Vegetative propagation of Anthurium scherzerianum Schott through callus cultures

A tissue culture method is described for the vegetative propagation of Anthurium scherzerianum Schott through callus induction, callus subculture, adventitious bud formation in callus and rooting of excised shoot cuttings. One genotype which has been propagated vegetatively in vitro, is already growing in the greenhouse.     

The inheritance of systemic resistance to the bacterial blight pathogen (Xanthomonas axonopodis pv. dieffenbachiae) in Anthurium andraeanum (Hort.)

The inheritance of resistance to systemic infection by bacterial blight disease (blight) caused by Xanthomonas axonopodis pv. dieffenbachiae in anthurium (Anthurium andraeanum Hort.) was investigated in 83 biparental crosses involving 39 anthurium cultivars, using a green fluorescent protein-based screening method. Genetic analysis was based on North Carolina mating design II analysis, combining ability analysis and parent–offspring regression analysis. The results showed a role for both additive and non-additive effects in the inheritance of systemic resistance to blight, although the former was predominant and between four- to five-fold larger than the latter. General combining ability (GCA) of the parents was able to fairly accurately predict the performance of progeny in 15 of the 17 M × N (‘M’ male cultivars crossed with ‘N’ female cultivars) mating designs. The highly resistant cultivars were the best general combiners, while the susceptible cultivars generally produced negative GCA values. Dominance was in the direction of susceptibility. Narrow sense heritability (h²) based on North Carolina mating design II analysis, was 42.5%, with broad sense heritability (H²) of 96.5%. The estimate of h² based on mid-parent–offspring regression analysis was slightly higher (58–62%). In conclusion, the results indicated that resistance to systemic blight is governed by both additive and non-additive genetic effects with the former playing a larger role. Clonal breeding for systemic resistance to blight using a family selection approach is suggested.     

Plantlet regeneration from root callus of different Prunus species

Caulogenesis was obtained in vitro on the calli of Prunus sp., P. dawyckensis, P. canescens and hybrid P. incisa x serrula. These calli were formed on the roots of plantlets derived from meristem culture on a medium containing 6-benzylaminopurine (BAP) (10^?3 g/l) and gibberellic acid (GA₃) (10^?4 g/l). The first buds appeared after a month. After transfer of the calli on the same medium, the caulogenesis continued. The degree of regenation varied according to the species.     

Cultivar differences in the deterioration of vase-life in cut-flowers of Anthurium andraeanum is determined by mechanisms that regulate water uptake

Vase-life, average daily water uptake rate and abaxial stomata density were evaluated in 17 anthurium (Anthurium andraeanum Hort.) cultivars over two trials. There were significant cultivar differences for vase-life (14–49 days), 5-day average water uptake rate and abaxial stomata density, in both trials. A progressively stronger correlation coefficient between average daily water uptake rates during the period 25–50 days after initiation of experiment (DAI) and vase-life was observed, indicating that cultivars with longer vase-life were able to maintain above average water uptake rate over a longer period of time and hence delay symptoms of water stress. This was evident in the water uptake rate curves as an inflection, resulting in the levelling off of water uptake rates. The cultivars with long vase-lives achieved steady state water uptake levels faster, and maintained a high steady state water uptake rate for longer durations. The role of vascular occlusion and other senescence factors on water relations and vase-life is discussed.     

Plantlet regeneration and stability from callus cultures of Freesia × hybrida Bailey cultivar ‘Royal’

True-to-type plantlets of Freesia × hybrida Bailey cultivar ‘Royal’ were generated from callus after 27 months of sub-culture in liquid medium. Callus was initiated from young flower pedicels cultured on semi-solid Linsmaier—Skoog (LS) medium supplemented with 5 mg/l of 2,4-D and 0.5 mg/l kinetin, and transferred to the same medium in liquid form without hormones and thereafter sub-cultured every 7–10 days. Liquid cultures with 2.4–4.3 g of callus per 25 ml medium produced largest increases in callus fresh weight. Callus generated the most shoots when cultured on LS medium supplemented with 5 mg/l of kinetin and incubated in the light, while fewer plantlets were produced when no growth regulator or GA₃ or PBA were used. Callus cultures incubated in continuous darkness did not form shoots.     

Change in flower morphology of Torenia fournieri Lind. induced by forchlorfenuron application

We induced various flower morphologies in torenia (Torenia fournieri Lind.) by the application of forchlorfenuron (CPPU). Those morphologies were the combination of four basic morphological changes, the development of serrate petals, incised petals, a paracorolla, and an increased number of floral organs. These morphological changes occurred systematically depending on the floral stage at the time of CPPU application. Serrate petals were induced when CPPU was applied during the stages of corolla development, whereas application at younger stages induced petal incision. The serrate petal margin resulted from preferential proliferation of cells around the vascular bundles, whereas petal incision likely resulted from the lateral outgrowths of petal. A paracorolla was induced at the adaxial petal face when CPPU was applied between the sepal development stage and early corolla development. The paracorolla appears to have arisen from the lateral outgrowths of the stamen. The numbers of stamens, petals, and sepals increased when CPPU was applied at and before the differentiation of sex organs and the corolla. Enlargement of the floral meristem probably caused this increase. Application of N⁶-benzylaminopurine and zeatin did not induce these morphological changes.     

In vitro somatic embryogenesis from Mangifera indica L. callus

The nucellus and globular adventitious proembryos were removed from 2-month-old fruits of mango (Mangifera indica L.) cultivars ‘Ono’ and ‘Chino’, and were cultured on sterile, solid Murashige and Skoog (MS) medium that had been modified as follows: half-strength major salts and chelated iron; 20% (v/v) coconut water (CW); 6% sucrose; 100 mg l^?1 ascorbic acid and 400 mg l^?1 glutamine. Embryogenic explants were sub-cultured after 4–6 weeks in liquid modified MS medium containing 2 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) instead of CW. Rapidly growing cultures were established and were sub-cultured monthly. Somatic embryogenesis was induced following sub-culture from MS medium with 2,4-D to MS without growth regulators and with or without activated charcoal (0.5%). Germination of somatic embryos appeared to be enhanced by 1 mg l^?1 benzyladenine (BA); however, most of the germinating embryos became embryogenic.     

Morphogenetic responses of Salpiglossis sinuata L. leaf explants and callus

Leaf explants of Salpiglossis sinuata L. were cultured on MS medium containing the auxins 2,4-D, IAA, NAA, or the cytokinins 6-BAP, 2ip, or K singly or NAA + 6-BAP combinations (0.01 to 10.0 mg/l) to determine their morphogenetic responses. IAA and NAA induced callus or roots at all levels except IAA at 0.01 mg/l. Callus varying in friability was obtained on MS + 2,4-D at various concentrations. Friable, uniform dividing callus was obtained on UM medium. Optimum adventitious shoot formation occurred on leaf sections and from callus cultures placed on MS + 2ip. Rooting-difficulties were encountered, but 75% rooting efficiency was obtained on shoots cultured in MS + 0.001 mg/l 2,4-D. Rooted plants were readily transferred to the greenhouse, and flowered.     

Flower formation in Calceolaria × herbeohybrida Voss

At high light intensity Calceolaria × herbeohybrida ‘Zwerg Meisterstück’ is a long-day plant with a critical day-length of 14–15 h. At low light intensity (e.g. as in winter) flowering will take place if the long-day treatment is preceded by a chilling period (10°C) or by short days at 15–20°C. During the chilling period day-length is of little influence. With increasing duration of the chilling period the requirement for long days decreases and the critical day-length becomes shorter. After a sufficient chilling period, flowering occurs both in long and short days. If the chilling period lasts approx. 40 days, however, flowering in short days is delayed, phyllody occurs and only a small number of flowers develop in comparison to long days. After at least 70–75 days of chilling plants show almost the same reaction in short and long days. Plants not chilled under conditions of high light intensity and short days flower either in naturally long days or by day extension with incandescent light. After chilling, fluorescent light of the type L 39 is also effective for day extension. A night break with incandescent light in a 16-h dark period induces flowering only after a chilling period. Incandescent but not fluorescent light causes a slight yellowing and more upright position of the leaves and an elongation of the internodes.     

Aerial plantlet formation in Asparagus officinalis L.

Removal of rhizome buds from asparagus plants grown in pots led to the formation of aerial plantlets from the nodes on the lower portion of shoots. A similar development was observed in vitro in cultured plantlets derived from bud explants if left for a sufficient length of time in culture. Aerial plantlets which developed either in vitro or in vivo could be detached and established in soil. These plants were morphologically indistinguishable from normal asparagus seedlings.     

Ploidy screening of anthurium (Anthurium andreanum Linden ex André) regenerants derived from anther culture

Anther culture was successfully developed in Anthurium andreanum Linden ex André cv. ‘Tropical’, but resulted in variation in morphology and growth response of regenerants. Ploidy levels varied in different morphological variants. Finding a convenient, rapid, reliable, practical and indirect method to screen and determine anthurium ploidy level is important not only for anthurium, but for ornamentals in general. Regenerants derived from anther culture showing three different ploidy levels (haploids, diploids, triploids) were used in this study. Five indirect methods were used to assess chromosome number: chloroplast number in stomatal guard cells (M1), stomatal length and width ratio (M2), stomatal density (M3), ratio of length and width of leaves (M4), and microspore number per anther (M5). These were compared to chromosome counting as the direct and control method (M6). Through simple regression correlation analysis, when compared to M6, M1 was the most convenient and reliable indirect method to determine the ploidy level. This method was highly and significantly positively correlated to anthurium ploidy level (r = 0.945; p < 0.01). This method could also be applied much faster than the conventional chromosome method.     

Propagation of Lilium hybrids. II. Production of plantlets from bulb-scale callus cultures for increased propagation rates

A method has been developed for the large-scale propagation of lilies from callus cultures. Bulb scales, being available at all times, are an ideal source of explants. Callus was induced throughout the annual cycle of all the 12 Oriental hybrid cultivars tested, by treating the bulb scales with a combination of 5 μM BA and 5 μM 2,4-D. Once initiated, this callus grew vigorously on media lacking exogenous growth substances. Callus induction by BA and 2,4-D, and the subsequent growth of such callus in the absence of exogenous growth substances, occurs within a broad range of cultivars and species within the genus. The callus could be maintained on a medium lacking ammonium salts, but the maximum growth rate was obtained at 10 mM NH₄NO₃. At low (0.2 mM) or zero levels of NH₄NO₃, light as compared with darkness was inhibitory to growth.The callus maintained its capacity for organogenesis. Maximum production of plantlets was obtained in continuous light on agar medium with 0.5 μM NAA.Potentially, the successive use of liquid and agar cultures can produce 6 × 10¹² plants per year from 1 g (dry wt.) of callus. Plantlets derived from callus were consistently diploid and could be readily transferred to soil.     

Factors affecting in vitro adventitious shoot formation on internode explants of Citrus aurantium L. cv. Brazilian

The in vitro formation of newly formed adventitious buds and shoots from internodal branch segments was studied on 12-month-old plants of Citrus aurantium L. cv. Brazilian. The effects of 6-Benzyladenine (BA) and α-Naphthalene acetic acid (NAA) treatments were evaluated on adventitious bud and shoot regeneration. High rates of bud initiation and shoot development were obtained both with BA supplemented medium, in the range from 1 mg L⁻¹ to 3 mg L⁻¹, and with 0.1 mg L⁻¹ NAA supplemented medium. NAA concentrations above 1 mg L⁻¹ significantly reduced bud initiation and shoot elongation. The results obtained using different in vitro culture vessels such as Petri dishes, tubes and glass culture jars were compared. The highest adventitious bud induction was observed in Petri dishes for internodes cultured in 2 mg L⁻¹ BA supplemented medium, with 95% responsive explants forming 9.0 ± 2.4 adventitious buds. The adventitious buds observed in Petri dishes reached a maximum height of 1 mm, with no further development, while some of the adventitious shoots cultured in tubes and glass culture jars grew over 1 cm in height. A shoot regeneration gradient of the internodes collected along the branch axis was noticed, with basal ones exhibiting higher regeneration frequency.     

Shoot production from cultured Allium porrum tissues

Large numbers of shoots have been obtained from the excised basal regions of leek plants, cultured on BDS medium. Shoots could be induced optimally upon media within the range of 6.0–8.0 mg 1^?1 6-(3-methyl-2-buten-1-ylamino)-purine (2iP) and 1.0–2.0 mg 1^?1 napthaleneacetic acid (NAA). Sub-culture of induced shoots onto fresh media, from the above hormone ranges, resulted in further multiplication. Sub-culture, onto fresh medium from the above hormone ranges, of the callus-like region around the bases of induced shoots and adjacent tissue, could also result in shoot production. Internally developing shoot primordia occurred in close proximity to meristematic regions, whereas superficially developing primordia did not show distinct affinities, although there was evidence of localised pockets of meristematic cells amidst senescing explant material at the surface of the culture. Such regions may represent early stages in the formation of the superficial primordia. The production of large numbers of leek shoots in vitro is mentioned in relation to present techniques of micro-propagation and cryopreservation.     

Regeneration of adventitious shoots from mature stored cotyledons of Japanese plum (Prunus salicina Lind1)

Adventitious shoot regeneration from mature stored cotyledons of Japanese plum (Prunus salicina Lind1) was achieved in vitro. The influences of the presence and absence of the light, different concentrations of thidiazuron (TDZ) and benzyladenine (BA) in the culture media, TDZ pretreatments and different basal salts on shoot regeneration were evaluated. TDZ was more effective in inducing shoot regeneration from mature stored cotyledons than BA. Dark incubation significantly increased the regeneration frequencies. Quoirin/Lepoivre (QL) basal salts stimulated shoot regeneration more than woody plant (WPM) or B5 salts did. The frequency of adventitious shoot formation varied among the varieties and the regeneration ability appeared to be genotype depended. The frequency of regeneration under the optimum tested conditions for ‘Bruce’, ‘Shiro’, ‘Redheart’, ‘Gladstone’ and ‘Early Golden’ cotyledons were 66.7%, 46.7%, 43.3%, 26.7% and 6.7%, respectively.     

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.     

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.     

Effects of chemical and organic fertilizers on the growth,flower quality and nutrient uptake of Anthurium andreanum, cultivated for cut flower production

Sustainable agriculture has become a concern, due to the pressures of the “energy crisis” and issues of “environmental protection”. The use of organic fertilizer made from agricultural waste regenerates natural resources and reduces the consumption of fossil energy as well as phosphorus (P) and potassium (K) deposits. There is scant information available concerning the use of organic fertilizer as the sole source of nutrients in flower production, especially in the cultivation of flowers in a soilless condition. The objective of this study was to develop an organic fertilization management system to replace the chemical fertilization management of the cut flower production of Anthurium andreanum Lind. cultivated under soilless conditions. Four fertilization treatments were carried out consisting of two chemical fertilizers [controlled release fertilizer (CRF) and a chemical nutrient solution (CNS)], and two organic fertilizers [pea and rice hull compost (PRHC) and cattle dung with tea leaf residue compost (CDTC)]. The effects of the various fertilizations on A. andreanum were evaluated based on plant growth, nutrient uptake, and cut flower quality during the 1-year experimental period. The results show that the growth, yield, and cut flower quality of plants receiving PRHC were the same as those receiving CNS and CRF, indicating that PRHC can substitute for CRF and CNS as a nutrient source for cut flower production of A. andreanum cultivated in soilless condition. The plants that received the CNS and PRHC treatments had a significantly increased leaf number and new leaf growth area than those that received the CRF and CDTC treatments. The plants receiving the CDTC showed the lowest increase in leaf area and number of flowers. The retardant growth of plants treated with CDTC has been explained as being due to less carbon (C) being assimilated, most likely as the result of an insufficient supply of nitrogen (N) and manganese (Mn) toxicity. The petiole and peduncle length of the plants receiving the CRF were the shortest, which might be due to the low level of potassium (K) accompanying the magnesium (Mg) deficiency. Even though there were significant differences in the N and K concentrations of the plants among the different treatments, no significant differences were observed in the cut flower quality. In short, the organic fertilizer PRHC can meet the nutrient requirements of A. andreanum cultivation for the cut flower production under soilless conditions.     

Priming abiotic factors for optimal hybrid Cymbidium (Orchidaceae) PLB and callus induction,plantlet formation,and their subsequent cytogenetic stability analysis

Abiotic factors affect the induction of PLBs and callus in hybrid Cymbidium Twilight Moon ‘Day Light’. The initiation and proliferation of new PLBs and callus could be achieved on NAA and kinetin, supplemented at 0.1 mg l⁻¹ each, respectively, both within 45–60 days. Bacto agar was found to be the most suitable solidifying agent for PLB induction, although a higher shoot fresh weight was obtained on Gelrite; a pH 5.3 was optimal while pH 4.5 caused 100% explant necrosis; coconut water, when supplied at 10–20% (v/v) resulted in a significant increase in the number of PLBs formed per PLB segment (23.1 versus 14.6 in controls) while a massive (almost four-fold) increase in fresh top weight occurred when PLB explants were placed in liquid culture, as a result of hyperhydricity; Fe-EDTA (1 mg l⁻¹) and activated charcoal (1 g l⁻¹) stimulated total fresh weight and PLB formation in the presence of PGRs; PLB formation decreased but total fresh shoot weight increased with the addition of niacin or myo-inositol, both vitamins. Dark-grown PLB-induced plants were etiolated and had longer internodes and higher fresh weight than light-grown control plants at 45 μmol m⁻² s⁻¹; at 15 μmol m⁻² s⁻¹ shoots were slightly etiolated, fragile, and PLB formation was scarce. RAPD and mtDNA analysis of all resultant PLBs, callus or plants showed them to be genetically identical, with comparable chlorophyll contents. Despite the detection of cytological variation between different plant parts, little variation resulted from abiotic factor treatment.     

High frequency in vitro embryogenic callus induction and plant regeneration from indiangrass mature caryposis

Indiangrass [Sorghastrum nutans (L.) Nash.] is native to the North America and is an important component of the original tall grass prairie. It is also an important ornamental and forage grass. Recently, it has been proposed as an ideal biomass producer for cellulosic ethanol production. Genetic transformation is an important tool for introducing important agronomic traits into plants, but an efficient and reproducible in vitro regeneration protocol is a prerequisite for successful genetic transformation. In this report, we used mature caryopses as explants and tested the effect of various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) (1–5) and kinetin (KT) (0, 0.1, and 0.2) on embryogenic callus induction using LS basal medium. Caryopses cultured on media supplemented with 2,4-D alone generally outperformed those cultured on media supplemented with both 2,4-D and kinetin for embryogenic callus induction. The best treatment is LS basal medium supplemented with 3 mg l⁻¹ 2,4-D. LS basal medium supplemented with KT of 0, 0.5, 1, 2 or 5 mg l⁻¹ were tested for regeneration efficiency which was shown to increase as the KT concentration increased. The quality of the shoots produced on the medium containing KT at 5 mg l⁻¹, which produced the highest regeneration frequency appeared to be lower as leaves become vitrified. Shoots were moved to a rooting medium containing either 0 or 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA). Rooted plantlets were then transferred to soil-containing pots and were placed in a mist room for 1 week before they are transferred to a normal greenhouse where they all survived. The reported regeneration protocol is very efficient and highly reproducible in spite of the heterogeneous nature of the tested cultivar; thus it should be suitable for genetic transformation.