In Vitro Conservation and Other Biotechnological Approaches

Synthetic Seeds

Synthetic seeds or artificial seeds in ginger were made by encapsulating in vitro-regen-erated shoot buds, somatic embryos, and calli in 5 percent sodium alginate gel. The beads were round, uniform in size, and also sufficiently strong for easy handling (see Figure 4.3c). The encapsulated synthetic seeds were stored up to 9 months when maintained aseptically in MS basal medium at 22±2°C. Such synthetic seeds germinated on MS medium supplemented with 1.0 mgl"1 BAP and 0.5 mgl"1 IBA into normal plants with 80 percent success (Sajina et al., 1997; Geetha, 2002; Swapna, 2002; and Peter et al., 2002).

Synthetic seeds form ideal source material for germplasm conservation and exchange. In addition, disease-free planting material can be moved from one place to another by using encapsulated propagules, especially in ginger, where major diseases are transmitted through infected rhizomes (see Figure 4.3c). Disease-free encapsulated shoot buds were produced in ginger by Sharma et al. (1994).

In Vitro Conservation and Cryopreservation of Germplasm

As in vitro techniques are becoming more important in crop improvement through somatic cell genetics, genetic stocks are assuming more variable forms from in vitro plantlets to protoplasts and DNA (Withers, 1985). With due precautions, the genotypes of plants propagated by node or shoot culture can be preserved without change. This type of in vitro culture can therefore be used to maintain genotypes over long periods. Fortunately, several ways have been found to reduce the rate of growth of cultured material so that it can be kept unattended for moderate lengths of time.

In Vitro Conservation

Modifying the constituents of culture medium by decreasing the carbohydrate/nutrient supply, changing the osmotic potential using the combinations of sucrose and mannitol and withdrawal of growth regulators from the culture medium, storage in reduced light, desiccation combined with cold treatment, reduced oxygen tension, and the use of growth regulators such as absisic acid induces slow growth in many crop species.

At the IISR, ginger plantlets could be successfully conserved for extended periods of over 12 months on half-strength MS basal medium supplemented with 15 gl-1 each of sucrose and mannitol. The cultures were sealed with aluminum foil and maintained at a temperature of 22±2°C. Thus, in ginger, minimal growth was induced by minimizing the evaporation loss using aluminum foil to seal the culture vessel, reduction of both the carbon source and the nutrients to half strength, and addition of mannitol. Ginger cultures grew well at 22±2°C but deteriorated when kept under lower temperatures of 5°C and 10°C. The rate of growth was higher when full-strength MS medium was used. High concentration of sucrose (30 gl-1) increased culture growth substantially, resulting in exhaustion of culture medium. When the concentration of sucrose was reduced to 20 gl-1 and nutrient concentration to half, the cultures could be maintained for a much longer period of 200 to 240 days with a survival percentage of 75 to 81 depending upon the closure used. Addition of mannitol (10 to 15 gl-1) and reduction of sucrose to lower levels (15 to 10 gl-1) induced slow growth, and subsequently 73 to 80 percent of the cultures could be maintained for a period of 360 days when the culture vessels were closed with aluminum foil (see Figure 4.3a and 4.3b). Full- or half-strength MS medium supplemented with 10 or 15 gl-1 each of sucrose and mannitol and 1/2 MS with 20 gl-1 sucrose and 10 gl-1 mannitol allowed the cultures to be maintained for 360 days (see Table 4.9) (Nirmal Babu, 1997; Nirmal Babu et al., 1999, 2000; Geetha, 2002; and Peter et al., 2002).

According to Balachandran et al. (1990), ginger cultures could be maintained up to 7 months without subculture by using polypropylene caps as culture vessel enclosures. Dekkers et al. (1991) reported that ginger shoots could be maintained for over 1 year at ambient temperatures (24 to 29°C) in a medium containing mannitol (25 gL-1) with an overlay of mineral oil.

At present, over 100 core collections of ginger are maintained at the IISR in vitro gene bank with yearly subculture. The small-sized plantlets kept in the conservation medium for over 5 years with yearly subculture when transferred to the multiplication medium (MS + 30 gl-1 sucrose and 1 mgl-1 NAA) gave normal growth with good multiplication rate. These plantlets were established easily with >80 percent success and developed into normal plants similar to the mother plants. Thus, the in vitro conservation technique is a safe alternative for a vegetativley propagated crop such as ginger for conservation and exchange of disease-free planting material.

Cryopreservation

Cryopreservation offers a better alternative when the base germplasm of any crop can be preserved for long durations with minimum effort. However, protocols for viable cryopreservation and post-thaw recovery of cryopreserved material are not available for ginger.

Geetha (2002) and Peter et al. (2002) reported that ginger in vitro-derived shoot tips could be successfully cryopreserved with limited success by pretreating the shoot tips with 0.75 M sucrose, desiccating for 1 hour, and plunging into liquid nitrogen (-185° C). Only 20 percent of the cultures developed into plantlets after cryopreservation. They also reported that encapsulated shoot buds of ginger (Synseeds) could be successfully cryopreserved after preculture on 0.75 M sucrose for 3 days and desiccated for 4 hours on laminar airflow. Plantlets could be regenerated from 20 percent of the cultures after cryopreservation.

Table 4.9 Effect of media components and culture vessel closures on induction of minimal growth in ginger cultures

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