Читать книгу Biotechnology of Fruit and Nut Crops - Группа авторов - Страница 202

Conserving papaya germplasm is important for conservation, breeding and genetic manipulation. Maintenance of breeding populations is costly, and the integrity of in vitro cultures can be altered by somaclonal variation (Larkin and Scowcroft, 1981). Genetic manipulation can be accelerated with a readily available source of target tissues.

Vitrified shoot tips from in vitro-grown plants demonstrated 70% regeneration of one genotype (Ashmore et al., 2001). Shoot tips from plants of three genotypes vitrified for 20 min in 100% vitrification solution (PVS2) (Sakai et al., 1991) prior to immersion in liquid nitrogen survived. Recovery from vitrification varied with genotype, but all genotypes recovered. The shoot tips of a wild species, V. pubescens, also recovered (Ashmore et al., 2007). The effect of cryopreservation on papaya DNA was examined. Twelve genotypes were vitrified and stored, and data on six regenerated plant lines were studied. There were DNA modifications (0–10.07%) and methylation modifications (0.52–6.62%) of detected markers. Somaclonal variations may result from these changes (Kaity et al., 2008).

Cryopreservation of embryogenic cultures was used to back up gene transfer studies. Long-term storage of tissues that retain morphogenic competence without loss of integrity would facilitate availability of tissues for genetic manipulation. The best results were achieved following treatment with 30% glycerol, 15% ethylene glycol and 15% DMSO (Lu and Takagi, 2000; Dhekney et al., 2003).

Seeds desiccated to <10% moisture and stored in liquid nitrogen for 24 h remain viable after thawing to room temperature, but the rate of germination and seedling growth were reduced (Chin and Krishnapillay, 1989). Seeds were desiccated to 5–40% fresh weight (FW) prior to plunging into liquid nitrogen (Azimi et al., 2005). Desiccated but non-cryopreserved seeds germinated at 70–90%. Cryopreserved seeds with 15–100% FW germinated with optimal germination (48%) for seeds desiccated to 10% FW. Germination was only 20% for seeds desiccated to 5% FW.

Fresh seeds showed low germination (Ashmore et al., 2009). Presoaking seeds in gibberellic acid (GA₃) resulted in increased germination. Seeds desiccated to 5–30% eRH (equilibrium relative humidity: relative humidity of the enclosed air at equilibrium with the moisture content in the grain) were stored in liquid nitrogen for 3 months. Dormancy was broken with treatments of 60 min GA₃ or 24 h KNO₃ followed by 2 mM GA₃ for 30 min, or 2–3 h 0.25 M KNO₃. Germination was 61–65% for ‘Solo’ and ‘QLD 007’, respectively. In contrast, recovery from seed bank control samples stored at −20°C was low with 1–36% germination. Growth differences in the field of cryopreserved and control seedlings were not observed (Azimi et al., 2005).

Vacuum infiltration vitrification (VIV) was attempted using papaya seeds (Nadarajan and Pritchard, 2014). An optimal PVS2 concentration of 60% resulted in a tenfold reduction in PVS2 exposure time, higher embryo viability and regrowth and greater throughput of material.