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.     

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.     

Genetic diversity of bitter gourd (Momordica charantia L.) genotypes revealed by RAPD markers and agronomic traits

Bitter gourd or bitter melon (Momordica charantia L.) is considered as minor cucurbitaceous vegetable in spite of having considerable nutritional and medicinal properties. Although some reports on genetic diversity based on morphological characterization are available, no work has been conducted to estimate genetic diversity using molecular markers in this crop. In the present study, 38 genotypes of M. charantia including few commercially cultivars collected from different parts of India based on agro-ecological zones were analysed for diversity study both at morphological and molecular levels. Genomic DNA was extracted from young healthy leaves following the procedure of Doyle and Doyle [Doyle, J.J., Doyle, J.L., 1990. A rapid DNA isolation procedure from small quantity of fresh leaf material. Phytochem. Bull. 119, 11–15]. Pair-wise comparison of genotypes was calculated as per the procedure of Jaccard [Jaccard, P., 1908. Nouvelles recherches sur la distribution florale. Bull. Soc. Vaud. Sci. Nat. 44, 223–270]. Dendrogram was constructed using the unweighted pair group method with arithmetic averages (UPGMA) and the computation for multivariate analysis was done using the computer programme NTSYS-pc Version 2.0 [Rohlf, F.J., 1998. NTSYS-pc Numerical Taxonomy and Multivariate Analysis System, Version 2.01. Exeter Software, Setauket, NY, USA]. Diversity based on yield related traits and molecular analysis was not in consonance with ecological distribution. Among 116 random decamer primers screened 29 were polymorphic and informative enough to analyse these genotypes. A total of 208 markers generated of which 76 (36.50%) were polymorphic and the number of bands per primer was 7.17 out of them 2.62 were polymorphic. Pair-wise genetic distance (GD) based on molecular analysis ranged from 0.07 to 0.50 suggesting a wide genetic base for the genotypes. The clustering pattern based on yield related traits and molecular variation was different.     

Genetic characterization of different demes in Prunus persica revealed by RAPD markers

DNAs of 180 accessions in 10 demes in Prunus persica were amplified with twenty-two, 10-base primers selected from 200 arbitrary primers using Randomly Amplified Polymorphic DNA (RAPD) technology. One hundred and eighty loci were observed and recorded. With statistical analyses of the data from the study, genetic diversity of the demes was expressed as follow: yellow peach group > honey peach group > flat peach group > red leaf peach group > crisp peach group > bitao group and juicy peach group > nectarine group > shouxingtao group > weeping peach group. Genetic variations among and within groups by AMOVA analyses were 11.9, 88.1%, respectively. Demes clustered by UPGMA modified from NEIGHBOR procedure of PHYLIP Version 3.5, the edible peaches of which were combined as a section, while the ornamental species were classified into separate sections. Through analyses of genetic diversity and genetic structure, the results could provide molecular biological evidence for conservation and utilization of P. persica germplasm.     

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.     

Weed competition in cauliflower (Brassica oleracea L. var. botrytis) in the Jordan Valley

A field experiment was carried out in 1996/1997 and repeated in 1997/1998 at the Jordan University Research Station located in the central Jordan Valley to determine the effect of weed competition on growth and yield of cauliflower Brassica oleracea var. botrytis cv. “White Cloud”. The treatments consisted of either allowing weeds to infest the crop or maintaining plots weed-free for increasing durations after transplanting. Results showed that longer periods of weed/cauliflower competition greatly reduced crop growth and head yield. Average reductions in shoot dry weight and head yield were 81% and 89%, respectively. Maintaining a weed-free crop for any period after transplanting increased cauliflower growth and head yield compared with the weed-infested control. Weed competition for 14 days after transplanting reduced cauliflower average head yield by 41%. To determine the critical period of weed competition and the influence of weed infestation on cauliflower head yield the Gompertz and logistic equations were fitted to data representing increasing duration of weed-free and weed-infested periods, respectively. Based upon an arbitrary 5% level of head yield loss, the critical period of weed competition occurred at 0–38 days after cauliflower transplanting which corresponded with the rapid increase in weed biomass. Results indicated that early weed removal is necessary to prevent yield loss.     

Genetic diversity in mainland and island populations of the endangered Pancratium maritimum L. (Amaryllidaceae) in Tunisia

Tunisian Pancratium maritimum L. populations are at present endangered and represented by scattered individuals as a result of coastal habitat destruction caused by urbanization and overharvesting for its significant ornamental interest. Nineteen populations growing in mainland and island habitats were sampled for allozyme diversity to assess their genetic diversity and structuration using seven isozymes revealed by starch gel electrophoresis. The species exhibited relatively high levels of genetic diversity (the mean A_p = 1.37, P = 37.4%, and H_e = 0.100), indicates a preferentially outcrossing mating system.     

Genetic diversity and population structure of an endangered Orchid (Dendrobium loddigesii Rolfe) from China revealed by SRAP markers

Dendrobium loddigesii Rolfe is an endangered perennial herb with ornamental and medicinal value. Due to habitat deterioration and human over-exploitation, it has suffered a significant decline in abundance. Determining the level of genetic diversity and pattern of population genetic structure of this species would be helpful for its conservation and management. In this paper, sequence-related amplified polymorphism (SRAP) markers were applied to assess the level and pattern of genetic diversity in seven populations of D. loddigesii. Seventeen SRAP primer combinations generated a total of 231 clear amplification bands encompassing 187 (80.95%) polymorphic bands. A high level of genetic diversity was detected (PPB = 80.52%, H = 0.2743, I = 0.4113) at the species level. There was a moderate genetic differentiation (G_st = 0.304) among populations. Two main clusters were detected by cluster analysis using the unweighted pair-group method with arithmetic average (UPGMA). Mantel test revealed that no significant positive correlation was found between genetic distances and geographic distances (r = 0.2302; P > 0.05). Recommendations for conservation of the endangered species resources are proposed.     

Use of ISSR markers to assess genetic diversity of African edible seeded Citrullus lanatus landraces

We used the twenty primers to evaluate the genetic variability of 80 individuals belonging to four accessions of edible seeded Citrullus lanatus originated from Côte d’Ivoire. Edible seeded C. lanatus, named “egusi” or “pistachio”, had a great importance in nutrition in West Africa. Nevertheless, due to its neglected status no study to our knowledge has been devoted to its genetic variability using DNA markers. The twenty ISSR primers generated 258 bands among which 252 were polymorphic (97.67%). On the whole, the bands generated revealed three types of profile sharply distinct from each other with minor differences within each type. One profile (P1) was most frequent with 65 individuals. Three accessions (NI084, NI127 and NI145) generated the three types of profile and had medium values of genetic diversity (GD = 0.246–0.275, respectively). On the opposite, the accession NI076 only contained individuals of the most represented type of profile (P1) and had the lowest genetic diversity (GD = 0.055 ± 0.017). The pairwise genetic distance between the 80 individuals varied from 0 to 0.61. The Factorial Component Analysis and the dendrogram clearly separated the 80 individuals into three clusters corresponding to the three types of profile. The results showed that clusters were well separated from each other whereas accessions were not. Our results suggest that high number of individuals should be taken into account for sampling missions and conservation strategies because accessions were not well differentiated from each other. Local agricultural practices consisting of frequent seeds exchanges between farmers and the conservation of harvested seeds for next year culture could be one explanation.     

Screening of cold-resistant seedlings of a Chinese wild grape (Vitis piasezkii Maxim var. pagnucii) native to loess plateau of eastern Gansu province,China, as rootstocks

Seedlings of a Chinese wild grape (Vitis piasezkii Maxim var. pagnucii) native to loess plateau of Eastern Gansu province, China, were evaluated to screen cold-resistant rootstocks in Lanzhou area. After 14-year investigation two selections of LDP-191and LDP-294 were screened as rootstocks for two table grape cultivars, ‘Fujiminori’ and ‘Red Globe’, respectively. The two graft unions demonstrated very high cold-resistance as well as good graft compatibility. Furthermore, they could survive through low temperatures in winter without soil coverage together with good fruit quality of the cultivars grafted.     

Genetic transformation of Prunus domestica L. using the hpt gene coding for hygromycin resistance as the selectable marker

The study and development of transformation technology with new selection schemes is important for various fundamental studies and for crop trait improvement via genetic engineering. Here we have shown that hygromycin resistance is an effective system for plum genetic transformation. Embryonic axes of mature seeds were co-cultivated with Agrobacterium strain LBA4404 containing the pC1381 plasmid carrying the hygromycin phosphotranferase gene (hpt) and β-glucuronidase (GUS) gene or with strain EHA105 containing the plasmid pC1301 carrying the same marker and reporter genes. The latter strain containing a pC2301 plasmid carrying the neomycin phosphotransferase gene (nptII) gene was used as a control. Infected explants were placed on shoot induction medium containing either 5 mg L⁻¹ hygromycin or 75 mg L⁻¹ kanamycin for selection. Green shoots developed from the explants under hygromycin pressure. These shoots showed continued and vigorous growth and development upon transfer onto fresh hygromycin medium. PCR using hpt sequence primers, and Southern blot analysis using a probe from the hpt gene, confirmed the presence of the transgenes and their stable integration in regenerated plants. Full transgenic plants were obtained in a greenhouse. Hygromycin selection was very effective and no escapes were observed. The study demonstrated that hygromycin resistance can be used as an effective selectable marker for plum transformation. The new system developed here is important and useful for multiple gene transformation in plum.     

Chemical compositions and insecticidal effects of essential oils isolated from Achillea gypsicola, Satureja hortensis, Origanum acutidens and Hypericum scabrum against broadbean weevil (Bruchus dentipes)

The essential oils of aerial parts of Achillea gypsicola Hub.-Mor., Hypericum scabrum L., Satureja hortensis L., and Origanum acutidens (Hand.-Mazz.) Letswaart were analyzed in this study by GC and GC–MS and their oils were tested for toxicity against broadbean weevil (Bruchus dentipes). A. gypsicola oil contained camphor (40.17%), 1,8-cineole (22.01%), piperitone (11.29%), borneol (9.50%) and α-terpineol (1.56%) as major components. A total of 74 components were identified by GC–MS in H. scabrum oil, including α-pinene (9.26%), terpinen-4-ol (5.12%), camphor (5.94%), δ-cadinene (4.52%), pulegone (4.45%), γ-muurolene (4.12%), pinocarvone (3.97%) and β-caryophyllene (3.42%) as predominant components. The essential oils of O. acutidens and S. hortensis were characterized by high contents of carvacrol (86.99% and 55.74%), γ-terpinene (0.71% and 20.94%), p-cymene (1.95% and 12.30%), α-terpinene (0.13% and 2.04%) and β-caryophyllene (1.30% and 1.08%). All of the essential oils were toxic to adults of B. dentipes and insect mortality increased with increasing concentration of each oil. The oils (20 μl dose) brought about 100% mortality in 36 h. Although desirable insecticidal activities against the pest were achieved with the oils from all four plant species, S. hortensis and O. acutidens oils were more effective, particularly after 6 h of treatment. The current results concluded that the essential oils, in particular O. acutidens and S. hortensis oils, may be used as potential botanical insecticides against B. dentipes.     

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.     

6-Benzylamino purine stimulates in vitro shoot organogenesis in Eucalyptus erythronema, E. stricklandii and their interspecific hybrids

In vitro bud and shoot organogenesis was investigated for the ornamental plants Eucalyptus erythronema var. erythronema, E. stricklandii and their interspecific hybrids cv. ‘Urrbrae Gem’ and ‘Hybrid 2.5’ by using 0.0, 0.1, 0.25, 0.5 or 1.0 μM BAP on apex and leaf explants. Callus developed on all explants and increased with all concentrations of BAP without significant differences between BAP concentrations. Buds formed on apex and leaf explants of E. erythronema and E. cv. ‘Urrbrae Gem’ especially with 1.0 μM BAP, but these buds rarely developed into shoots. Bud clusters formed on E. erythronema and E. cv. ‘Urrbrae Gem’ apex and leaf explants whereas E. stricklandii and ‘Hybrid 2.5’ produced fewer, individual buds on the explant. Shoots regenerated from apex explants of all genotypes with all levels of BAP, whereas few shoots of any genotype regenerated from leaf explants regardless of the number of buds formed. Shoots from apex explants could be multiplied successfully. Light microscopy showed meristems developed within the callus, and at the callus and bud surfaces. However, few shoots developed considering the level of bud and meristem formation. This report is the first for successful shoot organogenesis and multiplication in an ornamental eucalypt.     

Assessment of diversity in Podophyllum hexandrum by genetic and phytochemical markers

For successful conservation and domestication of a species, evaluation of its genetic diversity by different markers is important. Morphological characteristics, phytochemical variation and random amplified polymorphic DNA (RAPD) profiles were generated in different accessions of Podophyllum hexandrum in order to determine the genetic diversity. Random amplified polymorphic DNA (RAPD) analysis revealed a high degree of genetic diversity among the accessions used in the study. There was also high diversity in the concentration of marker compounds in the collected samples as revealed by HPLC analysis. It is shown that the approaches used in the work successfully discriminate between the accessions of this species and thus they constitute interesting tools to analyze molecular, biochemical and phenotypic diversity within this species. Similarity measurement using UPGMA followed by cluster analysis resulted in formation of many groups based on geographical distribution that generally reflected expected trends between the genotypes. There were also some important exceptions like PW-S, an accession from Wastoorwan, Khrew showing close resemblance to PG-S and PG-B collected from Gulmarg but grown at two different gene banks at Srinagar and Bonera. Further an accession PSH-B from Keller was significantly diverse from the rest of the native genotypes phytochemically, morphologically and at molecular level. RAPD data analysis was found to be significant predictor of phytochemical markers in cultivated P. hexandrum germplasm. Twelve accessions grown in gene bank repository were subjected to RAPD analysis and were assessed for content of podophyllotoxin and podophyllotoxin β-d-glycoside by HPLC. Individual regressions of podophyllotoxin and podophyllotoxin β-d-glycoside by RAPD analysis against HPLC has been found to determine linear values. Strong correlation and a strong association of values of the phytochemical variables and the DNA polymorphism data has been recorded.     

Genetic diversity and population's structure in Tunisian strawberry tree (Arbutus unedo L.)

A growing interest in the use of the Strawberry tree (Arbutus unedo L., Ericaceae) has been recently reported for industrial, pharmaceutical and chemical fields. However, the bulk material comes from natural populations because of the lack of selection of interesting cultivars. In Tunisia, A. unedo populations are severely destroyed due to deforestation and over-collecting. The species occurs in small scattered populations decreasing progressively in size. Yet, no conservation or improvement programs are attempted to preserve and promote the potential value of this resource. In this work, we assessed the genetic diversity of nine Tunisian populations of A. unedo L. from different bioclimates, using 65 polymorphic RAPD loci. The analysis of the genetic variation within and among populations is primordial to elaborate conservation and improvement programs. A low genetic diversity within a population estimated by both Nei's (He) and Shannon's diversity (H′) indices (0.155 < He < 0.248; 0.229 < H′ < 0.364) was observed due to genetic drift and selfing. At the species level, the amount of the within population variation estimated by Shannon's index (H_POP/H_SP = 0.686) and the molecular variance (80.67%) was higher than that among populations. A moderate genetic differentiation among populations (Φ_ST = 0.193 and G_ST = 0.314) which could be attributed to the long seed distance dispersal was detected. The UPGMA dendrogram based on Φ_ST values showed three clusters each including populations without relationship to bioclimatic or geographical origin indicating that differentiation occurs at a local space scale. The in situ protection measures should be made appropriately according to a population within bioclimates. The ex situ conservation and the selection of genotypes should involve extensive collection of seeds or cuttings from the within populations rather than among them.     

Comparative analysis of genetic diversity in Prunus L. as revealed by RAPD and SSR markers

RAPD and SSR markers were used for genetic diversity evaluations among 15 genotypes selected from the genus Prunus L. Altogether 40 RAPD primers and 21 primer pairs designated for microsatellite loci were applied on the whole group of genotypes.     

DNA profiling of Capsicum landraces of Manipur

Seven chilli landraces of Manipur belonging to three cultivated species of Capsicum (Capsicum annuum, Capsicum frutescens, and Capsicum chinense) form economically important food crops of the region. The genotypes were characterized using ten random amplified polymorphic DNA (RAPD) markers. The cluster analysis based on Jaccard's similarity coefficient calculated by UPGMA method differentiated the genotypes into two main cluster groups. One cluster represented the C. annuum genotypes while the other cluster represented the C. frutescens and the C. chinense genotypes. C. chinense genotypes were more close to C. frutescens genotypes. Genetic variation between the C. frutescens genotypes was more than among the C. annuum genotypes and the C. chinense genotypes were the least similar ones.     

Phylogenetic relationships in Origanum spp. based on rDNA sequences and intra-genetic variation of Greek O. vulgare subsp. hirtum revealed by RAPD

Origanum species are among the most widely spread herbs in the Mediterranean basin. Eventhough they are used as a spice, evaluation of their genetic diversity and evolution has only recently drawn attention. In order to study phylogenetic relationships, 14 ITS1-5.8S-ITS2 clones belonging to the most common Origanum species were sequenced and a parsimony tree was constructed, using the approximate likelihood ratio test. All Origanum species were clearly separated from allied genera of the Mentheae tribe while a clear distinction between the Greek and the Spanish accessions was revealed. In addition the germplasm variability of the most common Greek oregano (O. vulgare subsp. hirtum) was investigated using the RAPD markers. The use of 10 random decamers resulted in 133 unambiguous and reproducible bands detected across 27 entries. Two main groups were identified by the UPGMA clustering using Jaccard's similarity coefficient, and major genetic dissimilarities among Greek O. vulgare subsp. hirtum populations and O. onites/O. virens species were detected. Analysis of molecular variance revealed that genetic variability is distributed mainly within populations; however, significant Φ_st values were detected between different geographical localities, supporting noteworthy genetic differentiation among O. vulgare subsp. hirtum populations.     

Plant regeneration of an endangered medicinal plant Hydrastis canadensis L.

Goldenseal (Hydrastis canadensis L.) is an endangered medicinal plant used to treat sore eyes and mouths, cold and flu and also as a dye. The objective of this study was to develop an efficient in vitro propagation protocol for goldenseal. Significantly more shoots (26 shoots per leaf explants) were induced on a medium containing 2.5 μM thidiazuron (TDZ) and 5.0 μM 1-naphthaleneacetic acid (NAA) than any other treatment. Sub-culturing regenerated shoots on a medium with 5.0 μM 6-benzylaminopurine (BA) induced the maximum rate of shoot multiplication. Growth of the regenerated shoots in a temporary immersion bioreactor resulted in significant increases in biomass, shoot height and shoot multiplication. The regenerated shoots from the temporary immersion bioreactor formed roots when transferred onto a medium with 1.0–2.0 μM indole-3-butyric acid (IBA). Regenerated whole plantlets were acclimatized and maintained in standard greenhouse conditions for further growth. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of this rare, medicinally important species.