In Vitro Cellular & Developmental Biology - Plant

Recalcitrance to tissue culture is observed in some genotypes of Brassica napus. Several studies have confirmed that Pluronic F-68 has growth-promoting effects on numerous tissue types. This work investigated the effect of the non-ionic surfactant Pluronic F-68 at four concentrations (0.1%, 0.25%, 0.5%, and 1% (w/v)) on the responsiveness of recalcitrant B. napus lines to tissue culture. Microspores from seven populations of B. napus were cultured on Nitsch and Nitsch medium with this compound. The embryos obtained were plated on solid B5 medium supplemented with zeatin for shoot induction. Pluronic F-68 had a highly significant effect on the proportion of shoot regeneration (P < 0.05) in some of the recalcitrant populations. However, no strong dose–response effect was observed. The estimated probability of a shoot occurring in the absence of Pluronic F-68 ranged from 0.04 to 0.31 depending on the genotype, while in the presence of Pluronic F-68, it ranged from 0.07 to 0.53, respectively.

Previous investigations have demonstrated specific receptors and associated mitogenic actions for insulin and insulinlike growth factors I and II (IGF-I and II) in postnatal bovine aortic smooth muscle. Using fetal tissue we have observed different patterns of binding and action for these peptides. Smooth muscle cells isolated from near-term fetal bovine aortae were studied in early passage. Specific receptors for both IGF-I and IGF-II were identified. Specific binding averaged 5.7%/2.5×105 cells for IGF-I, and 16.2% for IGF-II, and 0.3% for insulin. High affinity K d for both IGF receptors were nanomolar. IGF-II was fivefold less potent than IGF-I in displacing IGF-I binding. IGF-I showed no affinity for the IGF-II receptor. Insulin, at physiologic concentrations, was incapable of displacing either IGF-I or IGF-II binding. Cellular incorporation of [methyl-3H]thymidine was stimulated at the lowest dose of IGF-I tested, 0.5 ng/ml. IGF-II showed no effect up to 100 ng/ml, after which a sharp increase in incorporation was noted. Insulin had a similar effect only at concentrations >0.5 μg/ml, with a maximal response noted at 5 to 10 μg/ml. Our results indicate that fetal bovine aortic smooth muscle cells have an abundance of IGF receptors but lack specific insulin receptors. In addition, IGF-II binding levels are three times higher than for IGF-I. These results are consistent with observations in other species, in which a predominance of IGF over insulin receptors has been demonstrated in fetal tissue, and provide further evidence for a role for the IGFs in embryonic cellular metabolism.

Nodal segment explants of Ilex paraguariensis, collected from greenhouse-grown plants, were found to contain endophytic bacteria. After culturing in bioreactors, 16 rRNA gene analyses and analytical profile index biochemical tests were used to identify these bacteria as Stenotrophomonas malthophilia. The presence of bacterial cells in the intercellular spaces of stem cortical parenchyma was detected in histological sections by scanning electron microscopy. A range of commercial isothiazolone biocides were tested for their ability to repress the growth of Gram-negative bacteria grown in liquid media during the micropropagation phase. The addition of 0.75 ml l−1 Delcide™ TG (5-chloro-2-methyl-4-isothiazolin-3-one + 2-methyl-4-isothiazolin-3-one, 1.05% and 0.45%, respectively) to the culture media resulted in 100% visibly clean cultures, with no suppression of shoot growth.

Allium tuncelianum (Kollman) Özhatay, Matthew and Şiraneci forms a single-cloved edible white bulb with a mild garlic (A. sativum) odor and taste. Its ability to form seeds makes it suitable for genetic improvement via classical and modern approaches. A detailed study was carried out to determine the gynogenic and somatic plant regeneration potential of two A. tuncelianum accessions (AT1 and AT2). Unopened flower buds of A. tuncelianum accessions were cultured in various BDS (Dunstan and Short, Physiol Plant 41:70-72, 1977) and MS (Murashige and Skoog, Plant Physiol 15:473-497, 1962)-based induction media. Accessions showed slight differences in their responses to gynogenesis and somatic shoot induction cultures. Gynogenic plantlets were obtained in six induction media (T2, T3, T6, T9, T12, and T15). Three of these (T2, T3, and T9) did not contain plant growth regulators (PGRs). Seventeen (0.09%) gynogenic plantlets were obtained from approximately 20,000 AT1 and AT2 flower buds used in gynogenesis induction experiments. The highest gynogenic plantlet production frequency (0.34%) in AT1 was achieved with buds cultured in T12 [MS-based medium with 1.0 mg L−1 α-naphthalene acetic acid, 8.0 mg L−1 isopentenyl adenine, and 100.0 g L−1 sucrose]. Flower buds of AT2 showed the best gynogenic response (0.44%) in T2 (PGR-free BDS–based medium with 50.0 g l−1 sucrose). Somatic regeneration was observed in six media (T5, T6, T11, T12, T14, and T15) containing at least 50.0 g L−1 sucrose and auxin- and cytokinin-type PGRs. Two hundred and thirty-three (1.18%) somatic plantlets were obtained from the cultured flower buds of AT1 and AT2. The highest somatic plantlet production frequencies for AT1 (4.44%) and AT2 (3.89%) were obtained from flower buds cultured in T5 [BDS-based medium with 2.0 mg L−1 2,4-dichlorophenoxyacetic acid, 2.0 mg L−1 6-benzylaminopurine, and 50.0 g L−1 sucrose]. Three induction media (T6, T12, and T15) provided both gynogenic and somatic regeneration in A. tuncelianum. Eight of 17 (46.06%) gynogenic plantlets and 33 of 233 (14.16%) somatic plantlets continued to grow, resulting in healthy plants with green leaves and well-established root systems. The remaining gynogenic plantlets had abnormal development. Flow cytometric analysis of the well-developed gynogenic plants showed that two were haploid (25%), four were diploid (50%), and two were mixoploid (25%) containing both haploid and diploid cells. The nine abnormal gynogenic plantlets were diploid. Results obtained from this study suggest that doubled haploid technology can be used in the production of homozygous A. tuncelianum inbred lines in breeding programs.

In a bid to better conserve endangered terrestrial orchids, we detail cryogenic research using a widely distributed terrestrial orchid, Caladenia latifolia, as a model species for development of cryopreservation for primary (seed generated) and secondary (adventitious) protocorms. Primary protocorm cryopreservation (using droplet vitrification) involved a number of experimental lines of inquiry: investigation of a suitable plant vitrification solution (PVS) by comparing three variants of a standard PVS (2, 3 and 4), determining the most suitable primary protocorm developmental stage for successful cryopreservation, testing the effectiveness of a preculture medium treatment prior to cryopreservation, and investigating temperature preconditioning at the preculture stage as well as different components of the recovery medium. Primary protocorms were generated using asymbiotic in vitro germination media developed by the authors specifically for the test species (half-strength MS macroelements and microelements with 5% (v/v) fresh filter sterilized coconut water). Secondary protocorms were propagated using an in vitro proliferation medium (½ MS with 5 μM α-naphthaleneacetic acid + 2 μM 6-benzylaminopurine). A modified preconditioning step was developed, involving incubation on ½ MS with 0.2 M raffinose for 48 h at 15°C instead of 20°C. The standard recovery medium (½ MS 1 μM zeatin + 0.5 μM gibberellic acid) was replaced after the first week following warming from liquid nitrogen (LN), with asymbiotic germination medium (½ MS + 5% (v/v) coconut water) for the remainder of the recovery phase. This new step increased the survival of primary protocorms from 68 to 85%. The average post-cryostorage regeneration of plants from primary protocorms increased from 17 to 48% after a 6-wk incubation. A similar protocol increased the survival of secondary protocorms from 63 to 84%. Regeneration of plants from secondary cryostored protocorms increased from 11 to 26% after 14 wk. The protocols developed here provide a useful template for advancing cryoconservation of other orchid taxa, particularly rare and threatened species.

The liver of rodents is sexually differentiated, i.e. the female liver differs from the male liver. This differentiation is largely controlled by the pattern of growth hormone (GH) secretion. We have attempted to maintain GH-dependent differentiation of cultured rat hepatocytes. We examined the level of alcohol dehydrogenase (ADH) activity, which responds to GH and is higher in female than in male liver, and the estrogen receptor, which is dependent on GH but is present in equal amounts in males and females. ADH activity was maintained in cells from male rats, but fell by 40% in cells from females in medium supplemented with insulin and dexamethasone. The estrogen receptor content of female cells fell dramatically to undetectable levels within 2 d of culture. Extensive supplementation of the medium failed to prevent the decrease in ADH activity in female cells; similarly, the addition of female sex steroids; rat serum; pituitary extracts; rat, human, or bovine GH; or ovine prolactin failed to maintain the enzyme activity. Insulin, dexamethasone, thyroid hormone plus GH or prolactin, or the combination of all five hormones also failed to prevent the loss of estrogen receptors. Short-term cultures of rat hepatocytes, although retaining the liver-specific expression of ADH at the male level, lose GH-dependent expression of the estrogen receptor and stimulation of ADH activity.

Anthranilate synthase (AS), a tetramer consisting of two α subunits and two β subunits, is the key control enzyme in the tryptophan (Trp) biosynthesis pathway. A naturally occurring α subunit of AS called ASA2 that is insensitive to Trp feedback inhibition was isolated from a tobacco suspension cell culture and has been extensively studied and used for both nuclear and plastid transformation. However, the obligate β subunit of AS had not been studied in tobacco. Therefore, the tobacco AS β subunit-encoding cDNA was cloned and its encoded protein was verified. Agrobacterium-mediated transformation of tobacco plants was performed, under the selection of the toxic Trp analogs, 7-methyltryptophan or α-methyltryptophan, with a construct containing both ASA2 and AS β subunit genes. Many transgenic plants overexpressing both subunits were identified and examined. Compared to the wild-type plants, the transgenic plants had higher levels of enzymatic activities for both holoenzyme and α subunit. The transgenic plants had 9 to 68 times the amount of free Trp as the wild-type plants, which was more pronounced than plants overexpressing ASA2 alone. This study demonstrates the potential of co-expressing AS α and β subunits as a robust plant transformation system as well as overcoming feedback inhibition to obtain high levels of Trp biosynthesis.

The kinds of data obtained in micropropagation studies are very often problematic, since they do not follow continous distribution and observations through culture vessels complicate measurement. Accordingly, standard analyses are often used, leading to misinterpretation of results. In this paper, we present a study of Viburnum opulus micropropagation using planned contrasts and fitting regression models in generalized linear models as an alternative statistical analysis of micropropagation results, and compare the results with that of traditional ANOVA. The advantages and possibilities of the alternative data analyses in plant tissue culture are discussed.