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Masanori Kadota, Takashi Hirano, Kiyotoshi Imizu, and Yoshiji Niimi

Effects of PA on in vitro shoot proliferation and root formation were investigated using shoot cultures of three Japanese pear (Pyrus pyrifolia Nakai) cultivars. PA inhibited shoot multiplication and promoted initiation and development of roots in the cultured shoots of three cultivars, resulting in increasing the proportion of rooted shoots. Chemical name used: pyroligneous acid (PA).

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Kathleen Heuss, Qingzhong Liu, Rosemarie Hammond, and Freddi Hammerschlag

As part of our program to develop transgenic peach cultivars with improved disease resistance, we showed that grafting of in vitro cultured `Suncrest' peach [Prunus persica (L.) Batsch] tips `onto decapitated stems of Prunus necrotic ringspot virus (PNRSV) infected `Suncrest' shoot cultures, resulted in consistent transfer of virus across grafts as demonstrated by RNA hybridization analysis, suggesting that such a system could be useful for measuring resistance to PNRSV in peach shoot cultures. We have extended these studies to include grafts of `Springcrest' and `Nemaguard' test tips onto `Suncrest' stocks. RNA hybridization analysis showed that PNRSV persists in shoot cultures for 18 months after initiation from PNRSV-infected `Suncrest' trees and after 16 weeks of treatment of 4°C in the dark, suggesting that a supply of infected shoot cultures could be maintained for repeated use. Graft success rates for grafts of `Springcrest' onto `Suncrest' and `Nemaguard' onto `Suncrest', equaled or exceeded success rates for `Suncrest' onto `Suncrest'. Virus was transmitted from infected stocks into `Suncrest', `Springcrest', and `Nemaguard' test tips by 2 weeks in most successful micrografts. There was no significant difference in the virus concentrations among the three scions at 2, 4, and 6 weeks after grafting, suggesting that there is equal efficacy of virus transfer through grafts from `Suncrest' to the three cultivars, and that no differences in resistance to PNRSV exist among these cultivars.

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Arthur M. Richwine and Robert D. Marquard

×Chitalpa tashkentensis (Chilopsis linearis × Catalpa bignonoides) is an attractive small tree producing lavender to white orchid like flowers. Micropropagation would allow for the rapid clonal propagation of new hybrids for testing cold hardiness and landscape performance. The rapid growth response of Chitalpa shoot cultures also makes it an excellent subject for the study of in vitro growth parameters of woody plants. Shoot cultures were initiated from shoot tips on Anderson's rhododendron medium with MS vitamins, 3% sucrose, 1 μm BA, pH 5.6 and solidified with 0.6% phytagar. Shoot cultures stabilized rapidly. Two-node microcuttings were placed on modified MS media (200.1 μm Na2 EDTA and 200.5 μm FeSO47H2O), MS vitamins, 3% sucrose, pH 5.6, 0.6% phytagar and supplemented with NAA (0 0.5, 1.5, or 3 μm) in combination with BA (0, 1, 5, 10, 15, 20, 30, or 40 μm). Cultures grown on media supplemented with 1 mm BA produced the longest shoots and the most nodes per shoot. Cultures grown on media supplemented with 10 μm BA produced the most shoots. Microshoots readily rooted on plant growth regulator free MS medium and were easily acclimated.

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Barbara M. Reed

Cold storage is important for managing in vitro germplasm collections. Strawberry shoot cultures can typically be held at 4 °C for 9 to 24 months before they require repropagation. Concentration of BA in the storage medium, pre-storage cold acclimatization (CA), and exposure to a photoperiod during storage were studied to determine conditions for improved strawberry culture storage. Fragaria shoot cultures stored at 4 °C were rated for plantlet condition on a 0-5 scale at 9, 12, and 19 months. Four species were CA and stored on medium with 0, 1, 2.5, or 5 μm BA either in darkness or under a 12-hour photoperiod. Mean ratings over all treatments and genotypes were best at 9 and 12 months (3.4) and declined at 19 months (2.2). BA in the storage medium significantly improved ratings for two species at 9 and 12 months, but ratings were not significantly different at 19 months. At 19 months of storage, shoot cultures stored with a photoperiod were rated significantly better (P ≤ 0.05) than those grown in darkness. Five Fragaria genotypes stored on medium without BA were used to study the effect of photoperiod and CA on ratings of stored plantlets. CA-shoot cultures stored for 9 or 12 months were rated significantly better than non-CA cultures. After 12 and 19 months storage, three of the five genotypes stored under a 12-hour photoperiod had significantly higher ratings than those stored in the dark (P ≤ 0.01), but by 19 months CA was nonsignificant. Overall, the addition of a photoperiod improved the condition of Fragaria shoot cultures stored at 4 °C. Chemical name used: N 6-benzyladenine (BA).

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Jessica D. Lubell-Brand, Lauren E. Kurtz, and Mark H. Brand

shoot cultures to maintain quality growth for an extended period of time ( Monthony et al., 2021 ). Microshoots from in vitro cultures are miniaturized, have altered physiology, and root easily ( Hartmann et al., 2002 ). Nursery producers use a process

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Suzanne M. Dethier Rogers and Sharon Banister

A micropropagation system was developed for micropropagation and rooting of Notholaena spp. Shoot cultures were initiated from mature sori on leaflets of the Notholaena cultivar Sun-Tuff and cultured on gelled Murashige and Skoog (MS) medium without hormones. Rooting and plant growth were evaluated on gelled MS, vermiculite moistened with liquid MS, or vermiculite moistened with water. Vermiculite wetted with MS was superior in promoting frond and root development. High humidity was not needed to acclimatize the plants ex vitro.

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X. Cao and F. Hammerschlag

As part of a program to develop transgenic highbush blueberry (Vaccinium corymbosum L.) cultivars, studies were conducted to determine optimum conditions for high-efficiency shoot regeneration from leaf explants of in vitro propagated, commercially important, tissue culture-recalcitrant `Bluecrop' shoot cultures. The effects of pretreatments, growth regulators, and age of explant source on shoot organogenesis were investigated. A maximum of 98% shoot regeneration and 10 shoots regenerating per leaf explant occurred when explants of 2-week-old shoot cultures were incubated in the dark (for a total of 14 days) on pretreatment medium #1 containing 2.6 μM NAA and 5 μM TDZ for 4 days, next on pretreatment medium #2 containing 2.6 μM NAA and 7 μM zeatin riboside for 3 days, then on regeneration medium containing 1 μM TDZ for 6 weeks, and last on medium without growth regulators for 10 days. No shoot regeneration occurred if explants were incubated without exposure to pretreatments before incubation on regeneration medium. There were no significant differences in percentage of regeneration or the number of shoots regenerating per explant from leaf explants derived from either 1-, 2-, or 3-week-old shoot cultures. Shoot production per explant on 1 μM TDZ was about three times that on either 0.5 μM TDZ or 20 μM zeatin riboside, and nine times that on 5 μM TDZ.

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K. Heuss, Q. Liu, F.A. Hammerschlag, and R.W. Hammond

As part of a program to develop transgenic peach (Prunus persica L. Batsch) cultivars with resistance to Prunus necrotic ringspot virus (PNRSV), we are testing a system for measuring virus in peach shoot cultures. Micrografting in vitro is used for inoculation and slot-blot hybridization, with a digoxigenin (DIG)-labeled cRNA probe complementary to the 5′ open reading frame (ORF) of PNRSV RNA 3, for detection. In this study, we investigated whether infected shoots maintain virus infection over long periods of culture at 4 °C and if PNRSV-infected `Suncrest' shoot cultures can serve as graft bases to transmit virus equally well into cultivars Nemaguard, Springcrest, and Suncrest. The results of RNA hybridization analysis showed that virus was present in extracts of leaf samples from 2-year-old PNRSV-infected `Suncrest' shoots that had been subjected to varying lengths of incubation at 4 °C in the dark, suggesting that infected shoots can be maintained for repeated use. Rates of graft success were higher in heterografts between `Suncrest' bases and tips of `Springcrest' or `Nemaguard' than in autografts between `Suncrest' and `Suncrest', and there was equal efficacy of graft inoculation from `Suncrest' into these three cultivars.

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Xiaoling Cao, Freddi A. Hammerschlag, and Larry Douglass

As part of a program to improve highbush blueberry (Vaccinium corymbosum L.) cultivars via tissue culture and genetic engineering, studies were conducted to determine optimum conditions for organogenesis from leaf explants of the previously recalcitrant cv. Bluecrop. The effects of a pretreatment, growth regulators, and age of explant source on shoot organogenesis were investigated. A maximum of 98% explants regenerated shoots with a mean of 11 shoots per leaf explant after 62 days when explants of 2-week-old shoot cultures were incubated on the following regime: pretreatment medium #1 containing 5 μm TDZ and 2.6 μm NAA for 4 days, pretreatment medium #2 containing 7 μm zeatin riboside and 2.6 μm NAA for 3 days, regeneration medium containing 1 μm TDZ for 6 weeks, and last on medium without growth regulators for 10 days. No shoot regeneration occurred if explants were incubated without exposure to pretreatment prior to incubation on regeneration medium. There were no significant differences in percentage of regeneration or the number of shoots regenerating per explant from leaf explants derived from either 1-, 2-, or 3-week-old shoot cultures. Shoot production per explant on regeneration medium containing 1 μm TDZ was about three times that on 0.5 μm TDZ or 20 μm zeatin riboside, and nine times that on 5 μm TDZ. Chemical names used: 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (thidiazuron, TDZ); 9-(β-D-ribofuranosyl)-6-(4-hydroxy-3-methyl-but-2-enylamino)purine (zeatin riboside).

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Yiqin Ruan and Mark H. Brand

Rhododendron `Montego' shoot cultures initiated from plants with and without tissue proliferation (TP and NTP) served as explant sources for all studies (Note: in vitro TP shoot cultures produce primarily dwarf shoots, some long shoots, and stem tumors). Calli induced from TP leaves and tumors and NTP leaves were cultured on woody plant (WP) medium containing NAA and 2-iP. During the first 4 weeks of culture, calli from NTP leaves had higher relative growth rates than calli from TP leaves or tumors. However, calli from TP leaves and tumors grew faster than calli from NTP leaves for all subculture periods that followed. Shoot tips (5 mm) were excised from TP dwarf shoots, TP long shoots, and NTP shoots and were cultured on WP medium with or without 15 μM 2-iP. Shoot tips from TP dwarf and long shoots multiplied on medium without 2-iP, averaging 18.4 and 1.7 shoots per shoot tip in 12 weeks, respectively. Shoot tips from NTP shoots only multiplied when maintained on 2-iP-containing medium. When placed on 2-iP-containing medium, both types of TP shoot tips produced clusters of callus-like nodules that gave rise to highly tumorized, short shoots or leafy meristems.