Improving the micropropagation efficiency of hybrid Dendrobium orchids with chitosan

The appropriate chitosan types and concentrations for enhancing the in vitro micropropagation of Dendrobium ‘Eiskul’ were studied using 70, 80 and 90% N-deacetylated polymeric (P-70, P-80 and P-90) and oligomeric (O-70, O-80 and O-90) forms of crab (Portunus pelagicus) chitosan. For the initial protocorm-like body (PLB) multiplication, the application of 10 mg/L of P-70 or 20 mg/L of P-90 was optimal, although 10 mg/L of P-80 and O-70 were also effective, and attained maximal PLB replication rates without increasing the detectable levels of somaclonal variation. However, during PLB-shoot induction, 10 or 20 mg/L of O-80 was the most appropriate chitosan and also induced further PLB formation. For plantlet regeneration, the addition of 10 mg/L of O-80 or P-80 gave the best quantity and quality, respectively, of plantlets. Finally, 20 mg/L of P-70 chitosan as a supplement during exflasking enhanced both the survival rate and the growth of the plantlets at one month after exflasking. Together, these data reveal a potentially beneficial and applicable protocol for commercial orchid micropropagation.     

High frequency plant regeneration through adventitious multiple shoot organogenesis in epicotyl explants of Indian gooseberry (Emblica officinalis Gaertn)

Shoots regenerated adventitiously on epicotyl segments from in vitro seedlings of Emblica officinalis var. ‘Kanchan’. Epicotyls derived from 2-week-old aseptic seedlings were most responsive and produced a maximum number of 303 shoots per explant in Murashige and Skoog (1962) medium (MS) augmented with 8.8 μM N⁶-benzyladenine (BA) + 1.425 μM indole-3-acetic acid (IAA). Shoots readily elongated in MS lacking growth regulators and rooted in half-salt-strength MS (1/2 MS) supplemented with indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). The highest rooting response was recorded in 1/2 MS containing 14.7 μM IBA. Plantlets were acclimatized inside the green house and 80% of the plantlets survived on transfer to garden soil.     

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.     

An improved protocol for adventitious shoot regeneration and plant formation in Psoralea corylifolia L.

An effective protocol was developed for in vitro regeneration of Psoralea corylifolia through enriched cotton moistened-liquid (CML) and solid culture systems. Prolific adventitious shoot buds were achieved from hypocotyl explants of 2-week-old cultures on enriched CML Phillips and Collins (L2) medium supplemented with different concentrations and combinations of thidiazuron (TDZ), benzyladenine (BA), kinetin (KIN), naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and bavistin (BVN). Combination of 2 μM TDZ, 0.5 μM BA, 100 mg l⁻¹ BVN and 2 μM NAA produced a greater number of adventitious shoots per explant (93.5) when transferred to half-strength enriched solid L2 medium. Regenerated shoots (40–50 mm in length) were exposed simultaneously for rooting as well as hardening in moistened (1/8-L2 basal salt solution with 5 μM IBA and 100 mg l⁻¹ BVN) soil mixture and vermiculite (3:1, v/v). The plants were subsequently established in the field. The survival percentage differed with seasonal variations.     

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.     

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.     

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.     

High frequency plant regeneration from microspore-derived embryos of ornamental kale (Brassica oleracea L. var. acephala)

The aim of this work was to study the effect of solid medium, developmental stage, embryonic age, cold treatment and additives to the medium on plant regeneration from microspore-derived embryos in four F₁ hybrids of ornamental kale (Brassica oleracea L. var. acephala). The results showed that all of the cultivars responded best when the embryos were cultured in solidified B5 medium with 1% agar. Optimal regeneration was gained when cotyledonary embryos were cultured for 25 days. Cold treatment significantly improved plant regeneration with a frequency of up to 79.0% under 4 °C for 2 d or 5 d. The addition of 3.0 or 5.0 mg/L silver nitrate (AgNO₃) increased the frequency of plant regeneration. In the Zhouyehongxin cultivar, the frequency of plantlet development reached 84.4%. The addition of activated charcoal reduced embryo hyperhydricity.     

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.     

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.     

Pollination with laser-irradiated pollens breaks cross-incompatibility between zicaitai (Brassica campestris var. purpurea) and ornamental kale (Brassica oleracea var. acephala) to produce hybrids with the aid of ovule culture

The objective of this study was to produce interspecific hybrids between an Ogura-cytoplasmic male sterile (CMS) line of zicaitai (Brassica campestris var. purupurea, 2n = 20) and cultivars of ornamental kale (Brassica oleracea var. acephala, 2n = 18) to develop a CMS system for hybrid seed production. Pollination with pollen grains of ornamental kales irradiated at a power output of 9.0 mW with a He–Ne laser for 3 min could overcome the cross-incompatibility between the species concerned. Intact hybrids could be efficiently produced from ovules cultured on Murashige and Skoog media supplemented by 0.2 mg l⁻¹ 6-benzyladenine. Chromosome number of hybrids was confirmed to be 2n = 19. Hybrids resembled ornamental kales in leaf morphology and in vernalization response. Pollens of hybrids had a sterile appearance. Moreover the hybridity of the putative hybrids was confirmed by RAPD data on a DNA fragment of 820 bp.     

Effects of explant type, culture media and growth regulators on callus induction and plant regeneration of Chinese jiaotou (Allium chinense)

To establish an efficient protocol of shoot regeneration from callus, effects of explant type, culture media and plant growth regulators on callus induction and shoot regeneration of Chinese jiaotou (Allium chinense) were evaluated. The results showed that basal plate was the best explant for callus induction (47.5%) when cultured on B5 medium supplemented with 0.1 mg l⁻¹ 6-benzylaminopurine (BA) and 1.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), and B5 was the best medium to induce callus formation with 49.3% of the explants forming callus. The highest callus induction (65.2%) was achieved culturing basal plate on B5 medium supplemented with 0.1 mg l⁻¹ BA and 1.0 mg l⁻¹ 2,4-D after 8 weeks of culture. The best callus proliferation was observed on B5 medium with 1.5 mg l⁻¹ 2,4-D. Shoots regenerated at the highest frequency of 58.8% with 4.5 shoots when calli were cultured on B5 medium with 0.1 mg l⁻¹ BA and 1.0 mg l⁻¹ a-naphthaleneacetic acid (NAA). This protocol provides a basis for future studies on genetic improvement and could be applied to large-scale multiplication systems for commercial nurseries of Allium chinense.     

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.     

Growth and flowering of black iris (Iris nigricans Dinsm.) following treatment with plant growth regulators

The effects of application method and concentration of gibberellic acid (GA₃), paclobutrazol and chlormequat on black iris performance were assessed. Plants (10 cm high, 4 ± 1 leaves) were sprayed with 125, 250, 375 or 500 mg L⁻¹ or drenched with 0.25, 0.5, 1 or 2 mg L⁻¹ GA₃. In a second experiment, the plants were sprayed with 100, 250, 500 or 1000 mg L⁻¹ or drenched with 0.25, 0.5, 1 or 2 mg L⁻¹ paclobutrazol. Other plants were sprayed with 250, 500, 1000 or 1500 mg L⁻¹ or drenched with 100, 250, 375 or 550 mg L⁻¹ chlormequat. In each experiment, the control treatment consisted of untreated plants. Results indicated that the tallest plants (37.3 cm) in the GA₃ experiment were those sprayed with 250 mg L⁻¹. The most rapid flowering (160 days after planting) occurred when a 375 mg L⁻¹ GA₃ spray was used, whereas flowering was delayed to 200 days using 1 mg L⁻¹ GA₃ drench. Drenching with 1 mg L⁻¹ GA₃ increased height of the flower stalk by 7 cm compared to the control. Though relatively slow to flower, plants drenched with 1 mg L⁻¹ GA₃ had long and rigid stalks, which were suitable as cut flowers. Number and characteristics of the sprouts were not affected by GA₃. All paclobutrazol sprays resulted in leaf falcation. A 500 or 1000 mg L⁻¹ paclobutrazol spray resulted in severe and undesirable control of plant height, drastic reduction in stalk height and weight, and delayed flowering. Plants drenched with 0.25 or 1 mg L⁻¹ paclobutrazol were suitable as pot plants. Chlormequat reduced plant height only at the highest drench concentration, which also reduced flowering to 70%. No leaf falcation was observed with GA₃ or chlormequat. Chemical names: ( ± )-(R*,R*)-beta-((4-chlorophenyl)methyl)-alpha-(1,1,-dimethylethyl)-1H-1,2,4,-triazol-1-ethanol (paclobutrazol); (2-chloroethyl) trimethylammonium chloride (chlormequat).     

Photosynthetic and growth responses of juvenile Chinese kale (Brassica oleracea var. alboglabra) and Caisin (Brassica rapa subsp. parachinensis) to waterlogging and water deficit

Chinese kale (Brassica oleracea var. alboglabra) and Caisin (Brassica rapa subsp. parachinensis) are leafy vegetable crops grown in south-east Asian countries where rainfall varies dramatically from excess to deficit within and between seasons. We investigated the physiological and growth responses of these plants to waterlogging and water deficit in a controlled experiment in a glasshouse. Juvenile plants were subjected to waterlogging or water deficit for 19 days in case of Chinese kale and 14 days in case of Caisin and compared with well-watered controls. Caisin tolerated waterlogging better than Chinese kale because it produced hypocotyl roots and gas spaces developed at the stem base. In Chinese kale, waterlogging reduced plant fresh weight (90%), leaf area (86%), dry weight (80%) and leaf number (38%). In contrast, waterlogging had no impact on leaf number in Caisin and reduced plant fresh and dry weights and leaf area by 60–70%. Water deficit reduced leaf area, fresh weight and dry weight of both species by more than half. Leaf number in Chinese kale was reduced by 38% but no effect occurred in Caisin. Water deficit increased the concentration of nitrogen in the leaf dry matter by more than 60% in both species and the leaf colour of water deficient plants was dark green compared with the leaf colour of well-watered plants. Soil water deficit delayed flowering of Caisin while waterlogging accelerated it. Thickening and whitening of the cuticle on the leaves of Chinese kale probably increased its ability to retain water under drought while Caisin adjusted osmotically and Chinese kale did not. Waterlogging and water deficit had strong effects on leaf gas exchange of both Brassica species. Water deficit closed the stomata in both species and this was associated with a leaf water content of 9 g g⁻¹ DW. In contrast, waterlogging reduced conductance from 1.0 to 0.1 mol H₂O m⁻² s⁻¹ in direct proportion to changes in leaf water content, which fell from 11 to 5 g g⁻¹ DW. This separation of the effects of water deficit and waterlogging on conductance was reflected in transpiration, internal CO₂ concentration and net photosynthesis. In conclusion, Chinese kale and Caisin showed rather different adaptations in response to waterlogging and water deficit. Caisin was more tolerant of waterlogging than Chinese kale and also showed evidence of tolerance of drought. There is genetic variation to waterlogging within the Brassica genus among the leafy vegetables that could be used for cultivar improvement.     

Influence of yeast extract and casein hydrolysate on callus multiplication and somatic embryogenesis of date palm (Phoenix dactylifera L.)

Complex organic additives are known to improve growth and differentiation of in vitro plant cultures. The present investigation was conducted to determine the effect of various concentrations of yeast extract (YE) and casein hydrolysate (CH) on callus growth and somatic embryogenesis in date palm cultivar Nabout Saif. Callus induced from shoot tip explants was grown on callus multiplication medium supplemented with either YE or CH at 0.0, 0.1, 0.25, 0.5, and 1 g l⁻¹. To induce somatic embryogenesis, callus was transferred to a hormone-free medium containing the corresponding concentration of additives. The results have shown that callus weight and the number of somatic embryos were directly proportional to increases in the concentration of organic additives tested. Callus growth was best achieved when 1 g l⁻¹ of either YE or CH was added to the culture medium. At this concentration of YE, callus growth was double that of the control medium. On CH-containing media growth was 2.3 times that of the control. This indicates that CH is more effective in enhancing callus growth. However, YE was more effective in enhancing somatic embryogenesis. The data show that the best somatic embryo formation was obtained on either 1 g l⁻¹ YE or 0.5 g l⁻¹ CH which produced 45 and 30 embryos per culture, respectively, as compared to 20 embryos produced in the control treatment. Resultant somatic embryos successfully rooted and regenerated plantlets which exhibited normal growth in the greenhouse. Enhanced plant regeneration, an essential criterion for commercial micropropagation, was achieved.     

The culture of isolated microspores of ornamental kale (Brassica oleracea var. acephala) and the importance of genotype to embryo regeneration

The culture of isolated microspores of kale (Brassica oleracea var. acephala) was studied including the importance of genotype to embryo regeneration, medium composition chiefly the sucrose concentration and the use of colchicine, simultaneously medium renovation. It was initiated using 29 different genotypes as donor plants. Embryos were induced from six of the kale genotypes and these corresponded to the more out-bred genotypes. Embryogenesis was achieved using four different combinations of culture media: (a) microspores initially cultured in NLN medium supplemented with 13% (w/v) sucrose (NLN-13) for 48 h, followed by transfer to fresh NLN-13 medium; (b) microspores cultured for 48 h in NLN-13 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-13 medium; (c) microspores cultured for 48 h in NLN-16 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-16 medium; (d) microspores cultured for 48 h in NLN-16 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-13 medium. The embryos obtained from four of the genotypes developed into plantlets and these regenerated plants have been successfully transplanted to soil.     

Effect of plant growth temperature on membrane lipids in strawberry (Fragaria × ananassa Duch.)

Changes in membrane lipid composition are important in the acclimation of plants. The influence of four day/night growing temperature combinations (18/12, 25/12, 25/22, and 30/22 °C) on membrane lipids of ‘Earliglow’ and ‘Kent’ strawberry (Fragaria × ananassa Duch.) were studied. The monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) were the major galactolipids in the strawberry leaves. ‘Earliglow’ contained a higher amount of galactolipids in the leaves than ‘Kent’. The major phospholipids in the strawberry leaves, roots, and fruit were phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidylethanolamine (PE). PC and PE were the two predominant phospholipids in the strawberry. The leaves and fruit of ‘Earliglow’ contained higher amounts of phospholipids compared to those of ‘Kent’, whereas ‘Kent’ strawberry roots had higher phospholipids. Palmitic (C16:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2), and α-linolenic (C18:3) acids were major fatty acids in galacto- and phospholipids of the ‘Earliglow’ and ‘Kent’ strawberry. PC is very rich in linolenic acid in leaves compared to the fruit and root tissues. PC had the highest ratio of unsaturated to saturated fatty acids among all phospholipids. There was a significant increase in the content of galactolipids (MGDG, DGDG) and phospholipids (PC, PI, PG and PE) and unsaturation of their fatty acids in the cool day/night growth temperature. Increasing day/night growth temperatures decreased MGDG/DGDG ratios. The shifts in saturation and composition of fatty acids observed with strawberry may be an adaptation response of plants to the temperature changes.     

Effect of air humidity on the growth and morphology of Hydrangea macrophylla L.

Hydrangea macrophylla cv. Leuchfeuer is grown in the greenhouse under two different conditions of relative humidity (vapour pressure deficits (vpd) of 1.6 and 0.6 kPa). The relative humidity does not affect the initiation or the release of new pairs of leaves. However, it modifies the size of the plant and the total leaf area. After 79 days of growth, a decrease in the relative humidity from 80% to 50% leads to a 38% decrease in height growth and a 30% decrease in leaf growth. The effect of relative humidity on the growth of H. macrophylla influences the management of the size of the plant and, therefore, the quality of the product marketed. It is likely that it will have an effect on floral development as well.