Premature deterioration and/or wilting of cut flowers such as roses (“bent neck”) has been attributed to vascular blockage within the cut stem. Vascular blockage has been attributed to both the proliferation of bacteria in the cut flower water and/or to products exuded by the stem. Separation of these causative agents is prevented by the inability to obtain intact microbe-free flowers. With the objective to produce microbe-free flowers, 36 miniature rose cultivars were screened for their capacity to flower in vitro. Stem segments containing single lateral buds were surface sterilized in 1.05% (v/v) sodium hypochlorite and rinsed three times in sterile distilled deionized water. Buds were established on medium consisting of Murashige and Skoog mineral salts, Woody Plant Medium organics, 3.0% (w/v) sucrose, 0.5 mg/liter benzyladenine, 0.1 mg/liter indole-3-acetic acid, and 50 mg/liter each citric and ascorbic acids. Medium was solidified with 1.5 g/liter gelrite and 4 g/liter TC® agar. Of the 36 cultivars screened, eight (22%) grew poorly in vitro. Of the 28 responsive cultivars, 14 (50%) produced flower buds in vitro However, only six cultivars produced open flowers in vitro.
Michael Kane, Nancy L. Philman, and Francis J. Marousky
Michael E. Kane, Nancy L. Philman, and Matthew A. Jenks
Only a few plants are suitable for reliably demonstrating rapid direct and indirect shoot organogenesis in vitro. A laboratory exercise has been developed using internodes of Myriophyllum aquaticum, an amphibious water garden plant. Stock shoot cultures are established and maintained in vitro from nodal explants cultured on agar-solidified medium consisting of half-strength Murashige & Skoog salts (MS) and 30 g·liter-1 sucrose. Students use these cultures as the source of internode explants. Explants are cultured on agar-solidified full-strength MS with 30 g·liter-1 sucrose, 100 mg·liter-1 myo-inositol, and 0.4 mg·liter-1 thiamine·HCL and factorial combinations of 0 to 10 μM 2iP and 0 to 1.0 μM NAA. Adventitious shoot development occurs directly from the explant epidermis within 4 days and is promoted in media supplemented with 2iP alone. Cytokinin-supplemented media amended with NAA induce organogenetic callus formation, but reduce 2iP promotion of direct shoot organogenesis. After 4 weeks, shoot organogenesis on the various media is quantified and can be analyzed statistically. Chemical names used: N-(3-methyl-2-butenyl)-1H-purin-6-amine (2iP); α-naphthaleneacetic acid (NAA).
Carmen Valero Aracama*, Michael E. Kane, Nancy L. Philman, and Sandra B. Wilson
A sea oats (Uniola paniculata L.) micropropagation protocol was previously developed for 28 genotypes that favored multiplication and rooting of shoots in vitro. However, microcutting size, morphology, and acclimatization ex vitro varied considerably among genotypes. In the present study we evaluated the effect of Stage III duration on in vitro morphology, biomass production, and ex vitro survivability of easy-(EK 16-3) and difficult-to-acclimatize (EK 11-1) sea oats genotypes. After 3, 6, and 9 weeks at Stage III, survivability of microcuttings was 85%, 96% and 98% for EK 16-3, and 2%, 27% and 40% for EK 11-1, respectively. After 9 weeks Stage III, EK 16-3 microcuttings had higher shoot dry weights but lower root dry weights than in EK 11-1. Moreover, roots in EK 11-1 were fewer but longer than in EK 16-3. Leaf production was similar in both genotypes. However, leaf elongation was significantly inhibited in EK 11-1, in which 95% of the leaves were ≤ 15 mm long in contrast with EK 16-3, with 50% leaves ≥ 16 mm long after 9 weeks Stage III. Light microscopy examinations showed anatomical similarities between EK 16-3 in vitro leaves and mature ex vitro leaves. Conversely, short in vitro leaves of EK 11-1 exhibited mesophyll disruption and reduced cuticle development. Conceivably, the short leaves had limited photosynthetic competency, thereby reducing ex vitro survival of rooted EK 11-1 microcuttings.
Carmen Valero Aracama, Michael E. Kane, Sandra B. Wilson, and Nancy L. Philman
Growth and development of two sea oats (Uniola paniculata L.) genotypes with differing acclimatization capacities when transferred from in vitro to ex vitro greenhouse conditions were compared as a function of the duration of shoot multiplication and rooting stages. Anatomical and morphological development differed between genotypes in vitro. After 4, 8, and 12 weeks of Stage II culture, leaf length and shoot number were significantly greater for the easy-to-acclimatize genotype (EK 16-3) than the difficult-to-acclimatize genotype (EK 11-1). Shoot dry weights in both genotypes were greatest after 4 weeks culture. Browning and dying of tissue steadily increased with time. Shoot number per plantlet increased from Week 4 to 8 in both genotypes but decreased after 12 weeks. Once transferred to Stage III culture for 6 weeks, significant differences in root architecture and morphology were observed between the two genotypes. EK 16-3 plantlets developed short but numerous roots and “grass-like” leaves with fully expanded blades. Conversely, EK 11-1 plantlets developed few long roots and “lance-like” leaves, which were short, thick, and without expanded blades. Anatomical and morphological development during Stage III differed between culture duration and genotypes. Shoot and root dry weights of both genotypes increased during 3, 6, and 9 weeks of culture. Shoot dry weights of EK 16-3 plantlets were lower at 3 weeks but higher at 9 weeks than EK 11-1. Conversely, root dry weights were higher for EK 11-1 than for EK 16-3 plantlets throughout Stage III culture. Anatomical observations of EK 11-1 plantlets using light and electron microscopy correlated poor ex vitro acclimatization and poor survival with abnormal tissue organization, stomatal aperture blockage, and thylakoid membrane disruption.