DEVELOPMENT OF in vitro REGENERATION AND Agrobacterium MEDIATED TRANSFORMATION SYSTEMS FOR Moringa oleifera PLANT

The present investigations were aimed to develop a high efficiency of in vitro regeneration and genetic transformation systems of Moringa oleifera Lam from nodal segments of young aseptically grown seedlings using Agrobacterium-mediated transformation approach. Frequency of responded explants and number of shoots per explant were recorded during the course of the regeneration experiment. Regeneration capacity of nodal segments was evaluated on Murashige and Skoog (MS) media supplemented with 18 different combinations of plant growth regulators of benzylaminopurine (BAP), Zeatine (Zea) and naphthaleneacetic acid (NAA). Application of 1.0 mg/l BAP individually was found to be superior in terms of highest number of responded explants (95.7%) as well as the highest average (6.6) of axillary shoot developments per explant with direct emerging of adventitious shoots escaping callus formations. Well developed shoots subjected to rooting media supplemented with IAA or IBA or their different combinations. The most successful rooting events (100%) for regenerated shoots were obtained on rooting media containing ½ MS salts and supplemented with 1.0 mg/l IBA along with 0.5 mg/l IAA within three weeks maximum. The plant transformation vector pBIN121 harbors both the uidA (GUS) and NPTII (kanamycin resistant) genes were used to establish the Agrobacterium-mediated transformation experiment. Number of Putative transformed young shoots that developed onto Kanamycin selective regeneration medium were recorded representing 39.6% transformation efficiency. PCR analysis was carried out to verify successful transformation and gene integration for both GUS and NPTll genes in randomly selected young shoots while Histochemical GUS assay confirmed the successful expression of GUS gene in different parts of the putative transgenic plantlets.

who utilized its seed oil. Moringa trees are usually propagated by seeds or cuttings. However, not enough seeds or cuttings are available for use as planting materials as moringa still has not been cultivated in commercial quantity in Egypt. Around 500,000 ha of land will be needed to produce moringa oil at a commercial scale (Marfori, 2010). At a planting distance of 1 m x 1 m, 5 billion seedlings will be needed as planting materials for 500,000 ha. Very limited information is available on genetic variability and availability of superior genotypes for commercial cultivation. The traditional way to improve the desired traits is by breeding techniques, but these techniques have limitations as they depend on sexual compatibility, and takes considerable time (10-15 years) to release a new variety. Alternatively, genetic manipulation tools, such as, genetic transformation methods are representing valuable tool for the functional study of genes as well as the production of genetically improved plants by introducing genes that responsible for increasing oil content and genes that regulate the production of endogenous plant antioxidants that leads to obtain plants with enhanced contents of agronomical, nutritional and M pharmaceutical traits. There are extensive reports that demonstrate successful attempts for the modification of plant oil composition via metabolic engineering as well (Abbadi et al., 2004;Hoffmann et al., 2008;Lopez et al., 2009;Cheng et al., 2010;Petrie et al., 2011;Lopez et al., 2012;Haslam et al., 2013;Lopez et al., 2014). Building on all previous considerations adding the tremendous potential opportunities with M. oleifera for sustainable agriculture and the development of cash crops in semiarid regions as Egypt, the plant was selected for this regeneration and transformation work. The aim of the present study was to optimize the regeneration and mass in vitro propagation of Moringa plants as well as to establish a reliable genetic transformation protocol as a preliminary request for further genetic improvements of moringa plant via genetic engineering approaches.

Plant material and explant preparation
Healthy uniformed seeds of M. oleifera were obtained from Faculty of Agricultural Science, Menofiya, Egypt. Seeds were surface sterilized inside the laminar flow hood with 75% ethanol for one minute followed by immersion in 30% sodium hypochlorite (v/v) for 10 min, followed by rinsing three times in sterile distilled water. Seed coats were removed aseptically and seeds were again surface sterilized by immersion in 20% sodium hypochlorite (v/v) for 5 min, followed by rinsing three times in sterile distilled water. Seeds were planted aseptically in MS basal medium (Murashige and Skoog, 1962) containing 30 g/l sucrose and solidified with 8 g/l agar (Himedia).
The pH was adjusted to 5.8, after which the medium was dispensed at 40 ml each in culture bottles and sterilized by autoclaving at 121C for 20 min. Seed cultures were maintained in the dark at 27 ± 1C for 15 days. Upon germination, seedlings were transferred under continuous light at 2,000-Lux intensity produced from cool white fluorescent tubes. Young, aseptically grown shoots (6 weeks old) were cut into nodal segment explants each bearing one or two axillary buds were inoculated on shoot induction media supplemented with different concentrations of growth regulators.

Bacterial strain and transformation vector
Agrobacterium tumefaciens strain EHA105 was used for plant transformation. The Agrobacterium cells were directly transformed with the Agrobacterium binary vector pBI121 that harbors both the uidA gene, coding for glucuronidase (GUS) (Jefferson et al., 1987) as a reporter gene under CaMV 35S promoter and neomycin phosphotransferase II (NPTII), kanamycin resistant gene (Bevan et al., 1983) as a selectable marker gene under NOS promoter in order to select for the transformants onto selective regeneration medium. Both Agrobacterium strain and plant transformation vector obtained from Clontech Laboratories.

Induction of multiple shoots
Nodal segment explants were pre-

Shoot elongation
Young developed shoots were transferred to shoot elongation media con-

Inoculation
The inoculium of Agrobacterium tumefaciens EHA105 cells harboring pBIN121 was prepared from a freshly streaked LB plate culture containing kanamycin and streptomycin 50 mg/l each. A loop from the plate culture was inoculated into 5 ml LB liquid medium containing the same antibiotics as LB plate with the same concentrations and grown for 16 h at 28C with vigorous shaking at 250 rpm. An aliquot of 100 μl from the culture was used to inoculate 10 ml of a LB broth that contained the same mentioned antibiotics as LB plate with the same concentrations in addition to 39.28 g/ml acetosyringone then incubated in a shaker incubator at 28C at 250 rpm for 3-4 hours. Optical density (OD) was measured by spectrophotometer at 660 m and the bacterial concentration was adjusted to approximately 510 8 cfu/ml by MS basal liquid medium prior to use. Nodal explants were immersed in the diluted bacterial solution for 5-10 minutes under sterilized conditions then the explants were blotted dry on sterile filter paper, then explants were kept for 2 days co-cultivation onto the same preculture medium in the incubator at 25C and cool white light. Light intensity was 45-50 m -2 s -1 under continuous light.

Selection and regeneration
After cocultivation, the nodal segment explants were subsequently transferred to the selective shoot induction plates containing suitable regeneration media supplemented with 50 mg/l kanamycin, to select for transformed shoots in addition to 500 mg/l carbenicillin and 250 mg/l cefotaxim to prevent Agrobacterium growth (all antibiotics were added as filter sterilized solutions after autoclaving) then cultures were incubated under the previous optimized conditions. After additional two weeks of incubation, the number of developed shoots was recorded. Will developed shoots were excised individually from the explants and subcultured on suitable rooting media that containing 50 mg/l kanamycin. The experiment had three replicates. Finally, Successful rooted plantlets were hardened in pots containing sterilized mixture of soil and sand v/v and were acclimatized in the greenhouse. The established putatively transgenic Moringa plants were designated as T 0 -generation plants.

Confirmation of successful transformation Polymerase chain reaction (PCR) analysis
Putative transgenic Moringa T 0 plantlets were verified for the presence of the transgenes by PCR analysis using GUS and kanamycin (Km). Gene specific primers. Total genomic DNA was isolated from leaves of randomly selected putative transgenic plants as well as the untransformed control plant using modified CTAB protocol (Puchooa, 2004). The GUS specific primer sequences; sense (GUS-F): 5'-CATGTCGCGCAAACTGTAAC-3' and anti-sense (GUS-R): 5'-AATCGCCTGTAA GTGCGCTTG-3') while the kanamycin specific primer sequences; sens (Km-F):5'-GCATACGCTTGATCCGGCTAC-3'and anti-sense (Km-R): 5'-TGATATTCG GCAAGCAGGCAT-3'), were used in the PCR analysis. PCR was performed in a total volume of 25 μl and the reaction mixture consisted of 10x PCR reaction buffer, 50 ng template DNA, 0.2 mmol/l dNTPs, 1.5 mM MgCl 2 , 0.2 mM of each primer and 1 unit of Taq DNA polymerase. The PCR reaction was started by an initial denaturing step at 94C for 2.5 min, followed by 35 cycles of the following profile: denaturing at 94C for 30 sec, annealing at 56C for 1 min, synthesis at 72°C for 1 min followed by an extension at 72C for 10 min. The amplification mixture was analyzed by electrophoresis in 1% agarose ethidium bromide gels.

Histochemical GUS assay
Histochemical analysis of GUS activity in putative transgenic Moringa plantlets was carried out using 5-bromo-4chloro-3-indolyl-b-d-glucuronide (X-Glu) as a substrate as described by Jefferson et al. (1987) using different parts of the plant. Following overnight tissue-staining with X-Glu at 37C, chlorophyll was re-

Influence of growth regulators on shoot regeneration and root induction
The percentages of responded explants as well as the mean number of axillary shoots per explant induced by BAP and Zea at different concentrations and their different 18 combinations with NAA after 3 weeks were represented in Table  (1). The SIM4 medium resulted in the highest number of responded explants as the application of 1.0 mg/l BAP gives rise of shoots on 95.2% of the inoculated explants with an average of 6.6 axillary shoots per explant with direct shoot development without any formation of callus structures (Fig. 1). SIM5 medium that contained a combination of BAP and NAA at 1.0 and 0.3 mg/l, respectively, enhanced axillary shoot proliferation at 93% of inoculated nodal explants with an average of 5.3 shoots per explants. The same concentrations of zea in SIM13 and SIM14 media was much less effective than BAP in respect of the responded explants which was only 57.3% and 52% with an average of 3.1 and 2.9 axillary shoots per explant, respectively with more tendency for callus formation than shoot proliferation. It was noticed that almost all media that were including both Zea or NAA or their combinations had developed more or less callus structures (data not shown). Higher concentration of BAP was inhibitory, inducing 2.7 axillary shoots per explants when used alone at 1.5 mg/l or in combination with 0.3 and 0.6 mg/l NAA inducing 1.4 and 0.7 axillary shoots per explants, respectively. Regenerated shoots were subcultured on the same shoot induction media supplemented with gibberellic acid (GA 3 ) at 0.5 mg/l to enable shoot elongation. Well developed shoots were transferred to root induction medium supplemented with IAA or IBA or their combination as well as free hormone medium, Figure 1D. Induction of roots was recorded as a percentage after two weeks from inoculation and data were represented in Table (2). Application of 1.0 mg/l IBA along with 0.5 mg/l IAA resulted in the highest percent of responded shoots (100%) with direct root developments and no callus formation ( Fig. 1 E1 & E2), while there were 95% responds among shoots when media containing IBA alone. However, application of IAA alone resulted in root induction (75%) with more tendencies for callus formation at the base of the young shoots ( Fig. 1 E3). The in vitro regenerated plantlets were successfully hardened and established on the soil with normal growth pattern (Fig. 1 F).
In vitro regeneration and multiplication of M. oleifera has been previously investigated using different growth regulators. Our results were in consensus with Stephenson and Fahey (2004) as they obtained 4.7 shoots per cultured seed in medium containing 1 mg/l BA with 1 mg/l GA 3 , while rooting was obtained in MS media containing 0.5 mg/l NAA. At similar result of ours Saini et al. (2012) reported that 1 mg/l BA was found to be optimal in producing maximum number of shoots per explants while efficient in vitro rooting of individual shoot culture was ob-tained in 0.5 mg/l IAA plus 1mg/l IBA treatment. Islam et al. (2005), demonstrated that using BAP at concentrations 1.0-1.5 mg/l was found to be best for shooting response, whereas rooting was efficient on MS basal medium. The efficiency of BA and NAA for organogenesis was further supported by similar reports. Riyathong et al. (2009) found that shoot multiplication were successfully carried out in the presence of BA 2.0 mg/l giving average number of 10.8 shoots per explant. In the same time they found that regeneration of in vitro explants was initiated by using the medium containing 0.5 mg/l NAA that produced the shoots and roots from callus culture. Marfori (2010) reported that of the three cytokinins tested, namely BAP, kinetin (Kin) and thidiazuron (TDZ), BAP at 0.5 mg/l was found to be optimal in inducing bud break, producing an average of 4.6 axillary shoots per explant after two weeks, whereas optimal rooting of individual shoot culture was obtained with application of NAA at 0.05 mg/l.

Statistical analysis for the efficiency of shoot regeneration
To compare the efficiency of the different treatments on explant responds and shoot regeneration statistical analysis has been conducted on the obtained results to evaluate the degree of significance between the different combinations of BAP, Zea and NAA. The data obtained were subjected to statistical analysis of variance as described by Steel and Torrie (1980). The treatment means were compared using LSD test at 0.05 level of significant.
The results of this analysis are summarized in Table (

Agrobacterium-mediated transformation
Nodal segment explants of young moringa seedlings were prepared as previously described and then were infected with A. tumeficiense strain EHA105 cells harboring pBI121. Then explants were maintained for two days cocultivation period with Agrobacterium then they were transferred to selective regeneration medium that contained SIM4 medium supplemented with 50 mg/l Kanamycin 500 mg/l carbincillin, and 75 mg/l cefotaxim and incubated until the putative transformed shoots were developed. It was found to be important that the explants must undergo in several subculture steps onto a fresh selection medium at least once after 10 days and in each time both abnormal structures and dead tissues must trimmed out to promote faster shoot formation, and to prevent Agrobacterium regrowth. Results showed that the surviving explants on kanamycin SIM4 medium ranged among the replica from 19 to 46 explants with a total number of 235, representing 39.6% transformation efficiency (Table  3). To the best of our knowledge and after extensive searches conducted via published article databases, we could not find articles on the transformation of M. oleifera. On the other hand there are many successful attempts to produce different transgenic oilseed crops that are successfully engineered to accumulate high levels of different fatty acids as well as to elevate the oil contents of its seeds (Abbadi et al., 2004;Lopez et al., 2009;Lopez et al., 2012;Lopez et al., 2014;Haslam et al., 2013).

Analysis of the putative transgenic moringa plantlets a. PCR analysis
The explants that were able to sur- and GUS genes, respectively, while they were absent in untransformed plant control (Fig. 2). Further PCR analysis could be conducted on large scale to evaluate the transgenic lines obtained.

b. Histochemical GUS assay
The uidA gene or gus gene is one of the most widely used reporter genes in plant transformation due to its accurate fluorimetric assays and precise histochemical localization of GUS in transgenic tissues. The explants that were able to grow on the selective media supplemented with Km were examined histochemically for GUS activity. Out of the total number of 93 Km-resistant shoots that were survived on selective shoot induction medium, only 20 young shoots were randomly selected and subjected to a histochmical GUS assay and visually compared with non-transformed plant materials. A percentage of 80 % of the tested plant materials developed blue color that indicating GUS expression in various parts of Moringa plantlets whereas no blue color was observed untransformed plants (Fig. 3).

SUMMARY
The present investigations were aimed to develop a high efficiency of in vitro regeneration and genetic transformation systems of Moringa oleifera Lam from nodal segments of young aseptically grown seedlings using Agrobacteriummediated transformation approach. Frequency of responded explants and number of shoots per explant were recorded during the course of the regeneration experiment. Regeneration capacity of nodal segments was evaluated on Murashige and Skoog (MS) media supplemented with 18 different combinations of plant growth regulators of benzylaminopurine (BAP), Zeatine (Zea) and naphthaleneacetic acid (NAA). Application of 1.0 mg/l BAP individually was found to be superior in terms of highest number of responded explants (95.7%) as well as the highest average (6.6) of axillary shoot developments per explant with direct emerging of adventitious shoots escaping callus formations. Well developed shoots subjected to rooting media supplemented with IAA or IBA or their different combinations. The most successful rooting events (100%) for regenerated shoots were obtained on rooting media containing ½ MS salts and supplemented with 1.0 mg/l IBA along with 0.5 mg/l IAA within three weeks maximum. The plant transformation vector pBIN121 harbors both the uidA (GUS) and NPTII (kanamycin resistant) genes were used to establish the Agrobacterium-mediated transformation experiment. Number of Putative transformed young shoots that developed onto Kanamycin selective regeneration medium were recorded representing 39.6% transformation efficiency. PCR analysis was carried out to verify successful transformation and gene integration for both GUS and NPTll genes in randomly selected young shoots while Histochemical GUS assay confirmed the successful expression of GUS gene in different parts of the putative transgenic plantlets. Values within a column followed by the same letter(s) are not significantly different at the P=0.05 level according to the least significant difference test as described by Steel and Torrie (1980).