tissue culture

Jose Faria, Lisa Hill, Christina Walters, Tree Seed Laboratory, Federal University of Lavras, Brazil, USDA-ARS, National Center for Genetic Resources Preservation, United States

Quercus imbricaria is included in the red oak group (Lobatae) and is broadly distributed in the Midwestern US. The embryonic axes are about 1 mg dry mass and have 0.68 g H2O/g dw in the acorn. Their small size and considerable desiccation tolerance made them ideal to compare various approaches for cryopreservation. Drying over a stream of nitrogen gas for 120 min reduced axis water content to 0.15 g/g, with 55% survival following liquid nitrogen (LN) exposure. To test whether addition of cryoprotectants could improve survival, axes were bathed in Plant Vitrification Solution #2 (PVS2) for 5-60 min and tested for viability before and after LN exposure. Axes submitted to PVS2 treatments and exposed or not to LN attained survival of 100% (or close to) after two weeks of tissue culture. Differential scanning calorimetry (DSC) was used to infer water freezing and melting patterns. In axes dried without PVS2, water melted at about 178 J/g H2O and melting transitions were not observed in axes dried to less than 0.34 g/g when standard methods of 10oC/min scanning rates were used. Using similar DSC methods, the water melting signal was no longer apparent in axes soaked in PVS2 for 45 minutes. To evaluate water melting behavior during fast cooling and warming, axes were plunged into LN and scanned at 300oC/min, revealing broad endothermic events between -120 to -40oC in axes that received no cryoprotectant, which we interpreted as crystal growth during warming. The enthalpy for melting transitions was reduced to about 70 J/g H2O, but there was no effect on the water content at which the melting signal was not detected. From our collective results, we suggest that PVS2 alters the rate of lethal ice crystal formation during rapid cooling and warming allowing for greater survival of axes exposed to LN.

Date Recorded: 
Thursday, July 25, 2019

Josette Tin, Taylor La Val, Sean Lahmeyer, John Trager, Raquel Folgado*, The Huntington Library, Art Collections and Botanical Gardens, San Marino, CA., United States, *Speaker

Succulent plants are significant to the horticultural industry, and they are also sources for food, fibers, medicines, and cosmetics. The main threats for the wild population of these often emblematic plants are human activities, such as over-collection in the wild. The Huntington Desert Garden holds one of the largest ex-situ collections of succulent plants. Besides the traditional propagation methods for the field collections and the cryopreservation of seeds, in vitro repositories have been created to assure the preservation of the clonal type plants, which often have historic and botanical value. Experiments of droplet-vitrification based techniques have been used to cryopreserve clonal accessions of aloes and agaves. Apical shoot tips of 1 mm size from 5-week- old in vitro plantlets (aloe or agave) were exposed to loading solution for 20 min at room temperature, dehydrated with Plant Vitrification Solution 2 (PVS2) for different times (from 0 to 90 min) at 0 °C, transferred to aluminum foil strips and directly plunged into liquid nitrogen. For re-warming, aluminum strips were rinsed in unloading solution for 20 min at room temperature. Explants were transferred to regeneration media and kept in the dark for one week. In additional experiments, shoot-tips excised from donor plants pretreated onto a sucrose- enriched medium for two weeks were also submitted to cryoprocedure. The pretreatment with sucrose- supplemented medium improved the regeneration of both aloe and agave cryopreserved explants. The optimized protocols that have been developed for Aloe fievetii and Agave sobria spp frailensis (70 % and 90 % of plants recovered after cryopreservation, respectively) are being tested for other Aloe and Agave species. Regenerated plants were acclimated to ex vitro conditions.

Date Recorded: 
Thursday, July 25, 2019

Maria Teresa Gonzalez-Arnao, Pedro Isidro-Adolfo, Maria F. NetzahualcoyotlMata, Lourdes G. Iglesias Andreu, Jaime Martínez-Castillo, University of Veracruz, INBIOTECA, CICY, 

In this study we used six selected ISSR primers (T05, T06, C07, UBC823, UBC825, UBC848) to evaluate the genetic stability during cryopreservation of vanilla (V. planifolia) shoot-tips subjected to a Droplet-Vitrification (D-V) protocol using PVS2 solution for dehydration. We studied the impact of tissues culture and cryopreservation, assessing the effect of multiplication cycles of donor plantlets, with few (less than 4) and multiples (more than 12) subcultures, from which were isolated the shoot-tips to be cryopreserved. It was also assessed the effect of treatments, which allowed growth recovery after immersion in liquid nitrogen. To apply D-V procedure, shoot-tips were dissected of motherplantlets with few or multiple subcultures, and then preconditioned for 1 day on MS semisolid medium supplemented with 0.3M trehalose, loaded in a solution of 0.4M sucrose + 2M Glycerol and exposed to PVS2 solution for 30 min at room temperature prior to the ultra-rapid cooling in liquid nitrogen placed on droplets of PVS solution over aluminium cryoplates. The analysis of electrophoretic profiles of in vivo plants and in vitro plantlets with different subculture numbers revealed a total of 153 bands and 21.2% of polymorphism. UPGMA dendrogram based on Nei’s genetic distance demonstrated that plants with multiple subcultures induced the greatest genetic variation but kept a high (0.90) similarity index. The electrophoretic profiles of all successive steps of D-V protocol revealed a total of 199 bands and 34.2% polymorphism. Treatment with PVS2 promoted the greatest variation and continued declining the level (0.816) of similarity compared to that of the plantlets with multiples subcultures. Plantlets regenerated after cryopreservation kept the same level (0.819) of those treated with PVS2, indicating that liquid nitrogen immersion did not induce a further genetic variation. Our studies revealed that both, subculturing number and the composition of vitrification solution influenced genetic variability.

Date Recorded: 
Tuesday, July 23, 2019

Gayle Volk, USDA ARS National Laboratory for Genetic Resources Preservation, United States

The USDA-ARS National Plant Germplasm System has over 30,000 clonally maintained accessions within its field, screenhouse, greenhouse, and tissue culture collections. These fruit, nut, tuber, and bulb crop collections are usually not duplicated at secondary locations and are vulnerable to bioticabiotic, and climatic threats. Only about 15% of the clonally maintained accessions are currently secured in long-term storage at the National Laboratory for Genetic Resources Preservation (NLGRP) in Fort Collins, Colorado. The labor required to cryopreserve the clonal collections at NLGRP exceeds that which is available, even when reliable, robust cryopreservation methods are available. We have sought to prioritize collection materials for cryopreservation and to identify methods that improve the efficiency of the shoot-tip cryopreservation procedure. In particular, we have used field-, screenhouse-, and growth-chamber harvested plant tissue as source material for shoot tip cryopreservation, rather than relying on in vitro grown cultures. This strategy has been particularly effective for garlic, citrus, and grape cryopreservation efforts. In addition, incorporation of antioxidants and shoot tip micrografting methods have made cryopreservation protocols widely applicable to diverse genetic resources for each crop.

Contributing Author(s): 
Date Recorded: 
Tuesday, July 23, 2019

Valerie Pence and Megan Philpott, Cincinnati Zoo & Botanical Garden

Crotalaria avonensis is a Florida endemic found in three populations and characterized by low seed production. In the late 1990s, CREW developed protocols for tissue culture propagation from field collected shoot cuttings as well as cryopreservation methods. In order to develop a genetically representative collection for conservation, in vitro lines were established from shoots collected at all three populations from 2008-2012. Plants were produced and sent to Bok Tower Garden for further growth and for use in an outplanting by Archbold Biological Station. The resulting collection of genotypes in culture at CREW provides an example of the challenges of a genetically diverse collection of an exceptional species. C. avonensis cultures require maintenance subculturing every 2-3 months. Only a low number of replicates could be maintained for each genotype, resulting in some loss of genotypes over time. Cryopreservation offered a solution to this challenge and over the course of 16 years, a number of lines were cryopreserved. In a study of lines stored for 5.5 Ð 16 years in liquid nitrogen, there was no change in average viability of the collection in storage, although specific survival differed by genotype. A cost estimation indicated that cryopreservation could decrease the cost of maintaining the collection over 20 years by at least 1/3. Genetic analysis of the collection and the wild populations is also underway in order to determine the genetic representation of the collection.

Contributing Author(s): 
Date Recorded: 
Friday, May 3, 2019

Joyce Maschinski, Center for Plant Conservation and Valerie Pence, Cincinnati Zoo & Botanical Garden

Crotalaria avonensis is an endangered legume endemic to Florida that produces few seeds. In vitro shoot cultures of multiple genotypes have been grown at CREW to provide genetic diversity for restoration and for tissue cryopreservation. These cultures harbor a bacterium, identified as Paenibacillus sp., which may be a natural endophyte in the species. The bacterium grows slowly and does not appear to inhibit the in vitro propagation of the species, but its effect on the recovery of shoot tips after the stress of cryopreservation was investigated. Samples banked using encapsulation vitrification and representing 63 genotypes were evaluated after 4 - 15 years in liquid nitrogen. The rate of recovery growth of samples with visible bacteria was significantly less than samples without bacteria. Similarly, when newly banked shoot tips of 15 genotypes were cryopreserved using an improved technique, droplet vitrification, and were recovered, the presence of antibiotic in the medium significantly increased the percent of shoot tips showing recovery growth. Whereas C. avonensis shoots can be propagated, rooted, and acclimatized in the presence of this bacterium, recovery after the stress of cryopreservation is reduced when the bacteria are present. An increasing number of plant species are being shown to have endophytes in the wild and removing such endophytes may not be possible or desirable in culture. These results with C. avonensis demonstrate the potential for controlling the negative effects of such microorganisms in vitro. This is one example of the particular challenges that may be presented in working with wild species and conserving endangered exceptional plants. Supported in part by grants from the Institute of Museum and Library Services.

Contributing Author(s): 
Date Recorded: 
Friday, May 4, 2018

Although many rare plants have seeds that can be stored by conventional methods, not all species have seeds that can live after drying or freezing. Sometimes called "Exceptional plants, " these species have a wide range of variation. Some produce few or no seeds, thus they cannot be seed banked, others have seeds or spores that will die if dried or frozen, while others have seeds that can tolerate drying, but not freezing, and another group have seeds that live less than 10 years at freezing temperatures. Recent studies suggest that there are many rare plant species that need alternative storage for conservation. This video describes a procedure for  testing whether rare plants can be stored in liquid nitrogen successfully.

Date Recorded: 
Friday, March 1, 2019

Peter Zale and Matt Taylor, Longwood Gardens

Several species of Spiranthes native to the Eastern U.S. are considered rare, threatened or endangered by federal and state agencies. Using the Pennsylvania endangered Spiranthes casei as a model species, experiments were designed to determine optimal conditions for in vitro seed germination and seedling development. Seeds were collected in November 2015 from 10 individual plants found in three subpopulations in Elk and McKean counties, Pennsylvania, and air-dried for six weeks. Seeds were surface sterilized for 10 or three minutes in a 10% bleach solution, then plated onto a commercially available terrestrial orchid seed germination media: P723, M551 or K400 (Phytotechnology Labs, Shawnee Mission, KS) with 5 replicate plates. Seed germination ranged from 24 to 60 % and occurred on all three media only with the 3-minute treatment. None of the seeds treated with bleach for 10-minutes germinated and visual inspection revealed badly damaged embryos. After shoot initiation, 150 seedlings were transferred to individual test tubes on one of two media (P723 or P658) and each was given one of the three 24-hour light/dark photoperiod treatments for 10 months: 24/0, 18/6, or 0/24. Seedling survival and growth occurred in all treatments, but seedlings on P723 with the 24/0 or 16/8 photoperiod treatments had a significantly greater fresh weight, leaf length, number of roots and root length than light treatments on P658 and dark treatments. Results indicate Spiranthes seeds can be damaged by extended chemical scarification times and the light is essential for optimal seedling growth.

Contributing Author(s): 
Date Recorded: 
Friday, May 4, 2018