Adventitious root formation in Anacardium occidentale L. in response to phytohormones and removal of roots

Despite advances in tissue culture techniques, propagation by leafy, softwood cuttings is the preferred, practical system for vegetative reproduction of many tree and shrub species. Species are frequently defined as ‘difficult’- or ‘easy-to-root’ when propagated by conventional cuttings. Speed of rooting is often linked with ease of propagation, and slow-to-root species may be ‘difficult’ precisely because tissues deteriorate prior to the formation of adventitious roots. Even when roots form, limited development of these may impair the establishment of a cutting.     

Hybridization among Epimedium (Berberidaceae) species native to China

The hybridization of fourteen plant populations belonging to seven Epimedium species native to China was studied by self-cross, infra-population cross, inter-population cross within species, and interspecific cross. Self-pollination studies on nine populations indicated high incompatibility; capsule-set rates were higher than zero in only three (4.61–6.76%). Interspecific cross-pollinations demonstrated high crossabilities in most cross combination (15.38–92.44% capsule-set rate), and the F₁ seeds possessed high germination rates (>20%). The F₁ hybrids of three interspecific cross combinations were raised to maturity. The morphology, karyomorphology of somatic cells, and pollen mother cell (PMC) meiosis of these F₁ plants revealed that they were all highly fertile (>76.10% in pollen viability), that there were few structural differences in chromosomes among species, and that most PMCs had 6 bivalents at MI. Abnormal chromosomal behaviors occurred in a minority, including chromosome bridges, unequal segregation of chromosome number, lagging chromosomes, and micronuclei. A series of experimental crosses provided strong evidence for an outbreeding system and a weak internal barrier to hybridizations in the taxa studied.     

Piriformospora indica promotes adventitious root formation in cuttings

Adventitious root formation in excised plant shoots is a crucial process in the vegetative propagation of many plant species, and insufficient rooting causes substantial losses in the propagation industry. Based on the various physiological effects on whole plants described for the basidiomycete Piriformospora indica, it was hypothesized that inoculation of the substrate with this endophyte should promote the generation and growth of adventitious roots in cuttings. Inoculation experiments were conducted to study the effects of P. indica on adventitious rooting in three plant species. Inoculation with P. indica dramatically enhanced the number and length of the adventitious roots in pelargonium and poinsettia. Root colonization parameters suggest that the interaction between the endophyte and cuttings had already occurred before physical contact. In contrast, petunia showed no rooting response to P. indica inoculation. Very fast root formation in this plant indicates that a minimum time period for the fungus–plant interaction is required for establishment of a promoting effect. P. indica-based biotechnology is proposed as a new tool for improving plant propagation systems of plant species or cultivars with low to moderate capacity of adventitious root formation.     

Analysis of adventitious root formation in isolated stem explants of Rhododendron

Factors affecting adventitious root formation were studied in isolated stem segments of three Rhododendron cultivars, ‘Catawbiense Album’, ‘Pink Pearl’ and ‘Van Weerden Poelman’, having different rooting ability. The experiments consisted of varying certain factors, while keeping the others constant.Root formation was always less in ‘Van Weerden Poelman’ than in the easy-to-root cultivars. Rooting usually occurred only in segments from young soft shoots. The length of the explants did not affect rhizogenesis. Removal of a strip of bark promoted adventitious root formation. Oxygen supply appeared to be essential since only inverted segments were capable of forming roots. Darkness was essential for rooting. The optimum temperature was 25°C. Root formation occurred only when a sugar was present in the medium. Rooting decreased with increasing osmotic pressure. Macroelements, boric acid and the pH of the medium did not play an essential role. Auxin was an absolute requirement for rooting, the kind and concentration depending on the cultivar used. Benzylamino purine at low concentrations increased root dry weight. GA₃ and abscisic acid decreased rooting.     

Effects of supplementary light to mother plants on adventitious shoot formation in flower peduncle segments of Begonia × hiemalis Fotsch in vitro

Plants of two Begonia × hiemalis cultivars (‘Schwabenland’ and ‘Nixe’) were grown in a greenhouse and continuously illuminated (24 h a day) from late October to early March, with light from warm white fluorescent lamps (L“WW”), high pressure mercury lamps (HPI/T) or high pressure sodium lamps (SON/T), each at three irradiance levels (5, 10 or 15 W m^?2). The control received light continuously (0.8 W m^?2) from incandescent lamps (INC). Every third week, plant material (flower peduncles) was collected for in vitro culture. The adventitious shoot formation was strongly dependent on cultivar, length of mother plant illumination and light source. The use of L“WW”, HPI/T or SON/T improved the regeneration ability of flower peduncle segments compared to control. Prolonged irradiation lowered the regeneration in ‘Schwabenland’, especially when HPI/T lamps were used.     

Variation in two Begonia × hiemalis clones after in vitro propagation

In order to study the variation of Begonia × hiemalis after in vitro propagation, one plant of ‘Aphrodite Pink’ and one of ‘Schwabenland Red’ were vegetatively propagated in four ways. One group of plants was obtained by propagation by means of cuttings. For the second group, the bacterial elimination system, as developed by Hakkaart and Versluijs (1983), was applied which includes an in vitro propagation step. The third and fourth groups were obtained by adding one and two cycles, respectively, of in vitro propagation after the bacterial elimination, so that one, two and three propagation cycles could be compared. One cycle, followed by bacterial elimination, gave nearly uniform offspring. However, when this was followed by one or two cycles of in vitro propagation, the variation increased. Variation also increased when the size of the plantlets obtained in vitro decreased. Thus, an important source of variation can be avoided by eliminating the smallest in vitro plantlets. It is recommended to flower and select the plants before propagation by conventional cuttings, whether or not a further in vitro propagation is applied after the bacterial elimination.     

ISSR variations in eggplant (Solanum melongena L.) and related Solanum species

Inter-simple sequence repeat (ISSR) analysis was performed in eight cultivars of eggplant (Solanum melongena) and 12 accessions in eight related Solanum species to evaluate the applicability of this analysis for assessing the phylogenetic relationships and identifying cultivars. A total of 552 polymorphic amplified bands were obtained from 34 of the 100 primers tested, and the percentage of polymorphisms was 99.1%. Cluster analysis based on the ISSR markers classified the Solanum species into seven groups: (i) S. melongena; (ii) S. aethiopicum and S. anguivi; (iii) S. incanum; (iv) S. violaceum and S. kurzii; (v) S. macrocarpon; (vi) S. virginianum and (vii) S. torvum. Combining the ISSR markers obtained by a few of the 34 primers was enough for distinguishing of the eight cultivars of eggplant. This ISSR analysis was demonstrated to be available for the phylogenetic study and the cultivar identification.     

The insertion of the Agrobacterium rhizogenes rolC gene in tomato (Solanum lycopersicum L.) affects plant architecture and endogenous auxin and abscisic acid levels

In the present paper we report on the effects of the insertion of the Agrobacterium rhizogenes rolC gene in the tomato (Solanum lycopersicum L., formerly Lycopersicon esculentum Mill.) cultivar Tondino. Several transgenic lines were successfully obtained, between which two clones, rolC1 and rolC3, were chosen for the analysis of morpho-productive traits as well as of the endogenous levels of auxin and abscisic acid. Consistent with the known phenotypic effect of this gene, the transformed tomato plants were significantly shorter than the corresponding controls. On the other hand, even if yield was not affected by the transformation in terms of average number of fruits produced, fruit weight was significantly lower in the transgenics with respect to the controls. Therefore, insertion of the rolC gene does not lead to an improvement in plant productivity.     

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.     

Effect of cutting date and position on rooting ability and fatty acid composition of Carignan (Vitis vinifera L.) shoot

The effects of cutting date and position (apical, basal and central) on rooting ability and fatty acid composition from Carignan (Vitis vinifera L.) shoot were determined. Root number and weight depended of the cutting date and position. Only in the case of the cutting in the basal position, there was a highly positive correlation between number and percentage of roots (r = 0.95) during sampling date. Concerning the influence of the cutting date, the root number by cutting oscillated in a saw tooth. The root weight and percentage showed a positive correlation for the three cutting positions and they increased with time. Cutting date and position had an irregularly effect on the contents of total lipid and different fatty acids. Independently of cutting position, the contents of oleic acid (C18:1) and palmitic acid (C16:0) correlated negatively with those of linoleic (C18:2) and linolenic acids (C18:3) during sampling date.     

Adventitious shoot regeneration in a bioreactor system and EST-PCR based clonal fidelity in lowbush blueberry (Vaccinium angustifolium Ait.)

Adventitious shoots were regenerated from leaf explants of three lowbush blueberry (Vaccinium angustifolium Ait.) clones: ‘QB1’, ‘QB2’ and ‘PB1’ by culturing on a gelled basal medium (BM) with 2.3–4.5 μM thidiazuron (TDZ) for four weeks followed by in a bioreactor system containing the same liquid medium but with 1.2–2.3 μM TDZ for another four weeks. Young expanding basal leaf segments with the adaxial side touching the culture medium and maintained for two weeks in darkness, produced the best results. Callus development and shoot regeneration were genotype dependent. Adventitious shoots were elongated in the liquid BM with 1 μM zeatin and rooted on a three peat: two perlite (v/v) medium. Acclimatized plantlets were grown actively in the greenhouse with an apparent normal leaf and shoot morphology. Ten random ‘QB1’ regenerated plants were screened using 14 expressed sequence tag-polymerase chain reaction (EST-PCR) markers and showed similar monomorphic amplification profiles confirming clonal fidelity of in vitro-derived ‘QB1’ plants. Results obtained suggested the possibility of adventitious shoot regeneration and true-to-type lowbush blueberry micropropagation using a bioreactor system combined with gelled medium.     

Cross-transferable polymorphic SSR loci in Prunus species

This work reports the transferability and polymorphism of previously reported SSRs in 10 Prunus species. The availability of a large number of SSRs in the genus Prunus makes marker choice random, while preventing comparison of results in fingerprinting studies. The availability of SSR markers, polymorphic in a wide sample of Prunus species, would facilitate marker choice, while allowing the comparison of results. In this work, microsatellite markers useful for analyzing 10 different Prunus species (P. persica, P. dulcis, P. armeniaca, P. domestica, P. insititia, P. salicina, P. cerasifera, P. avium, P. cersus and P. mahaleb) were searched through screening SSRs previously reported to be conserved and/or polymorphic in more than one Prunus species. A selected group of 13 SSRs, transferable to the 10 species, was analyzed in terms of their usefulness for analyzing these species. The amplification range, polymorphism and variability detected by these loci are reported. The information provided will be useful for Prunus genetic studies as well as conservation and management of Prunus germplasm resources.     

Influence of cutting position and temperature during rooting on adventitious root formation and axillary bud break of Stephanotis floribunda

Stock plants were grown in a glasshouse under standard growing conditions. Single-node leafbud cuttings were excised and numbered according to the position on the stock plant. Rooting took place at basal temperatures of 17,20 or 23°C and at different durations at 17 or 20°C followed by 23°C. The rooting period lasted 9 weeks.

The temperature of 17°C for 9 weeks completely suppressed root formation. A temperature of 20°C was decisive for root formation. The optimal rooting temperature was higher than 23°C. Temperature treatments of 17 or 20°C for 2–4 weeks only suppressed rooting slightly compared with the 23°C treatment. Cutting position on the stock plant affected the number of roots formed per cutting but not the rooting percentage. Best rooting was observed in cuttings from the middle part of the stock plant.

Axillary bud break was accelerated with increasing rooting temperature and decreasing duration of the lower temperatures. With increasing cutting position number (numbered from top to base), axillary bud break was considerably delayed.

Temperature treatments which delayed root formation also delayed axillary bud break. On the other hand, the cutting position on the stock plant, which had only a minor effect on the speed of root formation, had a pronounced effect on the speed of axillary bud break.     

Reproductive characteristics of Opisthopappus taihangensis (Ling) Shih,an endangered Asteraceae species endemic to China

Opisthopappus taihangensis is an endangered species endemic to China and represents an important genetic resource for chrysanthemum improvement. We describe here its basic reproductive characteristics. The anthers are tetrasporangiate and the anther wall is composed of an epidermis, endothecium, middle layer and tapetum. The middle layer is lost by the microspore tetrad stage, and the tapetum disintegrates at the trinucleate pollen stage. Meiosis in the microspore mother cells is of the simultaneous type, and the tetrad is tetrahedral in shape. Mature pollen grains have three germinal apertures, two sperm nuclei and one vegetative nucleus. The in vitro pollen germination rate is only ∼10%. The ovule is anatropous, dual-integument, tenuinucellatae and the development of the embryo sac follows the Oenothera pattern. The archesporial cell below the nucellus epidermis functions as the megaspore mother cell and forms a linear tetrad. The embryo passes through a globular, heart and torpedo stage before maturing into a cotyledon embryo. The endangerment of O. taihangensis may be associated with low reproductive capacity, as a consequence of poor pollen viability.     

Collection time,cutting age,IBA and putrescine effects on root formation in Corylus avellana L. cuttings

The effects of collection time, cutting age, indole-3-butyric acid (IBA) and putrescine application on the rooting of cuttings of Italian hazelnut cultivars ‘Tonda Gentile Romana’, ‘Tonda di Giffoni’ and ‘Nocchione’ were investigated. Samples collected during late June, late July and early September from newly formed and 1-year old part of twigs to be utilized to produce leafy cuttings, after being treated with 1000 and 2000 ppm IBA, respectively. In addition, the September cuttings were also treated, respectively, with 1000 and 2000 ppm IBA and 1600 ppm putrescine. Rooting ability was evaluated 2 months after planting for each treatment and collection time. Satisfactory rooting of hazelnut leafy cuttings was observed when collection time occurred in June and September, whereas leafy cuttings collected in July showed a limited capacity of rooting in all cultivars tested. On average, the rooting of the newly formed leafy cuttings was more than the 1-year old cuttings. Rooting was also promoted by IBA treatments, mainly in ‘Nocchione’ and ‘Tonda di Giffoni’. In contrast, young cuttings collected from ‘Tonda Gentile Romana’ in early September rooted poorly when treated with IBA alone, but showed the best rooting (∼80%) after the application of a combination of 1000 ppm IBA and 1600 ppm putrescine. The current findings confirm that putrescine can be a useful substance for increasing rooting percentage and root quality in cuttings of some hazelnut cultivars as obtained from ‘Tonda Gentile Romana’ cultivar.     

Development of novel chloroplast microsatellite markers for Dendrobium officinale, and cross-amplification in other Dendrobium species (Orchidaceae)

Nine pairs of polymorphic chloroplast microsatellite primers were developed for Dendrobium officinale Kimura et Migo, an endangered herb. Levels of polymorphism were tested across a total of 55 individuals from four natural populations (12–15 individuals per population). Allele numbers varied from two to four per locus, while the number of haplotypes ranged from four to six per population. Transferability of the nine polymorphic chloroplast microsatellite primers was checked on an additional set of 51 Dendrobium individuals (belonging to 17 different species). Three markers could be transferable to all the species tested, while the remaining six markers successfully cross-amplified in most species tested. Moreover, polymorphism of the nine chloroplast microsatellite primers was tested across Dendrobium moniliforme (L.) Sw. and Dendrobium loddigesii Rolfe. All of them were polymorphic in D. moniliforme, while seven of which were polymorphic in D. loddigesii. These polymorphic chloroplast microsatellite primers developed for D. officinale will be a useful tool for the study of genetic diversity, population genetic structure, evolution of D. officinale and establishment of effective conservation strategies.     

A method for evaluating bolting-resistance in Brassica species

Kale (Brassica oleracea var. acephala), Chinese cabbage (B. campestris ssp. pekinensis), chikale (B. campestris ssp. pekinensis × B. oleracea var. acephala hybrid) and turnip (B. campestris spp. rapifera) were studied. The proper plant size (number of true leaves) for the beginning of vernalization treatment was determined. All vernalization experiments commenced when the plants were 4 weeks old, with 5–7 true leaves. Plants were classified as bolters either through the visible appearance of flower buds at the apex, or by making longitudinal cuts through the apex if the flower buds were not visible. Cut plants with a pointed apex were classified as bolters. A definite relationship between the number of true leaves and bolting was observed for kale, the more difficult biennial species.     

Asymbiotic seed germination,mass propagation and seedling development of Vanda coerulea Griff ex.Lindl. (Blue Vanda): An in vitro protocol for an endangered orchid

The application of modern biotechnology for conservation of any endangered species requires an efficient in vitro regeneration protocol. In this study a reliable protocol was developed for in vitro seed germination, protocorm multiplication and subsequent plantlet regeneration of Vanda coerulea, an endangered orchid species. Among the four basal media evaluated for asymbiotic seed germination, Phytamax was found to be the best followed by Murashige and Skoog (MS). Phytamax was also found good for protocorm development. For protocorm like body (PLB) regeneration, protocorms were then further cultured on Phytamax media fortified with different phytohormones either individually or in combinations. The frequency of protocorm like body (PLB) regeneration significantly relied on kinds and concentrations of plant growth regulators used. A combination of 1-naphthaleneacetic acid (NAA) (5.36 μM) and 6-benzyle amino purine (BAP) (3.80 μM) was found to be suitable for maximum PLB regeneration. Healthy plantlets were induced from PLBs when cultured on same basal medium supplemented with activated charcoal (AC – 3.0 g/l). Plantlets with well developed leaves and roots were transplanted to pots filled with a mixture of charcoal, brick pieces and sphagnum moss and transferred to the greenhouse. This protocol will enable mass propagation and conservation of this exquisite orchid.     

Conservation of Zingiber germplasm through in vitro rhizome formation

The effects of various concentrations of maleic hydrazide (MH; 2, 4, 6, 8 mg/l) and three light treatments (16-h, 24-h, 0-h) on in vitro rhizome formation and conservation of ginger (Zingiber officinale Rosc. cv. Rio de Janeiro) were studied. In vitro rhizome formation occurred in all the above treatments. Addition of MH (2–8 mg/l) to the control medium (CM) comprising Murashige and Skoog's (1962) salts, 9% sucrose, 0.8% agar-agar, 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l N⁶-benzyladenine (BA), did not show any significant positive effects on rhizome formation as well as survival of cultures. A significant effect of light treatments was observed on survival of cultures but not on rhizome formation. More than 50% cultures survived up to 14 months on CM under 16-h and 24-h light conditions as compared to 20% cultures on same medium incubated under dark. A total of 33 genotypes of cultivated and wild species of Zingiber were subsequently tested for conservation through in vitro rhizome formation on CM under 16-h light condition. All genotypes produced rhizomes of varying size with numbers ranging from 3 to 15 per culture and were conserved for at least 12 months; some genotypes could be conserved even up to 16–20 months. Viability of rhizomes was determined by in vitro regeneration of shoots upon subculture and their subsequent establishment in soil. Following the protocol described in the present paper, some 160 genotypes of cultivated and wild species of Zingiber, collected from different geographical regions of India, are being conserved at In Vitro Genebank of National Bureau of Plant Genetic Resources, New Delhi.