Agrobacterium- mediated transformation of chickpea using mature embryos

��2007 ��ث �� ا��م ا� را� ا��د ��م R. J. of Aleppo Univ. Agricultural Science Series No. 2007

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Agrobacterium – mediated transformation of chickpea (Cicer arietinum L.) mature embryos

Fateh Khatib Dr. Michael Baum Ph. D. Student

in Department of Plant Protection Faculty of Agriculture

Aleppo University Aleppo, Syria

Biotechnologist in the International Centre for Agricultural

Research in the Dry Areas (ICARDA) Aleppo, Syria

Dr. Samir Koudsieh

Professor in the Department of Plant Protection

Faculty of Agriculture Aleppo University

Aleppo, Syria

Key words: Chickpea transformation, herbicide resistance, PPT, pCGP1258, histochemical assay, PCR

Abstract Chickpea is an important food legume, valued for its seeds with high protein content. Its production is bedeviled by major biotic and abiotic stresses. Plant transformation is a relatively new tool developed with potentially huge benefits for plant improvement. In this study, a transformation protocol based on the inoculation of decapitated mature embryos with the Agrobacterium tumefaciens strain AgL0 was used. Two chickpea lines, ILC482 and ICC12004 were transformed by the binary vector pCGP1258 which contains the bar and the gusA genes. Regenerated explants were selected on 10 mg/l of phosphinothricin. The putative transgenic explants were subjected to hormone-free media for rooting or were grafted on 5-day old non-transgenic rootstock. Transgene to chickpea was confirmed by the polymerase chain reaction (PCR). The expression of the bar gene encoding for the enzyme phosphinothricin acetyl transferase and confering resistance to the herbicide phosphinothricin (PPT)was detected by surviving plants after painting the leaves with 600 mg/l PPT. The activity of gus gene encoding for β-glucuronidase was confirmed by histochemical which revealed GUS-positive expression in T0 and T1 plants.

� ودRS .ود ��وم . ��T F. Khatib, M. Baum & S. Koudsieh

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1. Introduction Chickpea (Cicer arietinum L.) was domesticated in the old world in an area of present- day southeastern Turkey and adjoining Syria [1]. Currently, it is cultivated in over of 40 countries world wide and grown on 11 million hectares and have produced about 9 million tones in 2005. [2]. In Syria, chickpea is an important crop, valued for its nutritive seeds with high protein content (25.3 – 28.9 %), which is a relatively economic source of protein. Chickpea is also an important part of the agricultural rotation, and its residues are used as animal feed. Chickpea is mainly grown in the first and second stability zones (250 – 600 mm precipitation), and is sown in February or March as spring chickpea [3; winter cultivars are available and planted in November-December [4]. Chickpea production in Syria and in most growing regions is threatened by many limiting factors, as Ascochyta blight, Fusarium wilt, cyst nematode, insects, Orobanche sp., drought, salinity and cold. Conventional breeding has contributed to chickpea improvement; but many problems still need to be addressed. Genetic engineering might be a new approach that offers the possibility of introducing a desirable character from far or closely- related plants without associated deleterious genes. Agrobacterium –mediated transformation is one of the most commonly employed methods for gene transfer to dicot plants. Agrobacterium is a soil-borne bacterium, which causes crown gall disease on many dicots. Virulent Agrobacterium strains harbor a large plasmid (about 200 kbp) known as tumor –inducing (Ti) plasmid, which is necessary for tumor formation. Tumor cells contain a fragment of Ti plasmid called transfer/(ed) DNA (T-DNA). The T-DNA is flanked by 25 base pair (bp) repeats, which are the left and right borders. The T- DNA represents less than 10 % of the Ti plasmid (approximately 10 to 30 kbp in size). The T-DNA carries several genes conferring special properties to the tumor cells. These genes are all of the eukaryotic type with a typical plant promoter and terminator. As such, they do not function in bacteria. Virulence (vir) genes are located on the Ti plasmid and are necessary for T-DNA transfer [5, 6]. The principle underlying the use of the Agrobacterium plasmid as a vector is that any gene placed between the right and left border will be transferred to the plant genome. Like transformation in all organisms chickpea transformation requires a source of regenerated recipient plant cells, a mean for delivering the


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T-DNA in the target cells and a system for selecting the transformed cells (7). The susceptibility of chickpea to different wild strains of Agrobacterium tumefaciens was demonstrated [8]. Several attempts have been made to transform chickpea through Agrobacterium-mediation [9, 10, 11, 12, 13, 14] or by bombardment [15]. Special attention was given to plant regeneration [16], which is a very critical and necessary step towards the genetic engineering of plants. Reports concluded that legumes in general are recalcitrant to transformation, regeneration in vitro is highly genotype specific and slow and the transformation efficiency is low [7]. The objective of this study was to test the efficiency of the transformation by Agrobacterium tumefaciens strain Agl0 carrying the binary vector pCGP1258 which contains the bar and gus genes on two different chickpea lines. 2. Materials and Methods 2-1. Plant material Chickpea seeds of kabuli genotype ILC482 and desi genotype ICC12004 were used. The seeds were surface -sterilized with 75% ethanol (v/v) for 1 minute followed by 5 % sodium hypochlorite for 20 minutes with slow agitation, rinsed three times in distilled water and left to soak for 16 hours in 20 mM calcium chloride. 2-2. Bacterial inoculum Agrobacterium tumefaciens strain AgL0 is carrying the plasmid pCGP1258. The plasmid carries the bar gene coding for phosphinothricin acetyl transferase (PAT), which confers resistance to the herbicide phosphinothricin or glufosinate ammonium and uidA (gusA) gene coding for β-glucuronidase (GUS) used as reporter gene, both of which are driven by the CaMV 35S promoter. To prepare bacterial inoculum, the single colony of Agrobacterium strain was maintained in Petri dish on Luria Broth Agar (LB) medium containing 50mg/l tetracycline and incubated for one day at 28 ºC. A swab of bacterial cells was transferred to 25 ml liquid MG medium containing tetracycline, and grown in a rotary incubater overnight at 28 ºC and 150 rpm to reach the reading of 1.2 -1.4 optical density O.D600. (One


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liter of MG medium contains: 5 g tryptone, 2.5 g yeast extract, 5 g mannitole, 1 g glutamic acid, 0.25 g potassium phosphate, 0.1 g sodium chloride, 0.1 g magnesium sulphate and 1 µg biotin. pH was adjusted to 7.0 before autoclaving for 20 minutes at 120 º C). The bacterial cells were collected by centrifugation at 4000 rpm at 4 ºC for 10 minutes. the pellet was suspended in 20 ml Agro-suspension solution containing MS macro- and- micro basal salts [17] supplemented with the following extra amounts: (1L containing 12 mg Fe-Na-EDTA, 5 mg manganese sulphate, 2.6 mg zinc sulphate, 2 mg boric acid, 0.075 mg sodium molybdate, 0.0075 mg copper sulphate, 0.0075 mg cobalt chloride, 0.25 mg potassium iodide, 1mg nicotinic acid, 10 mg thiamine, 1mg pyridoxin, 100 µM acetosyrengone, 0.25 µM thidazuron (TDZ), 15 g sucrose and 15 g glucose). pH was adjusted to 5.7 before autoclaving at 121 ºC for 20 min. 2-3. Plant transformation procedures 2-3-1. Co-cultivation Embryos were dissected from the imbibed seeds under sterile conditions and injured in a drop of Agrobacterium suspension by cutting 1 – 2 mm of the apex and root apical meristem. Subsequently, 60 embryos at a time were incubated in 5 ml Agrobacterium suspension for 2 – 4 h, blotted dry on a sterile filter paper and co-cultivated on gerlite-solidified (0.2%) DKW (defined for in vitro propagation of Paradox Walnut rootstock) medium [18], supplemented with the same extra amounts used in Agro-suspension solution (micro salts, vitamins, 0.05 µM TDZ, sucrose and glucose). Co-cultivation was performed for 4 days at 22 ºC in the dark (fig. 1, A). Incurved roots were removed and the explants were rinsed three times with sterile distilled water, then immersed in 150 mg/l ticarcillin (Duchefa) for 20 minutes. Thereafter, ticarcillin was added to the medium to eliminate Agrobacterium from chickpea tissues. 2-3-2. Regeneration and elongation For regeneration, explants were subcultured for 7 – 9 days under indirect light conditions on regeneration medium (DKW medium containing 0.005 µM TDZ). Then, they were transferred to the elongation medium; DKW supplemented with 1g 2-(Morpholeno)ethanesulfonic acid (MES), 2 mg 6-Benzylaminopurine


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(BAP), and 0.01 mg Indole-3-butric acid (IBA) for 21 days at a light intensity of 75 µmol photon.m-2.s-1. 2-3-3.Selection Explants were sub-divided into three parts (shoots originated from apex and two axillary buds), and 8 explants per plate were cultured on selection medium (DKW supplemented with 5 mg/l PPT). Subsequently, selective pressure was increased to 10 mg/l, and 10 g/l Polyvinyl Pyrolidone (PVP) were added to the medium in the last three subcultures. The green surviving shoots were subjected by repeated excision of branches and their exposure to fresh selection medium for seven rounds with two weeks intervals, (fig.1, C). 2-3-4. Rooting Roots were produced by two methods: (i) the putative transgenic explants (fig. 1, D) were transferred to hormone-free DKW for one or two months, and (ii) small putative transgenic shoots were grafted onto 5- day-old root stocks (fig. 1, e) that were germinated on half strength MS medium as described by Krishnamurthy et al. [11]. After three weeks, successful events were transferred to pots containing a 2:1:1 (soil: sand: peat moss) soil mixture in a growth room adjusted to 22 ºC, 16/ 8 h photoperiod, and light intensity of175 µmol photon.m-2.s-1. For acclimatization, the plants were initially covered with a polyethylene bag for one week; then they were punctured to reduce the atmospheric humidity, and the bag was removed completely after 2 weeks (fig.1, F). 2-4. Functional assay 2-4-1. GUS expression The uidA or gusA gene that encodes the enzyme β-glucuronidase (GUS) is a widely used reporter for the identification of transformed cells or plants. This enzyme cleaves the substrate X-gluc (5-bromo-4-chloro-3-indolyl β-D-glucuronic acid) (Duchefa) resulting in the production of an insoluble blue color in those plant cells displaying GUS activity (19). For the GUS assay, 4 ml X-gluc (1mg/ml) was mixed with 6 ml GUS buffer [100 mM phosphate buffer, 10 mM Na2 EDTA, 0.5 mM K3 [Fe (CN) 6], 0.5 mM K4 [Fe (CN) 6]. 3H2O]. Random samples of the co-cultivated embryos were taken for GUS


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assay. Also, detached leaflets and flowers from plants in pots were tested. Samples were immersed in 200 µl solution and incubated for 16 h at 37 ºC. Green tissues were cleared over night with 100 % absolute ethanol [20]. 2-4-2. PPT assay The active ingredient glufosinate ammonium or phosphinothricin (PPT) is a non- selective herbicide that accumulates ammonia in plant cells up to toxic levels; if the plant cannot remove it, it dies. Plants resistant to PPT are produced when the bar gene is integrated in the plant genome. The gene product phosphinothricin acetyl transferase (PAT) inhibits the toxicity by adding ammonia with L-glutamate to make the amino acid L-glumatine. Transformants were tested for the expression of the bar gene by painting the upper surface of T1 leaflets with 600 mg/l PPT plus 0.1 % Tween 20. Results were scored after 7 days. 2-5. Polymerase Chain Reaction (PCR) analysis Genomic DNA was isolated from 0.5 g young leaves. Using a modified CTAB method (21). Leaves were macerated in 800 µl of CTAB buffer (3 % CTAB, 1.4 M NaCl, 0.2 % mercaptoethanol, 20 mM EDTA, 100 mM Tris-HCl (pH, 8.0), 0.5 % PVP), mixed at once and incubated for 30 minutes at 60 ºC. Followed by adding 800 µl chloroform-isoamyl alchohol (24:1); the tube content was gently mixed to avoid shearing of genomic DNA. The samples were centrifuged for 10 minutes at 14,000 rpm, and the liquid phase was transferred to a clean microfuge tube. 2/3 volume of pre-cold isopropanol was added to the liquid phase and mixed gently. The DNA was pelleted by centrifugation as mentioned above, the supernatant was discarded and the pellet washed in 200µl washing buffer (76 % ethanol, 10 mM ammonium acetate). The buffer was removed and the pellet was air-dried and resuspended in 200µl TE buffer (10 mM Tris-HCl (pH, 8.0), 1 mM EDTA) supplemented with 10mg/ml RNAse A; and incubated for 30 min at 37 ºC. Then 100 µl of 7.5 mM ammonium acetate and 750µl ethanol were added and mixed gently. The supernatant was discarded and the pellet was dried and resuspended in 200 µl of sterile distilled water. The primers used for the amplification of a specific bar sequence (264 bp) were 5'- GCAGGAACCGCAGGAGTGGA-3' and 5'-


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AGCCCGATGACAGCGACCAC-3'. PCR reaction was made with 0.25 µg /genomic DNA, 0.1 µM of each primer, 1X PCR buffer (100 mM Tris- HCl, 500 mM KCl, 15 mM MgCl2), 200 µM dNTPs and 0.8 unit of Taq DNA polymerase. The PCR conditions were 4 min initial denaturation at 94 ºC, followed by 30 cycles for 90 sec. denaturation (94 ºC), 90 sec. annealing (62 ºC), 30 sec. extension (72 ºC), and finally a 5 min extension step at 72 ºC. Also, the primers sequences 5'-CACAATCCCACTATCGTTCGC-3' and 5’-TCCGTCCACTCCTGCGGT-3' were used to amplify 294 bp of the CaMv 35 S promoter sequence with the following PCR conditions: 94 ºC (4 min), followed by 30 cycles for 1 min at 94 ºC, 1 min at 60 ºC, 2 min at 72 and finally 7 min at 72 ºC. 2.6. Electrophoresis PCR products were separated on 1.2% agarose gel. DNA fragments were visualized by 0.5 µg/ml ethidium bromide staining. And photographed in the presence of UV light. 3. Results and discussion 3.1. Tissue culture and plant transformation Two chickpea lines were tested for Agrobacterium-mediated transformation. For this purpose embryos were co-cultivated with the Agrobacterium strain AgL0. Explants were regenerated to produce 3-4 shoots (fig. 1, B). The cutting site was critical for trasgnic procedures because all the transgenic shoots were derived from the apices only. No one was derived from the axillary bud primordial on the cotyledonary nodes selection. Selection pressure was only applied after the shoot formation; otherwise it would inhibit the shoot formation by the elimination of untransformed cells [15]. The protocol used led directly to shoot formation without an intermediate callus phase, (fig. 1, B) which is an improvement of previous studies [9, 10]. In this study, a total of 8128 embryos were subjected to Agrobacterium-mediated transformation and the transformation efficiency ranged between 0.20 – 0.24 % (Table1). 3.2. Rooting Most of the putative transgenic explants were too small to produce roots on hormone- free DKW medium and died later. Recently, we


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demonstrated high rooting percentage in chickpea control explants (2 -4 cm length) by using half strength MS liquid medium supplemented with 3 mg/l IAA (results not shown). As an alternative we recommend the micro grafting for the small shoots as described by Krishnamurthy [11] which leads to high rooting percentage in chickpea control explants. About 61 % of the putative transgenic shoots were successfully grafted on non-transgenic rootstocks. Subsequent, plantlets developed during three weeks and were then transferred to soil. This process was faster than rooting shoots on DKW medium, which took about two months before plants could be transferred to pots. Furthermore, plants rooted on DKW medium produced fewer adventitious roots. Many plants died during the acclimatization period and we recovered only 11 plantlets from 48 putative transgenic plants that were planted in pots in a growth room. Three plants derived from independent lines produced 1, 2 and 20 T0 seeds. 3.3. Histochemical assay A total of 192 embryos were used for the GUS assay. Out of these, 47 % were stained blue after treatment with X-gluc. Staining was observed mainly at the apices. Primarily, this observation is necessary for the establishment of the protocol and for an assessment of the transformation efficiency in early phase. But it does not mean all this percentage will produce transgenic shoots. Many factors are involved specially, the ability of these transgenic cell to regenerate to entire plant. The histochemical X-gluc assay of leaflets and flowers (fig. 1, J and I)) detached from four independent lines revealed that two were GUS-positive and two were not. The absence of GUS-activity could be due to chimeric tissues in the putative transgenic plant produced through in vitro culture. In the same T0 plant (plants developed from the decapitated embryos which we start with) some leaves and branches could be transgenic and the others are not (tissues consisting of cells of different genotype). Histochemical assay of twenty T1 plants showed a positive result on 7 plants as faint greenish blue color in the entire flower and leaflets, or only at the base of the leaflet. Independent lines (shoots have originated from one embryo) that produce 1 or 2 T0 seeds did not express GUS activity or herbicide resistance in T1 plants. Up to twelve seeds derived from independent


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line for the different generations are needed to study the segregation of the gene. 3.4. Polymerase Chain Reaction All plants developed in the growth room (table 2) were tested by PCR using a specific primers produced a fragment of 264 bp and 294 bp for bar and 35 S sequences, respectively (fig. 2). 3.5. Herbicide resistance Functional tests were carried out for T1 plants (derived from T0 seeds) by painting leaves with 600 mg/l PPT; the herbicide resistance was scored on 7 plants derived from one line, which produced 20 T0 seeds. Some treated plants showed complete herbicide resistance and others revealed partial tolerance (fig. 1, G and H). The untransformed control plants were completely necrotic. PAT level in the transformed plant affects the ability of this enzyme to confer the herbicide resistance. In same situation, the PCR-positive plants may not express the new trait. That due to phenomena of gene silencing, which is defined as the inactivation of transgene expression. The prsence of multiple gene copies can correlate with transgene silencing or co-supression.Characterization of the transgene locus/loci at the molecular level (transmission of the transgene) as well as segregation analysis of the transgene-encoded phenotype (expression of the transgene) in the subsequent progenies would allow insight into the nature of transgene inheritance [22]. The demonstration of Agrobacterium potential for chickpea transformation was reported by Mohapatra and Sharma [12]. In this study we have shown that it is possible to transform our local chickpea lines with Agrobacterium tumefaciens. The protocol used does not differ substantially from the protocol used by Krishnamurthy [11], and Kiesecker [23] and the transformation efficiency was similar to those observed earlier. However, more efforts are needed to increase chickpea transformation efficiency. Acknowledgments: this work was supported by ICARDA; We would like to thank Ms. Nahed Al Sokhny and Mrs. Bianca Ghazal from biotechnology lab. (ICARDA). We are grateful to Dr. Heiko Kiesecker


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from DMSZ (Germany). Special thanks to Dr. Joanne Barton from CLIMA (Australia) for providing the plasmid. Table1. Transformation efficiency in different chickpea lines Line Total Number of Co-

cultivated embryos No. of Putative transgenic

Efficiency % *

ILC 482 6466 13 0.20 ICC 12004 1662 4 0.24 Total 8128 17 0.22

* The transformation efficiency was obtained by dividing the number of the independent clones × 100 with the total number of co-cultivated embryos.

Table2. In- vitro clones and recovering T0 and T1 plants

Line

No. of clones

Rooting on

DKW medium

No. of successful

grafts

No. of T0

plants

PCR test

No. of T0

seeds

ILC 482

2 2 - 2 + 0 1 1 9 2 + 1 1 1 1 1 + 0 1 1 - 1 + 0 1 1 2 1 + 0 1 1 0 1 + 0 1 1 5 1 + 20

ICC12004 3 3 12 3 + 2


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Fig. 1. A Co-cultivated embryos B Explants on regeneration medium. C Selection on sub culture supplemented with phosphinothricin. D Putative transgenic explants. E Transformed shoots grafted on non-transgenic root stocks. F Grafted shoots transferred to soil after acclimatization. G, H Herbicide resistant and susceptible leaves, respectively. I, J GUS activity in chickpea embryos and flower, respectively. Fig. 2. PCR analysis of T0 transgenic plants: (M) a marker for molecular weight, lanes 1 to 12: amplified 264 bp fragment of the bar gene, lanes 12, 13: negative control derived from wild type plants and water, the construct pCGP1258 served as positive control in lane 15.

AA BB CC DD

EE FF GG HH

II JJ

M


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LMBKآ

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� إ�� u� ب 3آ��¨p|¯و�³ ا ��§ �زر w� م��Tأselection ي آ��p�v ا��ي �� x�º�10أ ·�/ {p�PPT ��14ة ��tا��³ ا��ي آ� ا� �v��~ ¡� ع�tأ�

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��}از ا�x��p ��ا�R ا�tpSر ��T0 |��ح �� ا��t| xy �}u~�ت ا�� ~PCR ام زوج �� ا��}��ت�¨p�� «؛ وذ�Primers ª��~ xpوا� ��¨pا��

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Agrobacterium- mediated transformation of chickpea using mature embryos

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