, Plant Cell Rep, 1 5( 1996) 809.

13 Barcelo M, Iman El-Mansouri, Mercado J A,Quesada M A & Alfaro F P, Regeneration and transformation via Agrobacte­rium tumefaciens of the Strawberry cultivar Chandler. Plant Cell Tissue Organ Cult, 54 ( 1 998) 29.

14 Mertens M, Werbrouck S, Botelho dos Santos, Moreira da silva & Debergh P, In vitro regeneration of evergreen azalea from leaves, Plant Cell Tissue Organ Cult, 45(1 996) 23 1 .

1 5 Murashige T & Skoog F, A revised medium for rapid growth and bioassays with tobacco tissue culture, Physiol Plant, 5 1 ( 1 962) 473.

16 Hoekema A , Hirsh P R, Hooykaas P J & J Schilperoot R A, A binary vector strategy based on separation of vir and T­region of the Agrobacterium tumefaciens Ti plasmid, Nature, 303 ( 1 983) 1 79.

17 Jefferson RA, Assaying chimaeric genes in plants: the GUS gene fusion system, Plant Mol Bio Rep,5 ( 1987) 387.

18 Van Haute E, Joos H, Maes M, Warreng, Van Montagu M & Schell J, Inter genic transfer and exchange recombination of restriction fragments cloned in pBR322, a novel strategy for the reversed genetics of Ti plasmids of Agrobacterium tume­faciens, EM BO },2 ( 1 983) 4 1 1 .

1 9 Rogers S 0 & Benedich A J , Ribosomal genes i n plants, Variability in copy number and in the intergenic species, Plant Mol Bio Manual D, 1 ( 1 994) I .

20 Sambrook J, Fritsch E F & Maniatis T, Molecular cloning: A laboratory manual, (Cold Spring Harbour Laboratory Press, Plainview, New York) 1989.

2 1 Wehner T C & Locy R D , In vitro adventitious shoot and root formation of cultivars and lines of Cucumis sativus L, Hort Sci, 16 ( 198 1 ) 759.

22 Hong W & Debergh P, Somatic embryogenesis and plant re­generation in garden leak, Plant Cell Tissue Organ Cult, 43 ( 1 995) 2 1 .

2 3 Torregrosa L & Bouquet A , Adventitious bud formation and shoot development from in vitro leaves of Vitis X Mus­cadinia hybrids, Plant Cell Tissue Organ Cult, 45 ( 1 996) 245.

24 Marcotrigiano M, Mc Glew S P, Hackett G & Chawla B, Shoot regeneration from tissue cultured leaves of American cranberry (Vaccinium macrocarpum), Plant Cell Tissue Or­gan Cult, 44 ( 1 996)195.

25 Terresa K Orlikowska , Harwood J Cranston & William E Dyer. Factors influencing Agrobacterium tumefaciens­mediated transformation and regeneration of the safflower cultivar 'Centennial', Plant Cell Tissue Organ Cult. 40 ( 1 995) 85.