Effects of variety and growth year on the essential oil properties of lavender (Lavandula angustifolia Mill.) and lavandin (Lavandula x intermedia Emeric ex Loisel.)

Essential oil (EO) properties of six L. angustifolia (LA) and two L. x intermedia (LI) cultivars were evaluated during summer harvest periods of 2017 and 2018, The cultivars were grown in Dörgicse, Hungary. According to our results, the essential oil (EO) content was highly dependent on the varieties in both experimental years (2017: p < 0.0001 and 2018: p < 0.0001). In 2017, the EO content values of LI accessions were all higher than those of the LA cultivars. However, in 2018, the mean EO content value of ‘Budakalászi’ (LA) (8.2 mL/100g) exceeded that of ‘Grosso’ (LI) (7.7 mL/100g). The EO of ‘Mailette’ (LA) and ‘Budakalászi’ (LA) contained the highest linalool proportions (52.9% and 47.0%) while ‘Beate’ (LA) showed the lowest (18.1%); and this latter variety was richest in linalyl-acetate (58.9%). LI varieties produced lower linalyl acetate and total ester ratios in comparison to the LA cultivars. Concerning EO composition, only the UK varieties ‘Hidcote Blue’ (LA) and ‘Munstead’ (LA) met the requirements of the Ph. Eur.This study provided evidence that the effect of growth year on the EO content was significant only in two varieties out of the eight. However, in the more rainy year 2018, the accumulation of linalool and linalyl acetate was significantly affected in several cases, where linalool proportions decreased and linalyl acetate ratios increased.     

Differential accumulation of volatile terpene and terpene synthase mRNAs during lavender (Lavandula angustifolia and L. x intermedia) inflorescence development

Despite the commercial importance of Lavandula angustifolia Mill. and L. x intermedia Emeric ex Loisel floral essential oils (EOs), no information is currently available on potential changes in individual volatile organic compound (VOC) content during inflorescence development. Calyces were found to be the main sites of VOC accumulation. The 20 most abundant VOCs could be separated into three sub‐groups according to their patterns of change in concentration The three groups of VOCs sequentially dominated the global scent bouquet of inflorescences, the transition between the first and second groups occurring around the opening of the first flower of the inflorescence and the one between the second and third groups at the start of seed set. Changes in calyx VOC accumulation were linked to the developmental stage of individual flowers. Leaves accumulated a smaller number of VOCs which were a subset of those seen in preflowering inflorescences. Their nature and content remained constant during the growing season. Quantitative real time polymerase chain reaction assessments of the expression of two terpene synthase (TPS) genes, LaLIMS and LaLINS, revealed similar trends between their patterns of expression and those of their VOC products. Molecular and chemical analyses suggest that changes in TPS expression occur during lavender inflorescence development and lead to changes in EO composition. Both molecular data and terpene analysis support the findings that changes in biosynthesis of terpene occurred during inflorescence development.     

An improved protocol for the regeneration and transformation of tomato (cv Rio Grande)

An improved protocol for Agrobacterium tumefaciens-mediated transformation and regeneration of the tomato cultivar Rio Grande with Arabidopsis thaliana early flowering genes AP1 and LFY has been developed. The effect of different factors on the efficiency of regeneration as well as transformation has been studied. A. tumefaciens strain EHA 105 harboring either of the binary vectors pROKIIAP1GUSint or pROKIILFYGUSint, containing APETALA 1, LEAFY (LFY), β-glucuronidase (GUS), and neomycin phosphotransferase II (NPTII) was used for transformation. The effect of different regeneration media was studied and Murashige and Skoog (MS) medium containing Zeatin 1 mg/l and IAA 0.5 mg/l was found best, producing the highest percentage of regeneration, i.e., 30.4% and transformation, i.e., 84% (P < 0.05). The age of explants was studied and 1-week-old explants were found best producing highest regeneration (29.5%) and transformation (87.5%) values (P < 0.05). Three different types of explants were compared and best results were obtained from apical meristem explants (60.5% regeneration and 85% transformation) (P < 0.05). Transient and stable GUS expression was observed in the transgenic plants.     

An efficient genotype-independent method for regeneration of potato plants from leaf tissue

The regeneration of plants from leaf explants of a number of potato cultivars using a number of published one-, two- and three-step methods was assessed. A method using a pretreatment with high levels of auxin and cytokinin coupled with silver thiosulphate in the regeneration medium proved the most rapid and efficient for the eight cultivars examined.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IAA indoleacetic acid - MS Murashige and Skoog - NAA naphthaleneacetic acid - STS silver thiosulphate     

An efficient method for adventitious shoot regeneration from stem-segment explants of gypsophila

An efficient adventitious shoot regeneration procedure was developed for Gypsophila paniculata L. Using cultivar Arbel, shoot regeneration from the three upper internodes of the stem was monitored on MS media supplemented with different cytokinins (thidiazuron, benzyladenine, kinetin or zeatin) and an auxin (naphthaleneacetic acid). Thidiazuron was found to be the most efficient cytokinin, with up to 100% of the explants forming shoots, at an average of up to 19 shoots per explant being regenerated. The highest percentage of shoot formation was observed in the stem explants originating from the first internode, with all cytokinins tested. The adventitious origin of shoots regenerated from stem explants was confirmed by scanning electron microscopy. The regeneration procedure was found to be applicable to five other gypsophila cultivars (Perfecta, Golan, Gilboa, Flamingo and Tavor). Regenerating plants were successfully transferred to soil, and did not differ in flower color, size or shape from standard vegetatively propagated plants derived from cuttings. This revised version was published online in June 2006 with corrections to the Cover Date.     

An optimising protocol for protoplast regeneration of three peppermint cultivars ( Mentha x piperita)

An efficient protocol for plant regeneration from protoplasts of peppermint ‘Mitcham Digne 38’, ‘Mitcham Ribecourt 19’ and ‘Todd's#x2019; was developed by stepwise optimization of first cell division, microcalli formation and shoot differentiation. The rate of first cell divisions was strongly dependent on the addition of 2,4-D to callus induction medium. Best results were obtained with 1 μM 2,4-D in combination with NAA (2.5 μM) and BA (4 μM). Although liquid medium was more efficient to support first protoplast divisions, solid medium was clearly more suitable to sustain subsequent cell divisions leading to the formation of microcalli. Shoot organogenesis was induced from protoplast-derived calli by using reduced auxin concentration (0.5 μM NAA) and high concentration of cytokinins. Addition of 2.3 μM thidiazuron increased bud formation, allowing a regeneration frequency of more than 50% from calli of ‘Mitcham Digne 38’ and ‘Todd's’. Genotypic differences were noticed for regeneration capability and the pathway of shoot regeneration. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from protoplasts of wheat (Triticum aestivum cv. Hartog)

Morphologically normal green plants have reproducibly been regenerated from protoplasts of an Australian wheat (Triticum aestivum cv. Hartog). The protoplasts were isolated from fine embryogenic suspension cultures which were initiated from embryogenic callus. Protoplasts were incubated in a modified liquid MS medium containing half strength of the macroelements, 5 m 2,4-D and 0.6 M glucose. Colonies were formed at frequencies ranging from 0.1% to 5%. The frequency of colonies forming fully developed plants varied between 1% and 25%. More than eighty green plants with morphologically normal shoots and roots have been obtained and there was no difficulty in establishing these plants in soil. A cytological study of several randomly selected regenerated plants showed the normal chromosome complement for wheat (2n = 42).     

Direct and efficient plant regeneration from leaf explants of Solanum tuberosum l. cv. Bintje

Summary A two-step procedure was used for plant regeneration from in vitro grown leaf strips (2–3 mm wide) of cv. Bintje. Step I medium was designed with 2,4-dichlorophenoxycetic acid (2,4-D) at 0.0 or 9.0 M, in combination with 2.28 M kinetin (K), benzyl adenine (BA), zeatin (Z) or zeatin riboside (ZR). Step II media were 2,4-D-free media containing 5.78 M gibberellic acid (GA3) and growth regulators similar to those of step I media. Leaf explants cultured in medium I containing zeatin riboside or zeatin for 6 days and then subcultured in medium II containing zeatin riboside produced numerous shoots without callus formation. Zeatin riboside containing step I and II media caused shoot regeneration in a high number (97.5±2.2) of explants. Approximately, 33.7±8.4 shoots were regenerated from each leaf explant.Abbreviations BA benzyladenine - Z zeatin - ZR zeatin riboside (trans isomer) - 2,4-D 2,4-dichlorophenoxyacetic acid     

An efficient selection and regeneration protocol for Agrobacterium-mediated transformation of oriental melon (Cucumis melo L. var. makuwa)

An efficient selection and plant regeneration protocol for Agrobacterium-mediated transformation using cotyledon explants of oriental melon (Cucumis melo L. var. makuwa) has been developed. All six oriental melon cultivars evaluated in the study showed a >90?% shoot regeneration frequency and produced 1.8?C3.6 shoots per cotyledon explant when cultured on Murashige and Skoog (MS) medium supplemented with 1.0?mg?L^?1 benzyladenine and 0.01?mg?L^?1 indoleacetic acid. Kanamycin (Km) and geneticin (Gt) in the shoot induction medium (SIM) were compared both qualitatively and quantitatively for their efficiency as a selection agent for the selection and regeneration of transgenic plants after Agrobacterium-mediated transformation. Shoot formation was completely inhibited at 50?mg?L^?1 Km and 10?mg?L^?1 Gt. Relatively high concentrations of both Gt and Km (>100?mg?L^?1 Km and >25?mg?L^?1 Gt) were necessary because large numbers of non-transgenic shoots survived during the selection process. The incorporation of a selectable marker (neomycin phosphotransferase II) into the genome of transgenic plants was confirmed using ??-glucuronidase (GUS), PCR and Southern blot analysis. Shoot regeneration frequencies were 41.2?% at 100?mg?L^?1 Km and 15.2?% at 30?mg?L^?1 Gt 8?weeks after transformation, whereas the transformation frequencies based on the PCR were 2.9 and 7.1?%, respectively, 16?weeks after transformation. These results demonstrate that a large portion of the regenerated shoots on SIM supplemented with 100?mg?L^?1 Km consisted of non-transformed or escaped shoots, indicating that 30?mg?L^?1 Gt is the more suitable for the selection and regeneration of transgenic plants in oriental melon.     

Improvement of efficient in vitro regeneration potential of mature callus induced from Malaysian upland rice seed (Oryza sativa cv. Panderas)

A new and rapid protocol for optimum callus production and complete plant regeneration has been assessed in Malaysian upland rice (Oryza sativa) cv. Panderas. The effect of plant growth regulator (PGR) on the regeneration frequency of Malaysian upland rice (cv. Panderas) was investigated. Mature seeds were used as a starting material for callus induction experiment using various concentrations of 2,4-D and NAA. Optimal callus induction frequency at 90% was obtained on MS media containing 2,4-D (3 mg L⁻¹) and NAA (2 mg L⁻¹) after 6 weeks while no significant difference was seen on tryptophan and glutamine parameters. Embryogenic callus was recorded as compact, globular and light yellowish in color. The embryogenic callus morphology was further confirmed with scanning electron microscopy (SEM) analysis. For regeneration, induced calli were treated with various concentrations of Kin (0.5–1.5 mg L⁻¹), BAP, NAA and 0.5 mg L⁻¹ of TDZ. The result showed that the maximum regeneration frequency (100%) was achieved on MS medium containing BAP (0.5 mg L⁻¹), Kin (1.5 mg L⁻¹), NAA (0.5 mg L⁻¹) and TDZ (0.5 mg L⁻¹) within four weeks. Developed shoots were successfully rooted on half strength MS free hormone medium and later transferred into a pot containing soil for acclimatization. This cutting-edge finding is unique over the other existing publishable data due to the good regeneration response by producing a large number of shoots.Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; NAA, naphthaleneacetic acid; Kin, kinetin; MS, Murashige and Skoog; BAP, benzylaminopurine; TDZ, thidiazuron     

An efficient staining method for ultrathin sections.

A staining method to handle simultaneously as many as 20 electron microscope grids is described. The devices used are easily constructed of readily obtained inexpensive materials. The volumes of stain and wash water required are very small and drying grids is simplified.     

Plant regeneration in tissue cultures of pepper (Capsicum annuum L. cv. mathania)

Leaf, stem, hypocotyl, cotyledon, root, shoot tip and embryo explants of Capsicum annuum L. cv. mathania were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) or kinetin (Kin) alone or in combination with 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). BAP (5.0 mgl⁻¹) in the medium was found to be the best growth regulator for shoot bud differentiation. Shoot buds cultured on 5.0 mgl⁻¹ BAP increased in number but did not elongate. For obtaining complete plantlets, shoot buds were placed on a medium with IBA or NAA (0.1 mgl⁻¹). Histological evidence revealed direct differentiation of buds from cotyledons. Regenerated plants were normal diploids. Unorganized callus could not be induced to differentiate shoot buds.     

An efficient method for in vitro regeneration from immature inflorescence explants of Canadian wheat cultivars

Fertile, green plants were regenerated from immature inflorescence explants from each of four Canadian wheat cultivars. The cultivars were representative of four classes of Canadian wheat. Explants from immature inflorescences of three size ranges were cultured on two types of media: MSI/MSR, which contains 1650 mg l^-1 NH₄NO₃and sucrose as a carbon source, and BII/BIR, which contains 250 mg l^-1 NH₄NO₃and maltose as a carbon source. Regeneration from all cultivars was significantly better on BII/BIR media than on MSI/MSR media. On BII/BIR media, `AC Karma', `Plenty', and `Fielder' gave the highest number of shoots per 10 explants, where the explants were derived from immature inflorescences 5.1 to 10.0 mm in length. 'Columbus' did not regenerate on MSI/MSR medium, and regenerated poorly on BII/BIR medium. Differences were found between cultivars with regard to the number of regenerant plants produced with the best treatments: `Plenty' produced 16.1 shoots per 10 explants, `AC Karma' 12.4, `Fielder' 6.4, and `Columbus' 2.2.     

Tissue culture and plant regeneration of watermelon (Citrullus vulgaris Schrad. cv. Melitopolski)

Plants were regenerated from cotyledon and hypocotyl explants of watermelon (Citrullus vulgaris). The explants were cultured on a Murashige and Skoog's basal nutrient medium supplemented with auxin, cytokinin and auxin-cytokinin combinations. Green healthy nodular and compact callus was obtained in medium containing naphthalene acetic acid and benzylaminopurine. Shoot differentiation and root differentiation from the cotyledon and hypocotyl after callus formation in different media containing benzylaminopurine or naphthalene acetic acid, respectively. Shoot formation required benzylaminopurine. Kinetin proved ineffective in inducing shoot buds or shoots. Root differentiation occurred in a medium containing naphthalene acetic acid or indole acetic acid. There was a greater proliferation of roots on medium supplemented with naphthalene acetic acid. The regenerated shoots developed roots when transferred to medium containing naphthalene acetic acid and complete plantlets could be transferred to soil for further growth.Abbreviations BAP 6 Benzylaminopurine - NAA -Naphthalene acetic acid - MS Murashige and Skoog's medium - IAA Indole acetic acid - KN Kinetin     

An efficient method for producing alpha(1,3)-galactosyltransferase gene knockout pigs

We have reported relatively efficient methods for somatic cell nuclear transfer and for knocking out the alpha(1,3)-galactosyltransferase (alpha1,3-GT) gene in porcine fetal fibroblasts using a nonisogenic promoterless construct approach. Here we report the production of alpha1,3-GT gene knockout pigs using these procedures. Seven alpha1,3-GT gene knockout cell clones were identified by long-range PCR from 108 neomycin resistant (neo(R)) colonies, giving a 6.5% targeting efficiency. Three cell clones were used for nuclear transfer. Nuclear transfer was performed using a fusion before activation protocol using in vitro-matured adult oocytes. Between 51 and 110 fused couplets were transferred to 10 recipients synchronized 1 day behind the embryos. Parturition was induced on day 115, and piglets were delivered by caesarean section. Four recipients gave birth to a total of 18 live piglets. All pigs were female, and all three clones resulted in the birth of live pigs. alpha1,3-GT gene knockout pigs were identified by long-range PCR and confirmed by Southern blot analysis. The efficiency (embryos transferred/piglets born) of our cloning protocol was 1.9% for all transfers and 4.6% for animals that gave birth.     

A rapid and efficient method for regeneration of plantlets from embryo explants of cumin (Cuminum cyminum)

A new, simple and efficient method was developed for multiple shoot regeneration of cumin from imbibed embryo cultures. This method yielded a large number of shoots within short period of time (30–50 days) without any subculturing. The effects of different media, different embryo explants and various combinations of plant growth regulators (PGRs) on callus formation and shoot regeneration in cumin were investigated. Simultaneous callus formation and shoot regeneration was obtained. The best response for multiple shoot regeneration was observed on B5 medium containing 1.0 mg l^–1 BAP, 0.2 mg l^–1 NAA and 0.4 mg l^–1 IAA, with an average of 140 shoots per explant.     

Plant regeneration from embryogenic suspension cultures of Musa acuminata cv. Mas (AA)

Embryogenic callus was established using immature male flower of Musa acuminata cv. Mas. After 5–6 months of culture, embryogenic callus was obtained at 21.75±11.9 from 750 immature male flower clusters with translucent somatic embryos proliferated from the whitish friable callus. It was observed that flower clusters ranging from 4 to 11 responded to form embryogenic callus and out of which 3–10 somatic embryos were formed per flower cluster. Embryogenic callus were obtained at a percentage of 10.00±0.3 on M1 medium initially supplemented with 18 M 2,4-dichlorophenoxyacetic acid (2,4-D) for 3 months and subsequently transferred to the same media with reduced 2,4-D (9 M) for the next 2–3 months. Embryos developed into translucent spheres and slightly torpedo shaped embryos in suspension cultures. Plantlets were obtained on medium M4 supplemented with 0.8M BA, at an average regeneration rate of 13.00±0.58.     

An efficient in vitro regeneration protocol for Tagetes minuta

Tagetes minuta is a source of secondary products which are used as pharmaceuticals, pesticides and as flavour components in the food industry. Cotyledons and hypocotyls of T. minuta were cultured on MS medium with combinations of IAA or NAA and BA. Hypocotyl-derived callus developed adventitious shoots which failed to develop further. Cotyledon-derived callus, cultured on medium with IAA, regenerated adventitious shoots which developed into plantlets on MS medium or half-strength MS with 2.85 μM IAA. Cotyledons cultured on medium with 5.71 μM IAA + 44.4 μM BA and transferred to MS medium for shoot growth yielded the highest number of shoots. Nodal segments from developing shoots were micropropagated on half-strength MS medium with 2.58 μM IAA and 95% of plantlets produced adapted successfully to greenhouse conditions. In vitro plants micropropagated from nodes had many shoots whereas plants regenerated from shoot tips had only a single main stem. This difference in morphology was retained after two months growth in a greenhouse. There were no significant differences in leaf and shoot fresh and dry weights among the regenerated plants after two months growth. After six subcultures of cotyledon-derived callus on medium with IAA and BA all explants lost their ability to regenerate except those cultured on medium with 17.23 μM IAA and 44.4 μM BA. The methods of regeneration developed will facilitate selection of T. minuta plants more tolerant of environmental stress, their micropropagation, and the in vitro production of secondary products. This revised version was published online in June 2006 with corrections to the Cover Date.     

An efficient method for matching nucleic acid sequences.

A method of computing the fraction of matches between two nucleic acid sequences at all possible alignments is described. It makes use of the Fast Fourier Transform. It should be particularly efficient for very long sequences, achieving its result in a number of operations proportional to n ln n, where n is the length of the longer of the two sequences. Though the objective achieved is of limited interest, this method will complement algorithms for efficiently finding the longest matching parts of two sequences, and is faster than existing algorithms for finding matches allowing deletions and insertions. A variety of economies can be achieved by this Fast Fourier Transform technique in matching multiple sequences, looking for complementarity rather than identity, and matching the same sequences both in forward and reversed orientations.