Abstract: A rice chitinase gene (RCC2) multiplied in Agrobacterium strain (EHA 101), was simultaneously introduced into single buds of in vitro grown banana cultivar, Rastali (AAB). Plasmid pBI333-EN4-RCC2 contained a hygromycin phosphotransferase gene (hpt11) as the selectable marker to identify the transformants. Treatment A contained hygromycin at 25 mg L-1 and treatment B contained hygromycin at 50 mg L-1 in both MS medium supplemented 5 mg L-1 of BAP together with 2.7 g of gelrite agar. Single buds derived from multiple bud clumps (Mbcs), were the target explants for transformation. An assay was performed to identify the minimum concentration required for two antibiotics (carbenicillin and cefotaxime) that is most effective against Agrobacterium strain, EHA 101 and the effect on tissue regeneration capacity. Even though the transformation frequency based on hygromycin selection medium (treatment A) is higher, but there is no transformant could be confirmed based on PCR and Southern blot analyses, as compared using 50 mg L-1 hygromycin selection medium. Assay of protein extract from the transgenic plantlets showed an increased in chitinase enzyme activity over the untransformed plantlets. The present of Agrobacterium-mediated transformation reported here is suitable for using tiny meristem tissues to obtain fungal disease tolerant or resistant banana through genetic engineering.
INTRODUCTION
Banana were considered outside the host range of Agrobacterium, but an increasing amount of research reports indicates that banana can be transferred by Agrobacterium tumefaciens (May et al., 1995; Ganapathi et al., 2001; Sreeramanan et al., 2006). These reports include studies on transient expression of transferred genes, stable transformation, regeneration of transformed plants and heritability of transgenes.
Application of Agrobacterium-mediated transformation method in monocotyledons plants, with some minor modifications, resulted in the successful production of transgenic rice cultivars (Aldemita and Hodges, 1996; Dong et al., 1996; Rashid et al., 1996). In addition to these results obtained in rice, efficient Agobacterium-mediated transformation of maize (Ishida et al., 1996; Frame et al., 2002), Anthurium (Chen and Kuehnle, 1996), sugarcane (Arencibia et al., 1998), barley (Wu et al., 1999), orchids (Belarmino and Mii, 2000; Yu et al., 2003; Liau et al., 2003), sorghum (Zhao et al., 2000) and wheat (Wu et al., 2003) has been demonstrated. May et al. (1995) reported transformation of banana using meristems and corm slices explants from cultivar Grand naine (AAA).
Antioxidants used such as L-Cysteine during pre-culture, infection and during and after co-cultivation were reported to favor stable transgenic plants recovery in Agrobacterium-mediated transformation experiments (Enriquez-Obregon et al., 1999). Olhoft and Somers (2001) concluded that the increase in host-Agrobacterium compatibility mediated by the presence of antioxidants during transformation moderated the detrimental effect of hypersensitive response, which in turn lead to an increase in the survival rate of Agrobacterium tumefaciens-infected cells and a corresponding rise in stable transformation efficiency in soybean. Frame et al. (2002) reported that L-Cysteine in co-cultivation medium function to minimize cell death caused by the hypersensitive response of maize scutellum cells to Agrobacterium infection.
Generally, it is known that plants have defence systems which involve pathogenesis-related proteins such as chitinase (Nishizawa et al., 1999). Chitinase catalyzes the hydrolysis of β-1, 4 linkages of the N-acetyl-D-glucosamine polymer, chitin. The transgenic tobacco and canola which have been engineered with bean endochitinase gene were shown to exhibit resistance to Rhizoctonia solani (Broglie et al., 1991). The transgenic rice integrated with rice endochitinase driven by the 35S promoter also shown enhanced resistance to sheath blight (Lin et al., 1995) and the transgenic tobacco harboring rice endochitinase gene (Nishizawa et al., 1993) also possessed increased resistance against powdery mildew (Erysiphe cichoracearum).
This study describes a series of experiments that carried out using EHA 101 contained plasmid with chitinase gene (pBI333-EN4-RCC2). Comparisons between using hygromycin in two different concentrations were demonstrated in this study since both plasmid contained hpt11 gene. The inhibition of explants and bacterial growth by carbenicillin and cefotaxime were also presented here.
MATERIALS AND METHODS
Plant Materials
Corm slices of in vitro plantlets were cultured in MS (Murashige
and Skoog, 1962) medium supplemented with different concentrations of 6-Benzylaminopurine
(BAP) to obtain multiple bud clumps (Sreeramanan et al., 2002). Single
buds were used as a target tissue for transformation studies.
Agrobacterium Cultures
Disarmed Agrobacterium tumefaciens strain, EHA 101 (Hood et al.,
1986) and the binary vector (pBI333-EN4-RCC2) contains the hpt 11
gene linked to the nopaline synthase gene (nos). A binary vector, pBI333-EN4-RCC2
was constructed to replace the gusA gene of pBI121 (Clontech, USA)
with the cDNA (RCC2) of the rice chitinase gene (Nishizawa et al.,
1993) (Fig. 1). Plasmid pBI333-EN4-RCC2 was integrated
into Agrobacterium tumefaciens, EHA 101 by heat shock method.
Agrobacterium cultures were streaked out on LB medium supplemented with 50 mg L-1 of hygromycin and grown at 28°C for colonies to form. Single colony was transferred to 20 mL LB liquid contained the same antibiotic and the culture was allowed to shake at 28°C and 120 rpm for 20 h. After adjusting the Optical Density to 0.7 units at 600 nm (OD600), the bacterium culture was used for transformation.
| Fig. 1: | Diagram of T-DNA region in pBI333-EN4-RCC2 |
Explant and Bacterial Toxic Effects of Carbenicillin and Cefotaxime
For explant toxicity tests, single buds of 3 mm in size were transferred
to MS medium contain 5 mg L-1 of BAP and different concentrations
of carbenicillin and cefotaxime (0, 100, 200, 300, 400, 500 and 600 mg L-1).
In order to examine the bactericidal effects of carbenicillin and cefotaxime,
Agrobacterium strains EHA 101 (pBI333-EN4-RCC2) was used. After 30
min co-cultivation of explants in bacterial cultures, the percentage of single
buds with Agrobacterium strains were determined after three days using
various carbenicillin and cefotaxime concentrations.
Transformation Protocols of Single Meristematic Buds
Single buds (3 mm) were precultured for three days prior to Agrobacterium
infection. The explants injured mildly using scalpel. About 100 single buds
immersed in Agrobacterium suspension for 30 min together with an optimized
acetosyringone concentration at 100 μM. Acetosyringone is known to activate
the virulence genes of the Ti plasmid and to initiate the transfer of the T-DNA.
The explants were then blotted dry on sterile filter paper and cocultivated
for three days on hormone free MS medium (without CaCl2 during co
cultivation) designated as M1 medium containing 100 μM of acetosyringone,
60 mM D-(+)-glucose, 2 mM sodium phosphate and 40 mg L-1 of L-Cysteine
using Petri dish at 22°C temperature in the dark condition. After co-cultivation
period for three days, the buds were transferred on same medium but without
acetosyringone concentration together with 300 mg L-1 of carbenicillin
or 200 mg L-1 cefotaxime designated as M2 medium for 5 days and transferred
back on hormone free MS liquid medium with the same antibiotic concentrations
for another 5 days. Then, the buds were transferred to MS solid medium containing
10 mg L-1 of BAP with 25 or 50 mg L-1 of hygromycin for
2 weeks and transferred again on liquid medium with the same treatments for
5 days in 100 mL conical flasks. During this period, death buds were removed
before subcultured back on M2 medium. The transformed buds regenerated into
clumps which are known as multiple bud clumps (Mbcs). Single buds were separated
from multiple bud clumps and subcultured onto solid MS medium with 1 mg L-1
of BAP until the single plants were regenerated. All plants regenerated from
each putatively independent transformed bud line were maintained under in
vitro conditions.
Polymerase Chain Reactions (PCR)
Genomic DNA was extracted from putative transformants using an improved
and modified CTAB method adopted from Pasakinskiene and Paplauskience (1999).
PCR was done using the DNA Thermal Cycler 480 machine (Perkin-Elmer). The following
primers were used to amplify the transgenes and part of the regulatory sequences
(Table 1).
PCR amplications were carried out in 100 μl reactions volume containing template DNA (500 ng genomic DNA or 60ng plasmid DNA), 200 ng of each primer (forward and reverse), 0.2 mM dNTP mix, 1.5 mM MgCl2, 1X PCR buffer and 5 U Taq DNA polymerase (MBI Fermentas). Amplification of chitinase gene (RCC2) fragments was performed for 35 cycles at 94°C for 1 min, 55°C for 1 min and 72°C for 2 min, for denaturing, annealing and primer extention, respectively.
| Table 1: | Genes, primer, primer sequences and expected product length |
| 00 | |
Amplication of hpt11 gene fragments was performed for 35 cycles at 94°C for 1 min, 62°C for 1 min and 72°C for 2 min, for denaturing, annealing and primer extention, respectively. All amplified of PCR products were checked on 1.2% agarose gel.
Southern Blot AnalysesA nonradioactive method was used to confirm stable integration of chitinase (RCC2) transgene in the host banana genome. Genomic DNA (20 μg) from PCR positive transformants were subjected to digestion with Hind111 in buffer H (Promega). DIG DNA Labeling and Detection Kit (Roche) were used in this study.
Extraction and Determination of Total Acid Soluble Proteins
Tissue samples (0.25 g) were homogenised in 5 mL extraction buffer (0.1
M sodium buffer, 1 mM EDTA, 0.1% mercaptoethanol, pH 5.2), with 100 mg insoluble
polyvinyl polypyrroline. The homogenate was spun at 12,000 rpm for 20 min. The
supernatant was used as crude extract for determination of total acid soluble
protein, chitinase and β-1,3 glucanase activities. Protein was quantified
by Bradford (1976) methods, at wavelength 595 nm. Three replications were applied
to all treatments and extraction buffer was used as a blank reference. Total
acid soluble protein was expressed in mg g-1 fresh weight by using
bovine albumin as standard.
Chitinase Activity Assay
Methods of Tonon et al. (1998) were followed with a slight modification
in enzyme concentration. Chitinase activity was determined based on the rate
of N-acetylglucosamine production using chitin as the substrate. Amount of enzyme
catalyzing the formation of 1 nm N-acetlyglucosamine equivalent in one second
under assay conditions refers as 1 nkat (Anfoka and Buchenauer, 1997).
Statistical Analysis
Data were analysed using one-way ANOVA and the differences contrasted using
Duncan’s multiple range test. All statistical analyses were performed at
the level 5% using SPSS 10.0 (SPSS Inc. USA).
RESULTS AND DISCUSSION
Toxicity of Carbenicillin and Cefotaxime on Single Buds and Agrobacterium
Growth
For effective Agrobacterium-mediated transformation, the antibiotic
regime should control bacterial growth without inhibiting the regeneration of
the plant cells. The effect of carbenicillin and cefotaxime on the percentage
of single buds growth, were evaluated after four weeks of culturing on the solid
and liquid medium containing an appropriate concentrations (Fig.
2 and 3). No significantly differences were observed between
the cultures when the percentage number of single buds growth was counted.
However, using 600 mg L-1 cefotaxime in solid medium reduced average growth from 100% (control) to 30%. The percentage of growth in liquid medium using same concentration is lower 12% than solid medium. In comparison to carbenicillin, using 600 mg L-1 reduced average growth from 62% in solid medium and 54% in liquid medium. The percentage of growth is reduced to 8 and 12% in solid and liquid of 150 mg L-1 carbenicillin. However in cefotaxime, percentage of growth dropped tremendously to 82% in solid medium and 63% in liquid medium using the same concentration of 600 mg L-1.
| Fig. 2: | Percentage of single buds growth obtained after 4 weeks in MS media using different carbenicillin concentration. The data plotted were the means of four replicates. Different letters() indicate values are significantly different (p<0.05) |
| Fig. 3: | Percentage of single buds growth obtained after 4 weeks in MS media using different cefotaxime concentration. The data plotted were the means of four replicates. Different letter(s) indicate values are significantly different (p<0.05) |
| Fig. 4: | Percentage of single buds with Agrobacterium tumefaciens, EHA 101 growth using with different carbenicillin and cefotaxime concentrations. The data plotted were the means of four replicates. Different letter(s) indicate values are significantly different (p<0.05) |
In order to examine the bactericidal effects of carbenicillin and cefotaxime, Agrobacterium strains EHA 101 were inoculated with different concentrations of antibiotics. Using carbenicillin, the inhibition of cell growth started at a concentration of 100 mg L-1 for both strains, but complete inhibition obtained using 400 mg L-1 for EHA 101 (Fig. 4). For cefotaxime, the inhibition of cell growth started at 100 mg L-1 and complete inhibition of cell growth at 300 mg L-1 for Agrobacterium strains.
The results demonstrated that, Agrobacterium cells were more sensitive to cefotaxime than carbenicillin.
Although cefotaxime is more effective in inhibiting Agrobacterium cell growths, carbenicillin is the antibiotic of choice in Agrobacterium-mediated transformation because cefotaxime has shown high toxicity in many different plant tissues (Lin et al., 1994; Antunez de Mayolo et al., 2003). In cacao, the addition of the cefotaxime decreased somatic embryo production by 86% (Antunez de Mayolo et al., 2003). However, it has been reported that cefotaxime did not show negative effect in apple tissues (Maximova et al., 1998), adventitious buds and shoots induction in Pinus radiate (Holland et al., 1997) and chrysanthemum (Teixeira da Silva and Fukai, 2002). The other antibiotic, augmentin, timentin and moxalactam were also used after co-cultivation to kill the Agrobacterium as it was not reduce regeneration capacity (Park and Facchini, 2000; Antunez de Mayolo et al., 2003).
Therefore, different concentration of carbenicillin and cefotaxime required in solid and liquid medium of banana cultivar, Rastali (AAB) explants in order to avoid toxicity or overgrowth of Agrobacterium tumefaciens which could inhibit the explants growth and caused contamination which could reduces transformation efficiency.
Transformation of Single Meristematic Buds and Plant Regeneration
Single buds of banana cultivar, Rastali (AAB) were infected and co-cultivated
with induced Agrobacterium strains harboring different binary vectors,
following the condition described in materials and methods. Co-cultivated single
buds were selected on MS medium consisting of 10 mg L-1 of BAP containing
hygromycin at 25 mg L-1 (treatment A) or hygromycin at 50 mg L-1
(treatment B), during a period of 2 to 3 months, depending on the experiments.
Selection medium containing hygromycin at 50 mg L-1 produces lower
number of buds resistant to this antibiotic for both Agrobacterium strains,
EHA 101. However, none of the negative control plates did ever give rise to
surviving cell aggregates under any of the selective conditions used in this
experiment. In the absence of antibiotics, uninfected single buds proliferated
normally on 10 mg L-1 of BAP, whereas they turned to black and died
after two months under selection condition even at the lower concentration tested.
Percentage of transformation frequency was evaluated based on the number of explants regenerated. Transformation frequency of Agrobacterium, from treatment A is higher than treatment B. More than 30 cell clusters arose per individual co-cultivated sample after selection with many of which initiated the formation of multiple bud clumps, while the rest of untransformed cells which did not stand selection, blackened and died (Fig. 5).
After first level of selections, the remaining proliferation buds were subcultured into liquid medium containing 10 mg L-1 of BAP using the same concentration of antibiotics that had been used previously in first selections. After one week, the remaining healthy proliferating buds then selected in same concentration of selections in solid MS medium. Multiple bud clumps aggregates formed during the selective process were regenerated in BAP and antibiotics free medium. Single plantlets arising from hygromycin resistant cell clumps in hormone free (without BAP) formed shoots and root tips without any necrosis been observed. The observation patterns revealed that the tissue were fully transgenic without evidence of chimaeras. However, this is not true for explants regenerated in vitro using kanamycin as a selection marker in some of the transgenic plants experiment. Some of transgenic plantlets produced albinos and semi-necrosis growth in kanamycin selection which caused production of phenotypically abnormal plants confirmed observations in peas (Bean et al., 1997; Nadolska-Orcyzk and Orczyk, 2000). Similarly, kanamycin effect has been reported in transgenic papaya by Yu et al. (2003).
| Fig. 5: |
Production of transgenic banana cultivar, Rastali. (A) Two months in geneticin G-418 selection media; (B) Shoot proliferation from antibiotic resistant meristematic clumps in 5 mg LG1 of BAP concentration; (C) Some single bud develop into green multiple bud clumps in 5 mg LG1 of BAP concentration; (D) Shoots developed from green multiple bud clumps; (E) Multiple shoots proliferating; (F) Two single plantlet resistant to antibiotic selection; (G) and (H) Putative transgenic plantlet confirmed from PCR and Southern blot analyses |
In plant cells, kanamycin exerts its effect on mitochondria and chloroplasts by impairing protein synthesis, resulting chlorosis (Weide et al., 1989). For several Malus cultivars and rootstocks, kanamycin at 50 mg L-1 is pyhtotoxic and causes shoot chlorosis and necrosis (Yepes and Aldwinckle, 1994 b).
The single buds treated with L-Cysteine which is known as antinecrotic compounds in order to minimise the Agrobacterium-induced hypersensitive response. Enriquez-obregon et al. (1999) showed that the co-culture of sugarcane explants with Agrobacterium tumefaciens induces a rapid necrosis of the tissue. Similarly, Olhoft and Somers et al. (2001) demonstrated increased Agrobacterium-mediated transformation efficiencies in soybean cotyledonary-node cells using 400 mg L-1 of L-Cysteine. In addition, L-Cysteine and ascorbic acid has been reported decreased tissue necrosis of Japonica rice meristem cultures used for Agrobacterium-mediated transformation (Enriquez-Obregon et al., 1999).
The influence of calcium deprivation on the cell wall is considered during the interaction of Agrobacterium tumefaciens with explants by using CaCl2 free medium during cocultivation period. Ca2+ acts directly as an ionic cross-linkage of the carboxyl groups of linear macromolecules in the cell wall and directly as an inducer of changes in the cell wall composition through its influence on gene expression (Sander et al., 1999). Hence, lack calcium reduced cell wall matrix by modifying cell wall structure and made banana single buds more susceptible to Agrobacterium cell attachment. This was consistent with observations made on Arabidopsis thaliana tissues (Sangwan et al., 1992), Pisum sativum (de Katen and Jacobsen, 1995) and Hevea brasiliensis (Montoro et al., 2003).
Nevertheless, maintenance of cultures for long periods and with high antibiotics concentration for more than three phase selection resulted in reduced numbers of surviving cell aggregates. Irrespectively of the selective conditions used, average frequencies of plant regeneration ranged from 5 to 20% putatively independent lines per co-cultivation in all samples. This study demonstrated that the use of Agrobacterium tumefaciens as a transformation system for banana cultivar, Rastali (AAB) could be highly efficient when a rapid screening system for the identification of lines of interest developed before performing more detailed molecular analyses. Techniques based on the Polymerase Chain Reactions (PCR) are the best option for analyzing large amount amounts of transformants, since they are fast and demand low quantities of genomic DNA.
The use of highly regenerable single meristematic buds of banana cultivar, Rastali (AAB) in combination with Agrobacterium as a vector for DNA transfer has been not described elsewhere previously. However using other banana meristem tissue such as corm slices in Agrobacterium-mediated transformation may be limited application because of the risk of generating chimaeric plants even though the transformation frequency obtained with particle bombardment could be markedly improved (May et al., 1995). Besides being more efficient, Agrobacterium-mediated transformation is technically simpler than particle bombardment, only requiring basic microbiology facilities and generally results in high levels of expression due to a simple integration pattern of well defined DNA sequences into transcriptionally active regions of the plant genome, which make it the best option when both transformation systems are available (Hiei et al., 1997; Cheng et al., 1997).
Analysis of T-DNA Integration via PCR Amplification
Though Agrobacterium-mediated transformation is the most resorted
method for the generation of transgenic plants with single integration of a
precisely delimited DNA sequences (Smith and Hood, 1995; Lawrence and Koundal,
2001), the structure of the inserted T-DNA varies widely to include single or
multiple copies, individual or tandem repeats, at a unique or several loci in
the plant genome (Iglesias et al., 1997).
Total DNA isolated from the putative transformants was tested for the presence of the transgenes. The efficient and simplicity of the PCR analysis, enable screening for transformed plants in a shorter period. PCR amplification confirmed that the chitinase (RCC2) gene and hpt11 gene were present in a high proportion of the 50 mg L-1 hygromycin (medium B) compared to lower hygromycin selection medium (medium A). The PCR results of some transgenic plantlets are displayed in Fig. 6 and 7.
In all the experiments, no bands could be detected from DNA extracted from untransformed control. Eleven DNA samples were picked from hygromycin selection (medium A) and five from selection medium B were subjected for PCR analyses. In all experiments, no bands (RCC2 and hpt11 genes) were detected from plants using hygromycin selection (medium A).
Three out of the five DNA samples assayed successfully amplified the expected band size of 310 bp of chitinase gene (RCC2) from medium A (Fig. 6). Co-integration of the hpt 11 gene (900 bp) as expected, detect in these putative transformants (Fig. 7). Similarly to this result, all putative transgenic Agrostis palustris plants tested showed a band of expected size in PCR for the linked hph and gusA transgenes with transformation frequencies of 100% (Xiao et al., 1997). Three transgenic plantlets contained chitinase gene were differentiate according to plant code [B2Y (4); B2Y (16) and B3Y (11)].
Longer duration (more than eight cycles) of hygromycin in selection medium did not help to reduce the outcomes of chimeric tissue instead it decreases the survival of regenerating plants, appeared to effect fertility of the regenerated plants and surprisingly produced pink colour pigmentation in shoots and leaves. However, these pigmented plantlets return back to normal green plantlet when the selection removed after six cycles and maintained in hormone free MS medium.
The presence of hygromycin or basta during five cycles of oil palm embroyogenic callus clumps remained essential as otherwise some of the regenerated plants were not transgenic (Parveez et al., 1996). The fact that transgenic banana cells were selected during a longer period (four to six months) before regeneration is started probably explains the low number of transformants in higher hygromycin selection medium.
Southern Blot Hybridization Analysis
Southern blot analyses were carried out to evaluate further the transfer
and insertion of the chitinase gene (RCC2) in the genome of the transformed
banana cultivar, Rastali (AAB). Although two lines of evidence, (I) the antibiotic
resistant phenotype and (ii) the presence of the introduced genes in putative
transformants as shown indeed by PCR demonstrated that the regenerated plants
were transgenic, proof of stable integration of transgenes by Southern analysis
was required.
Hybridisation of chitinase gene (RCC2) to Hind111 digested genomic DNA from the transgenic plantlets was shown in Figure 8. The results confirmed that all three transgenic plantlets [Plant code: B2Y (4); B2Y (16) and B3Y (11)] derived from PCR positive results using Agrobacterium tumefacien, EHA 101 (pBI333-EN4-RCC2) contained chitinase genes in leaves and roots. Hind111 digestion of transforming plasmid, pRCC2, released an approximately 1.3 kb size fragment containing the p35S, RCC2 and nos terminator (Fig. 8 (lane 1)). Lane 1, 2, 4-5, 7-8 and 10-12 revealed bands with sizes larger than the RCC2 gene fragment (1.3 kb) were indicative of integration of the introduced plasmid into the host plant genome. The presence of bands with molecular weights different from the original transforming plasmid indicates that it is a possibility transgene rearrangement or multiple independent insertions had occurred and the observed bands represent plasmid-chromosome junction fragments (Janna et al., 2001).
Even though 3 transgenic plantlets tested were from different independent transformation events (treatment B), surprising 2 plantlets showed similar hybridisations patterns with except of root tissue from plant B3Y (11), which revealed an addition band in its hybridization pattern. DNA fragments smaller than expected size could be due to rearrangement of the transgene in the genome (Kohli et al., 1999).
| Fig. 6: | PCR analysis chitinase gene in transgenic banana plantlets selected on hygromycin 50 mg L-1. Lane M = Lambda DNA digested with Hind111 was used as molecular weight marker; Lane 1 = Transforming plasmid, pBI333-EN4-RCC2; Lane 2-9, 11 = Putative transformed plantlets showing the amplified 310 bp RCC2 gene and Lane 10 = Untransformed plantlet |
| Fig. 7: | PCR analysis hpt11 gene in transgenic banana plantlets selected on hygromycin 50 mg L-1. Lane M = Lambda DNA digested with Hind111 was used as molecular weight marker; Lane 1 = Transforming plasmid, pBI333-EN4-RCC2; Lane 2 = Untransformed plantlets; Lane 3-11 = Putative transformed plantlets showing the amplified 900 bp hpt11 gene |
| Fig. 8: | Southern blot analysis of the chitinase integration pattern in banana plantlets. Digested genomic DNA with Hind 111 using PCR amplified RCC2 gene (310 bp) as probe. Lane1=Transforming plasmid, pBI333-EN4-RCC2; Lane 2, 4-12 = genomic of putative transformed plantlets and Lane 3 and 9 = untransformed control plantlets |
| Table 2: | Chitinase enzyme activity in transgenic Pisang Rastali (AAB) plantlets transformed with chitinase gene (RCC2) |
| a One unit of chitinase activity: the amount of enzyme catalyzing the formation of 1 nm N-acetlyglucosamine equivalent in one second under assay conditions refers as 1 nkat. b Three replications were used for the assay to calculate the average activity. c Control: Untransformed banana cultivar, Rastali (AAB) | |
Analysis of Chitinase and β-1,3 Glucanase Protein Production in Transgenic Plantlets: The accumulation of chitinase activity in transformed banana cultivar, Rastali (AAB) plantlets was examined by enzyme assay. The chitinase activity in the whole plants of B2Y (4), B2Y (16) and B3Y (11) was measured (Table 2).
The chitinase activity of B2Y (4) and B2Y (16) was 3 to 4 fold higher than untransformed plantlet. Neuhaus et al. (1991) reported a similar observation in chitinase activity upon introducing the similar chitinase (chi 1) gene via Agrobacterium, LBA 4404 in transgenic tobacco. However, chitinase activity of B3Y (11) is slightly lower than B2Y (4) and B2Y (16) plantlets with only 2 fold higher than untransformed plantlet. The extra copy number of integrated RCC2 fragment might be related to the weak expression of chitinase activity on B3Y (11). Translation, mRNA expression and degradation might affect the differences in accumulation of rice chitinase protein in banana cell.
CONCLUSION
Single bud is more sensitive at higher cefotaxime concentrations compared to carbenicillin. DNA samples from transgenic banana cultivar, Rastali (AAB) obtained only from selection media contained higher hygromycin concentration (50 mg L-1) and were tested positive for the presence of the chitinase (RCC2 gene) and hpt11 coding sequences by PCR analyses. The results from PCR analyses suggested that the selective system protocol (solid media-liquid media-solid media) using higher hygromycin concentration as selection agent in banana is effective in reducing chimeras when compared to lower concentration (25 mg L-1). Integration of transgene and stable genetic transformation of Agrobacterium tumefaciens (EHA 101) using chitinase gene were assess by PCR amplification of 310bp of RCC2 gene (chitinase gene) and 900 bp of the hpt11 gene. Genomic Southern blot hybridization confirmed the incorporation of the RCC2 gene in host genome between one and two inserted copies. The accumulations of chitinase activities in transformed banana cultivar, Rastali (AAB) plantlets were higher than untransformed plantlets. The design of an adequate artificial environment to favor the interaction of Agrobacterium tumefaciens with the banana single buds is critical to the success of genetic transformation experiments. Oxidative burst, phenolization and subsequent cell death have been described as frequent phenomena during the interaction of Agrobacterium tumefaciens with monocot plant cells.
ACKNOWLEDGMENT
The generous gifts of a RCC2 (rice chitinase) plasmid by Dr Yokoshiwa from National Institute of Agrobiological Resourses, Japan is gratefully acknowledged. This research was supported by the Ministry of Science, Technology and Environmental funded this research through IRPA PR Research Grant (01-02-04-0000-PR0010/04)