Survival of sugarcane shoot tips after cryopreservation by droplet-vitrification

Nogueira, Gabriela Ferreira; Pasqual, Moacir; Scherwinski‐Pereira, Jonny Everson

doi:10.1590/s0100-204x2013001100014

Cited by 7 publications

(2 citation statements)

References 11 publications

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“…For this experiment, initial shoots from two varieties of Saccharum spp. (IAC862210 and RB855453) were established and multiplied in vitro for up to three subcultures, according to the protocol of Nogueira et al (2013).…”

Section: Methodsmentioning

confidence: 99%

An approach on the in vitro maintenance of sugarcane with views for conservation and monitoring of plant nuclear DNA contents via flow cytometry

Nogueira

Pio

Pasqual

et al. 2014

In Vitro Cell.Dev.Biol.-Plant

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In vitro conservation techniques can be utilized for germplasm maintenance. However, few reports on the in vitro conservation of sugarcane species are present in the literature. The objective of this study was to subject sugarcane plants to in vitro under minimal growth conditions and to evaluate the survival, regeneration, and the monitoring of nuclear DNA content levels of the plants. Shoots from 10 sugarcane varieties (Saccharum spp.) were introduced into two media: MC1, consisting of half-strength Murashige and Skoog (MS) salts and 3% sorbitol, or MC2, similar to the first formulation, but additionally supplemented with 3.8 μM abscisic acid (ABA). The shoots were maintained for up to 12 mo at 18°C in the presence of light. At the end of the period, the explants were inoculated onto multiplication medium containing 0.9 μM 6-benzylaminopurine (BAP) and 0.47 μM kinetin (Kin) for growth recovery. Flow cytometry analysis of shoots was verified at every 6 mo of storage. As a result, we found distinct behaviors of the varieties studied over the storage time, but in general, MC1 provided the greatest explant survival rates, with an average of approximately 80% cultures being able to recover. Once in the recovery media, the explant regrowth was fast, and the ability to multiply shoots was reestablished from the second 30-d subculture. However, by flow cytometry analysis, we observed a decrease in the estimated relative amount of DNA at 12 mo storage for most varieties examined, which was not observed when the monitoring was done at 6 mo. From these results, we conclude that sugarcane plants survived the minimal growth condition; however, maintaining the genotypes for extended periods in vitro may lead to variations in the estimated amount of nuclear DNA and, thus, be at risk of somaclonal variation.

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Section: Methodsmentioning

confidence: 99%

An approach on the in vitro maintenance of sugarcane with views for conservation and monitoring of plant nuclear DNA contents via flow cytometry

Nogueira

Pio

Pasqual

et al. 2014

In Vitro Cell.Dev.Biol.-Plant

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“…Published droplet-vitrification cryopreservation methods report the successful cryopreservation of sugarcane using PVS2 (Sakai et al, 1990) as the cryoprotectant. Nogueira et al (2013) found that 1-2 mm sugarcane shoot tips treated with a 20 min PVS2 exposure had survival levels of 20% (Nogueira et al, 2013). Kaya and Souza (2017) compared droplet-vitrification and encapsulation using three sugarcane cultivars and found that a 45-min treatment with PVS2 at 0°C resulted in regrowth levels of 63 to 76% after LN exposure.…”

Section: Introductionmentioning

confidence: 99%

Challenges in the development of a widely applicable method for sugarcane (Saccharum spp.) shoot tip cryopreservation

Volk¹,

Jenderek²,

Staats³

et al. 2019

Acta Hortic.

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The USDA-ARS National Plant Germplasm System (NPGS) maintains 946 accessions of sugarcane (Saccharum spp.) in the field at the Subtropical Horticulture Research Station in Miami, Florida. These accessions are particularly vulnerable to hurricanes, diseases, and other threats. We sought to identify a method whereby clonally propagated sugarcane accessions could be successfully introduced into tissue culture, multiplied, and then cryopreserved as shoot tips for long-term preservation at the National Laboratory for Genetic Resources Preservation in Fort Collins, Colorado. For many accessions, 70% isopropyl alcohol and 20% commercial bleach treatments, followed by three rinses of sterile water were sufficient to remove microbial contaminants during the introduction process. However, in some cases, cefotaxime was particularly effective for removing bacterial contamination. We found that antioxidant treatments of glutathione, glycine betaine, and ascorbic acid did not improve regrowth after liquid nitrogen exposure using either PVS2 or PVS3 as cryoprotectants in droplet vitrification cryopreservation methods. Exposure durations of PVS2 and PVS3 were optimized, with and without exposure to liquid nitrogen (LN), and shoot tip regrowth levels ranged from 0 to 37% after LN exposure. Published methods for encapsulation-dehydration and V-plate vitrification cryopreservation procedures were tested to determine if acceptable results could be obtained. Using these methods, shoot tip regrowth levels ranged from 0 to 50% after LN exposure. We conclude that the sugarcane cryopreservation methods that we tested are not yet ready for implementation in the NPGS.

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Long-term preservation of Lotus tenuis adventitious buds

Espasandin

Brugnoli

Ayala

et al. 2018

Plant Cell Tiss Organ Cult

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Encapsulation-dehydration, encapsulation-vitrification, and vitrification were tested for cryopreservation of Lotus tenuis (Fagaceae) adventitious buds clusters (ABCs) obtained by a direct regeneration system from leaves cultures. Among them, the PVS3-based vitrification procedure was found to be useful for survival and regrowth of the preserved explants. For vitrification, the ABCs were dehydrated in a solution containing 2 M glycerol + 0.4 M sucrose for 25 min at room temperature, submerged in PVS3 solution for 1 h at 0 °C, then immersed in liquid nitrogen for 48 h and rapidly rewarmed. Afterword, the explants were unloaded in MS liquid medium with 1.2 M sucrose for 30 min. The washed tissues were dried superficially on filter paper and cultured in semisolid hormone-free MS medium containing 0.1 M sucrose. All cultures were maintained at 25 °C in the dark for 10 days and transferred to the light conditions. With this procedure, 79 ± 5.3% survival and more than 80% of the plantlets displaying a phenotype similar to the non-treated control after acclimatization. The data settled from ISSR showed no genetic dissimilarities between in vitro regenerants derived from cryopreserved tissues and the non-treated plants. Thus, our results indicate that the use of vitrification-based PVS3 solution offers a simple, accurate, and appropriate procedure for the cryopreservation of L. tenuis adventitious buds.

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Survival of sugarcane shoot tips after cryopreservation by droplet-vitrification

Cited by 7 publications

References 11 publications

An approach on the in vitro maintenance of sugarcane with views for conservation and monitoring of plant nuclear DNA contents via flow cytometry

An approach on the in vitro maintenance of sugarcane with views for conservation and monitoring of plant nuclear DNA contents via flow cytometry

Challenges in the development of a widely applicable method for sugarcane (Saccharum spp.) shoot tip cryopreservation

Long-term preservation of Lotus tenuis adventitious buds

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