Hop Powdery Mildew (Podosphaera macularis) Spore Cryopreservation.

BACKGROUND: Hop powdery mildew (HPM), Podosphaera macularis, an important disease organism for hops, is an obligate parasite, requiring constant culture on living plant tissue for strain maintenance. OBJECTIVE: This study determined the parameters required to successfully cryopreserve HPM spores for the first time and reduce the need for constant culture. MATERIALS AND METHODS: Spores of an Oregon HPM strain, OSU C-100 were desiccated over silica gel for 2-10 h to determine the spore moisture content (MC). Regrowth of the hyphae before and after drying and liquid nitrogen exposure was determined on glass slides and leaf discs of several susceptible hop cultivars. A second mixture of strains was later tested with the protocol. RESULTS: Desiccation to an optimal 2-3 percent MC produced hyphal growth on slides and infection of leaf discs. The OSU C-100 HPM spore strain required 8-10 h desiccation to reach 2-3 percent MC while the mixed strains required 6-8 h due to slightly different MC when collected. CONCLUSION: HPM strains should be placed in cryovials, dried to 2-3 percent MC over silica gel, cryopreserved by direct immersion in liquid nitrogen. They can be rewarmed for 1 min each in 45C and 20C water and the viability tested on isolated leaf discs.

Growth medium alterations improve in vitro cold storage of pear germplasm.

BACKGROUND: Development of new fruit cultivars is dependent on genetic resource collections such as those at the Pomological Garden of the Institute of Horticulture and Viticulture near Almaty, Kazakhstan. The pear germplasm collection of the Pomological Garden contains 615 cultivars and three species. In vitro cold storage of the collection would provide additional security to the field collection. OBJECTIVE: This study was designed to improve medium-term in vitro storage of pear germplasm. METHODS: Shoots of seven pear cultivars (Pyrus communis L.) were stored in plastic five-section bags at 4 degree C and a 10-h photoperiod (7 µmol/m2/s). Treatments included medium with four carbohydrate sources (3% sucrose, 2% or 3% mannitol, or 2% sucrose + 2% mannitol) with 0.5 mg/l BAP and 0.1 mg/l IBA or without plant growth regulators (PGRs) and at three Murashige and Skoog (MS) nitrogen concentrations (100%, 50% or 25%). RESULTS: Pear shoots remained viable for 9 to 15 months without repropagation on the control MS medium with 3% sucrose without PGRs. There were significant impacts of cultivar and treatment on the duration of cold storage. Shoots of 'Mramornaya' remained viable (rating of ≥ 2) for 27 months with PGRs and 2% sucrose + 2% mannitol compared to 12 months for the PGR + 3% sucrose treatment. Talgarskaya Krasaviza stored for 18 months on 2% sucrose + 2% mannitol while all other treatments lasted only 6 to 9 months. Treatments with 0.5 or 1 mg/l abscisic acid (ABA) with 3% sucrose increased storage duration as did reducing the concentration of nitrogen in the medium to 25% without PGRs and with 3% sucrose.

Implementing cryogenic storage of clonally propagated plants.

Methodology for plant germplasm cryopreservation was created in the 1970s, expanded in the 1980s, and implemented at the end of the 20th century. Translating experimental techniques into routine cryostorage of a clonal collection requires attention to details beyond those normally required for methods development. Early decisions include the choice of accessions to be stored, number of each accession per storage unit, number of replicates, location of storage, viability testing, record keeping and proper control groups. Emphasis should be placed on selecting a secure storage site and compiling complete records needed for the recovery of plant material. Secure remote storage, duplicate locations, and secure, accurate records are all important in ensuring the safety and usefulness of base collections. Evaluation of cryostored collections should be initiated to determine the longevity of plants and stability of storage conditions. Collections of several clonal genera are now stored in liquid nitrogen and more are in progress worldwide. These base-storage collections of clonal germplasm provide security for safeguarding long-term access to genetic diversity that is vital to food security and to continued improvement of many clonally-propagated agricultural crops.

Extended alternating-temperature cold acclimation and culture duration improve pear shoot cryopreservation.

Meristems of many pear genotypes can be successfully cryopreserved following 1 week of cold acclimation, but an equal number do not survive the process or have very little regrowth. This study compared commonly used cold acclimation protocols to determine whether the cold acclimation technique used affected the cold hardiness of shoots or the regrowth of cryopreserved meristems. In vitro-grown pear (Pyrus L.) shoots were cold acclimated for up to 16 weeks, then either the shoot tips were tested for cold hardiness or the meristems were cryopreserved by controlled freezing. Cold acclimation consisted of alternating temperatures (22 degrees C with light/-1 degrees C darkness with various photo- and thermoperiods) or a constant temperature (4 degrees C with an 8-h photoperiod or darkness). Compared with nonacclimated controls, both alternating- and constant-temperature acclimation significantly improved postcryopreservation regrowth of P. cordata Desv. and P. pashia Buch. -Ham. ex D. Don meristems. Alternating-temperature acclimation combined with either an 8-h photoperiod or darkness was significantly better than constant-temperature acclimation. Alternating-temperature shoot acclimation for 2 to 5 weeks significantly increased postcryopreservation meristem regrowth, and recovery remained high for up to 15 weeks acclimation. Postcryopreservation meristem regrowth increased with 1 to 5 weeks of constant-temperature acclimation and then declined with longer acclimation. Shoot cold hardiness varied with the acclimation procedure. The LT(50) of shoots acclimated for 10 weeks with alternating temperatures was -25 degrees C; that with constant temperature was -14.7 degrees C; and that of the nonacclimated control was -10 degrees C. Less frequent transfer of cultures also improved acclimation of shoots. Shoots grown without transfer to fresh medium for 6-12 weeks had higher postcryopreservation recovery with shorter periods of acclimation than shoots with a 3-week transfer cycle.

Profiling cryopreservation protocols for Ribes ciliatum using differential scanning calorimetry.

DSC analysis was performed at three points in the cryopreservation process on encapsulated-dehydrated meristems of Ribes ciliatum. Meristems were excised from shoots pre-treated with either sucrose or glucose, encapsulated in alginate beads, dehydrated in sucrose solutions, air dried, and plunged in liquid nitrogen. Thermal analysis revealed glass transitions during cooling of air-desiccated meristems, however, on rewarming a small endothermic event was detected suggesting glass destabilization can occur. Interestingly, this did not occur in alginate beads or meristems when these components were cooled and rewarmed separately. The possibility exists that thermal and moisture gradients may arise within the alginate bead/tissue complex and we propose that the heterogeneous composition of the meristems and the surrounding alginate may promote ice nucleation on rewarming. The significance of this regarding the stabilization of glasses formed in alginate beads and their encapsulated meristems is discussed. This study also reports an approach to Ribes cryopreservation in which the pregrowth of shoots in 0.75M sucrose for 1 week can be used as a substitute for cold acclimation.

Cold acclimation improves recovery of cryopreserved grass (Zoysia and Lolium sp.).

Cold acclimation of Lolium L. and Zoysia Willd. Grass cultivars significantly increased regrowth of cryopreserved meristems. One wk of cold acclimation improved recovery following cryopreservation but extended acclimation (4-8 wk) resulted in the best regrowth. Cold acclimation also significantly increased the dehydration tolerance of both Zoysia and Lolium meristems. Lolium apices cold acclimated for 4 wk produced 60-100% regrowth following cryopreservation by slow freezing or encapsulation-dehydration. Cold-acclimated Zoysia had greater than 60% regrowth following encapsulation-dehydration when beads were dehydrated to less than 22% water content. Non-acclimated meristems of both genera had little or no regrowth. Thawed meristems grew quickly without callus formation and the plantlets produced were transplanted to pots in the greenhouse after 4 to 6 wk. Samples of each cultivar were stored in liquid nitrogen as part of the U.S. National Plant Germplasm System.

Improved shoot multiplication of mature hazelnut (Corylus avellana L.) in vitro using glucose as a carbon source.

Shoot cultures established from mature trees of hazelnut (Corylus avellana L.) cvs. Nonpareil and Tonda Gentile Romana were used to determine the effects of basal media, carbon sources and concentrations, pH and cytokinins on shoot multiplication. All factors except pH affected the multiplication rate. Shoot multiplication was the best on a modified Driver and Kuniyuki medium for Paradox walnut (DKW) supplemented with 6-benzylaminopurine (BA) (1.5-3 mg/l). Plants grown on 3% glucose or fructose medium produced more and longer shoots than those on sucrose. The general appearance and growth habit of shoots were better on medium with glucose than fructose. 'Nonpareil' shoots elongated better than those of 'Tonda Gentile Romana.' Changes in medium pH from 4.7 to 5.7 did not significantly affect the multiplication rate. More than 10 genotypes propagated well on modified DKW medium with glucose. This is the first report of the effect of carbon sources on shoot multiplication of hazelnut and provides a basis for further research in the improvement of hazelnut micropropagation.

Micropropagation of meadowfoam (Limnanthes spp.).

A rapid micropropagation system was developed for meadowfoam (Limnanthes spp. Brown) using four genotypes of three species. Murashige and Skoog (MS) medium supplemented with N(6) benzyladenine (BA) and indole-3-acetic acid (IAA) at 0, 0.1, 0.5, 1.0 and 2.0 mg/l was tested for multiplication, shoot elongation and rooting. Expiants were taken from pot-grown plants. The most useful level for shoot growth and multiplication of both floral induced and non-induced plants was 0.5 mg/l BA. IAA failed to affect shoot growth or multiplication. Expiants from non-induced plants multiplied at moderate to high rates on 0.5 mg/l BA, while those from induced plants multiplied slowly and tended to elongate and flower. Non-induced plants on 2 mg/l BA produced large numbers of tiny shoots; induced plants did not respond. Shoots of all genotypes rooted on MS medium without hormones and all plants grew normally after transplanting to soil. This system provides a new tool for the development of meadowfoam as a crop plant.

Application of gas-permeable bags for in vitro cold storage of strawberry germplasm.

This study reports the first use of gaspermeable, heat-sealable polyethylene bags for cold storage of plant tissue cultures. The bags were used to develop a new cold storage system for the in vitro strawberry collection at the National Clonal Germplasm Repository (NCGR), Corvallis. In vitro Fragaria plantlets of 96 different accessions (species and cultivars) were transferred to bags with basal medium without growth regulators, heat-sealed, grown for one week at 25°C, cold hardened for one week, and then stored in the dark at 4°C. These in vitro cultures were successfully stored for up to 24 months in polyethylene bags. Evaluations at three month intervals provided information on the condition of the diverse collection. Over 75% of the accessions originally stored remained in storage for 15 months and 47% remained for over 18 months. None of the 96 accessions studied was lost due to contamination or decline in vigor. Over 300 Fragaria accessions are currently stored using this system.