In Vitro Techniques for Shipping of Micropropagated Plant Materials

Abstract

Shipping of in vitro micro-cuttings in tubes or jars is a frequently used method as the plants are more likely to quickly reproduce and comply with quarantine regulations in plant germplasm distribution. However, these containers are fragile during transportation. To diminish the risk associated with the long-distance shipping of in vitro plants, a safe and widely applicable packing and conservation technique based on microplate and slow growth was developed in this study. Potato cultivar ZHB and ginger cultivar G-2 were used to optimize the system with microplates (96 wells), vacuum-sealed packaging, and slow-growth techniques. Under regular culture conditions, packing in vacuum-sealed microplates reduced the survival of ZHB and G-2 micro-cuttings to 85.8% and 20.0%, respectively, and regeneration to 61.8% and 0%, respectively. Reducing the temperature to 10 °C maintained the survival of ZHB and G-2 micro-cuttings in the range of 83.3–100% after 60 days. Exposure to darkness decreased the survival of G-2 and inhibited regrowth. Thus, conservation in darkness at 10 °C is suggested. The effects of iron concentration and plant growth retardants were further assessed. The addition of 1/4 MS medium combined with 100 mg/L chlormequat chloride (CCC) resulted in full survival and growth inhibition of plantlets, without malformation identified. Finally, incubation with 1/4 MS medium supplemented with 100 mg/L CCC in vacuum-sealed microplates at 10 °C in the dark resulted in high survival and suppressed germination. Sweet potato HXS was incubated as well to test the broad-spectrum applications of the technique; 100% survival and 6.7% germination was gained. Morphological indices of released cuttings recovered to control levels after two cycles of subculture in MS medium. A 0.1–0.2% genetic variation was detected by SSR and ISSR, suggesting genetic stability of the conserved samples. Finally, micro-cuttings were safely transported to cities located thousands of kilometers away without package and sample damage. Our results enable easy distribution of in vitro plant germplasms.

Response of Rowan Berry (Sorbus redliana) Shoot Culture to Slow Growth Storage Conditions

“…In general, woody plants of the temperate zone tolerate low storage temperatures well because they can go dormant to survive the winter [ 39 ]. Dark conditions can enhance the stability of in vitro culture via the inhibition of development of new tissues, as was observed during a 60-day storage of potato and ginger cultures [ 18 ]. Darkness can also enhance the vigor of shoots when stored up to 12 weeks, but in a longer storage period, illumination was found to significantly increase the regrowth ability of chokecherry shoots ( Prunus virginiana ) [ 40 ].…”

“…Those species that are propagated vegetatively because they do not produce seeds or those with offspring whose genetic identity cannot be ensured with seeds may be best suited to storage with biotechnological methods, including tissue cultures used for micropropagation, slow growth methods, or cryopreservation [ 14 , 15 , 16 ]. These methods are particularly suitable for the conservation of genetic resources of the forest trees and horticultural plants [ 17 ], and the pathogen-free in vitro cultures can also be used effectively for the distribution of germplasm [ 18 ].…”