The effect of endophytic bacteria on in vitro shoot growth of Prunus yedoensis and its identification and elimination Abstract Tissue cultures of Prunus yedoensis, a famous ornamental tree species, are important to mass-propagate true-to-type plants. Within in vitro cultures, bacterial contamination of explants hinders the propagation of sustainable cultures. Negative effects of endophytic bacteria vary from leaf yellowing to shoot-tip necrosis, ultimately resulting in shoot death. Explants with an overgrowth of endophytic bacteria on basal parts showed inferior growth than explants with no bacterial growth. Shoot growth and contamination became severe as the shoots were subcultured. Two endophytic bacteria, Pantoea spp. and Curtobacterium spp., were identified from the in vitro shoots using 16S rRNA gene sequences. Bacterial susceptibility to antibiotics was screened using different antibiotics. Two antibiotics, cefotaxime and tetracycline, effectively controlled bacteria growth. Although both antibiotics killed the bacteria, tetracycline adversely affected plant growth by reducing shoot growth. A broad range of cefotaxime concentrations had no toxic effects to the in vitro plants. However, concentrations between 50 and 150 mg/L showed no difference in eliminating bacteria. Shoot growth, induction, and elongation were greater in a medium supplemented with cefotaxime compared with a control (without antibiotics) and a medium amended with tetracycline.

Euclidean distance can recognize the Biojas ® concentration that produces the ideal physiological status of pineapple in vitro plantlets Abstract Biojas® is a fermented broth of Lasiodiplodia theobromae, a jasmonic acid–producing fungus characterized as a plant growth regulator and as biological control of phytopathogenic microorganisms and pests. The present work describes the use of Biojas® in in vitro culture of pineapple. On the other hand, plant scientists usually record multiple indicators in their experiments. The common statistical data evaluations involve univariate analyses such as t test, Mann-Whitney, and analysis of variance (ANOVA) followed by Tukey’s HSD. Such analyses do not evaluate integrally the effects of the experimental treatments because each indicator is analyzed independently. For this reason, we explored in this study the Euclidean distance combined with the data of the Biojas® treatment (0–2 mg l−1) on pineapple in vitro plantlets as an integrating indicator. Plant height; number of leaves; D leaf length, width, and area; diameter of the plant base; fresh and dry weights of the plant; levels of chlorophylls; transpiration rate; CO2 assimilation; and water use efficiency were recorded. Several statistically significant differences among Biojas® treatments were recorded. However, the most significant effects of Biojas® treatments were only noted in the plant height, length and area of D leaf, and water use efficiency. Variables mentioned above increased until 1.0 mg l−1 Biojas® and decreased with high levels of Biojas®. Calculation of the Euclidean distance from each Biojas® level to the ideal physiological status of the pineapple plantlets revealed that 1.0 mg l−1 Biojas® produced the pineapple plantlets with the best physiological status.

Modifying sugarcane mineral levels through sodium chloride and mannitol exposure in temporary immersion bioreactors Abstract Temporary immersion bioreactors (TIBs) have been shown to be useful for studying plant stress physiology and inducing chemical mutagenesis. This study describes the effects of exposure to NaCl (salt stress) and mannitol (osmotic stress) within TIB on sugarcane mineral levels in vitro. Shoots were exposed to concentration of NaCl (89.4 mM) and mannitol (123.1 mM) previously shown to result in a 50% reduction in multiplication rate for 30 d. Thereafter, shoot multiplication rate, shoot cluster fresh weight, and levels of selected minerals were measured. Using ICP-OES, the following minerals were quantified: Na, Ga, Mn, Cr, K, Zn, Ca, Li, Mg, Sr, Co, B, Fe, S, P, Al, Ba, and N. Both NaCl and mannitol decreased shoot multiplication by c. 53% and except for Al and Ba altered mineral levels significantly relative to the control: Na accumulation increased markedly (six-fold in NaCl treatment); levels of Ga, Mn, Cr, K, and Zn changed moderately; Ca, Li, Mg, Sr, Co, B, Fe, S, P, and N levels changed to a limited extent. In terms of the minerals that were most affected, Ga, Mn, K, and Zn levels declined under both stresses; Cr appears to be the only exception, having decreased under the salinity stress and increased under the osmotic stress. Our results suggest that both stresses not only affect growth in the same manner and degree but also appear to have similar effects on the physiological mechanisms that modulate mineral levels at the cellular level under stress conditions. Sugarcane growth inhibition appeared to be mainly due to turgor loss under mannitol-induced stress and the accumulation of Na ions under salt stress. Stress resistance in this species is most likely promoted by the retention of a “safe” water status, a high amount of K and Ca, and a low level of Na.

Growth and development of carnation ‘Dreambyul’ plantlets in a temporary immersion system and comparisons with conventional solid culture methods Abstract The aim of the current study was to compare the effects of the culture method—conventional solid medium culture and temporary immersion system (TIS)—on the growth and development of carnation ‘Dreambyul’ plantlets. At the same time, different immersion intervals and immersion durations of TIS culture were also tested to find the optimal setting for mass production of high-quality carnation plantlets in vitro. In the first experiment, the results showed that the shoot length, root length, and number of nodes of plantlets cultured in the TIS were highest when the immersion interval was 8 h. Compared with that of plantlets cultured in the conventional solid medium culture, the fresh weight of plantlets cultured in the TIS was at least 3 times greater. The greatest total chlorophyll content, stomata with normal shapes was observed for plantlets grown in the TIS with an 8-h immersion interval. The lowest H2O2 level was recorded in plantlets cultured with the 8-h immersion interval. In the second study, growth traits such as the shoot length, root length, and stem diameter, as well as the number of shoots and roots tended to increase with immersion durations, and reached their peaks when the immersion duration was 90 s. Excessive water accumulation in tissues and a higher incidence of hyperhydricity were observed in plantlets where the immersion duration was 120 and 150 s. These findings suggest that an immersion interval of 8 h, combined with an immersion duration of 90 s, could be the optimal setting for growth and development of carnation ‘Dreambyul’ plantlets cultured in the TIS.

Histo-morphological analysis of rice callus cultures reveals differential regeneration response with varying media combinations Abstract Genetic transformation of most indica rice (Oryza sativa) cultivars is hampered by poor in vitro culture performance and low regeneration potential. Histological study of primary calli can provide substantial information on their regeneration potential and can be used for early grading of calli expected to develop plantlets on regeneration media. The study was aimed to undertake histological analysis of primary calli derived from mature seeds of five indica rice cultivars viz. KSK-133, KS-282, Shaheen Basmati, Super Basmati, and DilRosh in order to assess their regeneration potential on different media combinations supplemented with various hormone concentrations (N6 + 2 mg/L 2,4-Dichlorophenoxyacetic acid; N6 + 2 mg/L 2–4 D + 2 mg/L Benzylaminopurine and MS + 2 mg/L 2,4-D). Calli with regeneration capability were subjected to histological assays by examining toulidine blue stained 5–8 μm thin sections for the presence of meristematic zones exhibiting embryogenic callus features. Based on our observations, formation of embryoids or embryoid-like structures was pronounced in KSK-133 and KS-282 calli. However, DilRosh, Super Basmati and Shaheen Basmati did not show these characteristic features. Three-week-old calli of all rice cultivars were transferred into regeneration medium (MS + 2 mg/L BAP + 1 mg/L Naphthaleneacetic acid). KSK-133 and KS-282 showed the highest regeneration potential (81% and 76%, respectively). These data were supported by histological observations where characteristic embryogenic units (EU) were noticed in these genotypes. These meristematic regions displayed high mitotic activity and stained relatively dark. The embryogenic calli cells were found heavily cytoplasmic with prominent nuclei and were located on the callus surface or inside surrounded by parenchymal cells.

Effects of sodium alginate elicitation on secondary metabolites and antioxidant activity of safflower genotypes under in vitro salinity stress Abstract The present study was aimed at investigating the effects of different concentrations of sodium alginate (NaAlg) (0.075 and 0.15% (w/v)) on the production of secondary metabolites (SMs) and antioxidant activity of seven safflower genotypes under in vitro salinity stress. The results showed that total phenolic content (TPC), total flavonoids (TFD), total flavonols (TFL), anthocyanin (Ant), total antioxidant capacity (TAC), phenylalanine ammonia-lyase (PAL), catalase (CAT) activity, and lipid peroxidation significantly increased under salinity stress consisting of the concentration of 1.5% (w/v) of NaCl, but callus growth traits decreased. The highest amount of TPC, Ant, and callus growth traits was observed under the elicitation of the sample with the concentration of 0.075(%) NaAlg under salinity stress, but the highest amount for TFD, TFL, CAT, PAL, and TAC was observed under elicitation of the sample with the concentration of 0.15% of NaAlg under salinity stress. This indicated the superiority of NaAlg for elicitation to increase SMs in safflower under salinity stress. Overall, the results showed that genotypes of Mex.22-191 and GE62918 could be processed to produce SMs by eliciting NaCl in safflower as an important medicinal plant at cellular level. A positive and significant correlation between CAT and TPC was observed and indicates that phenolic compounds are the major contributors to the antioxidant potential in safflower. This new elicitor introduced new ways to select and exploit the best NaAlg concentration to develop SMs that are of tremendous importance in terms of commercial purposes along with medical features in safflower at cellular level.

Morphogenesis and in vitro production of caffeoylquinic and caffeic acids in Baccharis conferta Kunth Abstract We established a protocol for the in vitro propagation of Baccharis conferta Kunth. This plant is used to treat gastrointestinal problems, cramps, pain, respiratory problems, and insect bites. A high rate of shoot multiplication was obtained from nodal segments on Murashige and Skoog (MS) culture medium. The shoots regenerated roots without exogenous plant growth regulators (PGRs). All explants of wild leaves on MS medium containing 5 μM of thidiazuron (TDZ) produced friable callus. An organogenic response was achieved after 3 wk of culture when callus segments were transferred to MS medium containing a combination of plant growth regulators (PGRs): either (i) 5 μM indole butyric acid (IBA) + 5 μM kinetin (KIN) or (ii) 0.5 μM IBA + 1.10 μM benzylaminopurine (BAP). The morphogenetic responses of callus were characterized by scanning electron microscopy. Shoots regenerated from callus and formed roots on MS medium without PGRs. The micropropagated plantlets and the organogenic callus showed similar chemical profiles in HPLC-mass spectrometry analyses. The main compounds present in the cultures were caffeoylquinic acids. Only plantlets contained small amounts of triterpenes (erythrodiol and ursolic acid). These findings will be useful for the micropropagation of this important native resource, and for further studies on its biology.

In vitro propagation and cryopreservation of Thuja koraiensis Nakai via somatic embryogenesis Abstract Korean arbor vitae (KAV; Thuja koraiensis Nakai) is a critically endangered coniferous tree in Korea. Here, we report the somatic embryogenesis (SE) and cryopreservation system that can be used for micropropagation of KAV and long-term storage of KAV cultures. To induce SE in KAV, the influence of the developmental stage of zygotic embryos and the effect of basal medium on embryogenesis induction were examined. The developmental stage of zygotic embryos had a significant effect on the embryogenesis induction (P < 0.0001). The highest frequency of embryogenesis induction occurred in megagametophytes with zygotic embryos at precotyledonary (P) and late embryogeny (L1) stage (36%). The highest frequency of embryogenesis induction was obtained on initiation medium containing IM basal salts with 2.2 μM 6-benzylaminopurine and 4.5 μM 2,4-dichlorophenoxyacetic acid (35%). The effect of abscisic acid (ABA) on production of somatic embryos was tested. The highest number of somatic embryos per 50 mg of embryogenic tissue was achieved on maturation medium with levels of 100 μM ABA (24.0 ± 2.4). The effect of cryopreservation treatment to embryogenic tissues on the maturation capacity of somatic embryos was also tested. No significant differences between noncryopreservation and cryopreservation treatment were observed (P = 0.1896), and the highest mean number of somatic embryo per 50 mg of embryogenic tissues was obtained in noncryopreserved cell line (28.17 ± 5.66). Finally, the genetic identities of the plantlets regenerated from non- and cryopreserved embryogenic cell lines were verified and there was no genetic variation in the regenerated plantlets from cryostored embryogenic cell lines. This study is the first report on SE and the successful cryopreservation of embryogenic culture of the genus Thuja.

A methodology for large-scale Athyrium sheareri gametophyte proliferation and sporophyte production using tissue culture Abstract Tissue culture methods using gametophytes are considered the easiest ways to mass-produce fern sporophytes. The aim of this study was to develop a practical propagation method for the ornamental fern, Athyrium sheareri. The gametophytes obtained from in vitro spore germination were used as experimental materials. We used the chopping method to investigate the culturing conditions for proliferating gametophytes and the blending method for evaluating the mass production of sporophytes in mixed soil. Gametophyte proliferation was determined via Knop medium, various concentrations of Murashige and Skoog (MS) basal medium (1, 1/2, 1/4), and media components (sucrose, nitrogen source, and activated charcoal). The fresh weight of the gametophytes increased by more than 24-fold in 1/2 MS medium. In addition, 1 g of gametophyte could produce a maximum of 255.3 sporophytes in a mixed soil of 7.5 cm2 area. Treating gametophytes with exogenous plant growth regulators promoted the formation and growth of sporophytes. The cultivated young sporophytes were acclimated and successfully grown in greenhouses. We developed a mass production protocol for A. sheareri sporophytes suitable for field application, which is expected to have commercial value.

Is the PLC pathway involved in the response to phenol treatment in tobacco hairy roots? Abstract Phospholipids and phospholipases play important roles in several cellular processes and responses to adverse growth conditions. However, the mechanism of action of phosphatidylinositol-specific phospholipase C (PI-PLC), and its resulting minor lipid function in response to pollutant-induced stress, remains to be elucidated. In this work, we studied the effects of phenol treatment on the PLC pathway, using two lines of hairy root cultures (HRs) from Nicotiana tabacum as plant model systems: wild-type (WT) and double-transgenic (DT) HRs. We quantified several product formations such as PIP, PIP2, DGPP, and PA, which are mainly synthesized by specific lipid kinases belonging to the PLC pathway. In both HRs, phenol treatment significantly increased the formation of these compounds in a differential manner. In WT HRs, PA formation was twofold higher than in control. PIP2 and PIP levels were about onefold higher than those of the controls while DGPP levels increased by 50%. In DT HRs, PIP levels were onefold higher than those of the controls while PIP2, DGPP, and PA levels increased by 120%. Phenol treatment also upregulated the PLC4 gene expression mainly in the first hours of exposure in both HRs, while the DGK1 gene expression was only upregulated in WT HRs after 24 h of treatment. These results show an active participation of the PLC pathway under phenol treatment suggesting that this signal pathway could be important in plant cell responses to phenol-induced stress.