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Mohamed F. Mohamed, P. E. Read, and D. P. Coyne

Regeneration in vitro from the embryonic axis in Phaseolus sp. has not been reported. Two embryo sizes, 0.3-0.4 mm and 0.6-0.7 mm long at 10-12 and 21 days after pollination, respectively, were excised from 4 P. vulgaris (P.v.) and 2 P. acutifolius (P.a.) genotypes. The embryonic leaves and radicale were removed, and 0.1-0.2 mm of the embryonic axis was cultured on Gamborg's B5 medium with 0, 5, 10 and 20μ MBA. The cultures were incubated in the dark at 25°C for 2 weeks followed by 1 week in continuous cool white light (25μ MS-1m2) before transferring to the second medium (0, 2μ MBA and 2μ MBA + 4μ MGA3). The tissues from the larger embryos initiated a single shoot without PGR in 30% of 1 P.v. explants and 30-60% in 2 P.a. The other 3 P.v. formed roots only. Multiple shoots were initiated in all P.v. (15-60%) and in 2 P.a. (60 and 70%) with 5 or 10μ MBA. The tissues from the smaller embryos had single shoots for all genotypes (30-60%) without PGR. Multiple shoots were initiated in 50-80% and 75-90% of the explants from P.v. and P.a., respectively, with 5 or 10μ MBA. Excess callus formed with 20μ MBA and regeneration decreased. After 3 weeks on the second medium, 6-8 shoot s/P. v. and up to 15-20 shoots/Pa. explants were observed.

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Bipul K. Biswas*, Nirmal Joshee, and Anand K. Yadav

Guava (Psidium guajava L.), also called `apple of tropics,' is immensely nutraceutical and horticulturally important. Being a tropical plant, it cannot stand temperatures below 25° F and needs frost protection to grow in temperate regions. To adapt in cold climate, cold hardy guava cultivars are needed. Conventional ways are uneconomic in time and efforts. Still, transgenic plants developed using biotechnological approaches of tissue culture and rDNA technology, appear to have great potential. Thus, protocols for in vitro propagation of guava were developed via organogenesis and somatic embryogenesis using nodal explants from mature trees and young zygotic embryos, respectively. Nodal explants induced multiple shoots when cultured on MS medium fortified with KIN, BAP and Ad.S. Adding a (NO3)2 to medium was useful to prevent in vitro shoot tip browning of adventitious shoots. Rocker liquid culture greatly increased growth of multiple shoots compared to the agar-based medium. It appears to be a good tool for woody plant tissue culture. Induction of somatic embryos in guava was also achieved on MS medium supplemented with IAA auxin. About 80% to 90% somatic embryos germinated normally. To achieve Agro-bacterium-mediated gene transfer in guava, on-going co-cultivation of organogenic tissues of guava is to optimize protocols for freeze tolerance gene (CBF1, CBF2, CBF3) transfer. Plasmid vectors containing selectable markers (nptII gene for antibiotic selection and GUS reporter gene as scorable gene mediated selection), with CaMV 35S promoter gene has been introduced into guava tissues and the resultant plants showed antibiotic resistance. Details of the experimental procedures and up-to-date results will be discussed.

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A. Raymond Miller and Craig K. Chandler

A protocol was developed for excising and culturing cotyledon explants from mature achenes of strawberry (Fragaria × ananassa Duch.). Cotyledon explants formed callus with multiple shoot buds on agar-solidified Murashige and Skoog media containing several combinations of hormones (1 μm 2,4-D; 10 μm 2,4-D; 1 μm BA + 1 μm 2,4-D; 1 μm BA + 10 μm 2,4-D; 5 μm BA; 5 μm BA + 1 μm 2,4-D; 5 μm BA + 10 μ m 2,4-D; 5 μ m BA + 5 μm NAA; 5 μ m BA + 15 μ m NAA). After three subcultures, only tissues maintained on the medium containing 5 μm BA + 5 μm NAA continued to form shoots. Tissues transferred to other media eventually died (1 μm 2,4-D; 1 μ m BA + 10 μ m 2,4-D; 5 μ m BA; 5 μ m BA + 1 μ m 2,4-D), became unorganized (1 μm BA + 1 μm 2,4-D; 5 μm BA + 10 μm 2,4-D; 5 μm BA + 15 μm NAA), or formed roots (10 μm 2,4-D). Whole plantlets were produced by transferring callus with buds to medium lacking hormones. The rapid regeneration of clonal plantlets from cotyledon explants may be useful for reducing variability in future developmental studies. Chemical names used: N-(phenylmethyl)-1H-purin-6-amine (BA); (2,4-dichlorophenoxy) acetic acid (2,4-D); and 1-naphthaleneacetic acid (NAA).

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James E. Faust and Royal D. Heins

Axillary buds of African violet develop vegetative shoots or reproductive inflorescences. Vegetative axillary development results in a multiple-shoot plant and reduces plant quality. We determined the effect of temperature and plantlet size on axillary bud development. Plantlets were removed from leaf cuttings, graded according to stem diameter, directly stuck into pots 10 cm in diameter, and placed in greenhouses at 18, 22, or 26C. Vegetative development was related to temperature, plantlet size, and nodal position. The number of vegetative axillary shoots per plant decreased from 3.7 to 1.3; that of leaves per vegetative axillary shoot decreased from 10.3 to 4.8 as temperature increased from 18 to 26C. The eight to 10 basipetal nodes developed vegetative shoots or were devoid of axillary development. The percentage of leaf axils in which inflorescences developed increased from 14 on node eight to 100 on nodes 12 and higher. The larger plantlets at the time of transplant had 20% fewer vegetative axillary shoots, whereas reproductive inflorescence development was not affected by plantlet size.

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Yan Ma, David H. Byrne, Jing Chen, and Amanda Byrne

Several rose species (Rosa rugosa, R. wichuraiana, R. setigera, R. laevigata, R. banksiae, R. roxburghii, R. odorata and hybrids) were employed to establish the appropriate nutrient media for shoot multiplication and root initiation of cultured shoots and to describe a procedure for the successful transfer to soil of plants obtained in vitro. Cultured shoot tips and lateral buds from different genotypes proliferated multiple shoots on a basal medium (MS salt, vitamins, glycine, sucrose and agar) supplemented with 0mg/l to 6mg/l 6-benzylamino purine (BA) and 0mg/l to 0.5 mg/l naphthalene acetic acid (NAA). Most rose species cultured in a modified MS medium supplemented with 2mg/l BA showed good growth and shoot proliferation. The buds nearest the apex exhibited the slowest rate of bud development. Root development was enhanced and shoot development inhibited by lowering the concentration of MS salts to quarter- and half-strength. With difficult-to-root species, rooting was improved by supplementing the media with auxin or giving them 3-7days of dark treatment.

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M.E. Oscar Mokotedi, M. Paula Watt, Norman W. Pammenter, and Felicity C. Blakeway

Multiple shoots of two Eucalyptus grandis Hill ex Maid. × E. nitens (Deane & Maid.) Maid. clones (GN121 and GN107) generated from axillary buds were used for in vitro rooting studies. The highest rooting rates in clones GN121 (75%) and GN107 (65%) were achieved on modified 1/4-strength Murashige and Skoog (MS) (1962) medium (Ca2+ and Mg2+ levels as for 3/4-strength MS), 0.5 μm IBA, 0.4 μm biotin, 0.2 μm calcium pantothenate, 0.04 m sucrose and 0.4% (w/v) Gelrite®. The optimal culture conditions were an initial 72-h dark incubation period followed by a 16-hour photoperiod at a photosynthetic photon flux density (PPFD) of 37 μmol·m-2·s-1 and 23 °C day/21 °C night for 7 days, after which the PPFD and temperature were increased to 66 μmol·m-2·s-1 and 27 °C day/21 °C night for 18 days. Plantlets were acclimatized with survival rates of 78% for GN121 and 58% for GN107 after 28 days. Chemical name used: indole-3-butyric acid (IBA).

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Sherry Kitto and Jeanne Frett

Hexastylis shuttleworthii is a highly ornamental shade-tolerant evergreen herbaceous plant native to the southeastern U.S. that is difficult to propagate using traditional methods. Micropropagation would make possible the wider distribution of selected clones. Seeds were surface-sterilized and germinated in vitro. Seedling clones were maintained on a MS basal medium containing 1 mg/L BA and were subcultured monthly. Proliferation of clones 2 and 3, maintained on media supplemented with 1, 2.5 or 5 mg/L BA for 6 months, increased slightly with increasing BA concentration; however, proliferation decreased slightly over the experimental period. Rooting medium (perlite, vermiculite, MetroMix 510, Bacto Growers Mix) did not effect microcutting root production or subsequent plant survival. Microcuttings rooted in vitro (67% survival) generated more leaves compared to microcuttings rooted under humidity domes with mist in the greenhouse (8% survival). After rooting in vitro, multiple-shoot clumps (95%) survived better than individual shoots (29%) under greenhouse conditions. Plants were easily established when planted in raised beds in a lath house.

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Ing-Jiun Tom Wu, G.L. Wheeler, and F.H. Huang

Scarification treatments (a control, a 10-minute vacuum, or a 1.5-minute ultrasound), different media (modified Norstog and Van Waes) and growth regulators [benzyladenine (BA) at 0, 1, 1.5, or 2 mg·L-1 and 6-(r,r-dimethylallylamino)-purine riboside (2iPR) at 0, 1, 1.5 or 2 mg·L-1] were used in combination to increase seed germination of Cypripedium calceolus var. parviflorum. Seeds treated with ultrasound had higher germination (58.0%) than those treated with vacuum (27.4%) or controls (19.2%). Germination rates increased with 2iPR level and reached a maximum between 1.5 and 2 mg·L-1. Seeds on Van Waes medium, which were not transferred to fresh medium after germination, had a severe browning problem causing many protocorms to die. Those on Norstog medium continued to grow into seedlings with less browning. Germination rates of Calopogon tuberosus × Calopogon `Adventure' and Liparis liliifolia were determined on the different media and growth regulator treatments. Multiple shoots of Calopogon developed from single seeds on media containing growth regulators. Flower buds formed in vitro on Calopogon in media containing 1 mg·L-1 or higher BA 5 months after germination. L. Iiliifolia seeds in Norstog medium had a higher proportion of germination than those in Van Waes medium.

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Don Waneck, H. Mathews, J. Stamp, and R. Bestwick

Zygotic embryo explants of grape cultivar AXR#1 were isolated from maw-e seeds and cultured on medium supplemented with naphthoxy acetic acid beta-(NOA) and benzylaminopurine (BA). Embryo explants dedifferentiated to form embryogenic callus. Globular stage embryos were visible in 9-10 months. On transfer 10 a growth regulator free medium supplemented with charcoal these globular embryos underwent further stages of embryo development. In a period of 30-40 days embryogenic tissues turned into clumps of somatic embryos displaying different stages of development Cotyledonary stage embryos were separated and transferred to basal medium. These embryos developed into complete plants. Cold and desiccation treatment of somatic embryos significantly enhanced the rate of plant conversion. Hypocotyl segments of elongated somatic embryos were good source explant for induction of shoot organogenesis. The hypocotyl-length and the proximity to-shoot-apex were found to influence the rate of shoot induction from hypotyl segments. Multiple shoot complexes which formed on hypocotyl segments were separated and individual shoots were grown on a root induction medium resulting in complete plant development. The possibility of both embryogenic and organogenic modes of plant regeneration make somatic embryos a highly versatile explant source for experiments on genetic manipulation.

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Young Goel Shon, Joong Choon Park, and Byoung Ryong Jeong

Effect of combination and concentration of growth regulators on the regeneration of pepper plant from different explant tissues was studied. Seedlings were grown aseptically in 400 ml glass bottles containing MS agar medium at 26±2C under a 16 h·d-1 photoperiod (2000 lux, florescent lamps). Explants taken from 4 week-old seedlings were cultured under these conditions on 40 ml of MS agar (8 g·liter-1) medium containing 3 g·liter-1 sucrose in a 400 ml glass bottle. Primary and subsequent leaves attached to petiole regenerated better than cotyledon and hypocotyl. Among the combinations of different concentrations of cytokinin and auxin added in the medium, a combination of 5 μM IAA with either 10 μM zeatin or 10 μM BA gave the best regeneration. With these combinations, regeneration frequency of multiple shoots from the primary and subsequent leaves was greater than 70%. Regenerated shoots rooted readily in MS agar medium containing 3 g·liter-1 sucrose and 0.5 μM NAA.