Methods to Transfer Foreign Genes to Plants

Genome sequencing of several organisms has resulted in the rapid progress of genomic studies. Genetic transformation is a powerful tool and an important technique for the study of plant functional genomics, i.e., gene discovery, new insights into gene function, and investigation of genetically controlled characteristics. In addition, the function of genes isolated using map-based cloning of mutant alleles has been confirmed by functional complementation using genetic transformation. Furthermore, genetic transformation enables the introduction of foreign genes into crop plants, expeditiously creating new genetically modified organisms. Gene transformation and genetic engineering contribute to an overall increase in crop productivity (Sinclair et al., 2004).


Introduction
Genome sequencing of several organisms has resulted in the rapid progress of genomic studies. Genetic transformation is a powerful tool and an important technique for the study of plant functional genomics, i.e., gene discovery, new insights into gene function, and investigation of genetically controlled characteristics. In addition, the function of genes isolated using map-based cloning of mutant alleles has been confirmed by functional complementation using genetic transformation. Furthermore, genetic transformation enables the introduction of foreign genes into crop plants, expeditiously creating new genetically modified organisms. Gene transformation and genetic engineering contribute to an overall increase in crop productivity (Sinclair et al., 2004).
This review outlines general methods for plant transformation and focuses on the development of the Arabidopsis transformation system.

Gene transformation
Several gene transformation techniques utilize DNA uptake into isolated protoplasts mediated by chemical procedures, electroporation, or the use of high-velocity particles (particle bombardment). Direct DNA uptake is useful for both stable transformation and transient gene expression. However, the frequency of stable transformation is low, and it takes a long time to regenerate whole transgenic plants.

Chemical procedures
Plant protoplasts treated with polyethylene glycol more readily take up DNA from their surrounding medium, and this DNA can be stably integrated into the plant's chromosomal DNA (Mathur & Koncz, 1997). Protoplasts are then cultured under conditions that allowed them to grow cell walls, start dividing to form a callus, develop shoots and roots, and regenerate whole plants.

Electroporation
Plant cell electroporation generally utilizes the protoplast because thick plant cell walls restrict macromolecule movement (Bates, 1999). Electrical pulses are applied to a suspension of protoplasts with DNA placed between electrodes in an electroporation cuvette. Short high-voltage electrical pulses induce the formation of transient micropores in cell membranes allowing DNA to enter the cell and then the nucleus. Fig. 1. Plant transformation process using particle bombardment includes the following steps: (1) Isolate protoplasts from leaf tissues. (2) Inject DNA-coated particles into the protoplasts using particle gun. (3) Regenerate into whole plants. (4) Acclimate the transgenic plants in a greenhouse.

Particle (microprojectile) bombardment
Particle bombardment is a technique used to introduce foreign DNA into plant cells (Birch & Franks, 1991;Christou, 1992Christou, , 1995Gan, 1989;Takeuchi et al., 1992;Yao et al., 2006) (Figure  1 ) . G o l d o r t u n g s t e n p a r t i c l e s ( 1 -2 µ m ) a r e c o a t e d w i t h t h e D N A t o b e u s e d f o r transformation. The coated particles are loaded into a particle gun and accelerated to high speed either by the electrostatic energy released from a droplet of water exposed to high voltage or using pressurized helium gas; the target could be plant cell suspensions, callus cultures, or tissues. The projectiles penetrate the plant cell walls and membranes. As the microprojectiles enter the cells, transgenes are released from the particle surface for subsequent incorporation into the plant's chromosomal DNA.

Transforming Arabidopsis thaliana
Arabidopsis thaliana, a small flowering plant, is a model organism widely used in plant molecular biology. The first in planta transformation of Arabidopsis included the use of tissue culture and plant regeneration (Feldmann & Marks, 1987). The Agrobacterium vacuum (Bechtold et al., 1993) and floral dipping (Clough & Bent, 1998) are efficient methods to generate transgenic plants. They allow for plant transformation without the need for tissue culture. The floral dipping method markedly advanced the ease of creating Arabidopsis transformants, and it is the most widely used transformation method. These methods were later simplified and substantially improved (Davis et al., 2009;Zhang et al., 2006), significantly reduced the required labor, cost, and time, as compared with earlier procedures.
However, these transformation methods have some problems. The floral dipping method involves dipping Arabidopsis flower buds into an Agrobacterium cell suspension, requiring large volumes of bacterial culture grown in liquid media. The large shakers and centrifuges, necessary to house the media, require sufficient experimental space. These factors limit transformation quantities. Here, we describe an improved method for Agrobacteriummediated transformation that does not require the large volumes of liquid culture necessary for floral dipping.

Improved method for Agrobacterium-mediated transformation
A. thaliana can be stably transformed with high efficiency using T-DNA transfer by Agrobacterium tumefaciens. Agrobacterium-mediated transformation using the floral dipping method is the most widely used method for transforming Arabidopsis. We have showed that A. thaliana can be transformed by inoculating flower buds with 5 µl of Agrobacterium cell suspension, thus avoiding the use of large volumes of Agrobacterium culture (Narusaka et al., 2010). Using this floral inoculating method, we obtained 15-50 transgenic plants per three transformed A. thaliana plants. The floral inoculating method can be satisfactorily used in subsequent analyses. This simplified method, without floral dipping, offers an equally efficient transformation as previously reported methods. This method reduces overall labor, cost, time, and space. Another important aspect of this modified method is that it allows many independent transformations to be performed at once.

Agrobacterium strains
The Agrobacterium strain GV3101 (C58 derivative) is frequently used to transform many binary vectors, e.g., pBI121, pGPTV, pCB301, pCAMBIA, and pGreen, into Arabidopsis. It carries rifampicin resistance (10 mg l -1 ) on the chromosome (Koncz & Schell, 1986). On the other hand, LBA4404 is a popular strain for tobacco transformation but is less effective for Arabidopsis.

Reagents
Eppendorf tube (1.5 ml) 1. Pellet 1.5 ml of overnight-grown Agrobacterium (GV3101) cells by centrifugation in an Eppendorf tube at 14,000 rpm for 1 min at 4°C. 2. Resuspend in 1 ml of ice-cold 20 mM CaCl 2 . 3. Recentrifuge at 14,000 rpm for 1 min at 4°C. 4. Resuspend in 200 µl of ice-cold 20 mM CaCl 2 . 5. Add binary vector DNA (500 ng or 5-10 µl from an alkaline lysis miniprep) to the suspension. Mix by pipetting. 6. Freeze the Eppendorf tube in liquid nitrogen for 5 min and thaw at 37°C in a water bath for 5 min. Repeat two times. 7. Cool on ice. 8. Add 1 ml LB liquid medium to the Eppendorf tube and incubate at 28°C for 2-5 hrs with gentle agitation (150 rpm; water bath). 9. Spread 50-200 µl of the cells onto LB agar medium containing appropriate antibiotics and incubate at 28°C for two days.

Selecting transformed Agrobacterium using polymerase chain reaction (PCR)
This method is designed to quickly screen for plasmid inserts directly from Agrobacterium colonies. Alternatively, the insert presence can be determined by DNA sequencing.

Simplified Arabidopsis transformation: Floral inoculating method
Until now, a limited number of constructs could be transformed into Arabidopsis because of difficulty growing large volumes of Agrobacterium. Therefore, we focused on improvements to the floral dipping method (Figure 3) (Narusaka et al., 2010). The problem of space and volume can be solved by using a small culture volume. Each plant is transformed using only 30-50 µl of bacteria grown in 2 ml of liquid culture. Our present method, as described below, is a simple modification of the method reported by Clough & Bent (1998). Recent papers (Liu et al., 2008;Zhang et al., 2006) illustrate the floral dipping process. Clough and Bent (1998) reported that neither Murashige and Skoog (MS) salts and hormones nor optical density (OD) makes a difference in transformation efficiency. An Agrobacterium cell suspension containing 0.01-0.05% Silwet L-77 (vol/vol) was used in the uptake of Agrobacterium into female gametes, instead of vacuum-aided infiltration of inflorescences.  (Zhang et al., 2006). We generally use the quick procedure, which is useful for rare seeds and seeds with low germination frequency. It is also used to retransform a transgenic line with a second construct.  1.a. Standard procedure (A): Suspend seeds in 0.1% (wt/vol) agar solution and keep in darkness for 2-4 days at 4°C to break dormancy. Spread seeds on wet soil (a mixture of peat moss and expanded vermiculite granules at a 1:2 ratio) in a 3-inch pot and grow under long-day conditions (16-hr light/8-hr dark) at 22°C. Thin to three seedlings per pot. Do not cover with a bridal veil, window screen, or cheesecloth. 1.b. Quick procedure (B): Sterilize seeds by treatment with 70% (vol/vol) ethanol for 1 min then immerse in sodium hypochlorite solution containing 1% available chlorine and 0.02% (vol/vol) Tween 20 for 7 min. Wash seeds five times with sterile distilled water. Place seeds on MS medium containing 0.8% (wt/vol) Bacto agar. Keep seeds in darkness for 2-4 days at 4°C to break dormancy. Grow under long-day conditions (16hr light/8-hr dark) for 3 weeks at 22°C. Transfer to pots per Step 1a. Do not cover with a bridal veil, window screen, or cheesecloth. 2. Clip primary bolts to encourage proliferation of secondary bolts ( Figure 4A). Plants will be ready approximately 4-6 days after clipping. 3. Prepare the Agrobacterium strain carrying the gene of interest. Spread a single Agrobacterium colony on an LB agar plate with suitable antibiotics. Incubate the culture at 28°C for two days. 4. Use feeder culture to inoculate a 2-ml liquid culture in LB with suitable antibiotics to select for the binary plasmid in a 15-ml Conical tube at 28°C for 16-24 hrs. Mid-log cells or a freshly saturated culture (Clough and Bent 1998) Figure 4E). Avoid excessive exposure to light. (Optional: For higher rates of transformation, inoculate newly forming flower buds with Agrobacterium 2-3 times at 7day intervals.) 8. Water and grow plants normally, tying up loose bolts with wax paper, tape, stakes, twist-ties, or other means. Stop watering as seeds become mature ( Figure 4F). 9. Harvest dry seeds. Though transformants are usually independent, independence can be guaranteed if seeds come from separate plants. 10. Surface-sterilize seeds by immersion in 70% (vol/vol) ethanol for 1 min, followed by immersion in sodium hypochlorite solution containing 1% available chlorine and 0.02% (vol/vol) Tween 20 for 10 min. Then, wash seeds five times with sterile distilled water.
11. Transplant putative transformants to soil per Step 1a. Grow, test, and use.

Screening of transgenic plants by PCR
Transgenes can be detected by plant genome DNA analysis with PCR ( Figure 5). Although transgenes can be distinguished from their surrounding host plant genome, their presence should be determined by DNA sequencing.
PCR-based transgene detection is a simple and highly sensitive process. Subsequent PCR tests are assessed by agarose gel electrophoresis, and results are visualized by the presence or absence of the appropriately sized DNA fragment. If PCR shows a positive result, the transgene may be present. Transgene presence is confirmed by incorporating it into the genome by DNA sequencing. In contrast, a negative PCR result implies that the transgene is not present.

Simplified DNA isolation method
A small plant leaf disc (3-4 mm diameter) can b e d i r e c t l y u s e d a s a P C R t e m p l a t e . Arabidopsis, tomato, Chinese cabbage, Komatsuna (Brassica rapa), and tobacco leaf discs are good template candidates.

Conclusion
The floral inoculating method resulted in 15-50 transgenic plants per three transformed A. thaliana plants (Table 1). The method can be satisfactorily used for subsequent analyses. This simplified method does not utilize plant inversion or floral dipping, which requires large volumes of Agrobacterium culture. It offers equally efficient transformation as previously reported methods with the added benefit of reduced labor, cost, time, and space. Of further importance, this modified method allows many independent transformations to be performed at once.