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Jeffrey Adelberg, Kazuhiro Fujiwara, Chalermpol Kirdmanee, and Toyoki Kozai

Two triploid clones of melon from the same tetraploid parent were grown in vitro with and without sugar, rooted without sugar in media both in a laboratory controlled environment chamber (in vitro) and a greenhouse acclimatization unit (ex vitro), and compared for subsequent nursery growth in the greenhouse unit. The clone `(L-14 c B) × L-14' produced more shoots in both photomixotrophic (with sucrose) or photoautotrophic (sugar-free) conditions. Both genotypes were equally likely to root in sugar-free media, and `(L-14 × B) × L-14' rooted as well from either photoautotrophic and photomixotrophic shoots but `(L-14 × B) × Mainstream' rooted less frequently from photoautotrophic shoots. Seventy-six percent (76%) of the shoots were able to root photoautotrophically in vitro, whereas 47% of the ex vitro shoots were rooted. About 85% of plantlets from all treatments survived after transfer to the nursery. After growth in the greenhouse nursery, the sizes of plants (fresh and dry weight, leaf area) were the same for either clone, from either photoautotrophic or photomixotrophic shoots. Also, after growth in the nursery, plantlets that had been rooted in vitro were larger than those rooted ex vitro. Photoautotrophic rooting demonstrates a concept for integrating micropropagation and plug-type vegetable transplant production.

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Mehmet Nuri Nas and Paul E. Read

Microshoots of four hazelnut genotypes grown in vitro on Nas and Read medium (NRM) containing various combinations of CuSO4 • 5H2O and myo-inositol were successfully rooted and acclimatized ex vitro without any need of in vitro hardening treatments. Dipping of shoot bases in 1000 ppm indole-3-butyric acid (IBA) solution for 5 or 10 seconds followed by placement of shoots in plant growth regulator free NRM gave rise to formation of roots as early as 8 days. Shoots treated for 5 and 10 seconds rooted similarly, and depending on genotype, 88% to 98% rooting was observed within 15 days after treatment with IBA. Ex vitro survival of shoots three months after in vitro-root induction was 73% when shoots were treated with IBA for 5 seconds and 66% when shoots were treated for 10 seconds. The highest ex vitro survival rate (97%) 3 months after root induction was observed when shoots were treated with IBA solution for 10 seconds, and then cultured directly in peat pellets. Shoots developed good roots, and grew up to 70 cm in height 3 months after root induction. The potential use of rooting and acclimatization protocol for commercial micropropagation of hazelnut is presented.

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Andres A. Estrada-Luna, Fred T. Davies Jr., and Jonathan N. Egilla

The role of mycorrhiza fungi during acclimatization and post-acclimatization of micropropagated chile ancho plantlets was characterized through physiological and plantlet development changes. Regardless of mycorrhizal colonization, the pepper plantlets had initially low photosynthetic rates and poor growth following transplanting ex vitro. During the first days of acclimatization, water deficits occurred as evidenced by drastic reductions in relative water content. Consequently, transpiration rates and stomatal conductance (gs) declined, confirming that in vitro formed stomata were functional, thus avoiding excessive leaf dehydration and plant death. Mycorrhiza had a positive effect on gas exchange as early as day 7 and 8, as indicated by increasing photosynthesis (A) and gs. Mycorrhizal plantlets had reduced levels of abscisic acid (ABA) during peak stress (6 days after transplanting ex vitro), which corresponded with subsequent increases in gs and A. During acclimatization, A increased in both non-colonized and colonized plantlets, with greater rates observed in mycorrhizal plantlets. During post-acclimatization, mycorrhiza colonized 45% of the roots of pepper plantlets and enhanced plant growth by increasing leaf area, leaf dry mass, and fruit number. Mycorrhiza also enhanced total leaf chlorophyll content, A, and nutrient uptake of pepper plantlets, particularly N, P, and K. Early mycorrhizal colonization produced important benefits, which helped ex vitro transplanted plantlets recover during acclimatization and enhance physiological performance and growth during post-acclimatization.

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Ribo Deng and Danielle Donnelly

Labeled (`“C) compounds were recovered from tissue disks taken from 14CO2-fed leaves of l-year-old greenhouse-grown plants and l-month-old ex vitro transplants of red raspberry (Rubus idaeus L.) by hot (boiling in 80% ethanol immediately after 14C exposure), delayed-hot (boiling in 80% ethanol after a 2- to 3-day ethanol soak), and room-temperature (RT) (2-to 3-day soak in 80% ethanol) extraction methods. The RT extraction method was simple but as effective for extracting 14C-labeled compounds from red raspberry leaf tissues as hot and delayed-hot extraction methods.

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Martine Korban and Danielle J. Donnelly

Mechanically induced stress (shaking stress) applied during shoot multiplication (Stage II) or rooting (Stage III) of micropropagated `Queen Elizabeth' rose was evaluated to determine its effects on in vitro hardening. Shaking during Stage II did not alter the growth responses of the shoots before transfer to Stage III. Shaking during Stage III, at 150 rpm for 15 min daily for 2 weeks, only caused a reduction in leaf dry weights before transfer to soil. Automated shaking stress during Stages II or III did not apparently promote hardening of cultured plants or improve their ex vitro performance.

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C.S. Walsh, F.J. Allnutt, G.R Welsh, and R.H. Zimmerman

A planting to compare budded apple trees (M7a, Ml11) and tissue-culture-(TC) propagated trees was established in 1985. `Golden Delicious' and `Gala' trees were more productive than other cultivars and appeared better-suited to micropropagation. High cumulative yields per tree were harvested regardless of rootstock. `McIntosh', `Delicious', `Mutsu', and `MacSpur' trees were less precocious and more responsive to size-controlling rootstocks. To control tree size prior to bearing and minimize propagation time, trees were set as containerized transplants in a subsequent trial begun in 1986. Small containerized trees were set directly into the orchard. Setting trees in this manner has restricted tree size without delaying bearing. `Oregon Spur II' trees and `Empire' trees are now about 4 m tall. Trees have wide branch angles and numerous spurs. To further control tree size, trees were root-pruned with a Vermeer tree spade in 1991. In the year following, treated trees flowered profusely but did not fruit. Since then, cropping has controlled tree size. Ten years ex vitro `Granny Smith', `Oregon Spur II', and `Empire' trees can be managed without ladders. The goals of this study were: 1) to avoid “short life” problems and 2) develop a management scheme that would allow rapid entry of “bioengineered” cultivars into commercial orchards. Based on our research, selecting precocious cultivars or spur-type clones, in combination with transplanting 3 to 4 months ex vitro and root pruning show promise toward accomplishing these goals.

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Hope Jones and Chieri Kubota

In vitro culture of orchid plantlets within conventional photomixotrophic micropropagation (PMM) systems (sucrose containing media in a non-enriched CO2 environment) often induces vigorous growth and multiplication. However, transition to ex vitro conditions frequently results in significant plantlet loss during the acclimatization process. Recent studies investigating micropropagation within photoautotrophic (PAM) systems (sucrose-free media in enriched CO2 conditions) have demonstrated improved plantlet survival during the acclimatization period due to greater root growth and stomata adaptation. Laelia purpurata var. alba, an orchid with many endangered relatives, was chosen as a model orchid species to investigate if plantlet culture within PAM in vitro systems has the potential to improve propagation success and ex vitro survival of endangered Laelia species. Protocorm-like bodies with developed two fully extended leaves were transferred into PMM (photosynthetic photon flux 50 μmol·m-2·s-1 under non-enriched CO2 conditions) and PAM (photosynthetic photon flux 150 μmol·m-2·s-1, CO2 level enriched to 1500 μmol·mol-1) systems. After 6 weeks, plantlet rooting within the PMM system was variable and inconsistent, while all PAM plantlets produced healthy robust root systems. Average fresh weights and percent shoot development were not significantly different between treatments. Induction of improved root growth by PAM systems may improve orchid plantlet survival rates during acclimatization and advance our ability to increase endangered orchid populations.

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Hazel Y. Wetzstein*, Allan M. Armitage, Gwen N. Hirsch, and Stephanie L. Anderson

Tissue culture is a useful means to clonally propagate new ornamental plant selections, particularly when plant material is limited and/or conventional propagation methods are ineffective. An efficient in vitro multiplication protocol was established to propagate a new goatsbeard hybrid (Aruncus dioicus, × A. aethusifolia). The hybrid is of interest because it exhibits a dwarf habit, delicate white flower panicles and fern-like leaves, yet is tolerant to heat and humidity. Experiments were conducted to evaluate explant type (nodes, stems, leaves, and floral parts), disinfestation procedures, and media formulations including varying concentrations of 6-benzylaminopurine (BAP) and naphthalene acetic acid (NAA). Rapid plant regeneration was obtained with a shoot organogenesis system using a half strength Murashige and Skoog medium supplemented with 4.4 μmol BAP, 0.54 μmol NAA, 30 g·L-1 sucrose, and 3.0 g·L-1 GelGro. Studies compared the performance and yield of plants rooted using different in vitro and ex vitro methods. Ex vitro rooting of shoots during greenhouse acclimatization under mist was most effective. Regenerated plants exhibited uniform and rapid growth, and performed well in greenhouse and field evaluations.

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Andres A. Estrada-Luna, Fred T. Davies Jr., and Jonathan N. Egilla

Micropropagated chile ancho pepper (Capsicum annuum L. cv. San Luis) plants were transferred to ex vitro conditions to study plantlet performance and selected physiological changes that occur during acclimatization and post-acclimatization. The physiology of the plantlets was characterized by measuring leaf gas exchange and water status. Plant growth was determined by assessing plant height, leaf number, total leaf area, relative growth rate (RGR), and leaf, root, and stem dry mass. Measurements were taken at 0, 1, 2, 3, 6, 12, and 24 days after transplanting. After initial transplanting ex vitro to liner pots with soilless media, plantlet wilting was observed that correlated with reduced leaf relative water content (RWC). Water stress was partially alleviated by a reduction in stomatal conductance (gs), confirming that the in vitro formed stomata were functional and able to regulate transpiration (E) to minimize desiccation losses. Because of this stomatal control, plantlets had minimal transplant shock, recovered, and survived. Prior to transplanting, micropropagated plantlets showed heterotrophic/mixotrophic characteristics as indicated by low photosynthesis [(A) 4.74 μmol·m2·s-1]. During acclimatization, RWC, gs, E, and A were significantly lower 2 days after transplanting. However, within 6 days after transplanting, plantlets recovered and became autotrophic, attaining high A (16.3 μmol·m-2·s-1), gs, and E. The stabilization and improvement of plantlet water status and gas exchange during acclimatization and post-acclimatization closely correlated with dramatic increases in plantlet growth.

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Fouad Mohamed, Harry Jan Swartz, and George Buta

In previous abstracts (HortScience 23:707;24:121), ABA when added throughout the in vitro production cycle, reversed the tissue culture-induced rejuvenation of the day neutral strawberry `Fern'. Compared to benzyl adenine (BA) proliferated plants, ABA treated tissue culture-produced plants flowered earlier and had more adult leaf patterns. In the present study, we analysed endogenous ABA concentrations in the apices and unexpanded leaves of BA treated tissue culture-propagated plants, selved seedlings and propagated adult runner tip plants at 3, 7 and 15 weeks ex vitro, after germination or after runner tip propagation. Using pentadeuterated standards and single ion monitoring, ABA concentrations in tissue culture produced and juvenile seedling plants were significantly lower than adult plants at 3 and 7 weeks. By 7 weeks, only the adult plants were flowering. At 15 weeks, no differences in ABA concentration were significant and all three types flowered.