Mesos components (CaCl2, MgSO4, KH2PO4) are critical for improving pear micropropagation

Pear accessions and species show a broad response to tissue culture media due to the wide genetic diversity that exists in the available pear germplasm. An initial study of mineral nutrition using a systematic response surface approach with five Murashige and Skoog medium mineral stock solutions indicated that the mesos factor (CaCl₂, MgSO₄, and KH₂PO₄) affected most plant responses and genotypes, suggesting that additional studies were needed to further optimize these three mesos components for a wide range of genotypes. Short stature, leaf spots, edge necrosis, and red or yellow coloration were the main symptoms of poor nutrition in shoot cultures of 10 diverse pear genotypes from six species. A surface response experimental design was used to model the optimal factor and factor levels for responses that included overall quality, leaf character, shoot multiplication, and shoot height. The growth morphology, shoot length, and multiplication of these pear shoots could be manipulated by adjusting the mesos components. The highest quality for the majority of genotypes, including five P. communis cultivars, P. koehnei, P. dimorphophylla, and P. pyrifolia ‘Sion Szu Mi’, required higher concentrations (>1.2× to 2.5×) of all the components than are present in Murashige and Skoog medium. ‘Capital’ (P. calleryana) required high CaCl₂ and MgSO₄ with low KH₂PO₄; for ‘Hang Pa Li’ (P. ussuriensis), low CaCl₂ and moderate to low MgSO₄ and KH₂PO₄ produced high-quality shoots. Suitable combinations of the meso nutrients produced both optimum shoot number and shoot length in addition to general good plant quality.     

Improving in vitro mineral nutrition for diverse pear germplasm

Mineral nutrition in the media used for growth of in vitro plants is often difficult to optimize due to complex chemical interactions of required nutrients. The response of plant tissue to standard growth media varies widely due to the genetic diversity of the plant species studied. This study was designed as the initial step in determining the optimal mineral nutrient requirements for micropropagation of shoot tips from a collection of genetically diverse pear germplasm. Five mineral nutrient factors were defined from Murashige and Skoog salts: NH₄NO₃, KNO₃, mesos (CaCl₂·2H₂0–KH₂PO₄–MgSO₄), micronutrients (B, Cu, Co, I, Mn, Mo, and Zn), and Fe-EDTA. Each factor was varied over a range of concentrations. Treatment combinations were selected using response surface methods. Five pears in three species (Pyrus communis ‘Horner 51,’ ‘Old Home?×?Farmingdale 87,’ ‘Winter Nelis,’ Pyrus dimorphophylla, and Pyrus ussuriensis ‘Hang Pa Li’) were grown on each treatment combination, responses were measured, and each response was analyzed by analysis of variance. The analyses resulted in the identification of the following factors with the single largest effects on plant response: shoot quality (mesos), leaf spotting/necrosis (mesos), leaf size (mesos), leaf color (mesos, NH₄NO₃, and KNO₃), shoot number (NH₄NO₃ and Fe), nodes (NH₄NO₃ and KNO₃), and shoot length (mesos and Fe). Factors with the largest effects (mesos and Fe) were similar among the genotypes. This approach was very successful for defining the appropriate types and concentrations of mineral nutrients for micropropagation of diverse pear genotypes.     

Plant regeneration from mesophyll protoplasts of Williams' Bon Chretien (syn. Bartlett) pear (Pyrus communis L.)

Leaf protoplasts of axenic shoot cultures of Pyrus communis L. cv. Williams' Bon Chretien (syn. Bartlett) underwent cell wall regeneration and division to give multicellular colonies in a modified Murashige and Skoog medium which lacked ammonium ions, but supplemented with 1-naphthaleneacetic acid (NAA), 4-indole-3yl-acetic acid, 6-benzylaminopurine (BAP) and casein hydrolysate. Protoplast-derived colonies gave callus on Murashige and Skoog salts medium with NAA and BAP and exhibited shoot regeneration on half-strength Murashige and Skoog medium supplemented with 0.2 mg 1^–1 4-indole-3yl-butyric acid, 2.0 mg 1^–1 BAP, 0.2 mg 1^–1 gibberellic acid, 50 mg 1^–1 casein hydrolysate and 10 mg 1^–1 Ca-pantothenate. Following rooting, protoplast-derived plants of pear were transferred to the glasshouse where they completed acclimatization.Abbreviations BAP 6-benzylaminopurine - FPE final plating efficiency - GA₃ gibberellic acid - IAA 4-indole-3yl-acetic acid - IBA 4-indole-3yl-but yric acid - IPE initial plating efficiency - NAA 1-naphthaleneacetic acid - f.wt. fresh weight - MES 2-N-morpholinoethane sulfonic acid - MS Murashige and Skoog (1962) - %PE % plating efficiency - PVP-10 polyvinylpyrrolidone (Av. MW 10,000) - FDA fluorescein diacetate     

Metabolic changes and improved growth in micropropagated red raspberry “Indian summer” are tied to improved mineral nutrition

Mineral nutrition is directly involved in plant metabolism and greatly affects growth and development. An initial study modeling Murashige and Skoog (MS) medium mineral components revealed that the quality of red raspberry shoot cultures was significantly affected by CaCl₂, MgSO₄, and KH₂PO₄ (mesos components). This study investigated the effects of increased mesos components on shoot growth and metabolism. Rubus idaeus L. “Indian summer” shoots grown on standard MS medium (1.0× MS mesos components) were compared to shoots grown with 1.5× and 2.5× MS mesos components. After 9 wk, shoots were evaluated for shoot quality, multiplication, elongation, and metabolic changes. Metabolic changes were determined by liquid chromatography (LC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). Shoots grown on increased mesos components had improved quality, shoot length, and leaf color compared to shoots grown on MS medium. Metabolomic analysis indicated that shoots grown on high mesos component medium had reduced amounts of some free amino acids (glutamine, arginine, histidine, and proline) and some secondary metabolites (epicatechin, quercetin, and ellagic acid) compared to shoots on MS medium, which indicated reduced stress. Shoots grown on high mesos component also had increases in fructose 1-phosphate and glutathione associated with biosynthetic pathways, plant defense mechanisms, and redox homeostasis. Another factor involved in improved growth responses may be that increased glutamine was also found in high mesos component treatments, possibly influenced by ammonium accumulated from photorespiration. These metabolic changes provide initial insights into medium optimization and in vitro mineral nutrition, and the impact of nutrients on plant growth and development in micropropagated red raspberry shoots.     

Genomic characterization of self-incompatibility ribonucleases in the Central Asian pear germplasm and introgression of new alleles from other species of the genus Pyrus

The Pyrus species exhibit the so-called S-RNase-based gametophytic self-incompatibility system, which is considered to be the most widespread self-incompatibility system among flowering plants. In this study, 57 Iranian pear (Pyrus communis L.) domestic cultivars and wild genotypes, plus 21 European pear cultivars used as references, were genotyped adopting a PCR-based genotyping assay using consensus and allele-specific primers. The results revealed traces of significant genetic contribution in the Iranian traditional varieties and genotypes from other Pyrus species; the genetic contribution of Japanese pear clearly emerged with the detection of some Pyrus pyrifolia S-RNase alleles. Moreover, our results highlighted the presence of three new S-RNase alleles (named S₁₂₆, S₁₂₇, and S₁₂₈) that were not previously identified in P. communis, possibly introduced in the germplasm of cultivated pear through gene transfer from other cultivated or wild species.     

Transcriptome-based single nucleotide polymorphism markers for genome mapping in Japanese pear (Pyrus pyrifolia Nakai)

The development of single nucleotide polymorphism (SNP) markers in Japanese pear (Pyrus pyrifolia Nakai) offers the opportunity to use DNA markers for marker-assisted selection in breeding programs because of their high abundance, codominant inheritance, and potential for automated high-throughput analysis. We developed a 1,536-SNP bead array without a reference genome sequence from more than 44,000 base changes on the basis of a large-scale expressed sequence tag (EST) analysis combined with 454 genome sequencing data of Japanese pear ‘Housui’. Among the 1,536 SNPs on the array, 756 SNPs were genotyped, and 609 SNP loci were mapped to linkage groups on a genetic linkage map of ‘Housui’, based on progeny of an interspecific cross between European pear (Pyrus communis L.) ‘Bartlett’ and ‘Housui’. The newly constructed genetic linkage map consists of 951 loci, comprising 609 new SNPs, 110 pear genomic simple sequence repeats (SSRs), 25 pear EST–SSRs, 127 apple SSRs, 61 pear SNPs identified by the “potential intron polymorphism” method, and 19 other loci. The map covers 22 linkage groups spanning 1341.9 cM with an average distance of 1.41 cM between markers and is anchored to reference genetic linkage maps of European pears and apples. A total of 514 contigs containing mapped SNP loci showed significant similarity to known proteins by functional annotation analysis.     

Response of the pear rootstock to boron and salinity in vitro

The effects of boron and NaCl induced salinity on growth and mineral composition of the pear (Pyrus communis L.) rootstock OH × F 333 shoots cultured in vitro were investigated. Shoots were grown in vitro for seven weeks on a Murashige and Skoog medium containing two B concentrations (0.1 and 2 mM) combined with five NaCl concentrations (0, 10, 20, 40, and 80 mM). The longest shoots were produced at 0.1 mM B and 80 mM NaCl, but highest number of shoots were produced at 0.1 mM B and 0–20 mM NaCl. Inclusion of 20 and 40 mM NaCl in the culture medium significantly increased fresh mass of cultures compared to 0 mM NaCl for all B concentrations tested. The concentrations of P, K, Ca, Mg, Na, Fe, Mn and Zn of plants were affected by B and NaCl concentration of the medium.     

<Emphasis Type="Italic">In vitro</Emphasis> biosynthesis of antioxidants from <Emphasis Type="Italic">Hemidesmus indicus</Emphasis> R. Br. cultures

Summary Shoot cultures and callus cultures from roots and leaves of Hemidesmus indicus R. Br (Asclepiadaceae) were established on Murashige and Skoog medium with various hormonal combinations. The production of antioxidants (lupeol, vanillin, and rutin) in shoot cultures, callus cultures derived from leaf cells and root cells, was compared with root and aerial portions of the parent plant. Shoot cultures and leaf callus cultures produced more antioxidants than root callus cultures. In vitro culture of this species might ofter an alternative method for production of these important pharmaccuticals, which would reduce the collection pressure on this rare plant.     

<Emphasis Type="Italic">Aloe vera</Emphasis> transformation: the role of Amberlite XAD-4 resin and antioxidants during selection and regeneration

A system for genetic transformation and subsequent plant regeneration via indirect organogenesis from callus was developed for Aloe vera. Young seedlings served as primary explants. Callus cultures were established on Murashige and Skoog (1962) medium supplemented with 3 mg l⁻¹ benzylaminopurine and 2 mg l⁻¹ indole acetic acid. A protocol was developed to switch from the differentiated stage, using in vitro shoots or young regenerated plants, back to the de-differentiated stage of the callus and vice versa. Long-term maintenance of this callus paved the way for genetic manipulation of Aloe vera. Calluses were bombarded with a plasmid containing uidA and hpt genes, both under the control of the 35S promoter. Dithiothreitol and gibberellic acid were found to play a major role in reducing tissue necrosis following bombardment. Transformed shoots were regenerated under stepwise selection in hygromycin-containing liquid medium supplemented with different antioxidants. Amberlite XAD-4 resin was embedded into alginate beads and added to the selection medium. Amberlite was best for adsorbing different phenolic compounds and blocking explant necrosis. Shoot initiation occurred after transfer of the transformed cells to Murashige and Skoog medium supplemented with 2.0 mg l⁻¹ thidiazuron and 0.1 mg l⁻¹ indole butyric acid. Murashige and Skoog medium supplemented with 1 mg l⁻¹ zeatin riboside promoted shoot elongation. Rooting and plant development were obtained on Murashige and Skoog basal medium supplemented with 15 mg l⁻¹ hygromycin lacking growth regulators. The transgenic nature of the regenerated plants was verified by histochemical GUS assay and Southern blot hybridization.     

Clonal propagation of someFreesia cultivars through tissue culture

A propagation method for someFreesia cultivars usingin vitro cultures is described. Bud and root formation was evoked in callus tissue culture on a modified Murashige and Skoog’s medium. Reconstituted plants were transferred into soil and cultivated in the glasshouse.     

GRAFT INCOMPATIBILITY BETWEEN PEAR AND QUINCE: THE INFLUENCE OF METABOLITES OF CYDONIA OBLONGA ON SUSPENSION CULTURES OF PYRUS COMMUNIS

The fresh weights of suspension cultures of pear (Pyrus communis) and quince (Cydonia oblonga) increased exponentially for 30 to 40 days after subculturing. Transferring pear cultures to media in which quince cultures had grown for 10 days resulted in a 70% inhibition of callus growth. Transferring quince cultures to media in which pear cultures had grown for 10 days resulted in less than a 20% inhibition of growth. Addition of the cyanogenic glycosides amygdalin and prunasin (as 50 ppm CN ^_) killed pear cultures, while growth of quince cultures was inhibited by only approximately 50%. Addition of 50 ppm CN- severely inhibited growth of both cultures. These results indicate that 1) suspension cultures of quince release factor(s) that significantly inhibit growth of pear cultures, 2) quince cultures are relatively unaffected by metabolites released by pear cultures, 3) the severe inhibition of pear growth by quince metabolites is mimicked by the addition of cyanogenic glycosides ubiquitous to vegetative portions of quince, 4) direct cellular contact is not necessary to elicit incompatibility between pear and quince, and 5) incompatibility between pear and quince need not be associated with any particular stage of graft development.     

Heat stress responses in cultured plant cells : effect of culture handling and age

The pipetting of pear (Pyrus communis cv Bartlett) suspension cultures was followed by a substantial but transient decrease in heat sensitivity. During a culture cycle, pear cells were most sensitive to heat at day 3, which coincided with the period of most active cell division. To minimize serious artifacts, the influence of culture handling and age on parameters such as heat sensitivity must be standardized.     

An efficient,widely applicable cryopreservation of Lilium shoot tips by droplet vitrification

We report a straightforward and widely applicable cryopreservation method for Lilium shoot tips. This method uses adventitious shoots that were induced from leaf segments cultured for 4 weeks on a shoot regeneration medium containing 1 mg/l α-naphthaleneacetic acid and 0.5 mg/l thidiazuron. Shoot tips (1.5–2 mm in length) including 2–3 leaf primordia were precultured on Murashige and Skoog (MS; 1962) medium with 0.5 M sucrose for 1 day and then treated with a loading solution containing 0.4 M sucrose and 2 M glycerol for 20 min, followed by a Plant Vitrification Solution 2 (PVS2) treatment for 4 h at 0 °C. Dehydrated shoot tips were transferred onto 2.5 µl PVS2 droplets on aluminum foil strips, prior to a direct immersion into liquid nitrogen for 1 h. Frozen shoot tips were re-warmed in MS medium containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for shoot regrowth. Shoot regrowth levels ranged from 42.5 % for L. longiflorum × Oriental ‘Triumphator’ to 87.5 % for L. Oriental hybrid ‘Siberia’, with a mean shoot regrowth level of 67.1 % across the six diverse Lilium genotypes tested. Histological observations found that the survival patterns were similar in cryopreserved shoot tips of ‘Triumphator’ and ‘Siberia’. Assessments using inter-simple sequence repeat markers found no differences in regenerants recovered from the control stock cultures and from cryopreserved shoot tips in ‘Triumphator’ and ‘Siberia’. This Lilium droplet-vitrification cryopreservation method is efficient, simple and widely applicable for the long-term conservation of lily genetic resources.     

Efficient,one step and cultivar independent shoot organogenesis of potato

An efficient, one step and genotype independent protocol of shoot organogenesis was developed from leaf and internodal explants taken from microshoots of different cultivars of potato (Solanum tuberosum L.). Initially, microshoots were cultured on basal Murashige and Skoog medium additionally supplemented with 10 µM AgNO₃ (MS1 medium) to achieve healthy shoot growth required to get the quality explants. Shoot organogenesis was induced from both types of explants (leaf and internodal) on MS1 medium variously supplemented with 6-benzyladenine (BA) and gibberellic acid (GA₃). Maximum explants were induced shoot organogenesis on MS1 medium supplemented with 10 µM BA and 15.0 µM GA₃ from both the cultivars namely ‘Kufri Chipsona 1’ and ‘Kufri Pukhraj’. Among the types of explants used, better response was observed from internodal segments as compared to leafs. This optimized medium combination was found to be equally effective for all the eight cultivars tested namely ‘Kufri Pukhraj’, ‘Kufri Chipsona 1’, ‘Kufri Chipsona 2’, ‘Kufri Jyoti’, ‘Kufri Surya’, ‘Kufri Chandramukhi’, ‘Kufri Khyati’ and ‘Desiree’. The clonal uniformity of the regenerated shoots was confirmed using random amplified polymorphic DNA and inter-simple sequence repeats markers.     

In vitro conservation and cryopreservation of mature pistachio (Pistacia vera L.) germplasm

As genetic erosion of pistachio (Pistacia vera L.) has been occurring in the Mediterranean, Central and West Asia and North Africa, experiments were conducted to conserve two cultivars (‘Atl?’ and ‘Siirt’) of mature pistachio germplasm by assessing both medium- and long-term conservation techniques. In medium-term conservation, our results showed that it was feasible to conserve both cultivars in the form of either microshoots or encapsulated shoot apices up to 12 months at 4°C in the dark. As regards long-term conservation, encapsulation-dehydration and droplet-vitrification techniques were assessed for cryopreservation of cold-hardened and osmoprotected shoot apices of mature ‘Atl?’ cultivar. Among the methods tested, 13.6% of regrowth was achieved with incubation of explants in the droplets of vitrification solution for 150 min at 0°C followed by direct immersion in liquid nitrogen (LN), rapidly thawed and then cultured on Murashige and Skoog’s (MS) medium containing 1 mg L^?1 BA and 0.5 mg L^?1 GA_3. The developed droplet-vitrification technique appeared as a promising procedure for long-term preservation of shoot apices of mature pistachio germplasm. Moreover, assesment of genetic fidelity by Random Amplified Polymorphic DNA analysis (RAPD) revealed out high levels of genetic stability between donor plant and cryopreserved plants (similarity indexes between 0.959 and 0.973) after they were subcultured for at least 3 months. The detected low level of genetic instability could be due to the toxic effect of PVS2 and regeneration phase. The optimized conservation techniques, especially slow growth storage, could be applied to preserve other Pistacia species.     

Plant regeneration from protoplasts of Felicia and Brachycome

Protoplasts were isolated from leaves of axenic shoot cultures of Felicia bergeriana (Kingfisher Daisy) and Brachycome iberidifolia (Swan River Daisy) and from callus cultures of Felicia. Plants were regenerated from all three sources and since both species are of ornamental value (blue flowered) the establishment of plant regeneration provides a basis for their incorporation in somatic hybridisation programmes involving important ornamentals such as Chrysanthemum.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - KIN 6-furfurylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) - FDA fluorescein diacetate - f. wt. fresh weight     

Genetic relationships between wild progenitor pear (Pyrus L.) species and local cultivars native to Georgia,South Caucasus

The genetic diversity of 108 individuals of wild pear species (Pyrus communis subsp. caucasica, P. balansae, P. salicifolia, P. syriaca, P. demetrii, P. bulgarica, P. ketzkhovelii, P. sachokiana) and 35 samples of local and introduced cultivated pears from the country of Georgia were compared to 73 individuals of wild P. communis subsp. caucasica and P. communis subsp. pyraster in the collection of USDA-ARS National Plant Germplasm System (NPGS). Pyrus communis subsp. caucasica from both Georgia and the NPGS, P. communis subsp. pyraster from the NPGS, and P. salicifolia from Georgia were differentiated, based on analysis of eleven microsatellite markers. In addition, accessions of P. communis subsp. caucasica from Georgia were genetically distinct from accessions of the same subspecies in the NPGS collection that originated from other European and Middle Eastern Asian countries. Local pear cultivars in Georgia were genetically similar to P. communis subsp. caucasica and P. balansae growing wild in Georgia suggesting that they may have originated from native pear trees that could serve as unique genetic resources for pear breeding programmes.     

Production of chrysanthemic acid and pyrethrins by tissue cultures ofChrysanthemum cinerariaefolium

Tissue cultures ofChrysanthemum cinerariaefolium were established, and then used to study the production of pyrethrin insecticides, and their precursor chrysanthemic acid. Callus cultures and root-differentiated cultures did not contain pyrethrins whereas shoot differentiated callus was found to produce the pyrethrins. Chrysanthemic acid was isolated by extraction from callus cultures, and feeding¹⁴C-labelled chrysanthemic acid to a cell suspension ofC. cinerariaefolium established that the acid accumulates largely as a glucoside ester.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - 1AA Indoleacetic acid - BAP 6-Benzylaminopurine - GC-MS Gas chromatography - mass spectrometry     

Zeatin and TDZ-induced Shoot Proliferation and Use of Bioreactor in Clonal Propagation of Medicinal Herb, Roseroot (Rhodiola rosea L)

A procedure for in vitro propagation of roseroots (Rhodiola rosea L), a medicinal plant, was developed using a RITA bioreactor system containing liquid medium, combined with a gelled medium. Wild roseroot clones: ‘RCi’, ‘RC2’ and ‘RC3’ were established on a basal medium (BM) from in vitro-germinated seedlings on half-strength Murashige and Skoog (MS) salts. TDZ at 2–4 μM supported shoot proliferation but inhibited shoot elongation of ‘RCi’ shoots on gelled medium. Clones differed significantly with respect to multiplication rate with ‘RCi’ producing the most shoots per explant on gelled BM with 2 μM zeatin. In a bioreactor system, TDZ supported rapid shoot proliferation at lower concentration (0.5 μM) but induced hyperhydricity at more than 0.5 μM. Bioreactor-multiplied hyperhydric shoots of all clones when transferred to gelled medium containing 1–2 μM zeatin produced normal shoots within 4 wk of culture. Shoots were rooted in vitro on BM void of growth regulators. Almost all (9U to 95%) in vitro plantlets survived when transferred to potting medium.