Effect of cold cathode fluorescent lamps on growth of Gerbera jamesonii plantlets in vitro

The goal of this study was to evaluate the effect of cold cathode fluorescent lamps (CCFLs) on the growth of Gerbera jamesonii var. ‘Rui Kou’ plantlets in vitro in six different light quality ratios: 100% red CCFL (R), 80% R + 20% blue CCFL (B), 70% R + 30% B, 60% R + 40% B, 100% B and white CCFLs (W). Control radiation was provided by conventional heat-generating plant growth fluorescent lamps (PGFLs). Plantlets under CCFLs showed better plantlet height, SPAD value (i.e., leaf chlorophyll content) and root activity (as assessed by root dehydrogenase activity) than those growing under PGFLs while all other growth parameters were comparable with plants under conventional lighting systems.     

Red:far-red light ratio and far-red light integral promote or retard growth and flowering in Eustoma grandiflorum (Raf.) Shinn

Night break treatment was applied to Eustoma grandiflorum ‘Nail Peach Neo’ using light sources with different red (R: 660 ± 30 nm): far-red (FR: 730 ± 30 nm) ratios or FR light intensities in order to investigate growth and flowering responses. Flower initiation and induction were promoted by night break treatment with a low R:FR light source, but was delayed by a high R:FR ratio. The promotion or delay of flower bud formation was accompanied by a decrease or an increase, respectively, in the number of nodes on the main stem at anthesis to the first floret. The difference between date of visible bud with plants under night break treatment and that of the control was approximated with high accuracy by a sigmoid function of the logarithms of R:FR ratio. The threshold R:FR ratio demarcating the promotion and delay of date of visible bud was about 5.3 under the experimental conditions used. The critical R:FR ratios for promotion or delay of visible bud would be about 0.5 and 50.0, respectively. In addition, the time from planting to visible bud was approximated with an exponential function of FR light intensity. The maximum acceleration of date of visible bud by long-day treatment would be 20 days, and the critical FR light intensity would be 2.0 μmol m⁻² s⁻¹. It is concluded that growth and flowering of E. grandiflorum can be regulated by long-day treatment using light sources with different R:FR ratios or FR light intensities.     

Cryopreservation of Limonium serotinum apical meristems from in vitro plantlets using droplet-vitrification

In this study in vitro shoot tips of a Sicilian genotype of Limonium serotinum were successfully cryopreserved using the droplet-vitrification technique. Growth recovery of cryopreserved shoot tips was possible only when samples were pretreated for 16 h in liquid medium with 0.3 M sucrose, then for 5 h in liquid medium with 0.7 M sucrose before performing the cryopreservation protocol. Optimal conditions included treatment for 20 min in a loading solution containing 1.9 M glycerol + 0.5 M sucrose, treatment with vitrification solution B5 (glycerol 40.0%, sucrose 40.0%, w/v) for 60 and 90 min or vitrification solution A9 (glycerol 30.0%, dimethylsulfoxide 20.0%, ethylene glycol 20.0%, sucrose 15.0%) for 20 min, rapid cooling in minute droplets of vitrification solution, rapid rewarming by immersion for 20 min in unloading solution containing 1.2 M sucrose. Under these conditions, 37% recovery of cryopreserved shoot tips was achieved. Regrowth of cryopreserved samples was slow but always direct, without callus formation.     

Light quality affects in vitro adventitious rooting and ex vitro performance of cherry rootstock Colt

The effect of light quality on in vitro rooting of cherry rootstock “Colt” was studied to evaluate adventitious root development, ex vitro acclimatization and plant growth performance. Adventitious rooting was dependent on the light quality to which the microcutting were exposed. Highest number of roots per microcuttings was recorded under dichromatic light (blue + red). This response could be ascribed to the control of a strong synergistic interaction between phytochromes and blue light photoreceptors. Red light was more effective on root elongation than blue light. In this response a strong synergistic interaction between phytochromes and blue light photoreceptors was suggested. The effect of light quality on the number of root/explant affected the plant during acclimation, scoring the highest level of plant survival in the blue-, red- and blue + red-exposed plantlets. The light quality effect was also observed under greenhouse culture conditions, as shown by growth parameters at the end of six months in plants growth. No specific role was observed for the photoequilibrium of phytochrome. The results reported in this work show that plantlets exposed to different light quality, during the in vitro rooting phase, retain the light quality effects in the subsequent plant acclimatization and greenhouse plant growth phase.     

Screening of vincristine yield in ex vitro and in vitro somatic embryos derived plantlets of Catharanthus roseus L. (G) Don.

Catharanthus roseus L. (G.) Don. (Apocynaceae) is an important dicotyledonous medicinal plant as it is the sole source of vincristine and vinblastine that are used against a variety of cancers. Quantitative estimation of vincristine was carried out using high performance liquid chromatography (HPLC) in various in vitro grown tissues; calluses (embryogenic and non-embryogenic callus), embryogenic stages (proliferated, matured and germinated embryos), somatic embryo derived plantlets (leaf, root and whole plant) and leaves of ex vitro developed plantlets. The yield in those in vitro and ex vitro-developed tissues was monitored for 30 weeks. Except at an early lag period, vincristine production was detected up to 20–25 weeks old plant samples. Vincristine content was very high in leaf callus and germinated embryos. Leaves of in vitro raised plants showed higher amount of vincristine when compared to ex vitro-developed leaves of similar age. Vincristine production was tissue specific and age dependent that was discussed in detail in this present communication.     

Chitosan specificity for the in vitro seed germination of two Dendrobium orchids (Asparagales: Orchidaceae)

The effects of different types of chitosan on seed germination and protocorm development were determined for two orchid species, Dendrobium bigibbum var. compactum and Dendrobium formosum. Six chitosan types derived from polymer or oligomer chitosan each with 70, 80 or 90% levels of deacetylation (P70, P80, P90, O70, O80 and O90, respectively), were evaluated as direct medium supplements at 0, 10, 20, 40 or 80 mg/L in modified VW medium by following seed germination and protocorm growth for 12 weeks. Chitosan of all six tested types and four concentrations were found to significantly enhance the proportion of D. formosum seeds that germinated, when compared to these germinated without chitosan. In contrast, chitosan caused no enhanced germination rate was noted for D. bigibbum var. compactum with all tested chitosans and doses tested. However, almost all types of chitosan at 10 mg/L, except O90, were able to significantly improve the growth of D. bigibbum var. compactum protocorms, whilst 10 or 20 mg/L of P70 chitosan was the best formula to enhance the growth of D. formosum protocorms. It is concluded that chitosan responses in seed germination and protocorm development were somewhat species and developmental stage dependent. Therefore, the appropriate chitosan application for each plant species should be evaluated first before use.     

The effects of temperature on in vitro pollen germination and pollen tube growth of Pistacia spp.

The pollen germination and pollen tube growth among nine Pistacia genotypes was quantified in order to identify differences in the tolerance of pollen to temperature variations. The effect of temperature on in vitro pollen germination and pollen tube growth was investigated in Pistacia vera (Uygur, Atli, Kaska, Sengel, Kavak), P. atlantica, P. khinjuk, P. terebinthus and P. palaestina. When pollen was incubated in a germination medium for 24 h in darkness, distinct differences were observed in pollen germination and pollen tube growth at different temperatures.     

The effect of nitrogen source and concentration,medium pH and buffering on in vitro shoot growth and rooting in Eucalyptus marginata

This work examined the effect of nitrogen source and medium buffering on the micropropagation of Eucalyptus marginata Donn ex Sm. The number of shoots was increased when media contained 2-(N-morpholino) ethanesulfonic acid (MES) but this increase was minor and only applied to one of the two clones tested. Highest root production was obtained when the medium contained 7.5 mM nitrogen in a ratio of 2NO₃⁻:1NH₄⁺ and was buffered with 10 mM MES. In the rooting medium the pH was influenced most significantly by the nitrogen source, and then whether the medium was buffered. The media pH remained relatively constant when nitrate was the sole nitrogen source and this was assisted by the addition of 10 mM MES. Lower concentrations (<10 mM) of MES were less effective in buffering media over a four-week culture period in both shoot multiplication and rooting medium.     

Elimination of a new ampelovirus (GLRaV-Pr) and Grapevine rupestris stem pitting associated virus (GRSPaV) from two Vitis vinifera cultivars combining in vitro thermotherapy with shoot tip culture

A new virus species designated as Grapevine leafroll associated virus-Pr (GLRaV-Pr), which is classified in a distinct phylogenetic group of the genus Ampelovirus (Closteroviridae), was recently characterized from Greek grapevine cultivars. Elimination studies of GLRaV-Pr were carried out in two grapevine cultivars, ‘Mantilaria’ and ‘Prevezaniko’, co-infected with Grapevine rupestris stem pitting associated virus (GRSPaV, Flexiviridae). Both viruses were detected by nested RT-PCR assays. Virus elimination was achieved by combining in vitro thermotherapy with meristem (≤0.2 mm) or shoot tip culture (≤0.5 cm). The survival and regeneration rate of meristems was very low. On the other hand, high survival rates were observed in the cultured shoot tips accompanied with high elimination rates for both viruses. Data obtained in this study indicate that virus elimination depends on the genotype of grapevine. The results confirmed that sanitation is easier for species of the Closteroviridae family than for GRSPaV, whereas it seems that eradication of GLRaV-Pr and GRSPaV is feasible even with larger plant tissue parts if combined with an appropriate thermotherapy profile in vitro.     

Regeneration and characterization of plants derived from leaf in vitro culture of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars

In the current work attempts were made to investigate culture of leaf explants derived from in vitro seedlings of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars, Bingtangcheng and Valencia. Effects of several factors, including culture medium, lighting condition, explant age and genotype on regeneration response were examined based on three parameters, percentage of explants producing shoots, mean number of shoots per explant and shoot forming capacity. Culture of the explants on shoot-inducing media (SIM) composed of MT salts supplemented with different growth regulators gave rise to disparate shoot regeneration, in which SIM₁ (MT + 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ Kinetin + 0.1 mg L⁻¹ NAA + 3% sucrose + 0.8% agar, pH 5.8) was shown to be the most effective medium for direct induction of shoots from leaf explants. Highly significant difference in the response of shoot bud regeneration was noted between the two cultivars, with Bingtangcheng being more responsive than Valencia. Culture of explants from fully developed leaves led to better shoot regeneration capacity in comparison to undeveloped ones. However, the two lighting conditions used herein did not cause significant difference in shoot regeneration. Phenotypic observation and randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants from both genotypes were genetically identical to their donor plants, suggesting absence of detectable genetic variation in the regenerated plants. The data presented here demonstrated that direct initiation of plants from leaf explants has been successfully accomplished. To our knowledge, this is the first report on direct regeneration of shoots from leaf explants in Citrus, which will provide an alternative source for citrus genetic manipulation in the future.     

Effect of carbohydrates on in vitro low-temperature storage of shoot cultures of apricot

Procedures for cold storage of in vitro cultures can delay subculturing, reducing production costs and risks of contamination and somaclonal variation. The present work investigates the effects of media with sorbitol (116.8 mM, medium SO) or sucrose (58.4 mM) alone (medium SU), or the latter in combination with mannitol (58.4 mM, medium M) on 7-month storage at 5 °C of apricot shoots, cv San Castrese and Boreale. Shoots in SO survived in lower percentages and grew less than in the other treatments during storage, and died in large numbers after transfer to standard culture conditions. In comparison to other treatments, survival was 100% in the presence of M and both shoot weight and number of surviving proliferated axillary shoots was increased. Moreover, M improved regrowth compared to SU under standard culture conditions. The SOD and CAT activity confirmed the higher stress of shoots stored in SO than controls, and in contrast, the low stress of shoots in M.     

Towards crop improvement in bell pepper (Capsicum annuum L.): Transgenics (uid A::hpt II) by a tissue-culture-independent Agrobacterium-mediated in planta approach

Agrobacterium tumefaciens-mediated transformation has been one of the methods used to generate transgenic plants in bell pepper. An alternate transformation method that avoids/minimizes tissue culture would be beneficial for the improvement of bell pepper due to its recalcitrant nature. In this report, transgenic bell pepper plants have been developed by a tissue-culture-independent A. tumefaciens-mediated in planta transformation procedure. In the present study, two open pollinated varieties viz., Arka Gaurav and Arka Mohini were used for transformation. Agrobacterium strain EHA105 harboring the binary vector pCAMBIA1301 that carries the genes for β-glucuronidase (uid A) and hygromycin phosphotransferase II (hpt II) was used for transformation. GUS histochemical analysis of T₀ and T₁ plants at various stages of growth followed by molecular analysis using PCR, Southern analysis and RT-PCR allowed selection of transgenics. The method resulted in 17.8% and 11.4% of the T₀ plants in Arka Gaurav and Arka Mohini being selected as chimeric and 35.0% and 29.7%, respectively, were identified as stable transformants in the T₁ generation based on PCR analysis.     

Plant regeneration from in vitro cultured leaves of Lanzhou lily (Lilium davidii var. unicolor)

Lanzhou lily (Lilium davidii var. unicolor) is one of the best lilies which are edible in China but the efficient shoot regeneration system has not been developed. The purpose of the present study is to establish an efficient and reproducible protocol for induction of shoots in vitro from L. davidii var. unicolor leaves. Shoot regeneration from in vitro cultured leaves of L. davidii var. unicolor was tested on the 26 media based on NN [Nitsch, J.P., Nitsch, C., 1969. Haploid plants from pollen grains. Science 163, 85–87] basal medium, containing different concentrations of thidiazuron (TDZ) in combination with different concentrations of α-naphthaleneacetic acid (NAA). Shoot organogenesis occurred directly from the leaves without forming callus. Shoot regeneration mainly occurred from the cuts across the midvein and the base of the leaf explants. The highest frequency of regeneration (93.3%) and the largest number of shoots per leaf (3.83) were obtained on NN basal medium supplemented with 0.5 mg l⁻¹ TDZ and 1.0 mg l⁻¹ NAA. All the regenerated shoots formed complete plantlets on half-strength MS [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473–497] basal medium containing 0.1–0.5 mg l⁻¹ indole-3-butyric acid (IBA) with in 30 days, and 92% of the regenerated plantlets survived in the soil. This study will be useful for Agrobacterium-mediated transformation and exploitation of somaclonal variation of Lanzhou lily.     

In vitro propagation using adventitious buds technique as a source of new variability in chrysanthemum

Eleven cultivars of Chrysanthemum × grandiflorum (Ramat.) Kitam.: ‘Richmond’ and its 10 radiomutants, representing the Lady group, were propagated in vitro with shoot tips and leaves as explants. The aim of this study was to investigate if the explant type used for micropropagation affects the genotype and phenotype of chrysanthemums. Plants grown from shoot tips and adventitious buds formed on leaves were rooted in vitro, acclimatized and cultivated in glasshouse up to full-flowering. The colour and shape of inflorescences of plants obtained from two different explant types were compared within the cultivars. All plants derived from shoot-tip explants showed the inflorescence colour and shape typical for the cultivars. Inflorescence colour of plants derived from adventitious buds were true-to-type in four cultivars: ‘Richmond’, ‘Lady Amber’, ‘Lady White’ and ‘Lady Yellow’. All plants of ‘Lady Apricot’ (originally: golden beet) and ‘Lady Salmon’ (salmon) propagated from adventitious buds technique showed altered inflorescence colour (respectively: purple gold; pink and white). ‘Lady Bronze’ (originally: reddish brown), ‘Lady Orange’ (orange brown) and ‘Lady Rosy’ (purple gold) propagated with adventitious buds had both typical and changed inflorescence colours (respectively: yellow; yellow and red; reddish pink). ‘Lady Vitroflora’ showed altered number of ligulate florets grown into tubes in inflorescence when propagated with shoot tips and leaves as explants. Those changes might be an effect of either chimeral structure or somaclonal variation of the plants investigated. The variation appears only if non-meristematical explants were used. The adventitious buds technique might be useful in chrysanthemum breeding as a source of a new variability.     

Effect of Glomus mosseae and Entrophospora colombiana on plant growth,production, and fruit quality of ‘Maradol’ papaya (Carica papaya L.)

The effect of inoculating ‘Maradol’ papaya plants with arbuscular mycorrhizal fungi (AMF) Glomus mosseae (GM) and Entrophospora colombiana (EC) was assessed. The results showed that both mycorrhizae species increased the number of fruits and yield in papaya plants by 41.9 and 105.2% for GM and 22.1 and 44.1% for EC, respectively, with respect to control plants. GM significantly increased plant height. Sugar content, firmness, color (°Hue), and ripening process of mycorrhized plant fruits were similar to those of the control. Weight loss of mycorrhized plant fruits was considerably less than that of the control. Inoculation of papaya with AMF is recommended, particularly with GM since it increases yield, and fruit weight (45.1%), furthermore, it reduced fruit weight loss during ripening.     

Aluminium-induced effects on growth,morphogenesis and oxidative stress reactions in in vitro cultures of quince

The effects of Al³⁺ [supplied as Al₂(SO₄)₃·18H₂O] addition to culture media (pH 4.0) on growth, morphogenesis (in leaf explants), and oxidative stress reactions in in vitro cultures of ‘BA 29’ quince were investigated. Aluminium (Al 0.5 mM) strongly inhibited shoot growth in the proliferation and rooting phases (Al 2.2 mM), reduced shoot proliferation (Al 1.1 mM), and induced tissue browning. Superoxide dismutase (SOD) activity was increased in shoot cultures supplemented with 2 mM Al. Malondialdehyde (MDA) content of shoots was strongly increased by Al during proliferation (starting from Al 1.7 mM) and rooting (already at Al 1.1 mM), thus serving as a good ‘marker’ for Al toxicity. Even a low concentration of Al (0.5 mM) in the shoot induction medium was found to inhibit shoot regeneration. When standard (Al 0) shoot induction medium was used, leaf explant growth was only reduced by 2.2 mM Al in the subsequent growth phases. Following a preliminary selection for their growth on Al-enriched media, 82 potentially Al-tolerant quince somaclones were selected for further trials.     

Auxin-dependent development and habituation of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures

The influence of IAA (1.0 mg dm⁻³), and IBA (1.16 mg dm⁻³), on the development of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures was examined. Depending on the kind of auxin and 2iP concentration in vitro cultures consisted of various number of axillary (AX) and adventitious (AD) shoots. Three different categories of AD shoots were found: leaf shoots (AD-L), node-adjoin shoots (AD-P), and base-adjoin shoots (AD-M, madshoots). The AX shoots were the least habituated (towards auxins, cytokinins and vitamins) whereas the AD-M shoots (madshoots) the most. In comparison to IAA, IBA caused dying or callusing higher number of initial explants. However, IBA generally suppressed development of AD shoots, especially madshoots whereas slightly weakened multiplication of AX shoots. IBA significantly enhanced elongation of AX shoots also. Axillary shoots obtained on IBA-media had relative long internodes and rigid, well-developed leaves. The adventitious shoots, especially base-adjoin (AD-M) ones, were easily distinguishable as were more thin and fragile, more or less vitrified, and had short internodes and smaller, sometimes unfolded leaves. Development of blueberry in vitro cultures on auxin-free and IAA-supplemented media was similar. AX shoots grown on such media resembled AD shoots. 2iP applied in higher doses along with IAA promoted much proliferation of AD than AX shoots. In contrast, 2iP applied in higher doses together with IBA stimulated significantly only growth of AX shoots whereas in general, development of adventitious shoots was not affected. Micropropagation carried out through routine method based on subculturing of shoot explants or shoot clumps on the medium supplemented with IAA (4 mg dm⁻³) and 2iP (10–15 mg dm⁻³) as well as stimulation of shoot elongation on the blank medium causes in fact the propagation of highbush blueberry through highly habituated adventitious madshoots. Replacement of IAA by IBA facilitates micropropagation of highbush blueberry cv. Herbert through axillary shoots.     

African spider flower (Cleome gynandra L./Gynandropsis gynandra (L.) Briq.) as a red spider mite (Tetranychus urticae Koch) repellent in cut-flower rose (Rosa hybrida L.) cultivation

Companion planting of Cleome gynandra, of Kenyan origin, in beds of cut-flower roses reduces significantly red spider mite (Tetranychus urticae Koch) infestation without any detrimental effect on productivity or flower quality. The level of reduction is dependent upon the density of the C. gynandra plants with 15 plants in a 1.8 m² bed (8.3 plants m²) being the most effective, planted either around the bed perimeter or within the rows of roses. The relatively high density of C. gynandra plants required may limit the direct application of this technology in export-focused, greenhouse rose production yet may be of significant value as a supplement to other mite-control strategies. The potential benefits of such companion planting for growers of field roses and those involved in some domestic markets are also evident. Research into the nature and extraction of the active, volatile mite-repellant components of C. gynandra is indicated.     

Effects of NaCl stress on red raspberry (Rubus idaeus L. ‘Autumn Bliss’)

The increasing interest in the cultivation of red raspberry (Rubus idaeus L.) in warmer climates and in the mild-Southern parts of Europe requires better understanding of the salinity effects especially in the primocane-fruiting type cultivars. The aim of this work was to study the response of the ‘Autumn Bliss’ primocane-fruiting cultivar in elevated NaCl concentrations.     

Asymbiotic seed germination,mass propagation and seedling development of Vanda coerulea Griff ex.Lindl. (Blue Vanda): An in vitro protocol for an endangered orchid

The application of modern biotechnology for conservation of any endangered species requires an efficient in vitro regeneration protocol. In this study a reliable protocol was developed for in vitro seed germination, protocorm multiplication and subsequent plantlet regeneration of Vanda coerulea, an endangered orchid species. Among the four basal media evaluated for asymbiotic seed germination, Phytamax was found to be the best followed by Murashige and Skoog (MS). Phytamax was also found good for protocorm development. For protocorm like body (PLB) regeneration, protocorms were then further cultured on Phytamax media fortified with different phytohormones either individually or in combinations. The frequency of protocorm like body (PLB) regeneration significantly relied on kinds and concentrations of plant growth regulators used. A combination of 1-naphthaleneacetic acid (NAA) (5.36 μM) and 6-benzyle amino purine (BAP) (3.80 μM) was found to be suitable for maximum PLB regeneration. Healthy plantlets were induced from PLBs when cultured on same basal medium supplemented with activated charcoal (AC – 3.0 g/l). Plantlets with well developed leaves and roots were transplanted to pots filled with a mixture of charcoal, brick pieces and sphagnum moss and transferred to the greenhouse. This protocol will enable mass propagation and conservation of this exquisite orchid.