Genetic Engineering

Abstract

A serious limitation of the traditional method of sexual hybridization to improve crop plants is reproductive isolation of two species. The rapid progress in understanding the plant processes during the past four decades has led to perfection of tools and techniques that allow transfer of specific genes without disrupting the elite phenotype of the recipient plant. This highly sophisticated technique of genetic modification of plants, variously referred to as recombinant DNA (rDNA) Technology, Genetic Transformation, Genetic Engineering (GE), Molecular Breeding or Precision Breeding, is not limited by sexual reproductive isolation. Required genes from any source, bacteria, viruses, fungi, animals or completely unrelated plants, can be inserted in a functional form into the genome of a well established plant cultivar, and their expression can be regulated so that it is expressed throughout the plant or in a specific organ or specific tissue of an organ. The genetically engineered crop plants are referred to as Transgenic Crops or Genetically Modified (GM) crops, and the gene introduced is termed transgene. The transgenic technology has emerged as one of the most powerful methods for crop improvement and has already produced several improved crop varieties by introducing a completely novel trait or manipulating a known biochemical pathway. Tissue or organ specific RNAi vectors have recently proved to be useful for targeted gene silencing in specific plant tissues and organs with minimal interference with the normal plant life cycle.

Appendix

• 1. Agrobacterium-mediated leaf disc method of transformation of tobacco. (after Horsch et al. 1985).

  • (i) Immerse tobacco seeds in 10 % ‘Domestos’ bleach solution for 20–30 min and rinse three times in sterile distilled water. Place the seeds on a 64 μm nylon mesh during surface sterilization for easy handling.

  • (ii) Sow the seeds on MSB medium containing MS salts, B₅ vitamins and 3 % sucrose and gelled with 0.8 % agar (20 mL medium/9 cm Petri plate). Incubate the plates at 24–28 °C, under 16 h photoperiod with a light intensity of 48 μmol m⁻² s⁻¹ (daylight fluorescent tubes) for 7 days, when the cotyledons are fully expanded.

  • (iii) Transfer individual seedlings to magenta boxes (one per box), each containing 40 ml of culture medium as in step (ii). Incubate the culture for 3–4 weeks under the same conditions as in step (ii) until plants have developed 4–5 leaves suitable for preparing leaf discs.

  • (iv) Excise leaves from the in vitro plants or 28 days old greenhouse grown plants and place them on a sterile tile. Using a cork borer or a scalpel, excise 1 cm diameter discs and transfer them, with abaxial surface down, onto MSB medium supplemented with 1 mg L⁻¹ BAP and 0.1 mg L⁻¹ NAA (MS104; 7–8 discs per plate). Place a single filter paper over the discs to help keep them flat on the medium. Incubate under conditions as in (ii).

  • (v) Take an overnight liquid culture of Agrobacterium tumefaciens and dilute it 10 times with MSB liquid medium (2 ml of bacterium culture and 20 ml MSB medium in 9 cm Petri plates).

  • (vi) Float the discs in bacterial suspension, making sure that the wounded surface of the explants is immersed in the suspension. After 5 min, blot the explants on a sterile filter paper, and transfer the explants back to MS104 medium and incubate at 24–26 °C at a low light intensity (24–48 μmol m⁻² s⁻¹) for 2 days.

  • (vii) Transfer the explants to the selection medium (MS104 + 500 mg L⁻¹ carbenicillin and 300 mg L⁻¹ kanamycin sulfate.

  • (viii) Shoots should be visible from the wounded edges of the discs after 18–21 days of inoculation. Putatively transformed shoots should emerge from kanamycin-resistant calli. After 28 days of inoculation, regenerated shoots should be large enough (ca 1 cm in height) to be excised and transferred to rooting medium (MSB + 500 mg L⁻¹ carbenicillin + 300 mg L⁻¹ kanamycin sulfate).

• 2. Floral-dip method of transformation of radish. (after Curtis and Nam 2001).

  • (i) Sow seeds (1 seed/3 cm²) in a deep seed tray (12 cm depth containing peat-based compost and maintain in a glasshouse under natural daylight, supplemented with 16 h photoperiod with a light intensity of 61 μmol m⁻² s⁻¹ (daylight fluorescent tubes) at 26 °C (day) and 18 °C (night).

  • (ii) Transfer 3 to 4-week-old seedlings, individually, to deep pots (20 cm diameter, 30 cm depth) containing new compost to encourage plants to develop long tap roots. Maintain the plants under glasshouse conditions for 10 days to aid recovery.

  • (iii) At the six leaf stage, transfer the plants to a cold chamber set at 4 °C, 16 h photoperiod and light intensity of 45 μmol m⁻² s⁻¹ (daylight fluorescent tubes) for 10 days to promote bolting. Return the plants to the glasshouse under conditions as in step (i).

  • (iv) Plants and with single thick stems and numerous immature floral buds are ideal for the transformation treatment.

  • (v) Four days before floral dip treatment, take a loop-full of a glycerol stock of Agrobacterium strain AGL1 carrying pCAMBIA3301 and streak onto the agar (14 g L⁻¹) solidified YEP medium (10 g L⁻¹ tryptone, 10 g L⁻¹ yeast extract, 5 g L⁻¹ NaCl) containing 50 mg L⁻¹ kanamycin and 100 mg L⁻¹ rifampicin. Incubate the cultures in the dark at 28 °C for 2 days.

  • (vi) Transfer a loop-full of bacteria to a 50 mL falcon tube containing 10 mL of bacterial culture medium (YEP medium + 50 mg L⁻¹ kanamycin sulfate + 50 mg L⁻¹ rifampicin). Place the culture on an orbital shaker at 1,800 rpm and maintain in dark at 28 °C overnight.

  • (vii) Transfer 10 ml liquid bacterial culture to a 1 L flask containing 500 ml of bacterial culture medium and incubate for 12–16 h as described in step (vi) until the OD reaches 1.0 at 600 nm.

  • (viii) Pellet the bacterial culture by centrifugation at 3,500 g, for 20 min at 4 °C. Resuspend the pellet in 500 mL of inoculation medium (50 g L⁻¹ sucrose + 0.05 % v/v Silwet L-77, pH 5.2).

  • (ix) Remove any floral buds which show petal color and carefully submerge the inflorescence into the inoculation medium and gently swirl for 5 s. Cover the inflorescence with a ploythene bag.

  • (x) Remove the bag next morning and allow the plants to grow under conditions as before. Hand pollinate all flowers using a fine paint brush to aid seed-set.