**5. Tissue culture-based transformation**

The success of *Agrobacterium*-mediated genetic transformation is largely due to the correct optimisation of culture conditions, each manipulated by media modification and environmental control. Four (4) main factors were recognised in this study for the improvement of genetic transformation under *in vitro* plant tissue culture. The specific factors are:

> 200 mgL−1 vancomycin), reduced co-cultivation period of 3-days and high concentrations of DTT and L-cysteine in the co-cultivation medium. These factors were modified to improve the transformation efficiency, particularly using aseptic culture conditions to stimulate proliferative capacity of explants and influence the explant viability. Given the current figure of less than 10% transformation efficiency so far achieved, there is no doubt that established

> **Table 2.** Modified Gamborg's B5 and MS basal culture media used for *in vitro Agrobacterium*-mediated genetic

The Role of Plant Genotype, Culture Medium and *Agrobacterium* on Soybean Plantlets…

http://dx.doi.org/10.5772/intechopen.78773

23

transformation of soybean using double cotyledonary-node explants derived from 10-day old seedlings.

The use of double cotyledonary-node explants derived from seedlings germinated on MS medium for *in vitro* regeneration and transformation of soybeans have been previously reported [38]. In this study, the coty-node explants were also prepared from 10-day old soybean seedlings developed from MS basal culture medium supplemented with 4.0 mgL−1 6-BA as indicated on **Table 2**. Seedlings of soybean cultivar Dundee, LS 677, LS 678, TGx 1740-2F, TGx 1835-10E and Peking were transversely cut on the hypocotyl segments, 5–8 mm beneath cotyledons and their epicotyls excised from the base, at cotyledonary junctions to produce double cotyledonary-node explants. The explants comprised of the embryonic axis, section of the hypocotyl and two cotyledons. All coty-nodes were placed with their abaxial side down

protocols still need to be improved.

**5.1. Double cotyledonary-nodes preparation and co-cultivation**


The establishment of aseptic *in vitro* culture conditions usually includes the culture medium with basal inorganic nutrient elements (both micro- and macro- elements), organic energy source (primarily sucrose) and the vitamins. Culture media are furthermore, semi-solidified with agar or other commercially available gelling agents, like the gelrite [36]. Precisely defined *in vitro* culture media and other conditions are required for exploitation in the initiation and induction of different cultures from genetically engineered tissues. In this study, defined media compositions were designed to assess the effects of media and *Agrobacterium* suspension on the regeneration of transformed soybean plantlets (**Table 2**). This was done for the seed cultures used to establish seedlings, to develop the double cotyledonary-node explants, infection of explants with *Agrobacterium*, co-culturing medium and regeneration of multiple adventitious shoots. Procedures used for the preparation of *Agrobacterium tumefaciens* inoculum and co-cultivation of explants was conducted as described by Paz et al. [37] with modifications.

Culture modifications included, reduced infection period (15 min immersion of explants in the infection medium), increased amounts of antibiotics used for washing of infected double coty-node explants following co-cultivation with *Agrobacterium* (200 mgL−1 cefotaxime and



stress, particularly drought. Drought refers to the absence of rainfall or irrigation for a period sufficient to deplete soil moisture to a level not sustaining plant growth [29]. The decrease in water content completely arrest cell functioning, including the support to plant cell and tissue metabolism, meiotic/mitotic division and differentiation. Some of the reports that have assessed the severity of drought on soybean growth include those of Heatherly [30], Desclaux et al. [31] and Brown et al. [32]. Studies continue to show that the GM soybean varieties have important applications, including the use in biodiesel production. Increased yield emanating from genetic improvement is required for the production of more soybean oil used for manufacturing of biodiesel. Besides the utilisation of soybean oil as a major feedstock for biodiesel production [33], the hull can be used to manufacture ethanol from the significant amount of carbohydrates it contains [34]. All these reports clearly indicate the importance of transformation in increasing yield quantity of high quality. According to Zia [23] and Yu et al. [35] transgenic soybean cultivars account for more than 85% of cultivars cultivated for commercial production worldwide, and the cultivars used still require traits improvements.

The success of *Agrobacterium*-mediated genetic transformation is largely due to the correct optimisation of culture conditions, each manipulated by media modification and environmental control. Four (4) main factors were recognised in this study for the improvement of

The establishment of aseptic *in vitro* culture conditions usually includes the culture medium with basal inorganic nutrient elements (both micro- and macro- elements), organic energy source (primarily sucrose) and the vitamins. Culture media are furthermore, semi-solidified with agar or other commercially available gelling agents, like the gelrite [36]. Precisely defined *in vitro* culture media and other conditions are required for exploitation in the initiation and induction of different cultures from genetically engineered tissues. In this study, defined media compositions were designed to assess the effects of media and *Agrobacterium* suspension on the regeneration of transformed soybean plantlets (**Table 2**). This was done for the seed cultures used to establish seedlings, to develop the double cotyledonary-node explants, infection of explants with *Agrobacterium*, co-culturing medium and regeneration of multiple adventitious shoots. Procedures used for the preparation of *Agrobacterium tumefaciens* inoculum and co-cultivation of explants was conducted as described by Paz et al. [37] with modifications.

Culture modifications included, reduced infection period (15 min immersion of explants in the infection medium), increased amounts of antibiotics used for washing of infected double coty-node explants following co-cultivation with *Agrobacterium* (200 mgL−1 cefotaxime and

genetic transformation under *in vitro* plant tissue culture. The specific factors are:

**5. Tissue culture-based transformation**

22 Transgenic Crops - Emerging Trends and Future Perspectives

• Culture medium conditions

• *Agrobacterium* strain and density

• Plant genotype • Type of explant **Table 2.** Modified Gamborg's B5 and MS basal culture media used for *in vitro Agrobacterium*-mediated genetic transformation of soybean using double cotyledonary-node explants derived from 10-day old seedlings.

200 mgL−1 vancomycin), reduced co-cultivation period of 3-days and high concentrations of DTT and L-cysteine in the co-cultivation medium. These factors were modified to improve the transformation efficiency, particularly using aseptic culture conditions to stimulate proliferative capacity of explants and influence the explant viability. Given the current figure of less than 10% transformation efficiency so far achieved, there is no doubt that established protocols still need to be improved.

### **5.1. Double cotyledonary-nodes preparation and co-cultivation**

The use of double cotyledonary-node explants derived from seedlings germinated on MS medium for *in vitro* regeneration and transformation of soybeans have been previously reported [38]. In this study, the coty-node explants were also prepared from 10-day old soybean seedlings developed from MS basal culture medium supplemented with 4.0 mgL−1 6-BA as indicated on **Table 2**. Seedlings of soybean cultivar Dundee, LS 677, LS 678, TGx 1740-2F, TGx 1835-10E and Peking were transversely cut on the hypocotyl segments, 5–8 mm beneath cotyledons and their epicotyls excised from the base, at cotyledonary junctions to produce double cotyledonary-node explants. The explants comprised of the embryonic axis, section of the hypocotyl and two cotyledons. All coty-nodes were placed with their abaxial side down on the culture media and used for establishment of callus and shoots cultures. A total of 60 double cotyledonary-node explants were prepared for each set of replicates, and the procedure was repeated four times for all transformation cultures.

Given the good and viable explants used in this study, the results obtained during callus cultures showed that, callus induction was achievable using the chosen MS medium and the different kinds of plant growth regulators (KI, IBA and NAA) used. However, the MS medium, together with the hormonal combinations resulted in significant amounts of callus cells in the controls than on explants infected with *Agrobacterium*. Variations in the amount/ size of the callus and calli phenotypes were also observed. Generally, very little callus cells were observed from cotyledonary nodes transformed with *Agrobacterium*. But, more compact, embryogenic and friable callus cells were observed from culture medium supplemented with different combinations of KI, IBA and NAA without explant co-cultivation with the bacterium. The infection of explants influenced explant survival and responsiveness in addition to the callus induction capacity as illustrated in **Table 3**. The results indicated that, MS media containing antibiotics also delayed callus initiation. This was confirmed when un-transformed explants were subcultured on a media containing antibiotics. Callus initiation was arrested for more than 3 weeks of culture. But later, swelling and traces of slightly white-yellow friable callus was observed on the explants. The few small masses of callus rapidly turned brownish to ultimately black in colour (**Figure 1**). Zhang et al. [41] made similar observations when investigating the effect of kanamycin in tissue culture and induction of somatic embryos in cotton. This direct organogenesis of callus reported reduction of callus initiation and growth on medium supplemented with 10 mgL−1 of kanamycin. The study furthermore, reported complete inhibition of callus formation in medium supplemented with 60 mgL−1 concentra-

The Role of Plant Genotype, Culture Medium and *Agrobacterium* on Soybean Plantlets…

http://dx.doi.org/10.5772/intechopen.78773

25

Paz et al. [37] reported prophylactic and proliferative effects in soybean culture using 100 mgL−1 cefotaxime and 50 mgL−1 vancomycin. Even though, Grzebelus and Skop [42] shared similar sentiments to Paz et al. [37] when investigating the effect of β-lactams antimicrobials on *in vitro* carrot protoplast culture, the formation of callus on medium amended with these antibiotics was negatively affected. They evaluated three types of β-lactams (cefotaxime, carbenicillin and timentin) at five different concentrations (100, 200, 300 or 500 mgL−1), which were higher than what was used in this study. The low levels of callus and poor explant competency observed in this study, on infected and un-infected explants, can be largely attributed to the presence of antibiotics in the medium used (**Figure 2**). However, a dual negative effect was exerted on the cultures by both *Agrobacterium* infection and media composition, particularly on the proliferation from cotyledonary-node explants. The level of explant competency on these cultures appeared profoundly negatively affected compared to nearly 100% callus induction efficiencies obtained in all genotypes for both proembryonic masses and large size formations in the controls (**Table 3**). According to **Figure 2**, the media, PGRs concentration selected and explants were successful in inducing callus as supported by Barwale et al. [43]

The media compositions directed the development of plant cells in the cultures by essentially influencing the plasticity and totipotency of the plant tissues used as explants. This observation is in line with many studies, especially demonstrating the synergic effects of auxin (IBA, NAA) and cytokinin (KI) combination used in this study. All cultivars produced some callus on MS medium containing 0.7 mgL−1 KI, 2.7 mgL−1 IBA and 3.2 mgL−1 NAA as indicated in **Table 3**, in relation to **Table 2**. The results observed on MS-A and MS-B culture medium formulations could also be linked to the soybean cultivars used. Plant tissue swelling and initiation of calli was observed within a week of culture in more than 50% replicates of all

tion of kanamycin.

and Franklin and Dixon [44].

## **5.2. Callus induction**

A full-strength MS culture media containing macronutrients, micronutrients and vitamins, 3% sucrose, as well as 0.25% gelrite were used in this study. The different concentrations and combinations of PGRs were added into the media from prepared stock solutions made by dissolving analytical reagent grade hormones with few drops of 1 N sodium hydroxide and making-up the final volume with distilled water. The medium used to test for the efficiency of callus initiation on double coty-nodes infected with *A. tumefaciens* containing the *oc-1* gene on pTF101.1 vector contained 0.70 mgL−1 kinetin (KI), 2.70 mgL−1 indole-3-butyric acid (IBA) and 3.20 mgL−1 α-naphthalene acetic acid (NAA). Another medium was supplemented with 0.50 mgL−1 KI, 1.20 mgL−1 IBA and 1.20 mgL−1 NAA. The media and hormonal compositions selected and reported in this chapter induced the best callus initiation and development, and were selected from findings made during preliminary studies.

*A. tumefaciens* strain EHA 101 constituting vector construct pTF101.1 used for transformation of cotyledonary explants was re-initiated, centrifuged and pelleted (OD650 = 0.6–0.8) as described by Paz et al. [37]. The bacterium was resuspended in liquid infection medium prepared as indicated on **Table 2**. Prepared soybean explants were then added into the infection medium and incubated at room temperature for 15-min with gentle shaking on an orbital shaker (Orbishake-Labotec). After infection, the explants were briefly rinsed and then placed on co-cultivation medium containing B5 chemicals (major salts, minor salts, vitamins and iron source), 30 gL−1 sucrose, 3.9 gL−1 MES, 4.25 gL−1 agar, at pH 5.4. Filter sterilised 0.25 mgL−1 GA<sup>3</sup> , 400 mgL−1 cysteine, 200 mgL−1 dithiothreitol (DTT) and 50 mgL−1 acetosyringone were added into the medium after autoclaving. Co-cultivation of explants was carried-out by incubation in a culture room for 3-days at 24 ± 2°C under 50–60 μmolm−2 s−2 light intensity with 16-h photoperiod. Later, these *Agrobacterium* infected explants were cultured on callus induction medium supplemented with different concentrations of growth regulators as indicated above.

### *5.2.1. Effect of culture media, explant infection and genotypes on callus induction*

The results obtained in this study indicated that aseptically developed seedlings were necessary for callus initiation. Seed germination as measured by protrusion of the root radicle was achievable from the 3rd day of incubation. Seed cultures were maintained in the culture room for 10-days to achieve more than 75% seed germination. Variations in the percentage germination were also observed, with cultivar LS 678 recording 98% germination followed by TGx 1835-10E, LS 677, Dundee, TGx 1740-2F and Peking with 95, 84, 80, 82 and 78% germination respectively. Successful seed germination has proved essential for many *in vitro* culture developments. This initial step has been reported by El-Kaaby et al. [39] and Bahry et al. [40] as a good indicator of seed viability and therefore, a determinant of the success to be achieved in plant tissue culture. According to the prescriptions by the International Seed Testing Association (ISTA), seeds should prove to be viable by ultimately achieving more than 60% overall germination percentage. This study, including other previous studies indicated that, reduced seed viability negatively affects seedling vigour required for successful initiation of shoots and callus cultures.

Given the good and viable explants used in this study, the results obtained during callus cultures showed that, callus induction was achievable using the chosen MS medium and the different kinds of plant growth regulators (KI, IBA and NAA) used. However, the MS medium, together with the hormonal combinations resulted in significant amounts of callus cells in the controls than on explants infected with *Agrobacterium*. Variations in the amount/ size of the callus and calli phenotypes were also observed. Generally, very little callus cells were observed from cotyledonary nodes transformed with *Agrobacterium*. But, more compact, embryogenic and friable callus cells were observed from culture medium supplemented with different combinations of KI, IBA and NAA without explant co-cultivation with the bacterium. The infection of explants influenced explant survival and responsiveness in addition to the callus induction capacity as illustrated in **Table 3**. The results indicated that, MS media containing antibiotics also delayed callus initiation. This was confirmed when un-transformed explants were subcultured on a media containing antibiotics. Callus initiation was arrested for more than 3 weeks of culture. But later, swelling and traces of slightly white-yellow friable callus was observed on the explants. The few small masses of callus rapidly turned brownish to ultimately black in colour (**Figure 1**). Zhang et al. [41] made similar observations when investigating the effect of kanamycin in tissue culture and induction of somatic embryos in cotton. This direct organogenesis of callus reported reduction of callus initiation and growth on medium supplemented with 10 mgL−1 of kanamycin. The study furthermore, reported complete inhibition of callus formation in medium supplemented with 60 mgL−1 concentration of kanamycin.

on the culture media and used for establishment of callus and shoots cultures. A total of 60 double cotyledonary-node explants were prepared for each set of replicates, and the proce-

A full-strength MS culture media containing macronutrients, micronutrients and vitamins, 3% sucrose, as well as 0.25% gelrite were used in this study. The different concentrations and combinations of PGRs were added into the media from prepared stock solutions made by dissolving analytical reagent grade hormones with few drops of 1 N sodium hydroxide and making-up the final volume with distilled water. The medium used to test for the efficiency of callus initiation on double coty-nodes infected with *A. tumefaciens* containing the *oc-1* gene on pTF101.1 vector contained 0.70 mgL−1 kinetin (KI), 2.70 mgL−1 indole-3-butyric acid (IBA) and 3.20 mgL−1 α-naphthalene acetic acid (NAA). Another medium was supplemented with 0.50 mgL−1 KI, 1.20 mgL−1 IBA and 1.20 mgL−1 NAA. The media and hormonal compositions selected and reported in this chapter induced the best callus initiation and development, and

*A. tumefaciens* strain EHA 101 constituting vector construct pTF101.1 used for transformation of cotyledonary explants was re-initiated, centrifuged and pelleted (OD650 = 0.6–0.8) as described by Paz et al. [37]. The bacterium was resuspended in liquid infection medium prepared as indicated on **Table 2**. Prepared soybean explants were then added into the infection medium and incubated at room temperature for 15-min with gentle shaking on an orbital shaker (Orbishake-Labotec). After infection, the explants were briefly rinsed and then placed on co-cultivation medium containing B5 chemicals (major salts, minor salts, vitamins and iron source), 30 gL−1 sucrose, 3.9 gL−1 MES, 4.25 gL−1 agar, at pH 5.4. Filter sterilised 0.25 mgL−1 GA<sup>3</sup>

400 mgL−1 cysteine, 200 mgL−1 dithiothreitol (DTT) and 50 mgL−1 acetosyringone were added into the medium after autoclaving. Co-cultivation of explants was carried-out by incubation in a culture room for 3-days at 24 ± 2°C under 50–60 μmolm−2 s−2 light intensity with 16-h photoperiod. Later, these *Agrobacterium* infected explants were cultured on callus induction medium supplemented with different concentrations of growth regulators as indicated above.

The results obtained in this study indicated that aseptically developed seedlings were necessary for callus initiation. Seed germination as measured by protrusion of the root radicle was achievable from the 3rd day of incubation. Seed cultures were maintained in the culture room for 10-days to achieve more than 75% seed germination. Variations in the percentage germination were also observed, with cultivar LS 678 recording 98% germination followed by TGx 1835-10E, LS 677, Dundee, TGx 1740-2F and Peking with 95, 84, 80, 82 and 78% germination respectively. Successful seed germination has proved essential for many *in vitro* culture developments. This initial step has been reported by El-Kaaby et al. [39] and Bahry et al. [40] as a good indicator of seed viability and therefore, a determinant of the success to be achieved in plant tissue culture. According to the prescriptions by the International Seed Testing Association (ISTA), seeds should prove to be viable by ultimately achieving more than 60% overall germination percentage. This study, including other previous studies indicated that, reduced seed viability negatively affects seedling vigour required for successful initiation of shoots and callus cultures.

*5.2.1. Effect of culture media, explant infection and genotypes on callus induction*

,

dure was repeated four times for all transformation cultures.

24 Transgenic Crops - Emerging Trends and Future Perspectives

were selected from findings made during preliminary studies.

**5.2. Callus induction**

Paz et al. [37] reported prophylactic and proliferative effects in soybean culture using 100 mgL−1 cefotaxime and 50 mgL−1 vancomycin. Even though, Grzebelus and Skop [42] shared similar sentiments to Paz et al. [37] when investigating the effect of β-lactams antimicrobials on *in vitro* carrot protoplast culture, the formation of callus on medium amended with these antibiotics was negatively affected. They evaluated three types of β-lactams (cefotaxime, carbenicillin and timentin) at five different concentrations (100, 200, 300 or 500 mgL−1), which were higher than what was used in this study. The low levels of callus and poor explant competency observed in this study, on infected and un-infected explants, can be largely attributed to the presence of antibiotics in the medium used (**Figure 2**). However, a dual negative effect was exerted on the cultures by both *Agrobacterium* infection and media composition, particularly on the proliferation from cotyledonary-node explants. The level of explant competency on these cultures appeared profoundly negatively affected compared to nearly 100% callus induction efficiencies obtained in all genotypes for both proembryonic masses and large size formations in the controls (**Table 3**). According to **Figure 2**, the media, PGRs concentration selected and explants were successful in inducing callus as supported by Barwale et al. [43] and Franklin and Dixon [44].

The media compositions directed the development of plant cells in the cultures by essentially influencing the plasticity and totipotency of the plant tissues used as explants. This observation is in line with many studies, especially demonstrating the synergic effects of auxin (IBA, NAA) and cytokinin (KI) combination used in this study. All cultivars produced some callus on MS medium containing 0.7 mgL−1 KI, 2.7 mgL−1 IBA and 3.2 mgL−1 NAA as indicated in **Table 3**, in relation to **Table 2**. The results observed on MS-A and MS-B culture medium formulations could also be linked to the soybean cultivars used. Plant tissue swelling and initiation of calli was observed within a week of culture in more than 50% replicates of all


**Table 3.** The response of un-infected coty-node explants and *Agrobacterium tumefaciens* infected double cotyledonarynode explants on callus induction in soybean.

to the culture conditions than any other genotypes. Furthermore, MS-A induced the largest amount of callus in cultivar LS 677, LS 678, Dundee, TGx 1740-2F, TGx 1835-10E and Peking

**Figure 2.** Callus induction capacity on double cotyledonary-node explants on MS medium A (KI- 0.70 mgL-1, IBA-2.70 mgL-1 and NAA-3.20 mgL-1) and MS medium B (KI-0.50 mgL-1, IBA-1.20 mgL-1 and NAA-1.20 mgL-1). (a) callus induction on un-infected explants subcultured on MS-A (black bar) and MS-B (grey bar) containing antibiotics. (b) callus induction

The Role of Plant Genotype, Culture Medium and *Agrobacterium* on Soybean Plantlets…

http://dx.doi.org/10.5772/intechopen.78773

27

TGx 1835-10E produced the largest amount of chlorophyll callus and exhibited considerably high frequency of shoot and root organogenesis than TGx 1740-2F. Cultivar Peking on the other hand, produced clumps of callus cells on the cotyledon margins, junction and exhibited

The cotyledonary-node explants exhibited progressive chlorosis and necrosis after 3 weeks of culture for both MS medium A and B. Generally, inhibitory effects on explant swelling for formation of plant callus tissues was more pronounced on infected tissues and explants subcultured on media containing antibiotics. These results clearly demonstrated the effects that all factors examined have on callus initiation and development, as well as morphogenesis,

some browning of the cells at the bases of the hypocotyls as indicated in **Figure 3**.

on infected explants subcultured on MS-A (grey bar) and MS-B (black bar) containing antibiotics.

including potential organogenesis in *in vitro* culture of soybean.

consecutively than MS-B culture medium.

**Figure 1.** Double cotyledonary-nodes showing callus induction on infected and un-infected explants. (a, d) Failure of callus formation on a coty-node infected with *A. tumefaciens*. (b) White-yellow and brownish callus induced on MS medium containing antibiotics using un-infected explants. (c) Callus and shoot formed on un-infected explant subcultured on MS medium containing KI (0.70 mgL−1), IBA (2.70 mgL−1) and NAA (3.20 mgL−1). (e, f) Callus formation on un-infected explants subcultured on MS medium containing KI (0.50 mgL−1), IBA (1.20 mgL−1) and NAA (1.20 mgL−1).

genotypes. The induced callus culture grew into significantly large amount of callus, particularly in the controls, while maintaining their morphological potential. Callus initiation and cell multiplication for example; in soybean cultivar LS 678 and Peking responded rapidly

The Role of Plant Genotype, Culture Medium and *Agrobacterium* on Soybean Plantlets… http://dx.doi.org/10.5772/intechopen.78773 27

**Figure 2.** Callus induction capacity on double cotyledonary-node explants on MS medium A (KI- 0.70 mgL-1, IBA-2.70 mgL-1 and NAA-3.20 mgL-1) and MS medium B (KI-0.50 mgL-1, IBA-1.20 mgL-1 and NAA-1.20 mgL-1). (a) callus induction on un-infected explants subcultured on MS-A (black bar) and MS-B (grey bar) containing antibiotics. (b) callus induction on infected explants subcultured on MS-A (grey bar) and MS-B (black bar) containing antibiotics.

to the culture conditions than any other genotypes. Furthermore, MS-A induced the largest amount of callus in cultivar LS 677, LS 678, Dundee, TGx 1740-2F, TGx 1835-10E and Peking consecutively than MS-B culture medium.

TGx 1835-10E produced the largest amount of chlorophyll callus and exhibited considerably high frequency of shoot and root organogenesis than TGx 1740-2F. Cultivar Peking on the other hand, produced clumps of callus cells on the cotyledon margins, junction and exhibited some browning of the cells at the bases of the hypocotyls as indicated in **Figure 3**.

The cotyledonary-node explants exhibited progressive chlorosis and necrosis after 3 weeks of culture for both MS medium A and B. Generally, inhibitory effects on explant swelling for formation of plant callus tissues was more pronounced on infected tissues and explants subcultured on media containing antibiotics. These results clearly demonstrated the effects that all factors examined have on callus initiation and development, as well as morphogenesis, including potential organogenesis in *in vitro* culture of soybean.

genotypes. The induced callus culture grew into significantly large amount of callus, particularly in the controls, while maintaining their morphological potential. Callus initiation and cell multiplication for example; in soybean cultivar LS 678 and Peking responded rapidly

**Figure 1.** Double cotyledonary-nodes showing callus induction on infected and un-infected explants. (a, d) Failure of callus formation on a coty-node infected with *A. tumefaciens*. (b) White-yellow and brownish callus induced on MS medium containing antibiotics using un-infected explants. (c) Callus and shoot formed on un-infected explant subcultured on MS medium containing KI (0.70 mgL−1), IBA (2.70 mgL−1) and NAA (3.20 mgL−1). (e, f) Callus formation on un-infected explants subcultured on MS medium containing KI (0.50 mgL−1), IBA (1.20 mgL−1) and NAA (1.20 mgL−1).

**Table 3.** The response of un-infected coty-node explants and *Agrobacterium tumefaciens* infected double cotyledonary-

node explants on callus induction in soybean.

26 Transgenic Crops - Emerging Trends and Future Perspectives

**Figure 3.** Examples of root and shoot organogenesis and swollen nodes, on explants subcultured for callus induction using MS-B. (a) Rooting and shoot initiation occurring during callus culture. (b) Induced friable callus. (c) Swollen cotyledons. (d) Swelling on hypocotyl segments.

medium without antibiotics (**Table 4**). The suppression of shoot initiation and growth clearly appears to be instigated by the presence of antibiotics in the medium than the genotype factor. These observations confirmed the findings made in the previous section on callus induction. The initiation and proliferation of callus evidently relied upon the presence of antibiotics in the culture medium. Similar results were obtained by Yu et al. [45], when assessing the effects of carbenicillin and cefotaxime on callus and somatic embryogenesis from adventitious roots of papaya, as previously indicated. The report indicated extreme inhibitory effect of these antibiotics on callus growth, and reported abnormalities on somatic embryos generated on

**Table 4.** Comparison of the effect of MS basal culture media with or without antibiotics and *Agrobacterium* infection of double cotyledonary-node explants on average number of shoots induced per explant after 30 days of culture.

The Role of Plant Genotype, Culture Medium and *Agrobacterium* on Soybean Plantlets…

http://dx.doi.org/10.5772/intechopen.78773

29

The cotyledonary-nodes subcultured on MS basal medium with only 2.0 mgL−1 6-BA, without antibiotics induced the highest significant number of shoots within 2 weeks of culture (**Table 4**). Vigorous shoot growth, as indicated in **Figure 4a** and **b**, was obtained on this medium and the initiated microshoots reached elongation, rooting and acclimatisation stages successfully. Generally, variations in culture were observed among the basal media and infected cultures in the percentage of explants forming shoots and mean number of shoots induced per explant. Shoot induction dynamics similar to this observation were reported by El-Siddig et al. [46] and Yan et al. [47]. The transformation trials in these studies revealed great differences in the regeneration frequency, which mostly depended on the explant type and the culture conditions, particularly the culture media compositions. In this study, the results showed that morphogenetic processes are strongly influenced by the culture media, predominantly the presence of antibiotics in the medium.

The requirement of suitable explants for soybean regeneration *in vitro* is one of the main goals leading to efficacious genetic transformation. The use of double cotyledonary-nodes still shows superiority, with greater potential on the development of a simple and effective genetic manipulation protocol. These explants withstood the culture conditions used and provided a feasible mean of culture establishment. The double coty-node explants in addition, offer prolonged

culture medium supplemented with 250–500 mgL−1 carbenicillin and cefotaxime.

#### **5.3. Shoot multiplication**

The development of an efficient protocol for *Agrobacterium*-mediated genetic transformation in soybean is important for improving the genetic pool of this crop. Soybean is considered recalcitrant to genetic manipulation, and its *in vitro* based protocols are faced with many challenges. The following results summarises the influence that factors such as; the genotype, explant type, culture media and *Agrobacterium* infection have on the optimisation of efficient shoot induction during *in vitro* transformation procedures.

#### *5.3.1. Response of explants to culture medium and Agrobacterium infection*

Shoot induction was achievable on the MS media compositions used in this study. More than 60% of the coty-node explants proliferated adventitious shoots within 2-weeks of culture on MS medium supplemented with 2.0 mgL−1 6-BA. According to the results, the highest number of shoots was obtained on all MS media containing 2.0 mgL−1 of 6-BA and un-infected explants used as a control. MS medium further supplemented with cefotaxime and vancomycin (**Table 2**) also successfully induced a significant number of multiple shoots. Generally, sufficient shoot induction was achievable using cotyledonary nodes without co-cultivation with *Agrobacterium* on MS medium supplemented either with antibiotics or without antibiotics (**Table 4**). Shoot formation on transformed explants was highly reduced (**Table 4**), probably because of the combined inhibitory effects of bacterial infection and antibiotics as described during callus induction.

A small number of shoots on un-infected explants was induced on medium containing antibiotics compared to the MS medium without antibiotics. Most of these explants initiated highly reduced shoots and multiple buds without promotion to further growth. In contrast, intact and elongated multiple shoots were observed, ranging between 3 and 5 shoots on average for MS The Role of Plant Genotype, Culture Medium and *Agrobacterium* on Soybean Plantlets… http://dx.doi.org/10.5772/intechopen.78773 29


**Table 4.** Comparison of the effect of MS basal culture media with or without antibiotics and *Agrobacterium* infection of double cotyledonary-node explants on average number of shoots induced per explant after 30 days of culture.

medium without antibiotics (**Table 4**). The suppression of shoot initiation and growth clearly appears to be instigated by the presence of antibiotics in the medium than the genotype factor. These observations confirmed the findings made in the previous section on callus induction. The initiation and proliferation of callus evidently relied upon the presence of antibiotics in the culture medium. Similar results were obtained by Yu et al. [45], when assessing the effects of carbenicillin and cefotaxime on callus and somatic embryogenesis from adventitious roots of papaya, as previously indicated. The report indicated extreme inhibitory effect of these antibiotics on callus growth, and reported abnormalities on somatic embryos generated on culture medium supplemented with 250–500 mgL−1 carbenicillin and cefotaxime.

**5.3. Shoot multiplication**

cotyledons. (d) Swelling on hypocotyl segments.

28 Transgenic Crops - Emerging Trends and Future Perspectives

The development of an efficient protocol for *Agrobacterium*-mediated genetic transformation in soybean is important for improving the genetic pool of this crop. Soybean is considered recalcitrant to genetic manipulation, and its *in vitro* based protocols are faced with many challenges. The following results summarises the influence that factors such as; the genotype, explant type, culture media and *Agrobacterium* infection have on the optimisation of efficient

**Figure 3.** Examples of root and shoot organogenesis and swollen nodes, on explants subcultured for callus induction using MS-B. (a) Rooting and shoot initiation occurring during callus culture. (b) Induced friable callus. (c) Swollen

Shoot induction was achievable on the MS media compositions used in this study. More than 60% of the coty-node explants proliferated adventitious shoots within 2-weeks of culture on MS medium supplemented with 2.0 mgL−1 6-BA. According to the results, the highest number of shoots was obtained on all MS media containing 2.0 mgL−1 of 6-BA and un-infected explants used as a control. MS medium further supplemented with cefotaxime and vancomycin (**Table 2**) also successfully induced a significant number of multiple shoots. Generally, sufficient shoot induction was achievable using cotyledonary nodes without co-cultivation with *Agrobacterium* on MS medium supplemented either with antibiotics or without antibiotics (**Table 4**). Shoot formation on transformed explants was highly reduced (**Table 4**), probably because of the combined inhibitory effects of bacterial infection and antibiotics as described during callus induction.

A small number of shoots on un-infected explants was induced on medium containing antibiotics compared to the MS medium without antibiotics. Most of these explants initiated highly reduced shoots and multiple buds without promotion to further growth. In contrast, intact and elongated multiple shoots were observed, ranging between 3 and 5 shoots on average for MS

shoot induction during *in vitro* transformation procedures.

*5.3.1. Response of explants to culture medium and Agrobacterium infection*

The cotyledonary-nodes subcultured on MS basal medium with only 2.0 mgL−1 6-BA, without antibiotics induced the highest significant number of shoots within 2 weeks of culture (**Table 4**). Vigorous shoot growth, as indicated in **Figure 4a** and **b**, was obtained on this medium and the initiated microshoots reached elongation, rooting and acclimatisation stages successfully. Generally, variations in culture were observed among the basal media and infected cultures in the percentage of explants forming shoots and mean number of shoots induced per explant. Shoot induction dynamics similar to this observation were reported by El-Siddig et al. [46] and Yan et al. [47]. The transformation trials in these studies revealed great differences in the regeneration frequency, which mostly depended on the explant type and the culture conditions, particularly the culture media compositions. In this study, the results showed that morphogenetic processes are strongly influenced by the culture media, predominantly the presence of antibiotics in the medium.

The requirement of suitable explants for soybean regeneration *in vitro* is one of the main goals leading to efficacious genetic transformation. The use of double cotyledonary-nodes still shows superiority, with greater potential on the development of a simple and effective genetic manipulation protocol. These explants withstood the culture conditions used and provided a feasible mean of culture establishment. The double coty-node explants in addition, offer prolonged

**Figure 4.** Shoot induction from double-cotyledonary node explants on MS culture medium supplemented with 2.0 mgL−1 6-BA. (a, b) Shoot multiplication on un-infected coty-node explants cultured on MS medium used as control without antibiotics. (c) Callus formation and senescence from cotyledonary-nodes cultured on MS medium containing cefotaxime and vancomycin antibiotics. (d) Shoot buds initiated on explants infected with *Agrobacterium*. (e) Shoots initiated with on explants cultured on medium containing antibiotics without *Agrobacterium* infection.

support to shoots and buds initiated on the cotyledonary junction and exhibit minimal deficiency symptoms on their cotyledons. Even though, these explants show little sensitivity to culture conditions compared to the hypocotyl segments or immature cotyledons as indicated by Zhang et al. [41], their response was much better, especially subsequent *Agrobacterium* infection. The shoots and buds appeared to be well and effectively supported by these explants. Double cotyledonarynode explants produced better responses than the use of single coty-node explants. These single coty-nodes are prepared by longitudinally splitting the double coty-nodes at the cotyledonary junction into two single nodes [38]. Even if high proliferation of shoots can be achieved on other types of explants by directly stimulating pre-existing axillary meristems and induce rapid cell division because of severe wounding, tissue senescence due to bacterium overgrowth and callus formation instead of shoots still make double coty-nodes preferable than single coty-nodes.

of explants exhibiting shoot growth inhibition due to oxidative browning induced compact callus. This had negative effects on the rate of shoot and thus, the regeneration of transformed microshoots was dramatically decreased in all cultures tested for *Agrobacterium*-mediated genetic transformation. All soybean genotypes used in this study had been affected by oxidative browning, despite modifying the medium according prescriptions made in the literature. Several approaches have been reported, such as; the addition of dithiothreitol, polyvinylpyrrolidone (PVP), activated charcoal and other antioxidant mixtures like ascorbic acid. Jones and Saxena [48] report a novel approach of introducing PAL inhibitor, inhibiting the activity of phenyl-alanine ammonia lysate (PAL) enzyme. This enzyme catalyses the formation of phenolic compounds through phenylpropanoid metabolic pathway. However, shoot induction frequency of all cultivars ranged between 0.0 and 20% as indicated in **Table 5**. Only 0.31% regeneration frequency was obtained in LS 677 (**Table 5**). The detrimental effect of oxidative stress on the number of shoots induced per explants, induced shoots growth and transformation efficiency was also reported by Li et al. [49]. This problem has been correspondingly reported to have affected the efficiency of many cultures in plant tissue organogenesis and embryogenesis. For example; Hartmann et al. [50] reported failure to achieve *in vitro* plantlets

**Table 5.** The response of soybeans to modified MS culture media and infection with *Agrobacterium tumefaciens* constituting

The Role of Plant Genotype, Culture Medium and *Agrobacterium* on Soybean Plantlets…

http://dx.doi.org/10.5772/intechopen.78773

31

regeneration during mass propagation of non-transgenic plants.

a pTF101.1 vector.
