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Physiological and anatomical basis for differences in growth performance during in vitro and ex vitro culture of Sea oats (uniola paniculata l.) genotypes [electronic resource] /
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ABSTRACT: Sea oats (Uniola paniculata L.), a dune species native to the southeastern U.S., is commonly used for dune stabilization and restoration in Florida. A micropropagation protocol was developed for sea oats commercial production. However, significant variability in shoot production, rooting and ex vitro survival among sea oats genotypes was observed. Understanding the morphological, anatomical, and physiological basis for differences among genotypes would allow development of efficient micropropagation protocols to produce diverse sea oats genotypes for dune stabilization. Growth and development of two sea oats genotypes with differing acclimatization capacities were compared at morphological and anatomical levels as a function of the duration of multiplication and rooting stages using light and electron microscopy. During in vitro and ex vitro stages, changes in photosynthetic rates were monitored, and carbohydrate levels and photosynthetic enzymes activities were measured. Additionally, sea oats photosynthetic capacity in vitro and ex vitro and during acclimatization was evaluated using in vitro photoautotrophic and photomixotrophic culture conditions. During the rooting stage, the easy-to-acclimatize genotype (EK 16-3) developed short but numerous roots and grass-like leaves with fully expanded blades. Conversely, the difficult-to-acclimatize genotype (EK 11-1) developed few long roots and short and thick lance-like leaves without expanded blades. During in vitro development of grass-like leaves, EK 16-3 plantlets exhibited increases in activities of the two photosynthetic enzymes phosphoenolpyruvate carboxylase (PEPC) and ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco), and chlorophyll and total soluble protein content. These increases were correlated with higher net photosynthetic rates in EK 16-3 than EK 11-1 plantlets after ex vitro transfer. Both genotypes accumulated carbohydrate and starch reserves during in vitro rooting, which were depleted during the transition from photomixotrophic to photoautotrophic mode of nutrition. However, due to the lack of production of photosynthetically competent leaves on EK 11-1 plants coupled with rapid exhaustion of carbohydrate reserves, acclimatization and survival ex vitro for this genotype were low. Conversely, rapid production of photosynthetically competent leaves resulted in 100% survival ex vitro in EK 16-3 plantlets. In conclusion, the major cause for low acclimatization was the in vitro formation of leaves with abnormal anatomy correlated with limited photosynthetic capacity. Text (Electronic thesis) in PDF format. System requirements: World Wide Web browser and PDF reader. Mode of access: World Wide Web. Title from title page of source document. Document formatted into pages; contains 173 pages. Thesis (Ph.D.)--University of Florida, 2005. Includes vita. Includes bibliographical references.
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... Greater decline in starch reserves in EK 16-3 over EK 11-1 after 3 wk in vitro may have resulted from the more rapid and earlier initiation and growth of EK 16-3 roots and shoots. This observation is supported by comparative growth measurements of both genotypes during Stage III culture (Valero-Aracama, 2005). Our studies indicated that sea oats genotypes stored starch and contained soluble sugars in shoots and roots throughout Stage III. ...
... During weeks 6 and 9 of Stage III culture, the activities of PEPC and rubisco and the contents of chlorophyll and TSP were significantly greater in EK 16-3 than EK 11-1. Histological sections of in vitro-produced leaves of EK 11-1 and EK 16-3 (Valero-Aracama, 2005) indicated that after 6 wk in Stage III, EK 16-3 leaves exhibited more typical Krantz anatomy of C 4 plants than EK 11-1 leaves. After 6 wk in Stage III, the fixation of CO 2 by PEPC and the CO 2 concentrating mechanism in EK 16-3 might have reduced some of the inefficacy of rubisco under the stress caused by in vitro conditions, enabling the maintenance of photosynthesis (Rodríguez et al., 2003). ...
... Additionally, in vitro sugarcane leaves exhibited minimal Krantz anatomy compared with the more typical Krantz anatomy exhibited by ex vitro leaves (Rodríguez et al., 2003). This increase in enzymatic activity, chlorophyll and soluble protein content after 6 wk in Stage III correlates with the initial decrease of starch and soluble carbohydrates in shoots of both genotypes during the same time period, and with the formation of the first fully expanded leaves with typical Krantz anatomy in EK 16-3 (Valero-Aracama, 2005)7. A, Rubisco activity per shoot gram fresh weight; B, PEPC activity per shoot gram fresh weight; and C, PEPC/rubisco ratio of EK 11-1 and EK 16-3 sea oats genotypes during in vitro Stage III (left) and after microcuttings were rooted for 6 wk, Stage III and transferred to Stage IV (right). ...
The photosynthetic and carbohydrate status of an easy-to-acclimatize (EK 16-3) and a difficult-to-acclimatize (EK 11-1) genotype
of Uniola paniculata L. (sea oats), a native dune species of the southeastern US, were evaluated during in vitro culture and ex vitro acclimatization. Net photosynthetic rate was eight times greater for EK 16-3 than EK 11-1 plantlets after ex vitro transfer. In vitro-produced leaves were morphologically similar to ex vitro-produced leaves and exhibited similar photosynthetic competence. EK 11-1 plantlets exhibited greater transpiration rates
at the time of ex vitro transfer than EK 16-3 plantlets. However, the small magnitude of this difference, although significant, indicated that control
of water loss was probably not the main cause for poor acclimatization of EK 11-1 plantlets. Carbohydrate analysis in vitro revealed that EK 16-3 plantlets utilized leaf starch reserves more rapidly than EK 11-1 plantlets. Starch utilization correlated
with the development of leaves with expanded leaf blades during in vitro rooting in EK 16-3 plantlets. After ex vitro transfer, both genotypes exhibited significant decreases of starch and soluble sugar content in shoots and roots. However,
the higher photosynthetic ability of shoots in EK 16-3 resulted in greater accumulation of shoot soluble sugars than EK 11-1
after 2-wk ex vitro culture. After 6-wk in vitro rooting, there were significantly higher chlorophyll and soluble protein contents, ribulose 1,5-bisphosphate carboxylase
(rubisco) and phosphoenolpyruvate carboxylase activities in EK 16-3 than EK 11-1 shoots. These differences also correlated
with the development of anatomical and morphological leaf features in EK 16-3 similar to those of greenhouse-produced leaves.
Seeds of southern seaoats (Uniola paniculata L.) were removed from storage at 4C (39F) and treated with the following selected surface disinfestants, fungicides, or combinations of these chemicals: nontreated (control), 1.3% sodium hypochlorite [NaOCl (chlorine bleach)], 2.6% sodium hypochlorite, RTU® (12.6% thiram + 0.34% thiabendazole), RTU®-PCNB (24% pentachloronitrobenzene), 1.3% sodium hypochlorite and RTU®, 2.6% sodium hypochlorite and RTU®, 1.3% sodium hypochlorite and RTU®-PCNB, or 2.6% sodium hypochlorite and RTU®-PCNB. Following treatment, seeds were germinated at an 8/16 hr thermoperiod of 35/20C (95/68F). The seed treatments and germination thermoperiod utilized were based on three preliminary trials that investigated the influence of selected surface disinfestants, fungicides, and temperature on seed germination of the species. Germination was recorded every 3 days for 30 days. Seed treatment was highly significant (P = 0.0001) for both total percentage germination and total percentage of decayed seeds. Germination of nontreated seeds was 45%, and four treatments resulted in germination > 80% [RTU®-PCNB (81%), 2.6% sodium hypochlorite and RTU® (83%), 1.3% sodium hypochlorite and RTU® (87%), and 1.3% sodium hypochlorite and RTU®-PCNB (89%)]. A subsequent experiment investigated the effects of the aforementioned treatments with the exception of 1.3% sodium hypochlorite and RTU®, both used alone, on initial seedling growth of the species. Following treatment, seeds were sown in containers filled with a peat-based medium and the containers placed in a growth chamber maintained at an 8/16 hr thermoperiod of 35/20C (95/68F) with long day conditions. Emergence data were recorded every 3 days for 45 days. After 45 days, the study was terminated and additional data recorded to include plant height (height of main stem), leaf number, length and width of the two longest leaves, and top and root dry weights. Surface disinfestant, fungicide, and combination treatments were highly significant (P = 0.0004). Percentage emergence of nontreated seeds was 35% and five of the seven treatments resulted in emergence ≥ 75% [2.6% sodium hypochlorite (75%), 1.3% sodium hypochlorite and RTU® (75%), 1.3% sodium hypochlorite and RTU®-PCNB (76%), 2.6% sodium hypochlorite and RTU®-PCNB (81%), and 2.6% sodium hypochlorite and RTU® (83%)] with negligible effects on seedling growth. There were significant treatment differences regarding some of the variables used to evaluate seedling growth. In most cases these differences were due to seedlings from nontreated seeds having lower values for each measured variable than values for the same variables from treated seeds. Results of both experiments demonstrate the potential value of chemical seed treatment during production of seedling transplants of U. paniculata.
Seeds of southern seaoats (Uniola paniculata L.) were removed from storage at 4C (39F) and stratified (moist-prechilled) for 0, 15, or 30 days at 4C (39F). Following stratification, seeds were germinated at 25C (77F) or 30C (86F) or at 8/16 hr thermoperiods of 30/20C (86/68F) or 35/25C (95/77F) with daily photoperiods at each temperature of 0 (total darkness), 2, 4, 8, 12, or 24 hr. Germination was recorded every 3 days for 30 days. Light had no effect on germination. Regardless of photoperiod the influence of light was nonsignificant (P = 0.45). On the other hand, temperature and stratification were significant (P = 0.0001) and there was a significant interaction (P = 0.001) between the two parameters. Averaged across all treatments, the highest total germination was realized at 35/25C (95/77F) (60%) followed by 30/20C (86/68F) (48%), 30C (86F) (37%), and 25C (77F) (31 %). Stratification was not a requirement for germination but stratification for 15 days increased the rate of germination but not total germination. However, stratification for 30 days decreased germination due to seed decay caused by fungal growth despite seed treatment with 1.3% sodium hypochlorite prior to stratification. Seed decay during germination was observed and treatments to reduce decay should be investigated since viability tests with 2,3,5-triphenyltetrazolium chloride (TTC or TZ) indicated that initial seed viability was >95%.
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.
Beach stabilization by replanting dune species such as Uniola paniculata L. (Sea Oats), is an accepted practice to control erosion in the southeastern United States. Increased restrictions on collection of sea oat seed and plant material for propagation is of increasing concern. Development of micropropagation protocols for establishment and production of sea oats from donor plants of known phenotype would be useful for selecting and producing plants with commercially valuable characteristics. Terminal and lateral shoot tips (3 mm wide and 4 mm high) from containerized plants were surface sterilized and established on Linsmaier & Skoog mineral salts and organics supplemented with 87.6 mM sucrose, 2.2 μM benzyladenine solidified with 0.8% TC® Agar. Terminal tiller shoot tips were more responsive than lateral shoot tips. Four monthly subcultures were. required for stabilized shoot multiplication from culture lines established from terminal tiller shoot tips. Shoot organogenesis frequently occurred from the cut leaf surfaces of subcultured shoot clusters. Microcuttings were established ex vitro in plug cells containing sand or vermiculite.
Many wetland plant species used for aquascaping and wetland revegetation projects are collected from donor wetland sites for planting elsewhere. Increased demand for wetland plants has lead to over-collection and subsequent environmental damage to these donor sites. Micropropagation provides an ecologically sound alternative to field collection and allows for production of under utilized wetland species and genotypes that are either slow-growing or difficult to propagate using conventional methods. Sagittarialatifolia Willd. (Duck-potato), a rhizomatous herbaceous wetland species, was established in vitro from surface-sterilized lateral and terminal rhizome shoot-tips cultured in liquid basal medium consisting of half-strength Murashige and Skoog mineral salts, 0.56 mM myo -inositol and 1.2 μM thiamine supplemented with 87.6 mM sucrose. Prior to multiplication, responsive Stage I cultures were indexed for cultivable bacteria and fungi. Shoot multiplication occurred in vitro through formation of multiple node rhizomes bearing terminal shoots. Duck-potato exhibited a high sensitivity to relatively low benzyladenine (BA) levels. Maximum rhizome and shoot production occurred from single shoot explants initially cultured on agar-solidified BM supplemented with 4.0 μM BA for 28 days. However, repeated subculture on BM supplemented with greater than 2.5 μM BA resulted in increased mortality, reduction in multiplication rate, or production of dormant corms. Consistent shoot multiplication (four to five shoots/explant) was possible in the presence of 1.5 μM BA. Maximum (100%) acclimatization and rooting was attained by direct sticking of Stage II microcuttings in soilless growing medium contained in 38 cell plugs. Production of salable plants bearing multiple rhizomes was possible within 6 weeks post-transplant. Preliminary observations indicate that corm formation in Sagittaria latifolia may be mediated by photoperiod.
The endemic Hawaiian species of Scaevola and Euphorbia grow in a wide variety of native habitats and exhibit a wide range of variation in photosynthetic responses. Light-saturated photosynthetic capacities range from 12.0 to 24.7 μmol CO2 m⁻⁻² s⁻⁻¹ in the Scaevola species and from 18.2 to 51.4 μmol CO2 m⁻⁻² s⁻⁻¹ in the Euphorbia species. Within each genus, differences in light-saturated photosynthetic capacity are paralleled by differences in mesophyll and leaf conductances to CO2. Within each habitat, the C4Euphorbia species exhibits a significantly higher photosynthetic capacity and a significantly higher mesophyll conductance than the corresponding C3Scaevola species. These differences are greatest in the dry scrub habitat and least in the wet forest habitat. One photosynthetic characteristic that exhibits little variation among the species within each genus, yet that exhibits a consistently large difference between the species within each habitat, is photosynthetic water-use efficiency. The C4Euphorbia species possess water-use efficiencies that are 2–3½ times as high as those of the C3Scaevola species, regardless of whether these species are native to very dry or very wet habitats. At present, the ecological significance of this large inherent difference in photosynthetic water-use efficiency is unknown. Indeed, it appears that neither photosynthetic pathway has imposed any major inherent constraints on the ability of the Scaevola and Euphorbia species to diversify into a wide variety of habitats.
Germination response following various periods of cold treatment and seedling response to temperature, daylength, and salinity were studied for several Atlantic and Gulf coasts populations of Uniola paniculata L. Results indicated that Atlantic coast Florida populations did not require cold treatment prior to germination at 95–65 F, but that populations from Virginia and North Carolina did. Gulf coast populations exhibited a germination response intermediate between those just mentioned. Seedling studies revealed that alternating diurnal thermoperiods with daytime temperatures of 80 F and above produced good vegetative growth in all populations with little preference for either short- or long-day conditions. Gulf coast populations produced the most biomass under all treatment conditions. Seedlings from a North Carolina and a Florida population indicated no difference in substrate salt tolerance. Salt tolerance was reduced in the higher temperature thermoperiod for both populations. Seedlings from these two populations produced more biomass in a salt spray treatment than in substrate salinity treatments.
Sea oats, Uniola paniculata, is a characteristic and often dominant perennial dune grass of the southeastern Atlantic and Gulf coasts of the United States. However, U. paniculata loses its dominance and becomes almost nonexistent along a relatively narrow portion of its range, from west of the Mississippi River delta, Louisiana, through northeastern Texas. In Louisiana, U. paniculata is abundant on the Chandeleur Islands east of the delta, but along the barrier island chain west of the delta only three sizable populations have been identified, all on the Caminada-Moreau coast. We measured seed production and germination response to various moisture and temperature combinations in these populations. Seed production was found to range from one to three orders of magnitude less than what has been reported in areas where U. paniculata is dominant. Rates ranged from 0.091 seeds/spikelet on the Chandeleur Islands to only 0.002 seeds/spikelet on the Caminada-Moreau coast; one population failed to produce any seeds at all. Seed germination in an 18.3–35.0 C alternating thermoperiod was high (65–88%) after exposure of the seeds to a variety of pregermination moisture and temperature conditions. Exposure to 4.4 C during imbibition did not increase percentage germination, but did significantly increase the rate of germination over the pregermination treatments that did not include moisture. The very low seed production rates of these populations could explain the limited distribution of this species along the Louisiana coast, where overwash events are frequent, and rates of coastline retreat often exceed the highest measured rates of vegetative lateral spread.