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.     

Phenotypic variability and genetic structure in plum (Prunus domestica L.), cherry plum (P. cerasifera Ehrh.) and sloe (P. spinosa L.)

In the present study, phenotypic variability of 80 plum (Prunus domestica L.) varieties maintained in the French National Plum Collection was evaluated with 19 quantitative traits. In addition, genetic diversity and genetic structure was studied in three plum species (P. domestica L., Prunus cerasifera Ehrh. and Prunus spinosa L.) using chloroplast DNA (cpDNA) markers and five single sequence repeat (SSR) loci. Based on phenotypic traits, some varieties, such as mirabelle plums, grouped together. Bayesian structure analysis was used to identify different genetic groups, whereby damson plums were clearly distinguished from greengage plums. When examining the three species together, a higher level of cpDNA allelic richness was found in P. cerasifera and in P. spinosa than in P. domestica where only five cpDNA haplotypes were detected in the national plum collection, with one main haplotype that accounted for 80% of the varieties studied. P. domestica cpDNA haplotypes tended to group together with P. cerasifera haplotypes whereas most of P. spinosa haplotypes formed a separate cluster. SSR markers were somewhat able to distinguish the three species. These results provide some clues as to the origin of plum and the various plum varieties. Our results also provide useful information for the management of plum genetic resources.     

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.     

Genetic structure of Buxus sinica var. parvifolia, a rare and endangered plant

Buxus sinica var. parvifolia, a rare and endangered tree species in some semitropics alpine areas of China, plays an important role in the maintenance of the landscape and ecosystem. In this study, RAPD and ISSR markers were used to investigate the genetic diversity and structure of five natural populations and one tamed population of B. sinica var. parvifolia. 21 RAPD primers amplified 209 bands with 167 (79.90%) polymorphic and 21 ISSR primers amplified 518 bands with 467 (90.15%) polymorphic. The genetic diversity, estimated by Shannon’ index, was 0.4343 (by RAPDs) and 0.3661 (by ISSRs). Both RAPD and ISSR analyses revealed a high level of genetic diversity in natural populations of B. sinica var. parvifolia. Furthermore, analysis of molecular variance (AMOVA) was used to apportion the variation within and between populations. The proportion of variation attributable to within-population differences was very high (69.2% by RAPDs; 84.51% by ISSRs). Moderate differentiation was detected among populations using RAPDs (30.80%), while only a small amount of variation (15.49%) was detected among populations using ISSRs. We suggest that the present genetic structure is due to high levels of environmental variability and gene flow, which still need further study to confirm. Conservation measures are suggested, including in situ and ex situ strategies, based on the observed population genetic information.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

Somatic embryogenesis and plant regeneration in Opuntia ficus-indica (L.) Mill. (Cactaceae)

We present here a method for the regeneration of the prickly-pear (Opuntia ficus-indica (L.) Mill.) through somatic embryogenesis (SE). Direct SE was induced by cultivating shoot apices devoid of leaf primordia on semisolid Murashige and Skoog (MS) basal medium supplemented with 4-amino 3,5,6-trichloropicolinic acid (picloram) at 4 mg l⁻¹. Somatic embryogenesis was influenced by the type, age, physiological and developmental stage of the explants, and also by light conditions, wounding, and the sucrose concentration in the induction medium. A histological analysis confirmed SE by revealing the presence of a closed vascular system in the developing embryos and the absence of a vascular connection between the somatic embryos and the explant.     

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.     

Callus induction and plant regeneration from embryonic axes of Kosteletzkya virginica

Kosteletzkya virginica, a perennial dicot halophytic species of the Malvaceae, is native to American salt marsh. It was introduced into China as a potential species to improve coastal wetlands and to develop ecologically sound saline agriculture. K. virginica adapts excellently to the tidal-flat habitats in China's east coast, with multiple eco-benefits; in particular, its seed oil could be used to produce biodiesel. The purpose of this study was thus to develop a standardized protocol to induce a high frequency of callus and subsequent plantlet regeneration system for a K. virginica breeding program with the final objective of applying transgenic techniques to improve seed oil yield. The embryonic axes of K. virginica were used as explants for callus induction, shoot induction from the callus and then adventitious root induction from the shoots on nine culture media with different hormone combinations. The best results were achieved on the following media: (1) 93.94% callus induction on MS medium supplemented with 1.0 mg L⁻¹ indole-3-acetic acid (IAA), 0.3 mg L⁻¹ kinetin, 30 g L⁻¹ sucrose and 8 g L⁻¹ agar; (2) 65.83% shoot induction on 1/2MS medium supplemented with 0.1 mg L⁻¹ IAA, 0.5 mg L⁻¹ zeatin, 30 g L⁻¹ sucrose and 8 g L⁻¹ agar; (3) 96.67% rooting on MS medium containing 30 g L⁻¹ sucrose and 8 g L⁻¹ agar. The survival rate of plantlets by organogenic regeneration was 85% after being transplanted into potting soil in flowerpots and placed in the greenhouse. This experiment indicates that we established successful callus induction and plant regeneration protocols for K. virginica.     

In vitro shoot regeneration from immature seeds of Epimedium alpinum induced by thidiazuron and CPPU

In vitro propagation of Epimedium alpinum L. was carried out using immature seed explants. The effects of various concentrations of thidiazuron (TDZ) and 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), on the induction of organogenic callus, were evaluated. Organogenesis occurred most efficiently when explants were transiently exposed (48 h) to 20 μM CPPU or 80 μM TDZ followed by culture on hormone-free woody-plant medium (WPM). Organogenic callus consisting of white, compact clumps of tissue proliferated slowly on hormone-free WPM. To promote adventitious shoot induction, the effects of different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA) were investigated. The highest per cent shoot regeneration, 66.7% of explants, and the maximum mean number of shoots, 2.6 per explant, were obtained on WPM containing 1.1 μM 2,4-D and 22 μM BA. Shoots were rooted on hormone-free WPM and well-developed plantlets were successfully transferred to soil.     

Assessment of genetic diversity of Latvian and Swedish sweet cherry (Prunus avium L.) genetic resources collections by using SSR (microsatellite) markers

Three previously described highly polymorphic SSR (microsatellite) primer pairs were tested on 126 sweet cherry (Prunus avium L.) accessions to adapt a fast, reliable method for preliminary screening of sweet cherry germplasm collections and to compare two sweet cherry germplasm collections: at the Latvia State Institute of Fruit-Growing, Dobele (LIFG-Dobele) and at the Division of Horticultural Genetics and Plant Breeding at Balsgård, Department of Crop Sciences, Swedish University of Agricultural Sciences (SLU-Balsgård). The SSR loci were highly polymorphic with 4–10 different alleles and 5–18 genotypes. Heterozygosity values ranged from 0.431 to 0.809, gene diversity (PIC) values ranged from 0.400 to 0.753, and the discriminating power of each locus varied from 0.631 to 0.894. The combined discriminating power of all loci was highly effective (0.996). Sixteen identical accession groups with the same allele profile were discovered in both collections. This study demonstrated that SSR fingerprinting with the three primer pairs tested, can be used for preliminary characterization of sweet cherry germplasm collections.     

Micropropagation of Capsicum frutescens L. using axillary shoot explants

A novel micropropagation protocol was established for Capsicum frutescens L. cv. ‘Uchithi’, a pungent chilli cultivar, through induction of axillary shoot proliferation of in vitro raised plantlets by decapitation and using the axillary shoots as explants for multiple shoot bud induction. About 2–6 axillary shoots were induced within 2 weeks when 4-week-old in vitro raised plantlets were decapitated. The axillary shoot-tip explants produced multiple shoot buds when cultured on Murashige and Skoog's (MS) medium containing 8.8–44.4 μM 6-benzylaminopurine (BAP) or 9.3–46.7 μM kinetin alone or 8.8–44.4 μM BAP with 4.6 μM kinetin or 5.7 and 28.5 μM indole-3-acetic acid (IAA). Maximum number of shoots (5.6) were induced on medium containing 22.2 μM BAP in combination with 4.65 μM kinetin. The separated shoots rooted and elongated on medium containing 2.8 μM IAA or 2.4–4.9 μM indole-3-butyric acid (IBA). Rooted plantlets were successfully established in the soil. Efficient mass multiplication of this important food crop was achieved.     

Agrobacterium × plant factors influencing transformation of ‘Joseph's coat’ (Amaranthus tricolor L.)

A protocol is developed for Agrobacterium-mediated genetic transformation of Amaranthus tricolor via explant co-cultivation with Agrobacterium rhizogenes. Bacteria-plant specific factors which influenced transformation were optimized. Of the two Agrobacterium strains employed, LBA9402 was more infectious compared to A4. Bacterial suspensions grown overnight with 100 μM acetosyringone and experiencing O.D.₆₆₀ = 0.6 followed by dilution to a density of 10⁹ cells ml⁻¹ were the most effective. Explants from garden-grown plants were more responsive than those from in vitro cultures; stem internodes being better than leaves. Immersion of the pre-pricked explants in bacterial suspension resulted in a markedly higher transformation frequency compared to the direct injection method. The infection of internode explants with the LBA9402 strain followed by co-cultivation on growth regulator-free MS medium (MS0) for 5 days resulted in emergence of hairy roots up to a maximum frequency of 97.22%. Roots were individually cultured in MS0, but fortified with bactericidal antibiotic (500 μg ml⁻¹ cefotaxime). Rhizoclones showing prolific growth were renewed through successive subcultures in MS0. Opine gene expression was revealed by positive agropine and mannopine synthesis in all selected transformed rhizoclones. Shoot regeneration from root clones, capable of auxin-independent growth and opine proficiency, was stimulated in MS augmented with 2.0 mg l⁻¹ zeatin. pRi TL–DNA rolB and pRi TR–DNA man2 ORF were detected in leaf tissues of regenerated plants from selected hairy root clones through PCR amplification. The implication of such findings is discussed on the possibility of conferring protection to crop amaranths against biotic stress challenges, particularly due to insects, viruses or fungal pathogens.     

Identification of Italian landraces of bean (Phaseolus vulgaris L.) using an image analysis system

Colour, shape and size of whole seeds and their spots of some Italian landraces of bean (Phaseolus vulgaris L.) were measured using a specifically developed macro, based on image analysis library KS-400 V 3.0 (Carl Zeiss, Germany).     

Factors affecting mango (Mangifera indica L.) protoplast isolation and culture

The major factors influencing protoplast isolation and culture of mango (Mangifera indica L.) cv. Kensington Pride were investigated. The resultant protocol was used to compare plating efficiency among 4 mango cultivars. Most responses differed between proembryonic masses (PEMs) and leaf sources. Protoplast yields of 15.22 × 10⁶ g⁻¹ from PEMs and 8.68 × 10⁶ g⁻¹ from greenhouse-derived leaves were obtained in a solution of 0.7 M mannitol CPW plus 1.5% cellulase, 1% hemicellulase and 0.75% macerozyme for PEMs or 0.5 M mannitol CPW plus 1.5% cellulose, 1% hemicellulase and 1.5% macerozyme for leaves. Culture in Ca-alginate beads with initial plating densities (IPD) of 2.5 × 10⁴ Pp mL⁻¹ for PEMs and 2.5 × 10⁵ for leaves gave the highest plating efficiencies (FPE). For PEMs 1 mg L⁻¹ 2,4-d and 3.5 mg L⁻¹ kinetin gave an FPE of 2.85% whereas lower kinetin (2 mg L⁻¹) plus 0.5 mg L⁻¹ 6-BAP was most effective for leaves (FPE of 2.12%). Most protoplast mortality occurred during the first week of culture and was more severe in liquid culture. In Ca-alginate beads, protoplast survival at 14 days was higher for PEMs (30%) than leaf (21%) as was the frequency of cell division (17.6% compared to 13.6%). PEMs protoplasts continued development through embryogenesis to in vitro plantlet regeneration whereas leaf protoplasts underwent cell division up to 40-cell colonies but failed to proceed further. For PEMs, polyembryonic cvs. Kensington Pride and Keow Savoey produced higher FPE (1.95%) than monoembryonic cvs. Tommy Atkins and Keitt (1.75%). There was no effect of cultivar for leaf protoplasts.     

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.     

Molecular polymorphism of cytoplasmic DNA in Ficus carica L.: Insights from non-coding regions of chloroplast DNA

The trnL (UAA) intron and the intergenic spacer between the 3′ exon of trnL (UAA) and trnF (GAA) sequences were used as genetic markers for differentiating Ficus carica cultivars and establishing refined genetic relationships. The study was based on 20 fig cultivars, collected from south and centre of Tunisia. Since, the intron was thought to be more variable among close relatives than is the chloroplast spacer. The size of these non-coding regions varied from 554 to 589 and from 989 to 1022 bases pairs for the intron and the combined sequences correspondingly. The average of GC content was 33.9% and 34.6% in the intron and the combined intron and spacer respectively. High values of A + T contents were detected in both data sets and may explain the high proportions of transversions founded. The observed variation pattern of plastid DNA provides evidence of an important genetic diversity. The overall transition/transversion bias (R) was 0.202 in the intron and 0.27 in the combined regions. The RI index of 0.592 indicates that these combined sequences have clearly more homoplasy then the intron (RI = 0.705) and spacer (RI = 0.777) sequences separately. Phylogenetic trees were generated based on maximum parsimony (MP) and neighbor-joining (NJ) analysis of the chloroplast sequences data. Results proved that a typically continuous genetic diversity characterizes the local fig germplasm. In fact, relationships inferred from the cpDNA analysis suggest several clades, which do not show geographical or tree sex correspondence. Although the level of apparent diversity is considerable, we may conclude that non-coding regions of chloroplast genome provide a new and practical opportunity to evaluate genetic diversity and to discriminate fig cultivars. Revealed cytoplasmic DNA markers are reliable to elaborate a molecular data base to conduct management and breeding programs on local fig germplasm.     

In vitro germination of walnut (Juglans regia L.) embryos

The present studies were undertaken with a view to standardize the medium and culture conditions for embryo culture of five cultivars of walnut viz., ACO 38853, Netar Akhrot, Gobind, Solding Selection and Blackmore. Embryos from mature fruits were aseptically excised and cultured on MS medium supplemented with different combinations of BAP, kinetin and GA₃. Best performing medium was MS with 0.5 mg l⁻¹ kinetin, 0.5 mg l⁻¹ BAP and 2 mg l⁻¹ GA₃ yielding 66.6% germination in Netar Akhrot after 12 days of culturing. Percent germination of excised embryos was higher when GA₃ and cold treatments were simultaneously applied as compared to those when applied separately. Netar Akhrot was found to be the best responding cultivar, which had a range of 25–66.6% embryo germination under different culture conditions. Plantlets with shoots and roots have been obtained in Netar Akhrot and ACO38853 and are transferred to soil after hardening.     

A SCAR marker linked to the N gene for resistance to root knot nematodes (Meloidogyne spp.) in pepper (Capsicum annuum L.)

The root-knot nematodes (Meloidogyne spp.) are important nematode pests and cause serious diseases in pepper in the world. No molecular markers linked to the nematodes resistance N gene have been reported. In this paper, ‘Carolina Wonder’ (Capsicum annuum L.), a sweet pepper line resistant to root-knot nematode with N gene, ‘20080-5-29’ (C. annuum L.), an inbred line susceptible to root-knot nematode with good horticultural characteristics, and their F₂ progeny with 320 individuals were used as materials. Evaluation of resistance and susceptibility of parental lines, F₁ and F₂ progeny inoculated with root-knot nematodes (Meloidogyne incognita) were carried out. ‘Bulked segregant analysis’ method was used to search for polymorphic markers from 512 pairs of AFLP primers. Based on the assessment of resistance and susceptibility and polymorphism of the AFLP marker in F₂ population, the genetic linkage distance between the AFLP marker and the N gene was estimated. One AFLP marker E39/M41-339 was obtained and transferred to a SCAR marker amplifying a 315 bp DNA fragment linked to the N resistant allele and a 331 bp fragment linked to the N⁺ susceptible allele. The distance between the molecular marker and the nematodes resistance N gene is 6.3 cM. This research delivered a valuable tool for the marker assisted selection of nematodes resistance in pepper.     

Agrobacterium tumefaciens mediated genetic transformation and regeneration of Morus alba L.

An efficient method was established for genetic transformation of Morus alba clone M5 using Agrobacterium tumefaciens mediated gene transfer. Cotyledon explants from in vitro grown seedlings were co-cultivated with disarmed strain LBA 4404 harbouring the binary vector p^BI121 carrying chimeric β-glucuronidase (GUS) and neomycin phosphotransferase (npt II) genes. Maximum transformation frequency of 18.60% was recorded with 48 h of pre-conditioning followed by co-cultivation for the same duration. Expression and presence of transgene was confirmed by histochemical test and polymerase chain reaction. The transgenic plants were micropropagated and successfully acclimatised.