A simple and efficient method for clonal propagation of Casuarina sumatrana (de Vriese) L. Johnson

A new and efficient method for clonal propagation of Casuarina sumatrana by rooting stem cuttings is described. High percentage (about 60–70%) of rooting was achieved with mature softwood stem cuttings. A quick-dip of 5 s in NAA (1–10mM) solution followed by sand culture under high humidity were required for a high rate of survival and rooting of stem cuttings. A simple, closed chamber propagation system, using fluorocarbon polymer (tetrafluoroethyleneperfluoroalkyl vinyl ether) film (Neoflon PFA film), was successfully developed for the rooting of stem cuttings without mist. Rooted cuttings inoculated with Frankia were easily transplanted and established in field conditions with very low (about 3%) mortality. The significance of these findings for mass clonal propagation of C. sumatrana is discussed.Abbreviations IBA indole-3-butyric acid - NAA -naphthaleneacetic acid     

Delayed flowering and reduced branching in micropropagated mature wild cherry (Prunus avium L.) compared with rooted cuttings and seedlings

The development of micropropagated wild cherry (Prunus avium L.) was compared in the nursery and the field with cuttings and seedlings, over a total period of 6 years. Summer semi-hardwood cuttings tended to produce moderate numbers of branches in the season following propagation, whereas micropropagules and seedlings produced significantly fewer or none at all. Removal of branches from cuttings resulted in taller trees. A greater proportion of cuttings than micropropagules flowered in the first year in which trees produced flowers. In the second flowering year, there were no differences in flowering habit between cuttings and micropropagules. Propagation by cuttings or micropropagation did not consistently affect increments in stem diameters or heights. These results are discussed in terms of the suitability of micropropagation and cuttings to produce clonal wild cherry. Received: 23 February 1997 / Revision received: 3 January 1998 / Accepted: 5 February 1998     

Field performance of <Emphasis Type="Italic">Theobroma cacao</Emphasis> L. plants propagated via somatic embryogenesis

Somatic embryogenesis is an in vitro clonal propagation method with potential to contribute to the improvement of cacao varieties. Before using this technology for commercial production, it is essential that somatic embryogenesis-derived plants be tested in field conditions. Therefore, we established a field test at Union Vale Estate, Saint Lucia. Thirty- to 50-yr-old trees were selected for clonal propagation as potentially high yielding based on local farmers observations. Clonal plants were propagated in vitro from immature flowers by embryogenesis and micropropagation. Multiple plants from nine genotypes were acclimated to greenhouse conditions then returned to Saint Lucia and planted in a field. Orthotropic rooted cuttings and locally propagated open pollinated seedlings were also planted for a total of 214 trees. Growth data were collected every 4–6 mo. including: stem diameter, stem height, length of the longest jorquette branch, number of jorquette branches, and dates of first flowering and fruiting. At 4.5 yr after planting in the field there were no major differences in all growth parameters among the propagation methods evaluated with exception of the orthotropic rooted cuttings. Trees grown from seeds were slightly taller then trees propagated by the other methods. Trees propagated as orthotropic rooted cuttings exhibited smaller average stem diameters, shorter stem heights to the jorquette, and shorter jorquette branches. We concluded that somatic embryo-derived plants demonstrated normal phenotypes in field conditions and have growth parameters similar to plants propagated by traditional methods.     

Dormant Buds and Adventitious Root Formation by Vitis and Other Woody Plants

Viticulture has historically depended upon clonal propagation of winegrape, tablegrape, and rootstock cultivars. Dependence on clonal propagation is perpetuated by consumer preference, legal regulations, a reproductive biology that is incompatible with sustaining genetic lines, and the fact that grapevine breeding is a slow process. Adventitious root formation is a key component to successful clonal propagation. In spite of this fact, grapevine has not been a centerpiece for adventitious root research. Dormant woody canes represent complex assemblages of tissues and organs. Factors that further contribute to such complexity include levels of endogenous plant growth regulators, the extent and duration of dormancy, carbohydrate storage, transport, the presence or absence of dormant buds or emergent shoots, and preconditioning treatments. For the above reasons, the mechanisms driving adventitious root formation by grapevine and other woody cuttings are poorly understood. We present results indicating that the dormant bud on cane cuttings from a non-recalcitrant to root Vitis vinifera cultivar, cv. Cabernet Sauvignon, slows or inhibits adventitious root emergence. In contrast to Cabernet Sauvignon, removal of the dormant bud from cane cuttings of a recalcitrant to root hybrid rootstock (V. berlandieri × V. riparia cv. 420A) and an intermediate to root hybrid rootstock (V. riparia × V. rupestris cv. 101-14) had no influence on adventitious root emergence. Reciprocal transplanting of nodes containing dormant buds among all three cultivars did not affect rooting behavior. Our results indicate that the commonly held belief that bud removal diminishes adventitious root emergence is not true.     

Dormant Buds and Adventitious Root Formation by <Emphasis Type="Italic">Vitis</Emphasis> and Other Woody Plants

Viticulture has historically depended upon clonal propagation of winegrape, tablegrape, and rootstock cultivars. Dependence on clonal propagation is perpetuated by consumer preference, legal regulations, a reproductive biology that is incompatible with sustaining genetic lines, and the fact that grapevine breeding is a slow process. Adventitious root formation is a key component to successful clonal propagation. In spite of this fact, grapevine has not been a centerpiece for adventitious root research. Dormant woody canes represent complex assemblages of tissues and organs. Factors that further contribute to such complexity include levels of endogenous plant growth regulators, the extent and duration of dormancy, carbohydrate storage, transport, the presence or absence of dormant buds or emergent shoots, and preconditioning treatments. For the above reasons, the mechanisms driving adventitious root formation by grapevine and other woody cuttings are poorly understood. We present results indicating that the dormant bud on cane cuttings from a non-recalcitrant to root Vitis vinifera cultivar, cv. Cabernet Sauvignon, slows or inhibits adventitious root emergence. In contrast to Cabernet Sauvignon, removal of the dormant bud from cane cuttings of a recalcitrant to root hybrid rootstock (V. berlandieri × V. riparia cv. 420A) and an intermediate to root hybrid rootstock (V. riparia × V. rupestris cv. 101-14) had no influence on adventitious root emergence. Reciprocal transplanting of nodes containing dormant buds among all three cultivars did not affect rooting behavior. Our results indicate that the commonly held belief that bud removal diminishes adventitious root emergence is not true.     

Effects of propagule type on genetic parameters of wood density and growth in a loblolly pine progeny test at ages 10 and 11?years

Nine full-sib families of loblolly pine (Pinus taeda L.) were produced by a 3 × 3 factorial mating design. Rooted cuttings and seedlings of full-sib families were tested together in two field locations. Twelve-millimeter wood increment cores were collected from 10- and 11-year-old test trees. On each of the two sites, there were six blocks and a split-plot design, with propagule type as the whole plot and family as the sub-plot. In addition to the collection of wood samples, height and diameter of 1,600 trees were measured. No significant differences were found between cuttings and seedlings for wood density and growth traits. Significant family variation was found for growth and wood density. Genetic parameters estimated for wood density and growth traits using seedlings and rooted cuttings showed that individual-tree and family heritability estimates from rooted cuttings were similar to or higher than those from seedlings for all traits. Half-sib breeding values for parents were highly correlated based on seedling and rooted cutting estimates for height (0.95) and wood density (0.99) but not for diameter (0.56), which suggests that wood density and height breeding value estimates from rooted cuttings in clonal progeny tests can be estimated by traditional seedling tests, but not for tree diameter.     

Evaluation of aeroponics for clonal propagation of Caralluma edulis,Leptadenia reticulata and Tylophora indica – three threatened medicinal Asclepiads

The present study explores the potential of aeroponic system for clonal propagation of Caralluma edulis (Paimpa) a rare, threatened and endemic edible species, Leptadenia reticulata (Jeewanti), a threatened liana used as promoter of health and Tylophora indica (Burm.f.) Merill, a valuable medicinal climber. Experiments were conducted to asses the effect of exogenous auxin (naphthalene acetic acid, indole-3-butyric acid, indole-3-acetic acid) and auxin concentrations (0.0, 0.5, 1, 2, 3, 4 or 5gl⁻¹) on various root morphological traits of cuttings in the aeroponic chamber. Amongst all the auxins tested, significant effects on the length, number and percentage of rooting was observed in IBA treated nodal cuttings. Cent per cent of the stem cuttings of C. edulis rooted if pre-treated with 2.0 gl⁻¹ of IBA for 5 min while 97.7 % of the stem cuttings of L. reticulata and 93.33 % of stem cuttings of Tylophora indica rooted with pre-treatment of 3.0 gl⁻¹ of IBA for 5 min. Presence of at least two leaves on the nodal cuttings of L. reticulata and T. indica was found to be a prerequisite for root induction. In all the species, the number of adventitious roots per cutting and the percentage of cuttings rooted aeroponically were significantly higher than the soil grown stem cuttings. Shoot growth measured in terms of shoot length was significantly higher in cuttings rooted aeroponically as compared to the cuttings rooted under soil conditions. All the plants sprouted and rooted aeroponically survived on transfer to soil. This is the first report of clonal propagation in an aeroponic system for these plants. This study suggests aeroponics as an economic method for rapid root induction and clonal propagation of these three endangered and medicinally important plants which require focused efforts on conservation and sustainable utilization.     

Genetic variation in the horsetail Equisetum variegatum Schleich., an endangered species in the Parisian region

Equisetum variegatum Schleicher is a circumboreale species of horsetail. In France, it typically grows at high elevations but is very rare in lowlands. The genetic variation of these populations is described using isozyme electrophoresis and PCR-RFLP of chloroplast DNA. Sampled sites were chosen to represent central vs. marginal and/or endangered parts of the distribution area. Extensive clonal multiplication of plants together with the absence of local recruitment by sexual reproduction seem to be responsible for the low genetic diversity observed within populations. Since adaptive response to environmental changes ultimately relies on the presence of genetic variability, clonal populations of E. variegatum may be particularly vulnerable to disturbance. Moreover, in lowland populations, isolation gives no chance to recover new genotypes through migration events. The preservation of the two endangered populations is proposed by propagation by cuttings of all extant genetic individuals. In the case of a disappearance of one genotype in the field, a replacement will be possible. This plan may be sufficient to preserve E. variegatum in the French lowland for several years.     

Microevolution in agricultural environments: how a traditional Amerindian farming practice favours heterozygosity in cassava (Manihot esculenta Crantz, Euphorbiaceae)

We demonstrate a novel case of selection for heterozygosity in nature, involving inadvertent human selection on a population of domesticated plants. Amerindian farmers propagate cassava (Manihot esculenta) clonally by cuttings. Seedlings also appear spontaneously in fields, and farmers allow them to grow, later using some for cuttings. These ‘volunteers’ contribute new genotypes. However, many are inbred, whereas multiplied clones are highly heterozygous. We demonstrate the selective retention of heterozygous volunteers. When farmers weeded fields, they killed small volunteers, but retained large ones. Plant size and heterozygosity were correlated, and both increased after weeding. The process we document allows maintenance of genotypically diverse and heterozygous clonal stocks. Demonstrating heterosis in nature usually requires large sample sizes, but novel features of our system allowed escape of this constraint. Traditional agroecosystems offer unusual opportunities for the microevolutionary studies required to give on‐farm conservation of genetic resources a solid scientific basis.     

The community of needle endophytes reflects the current physiological state of Norway spruce

This study investigated fungal endophytes in the needles of Norway spruce (Picea abies) cuttings in relation to host tree growth. We also determined the prevalence of endophytes in needles incubated for six months. The cuttings originated from clonal origins showing slow- and fast-growth in long-term field trials but the heritable differences in growth rate were not yet detected among the studied cutting. Endophytes were isolated from surface-sterilized needles with culture-free DNA techniques. No significant differences were observed between endophyte communities of slow- and fast-growing clonal origins. However, the endophyte community correlated with the current growth rate of cuttings suggesting that endophytes reflect short- rather than long-term performance of a host. The concentration of condensed tannins was similar in slow- and fast-growing clonal origins but it showed a negative relationship with endophyte species richness, implying that these secondary compounds may play an important role in spruce tolerance against fungal infections. More than a third of endophyte species were detected in both fresh and decomposing needles, indicating that many needle endophytes are facultative saprotrophs. Several potentially pathogenic fungal species were also found within the community of saprotrophic endophytes.     

Causes of Centrifugal Root Promotion

Role of water in centrifugal root promotion, effect of centrifugation on ethylene concentration of cuttings, and effect of ethylene on root formation were investigated using Salix jragilis softwood cuttings. When cuttings were centrifuged with water, more roots formed with increasing water depth. Soaking of cuttings upright in water for 24 h stimulated root formation and produced more roots as the depth of water was increased, with submerged cuttings producing the highest number of roots. Soaking of cuttings upright in hot water for 1 h also stimulated root formation with the best root formation occurring at 40°C. Submerging in, or centrifuging with water increased ethylene concentration in cuttings. Ethylene gas and Ethrel treatments stimulated root formation. It is suggested that submerging in water increases the ethylene concentration in Salix fragilis softwood cuttings which in turn stimulates root formation of the cuttings. Centrifuging cuttings with water increased water content of the cuttings. It is suggested that this increase in water content plays a role similar to the submersion of cuttings as described above.     

Use of RAPD patterns for clone verification and in studying provenance relationships in Norway spruce (Picea abies)

 We have used the RAPD technique to analyse samples of Picea abies obtained from an improvement forestry station. Two types of plant material were harvested, the first being clones and the second provenances from various regions. We first checked the clonal identity of elite tree cuttings and clones; some differences in the RAPD patterns resulting from mis-planting or mis-labelling of cuttings were found. We also established a reference library of RAPD fingerprints for 96 clones, which will serve as a reference source in cases of litigation concerning clone identity. The RAPD technique was also used to study the genetic relationship between nine European provenances of Norway spruce. A dendogram was obtained by individual pairwise comparison of 42 RAPD bands, which separated the nine provenances into two major groups, one containing the Nordic provenances (Sweden and Bielorussia) and another the Alpine provenances (France, Austria, Germany and Belgium). The Belgian provenance, which is not indigenous, is most closely related to the German provenance. We conclude that the RAPD technique is a useful tool for forestry stations in managing propagation operations. Received: 15 June 1996 / Accepted: 11 October 1996     

Developmental quantitative genetics of growth in Populus

 Nursery growth and dry weight were analyzed for F₂ genotypes derived from Populus trichocarpa×P. deltoides that have been field tested with clonal replicates in three different environments. The correlations between nursery and plantation performance differed among the environments, with higher values at Boardman and Clatskanie (both planted with rooted cuttings) than Puyallup (planted with unrooted cuttings). At Puyallup, nursery height was more strongly associated with plantation growth than were nursery diameter and dry weight. Yet, this finding was not supported by QTL mapping. A single overdominant QTL on linkage group G affected the stem height of both seedlings and resprouts in the nursery but showed nonsignificant LOD scores for plantation height from ages 1 to 5 at Puyallup. A total of four QTLs were identified for nursery diameter, one of which on linkage group O also controlled plantation basal area at all ages. Two important nursery QTLs on linkage groups B and G were used to estimate the relative efficiency of marker-assisted selection for plantation productivity. Despite the fact that they were not detected in the plantation stage, these two QTLs could significantly increase the proportion of the phenotypic variance explained by plantation QTLs. Received: 5 February 1998 / Accepted: 1 April 1998     

Comparison of Stevia plants grown from seeds,cuttings and stem-tip cultures for growth and sweet diterpene glucosides

The growth and sweet diterpene glucosides of Stevia plants propagated by stem-tip cultures were compared with those of the control plants propagated by seeds. There was no significant difference between the two groups both in growth and in chemical composition. As for the contents of sweet diterpene glucosides, however, the clonal plants showed significantly smaller variations than the sexually propagated plants; they were almost as homogeneous as the plants propagated by cuttings. These results suggest that the clonal propagation by stem-tip culture is an effective method of obtaining a population of uniform plants for the production of sweet diterpene glucosides.     

Antagonistic effects of soil bacteria onFusarium oxysporum Schlecht f.sp.dianthii (Prill and Del.) Snyd. and Hans.

Summary Thirty two bacteria antagonistic to a number of phytopathogenic fungi were isolated from soil samples. One bacterial strain, designated as M 51, appeared to be particularly active towardsF. oxysporum f. sp.dianthii, in vitro andin vivo and it was inhibitoryin vitro to three otherFusarium spp. used. Tests to find if there was protection against fusarium wilt were carried out by three different methods of inoculation of the cuttings: a) dipping of cuttings for ten minutes in bacterial suspension; b) spraying of suspension on perlite where the rooted cuttings were planted; c) spraying the greenhouse bench rooting boxes, where the non-rooted cuttings were planted, with bacterial suspension. Following this all the cuttings were transplanted into soil naturally highly infested withFusarium oxysporum f. sp.dianthii (3000 units/g). Good protection against fusarium wilt was obtained for cuttings inoculated by method (b). However protection decreased gradually about 60 days after they were transplanted; both control and inoculated cuttings showed a comparable mortality rate. Method of inoculation and the development of the protective effect are discussed.     

Effects of growth substances on male and female cone initiation in conifers

Current knowledge about effects of exogenous PGR on cone initiation in coniferous trees is briefly reviewed. Long life-cycles, large tree size and great irregularity in coning have imposed severe restraints, but experimental study of reproduction can now be achieved in small plants of some species. In adult, clonal cuttings ofThuja plicata, standard injections of 50–250 (μg GA₃ induce substantial male and female coning, and have been used to test effects of other PGR on cone initiation and development. In thePinaceae, propagation of naturally heavily-coning genotypes has facilitated research on the effects of PGR.     

Application of Ectomycorrhizal Fungi in Vegetative Propagation of Conifers

In forestry, vegetative propagation is important for the production of selected genotypes and shortening the selection cycles in genetic improvement programs. In vivo cutting production, in vitro organogenesis and somatic embryogenesis are applicable with conifers. However, with most coniferous species these methods are not yet suitable for commercial application. Large-scale production of clonal material using cuttings or organogenesis is hindered by rooting problems and difficulties in the maturation and conversion limit the use of somatic embryogenesis. Economically important conifers form symbiotic relationship mostly with ectomycorrhizal (ECM) fungi, which increase the fitness of the host tree. Several studies have shown the potential of using ECM fungi in conifer vegetative propagation. Inoculation with specific fungi can enhance root formation and/or subsequent root branching of in vivo cuttings and in vitro adventitious shoots. Germination of somatic embryos and subsequent root growth can also be improved by the use of ECM fungi. In addition, inoculation can increase the tree's ability to overcome the stress related to ex vitro transfer. A specific interaction between a fungal strain and tree clone occurs during root induction and germination of somatic embryos. Multiple rooting factors exist in this interaction that complicate the predictability of the response to inoculation. Fungal-specific factors that influence rooting responses to inoculation may include plant growth regulator production, modification of the rooting environment, and interactions with beneficial microbes. A combination of these factors may act synergistically to result in positive responses in tree genotypes that are compatible with the fungus.     

Resprouting Ability of Dry Forest Tree Species after Disturbance Does Not Relate to Propagation Possibility by Stem and Root Cuttings

Tropical dry forest tree species are recognized for their high resprouting ability after disturbance. We tested whether species that commonly produce root and stem suckers can be propagated by large stem and root cuttings, a useful method for landscape restoration programs. We performed four experiments: (1) In a greenhouse, we tested the propagation of six species using large stem cuttings collected from early successional sites. We used the following treatments: (i) dry season collection and planting; (ii) dry season collection, storage in humid soil, and wet season planting; (iii) wet season collection and planting; and (iv) wet season collection and planting after treatment with commercial NAA auxin. (2) Stem cuttings of Myracrodruon urundeuva were planted in a pasture during the rainy season after either NAA, IBA, or no auxin treatment. (3) As a control experiment, we also planted cuttings of Spondias mombin, a species known for successfully regenerating from cuttings. (4) Root cuttings of six species were collected in recently plowed pastures and planted in the greenhouse with and without treatment with NAA auxin. No root cuttings rooted. Only M. urundeuva and Astronium fraxinifolium stem cuttings rooted. Maximum success was obtained for stem cuttings collected and planted in the dry season (23%). Only 13% of M. urundeuva had sprouted by the 15th month of the field experiment. As a result, large cuttings are not recommended for propagation of the studied species. Future studies should include development of suitable methods of root harvesting and prospection of traditional knowledge for species selection.     

Effects of auxin and irradiance on the rooting of cuttings of Pinus sylvestris

Abstract Seedlings of Pinus sylvestris L. were grown under controlled conditions (temperature 20°C, photoperiod 17 h) at two irradiances, 8 or 40 W m^-2. Hypocotyl cuttings were excised and rooted at different irradiances in tap water solutions of indolebutyric acid (IBA). The fastest rooting and highest rooting percentage were obtained with cuttings from stock plants grown at 8 W m^-2 and treated with 10^-5M IBA for 21 days. The concentration of 10^-4M IBA inhibited root formation. In comparable treatments rooting was always better in cuttings from stock plants grown at 8 W m^-2 than in cuttings from stock plants grown at 40 W m^-2. The irradiance during the rooting period had only a minor influence on rooting. When cuttings from plants irradiated with 40 W m^-2 were treated with 10^-5M IBA for 21 days the rooting percentage almost reached the same level as in untreated cuttings from stock plants given 8 W m^-2. In cuttings treated with IBA during the whole rooting period, rooting was depressed in comparison to untreated cuttings. Aeration of the 10^-4M IBA solution increased the rooting percentage, but aeration had no effect on untreated cuttings and on cuttings treated with lower IBA concentrations.     

Effects of combinational treatment with ethephon and indole-3-butyric acid on adventitious rooting of <Emphasis Type="Italic">Pinus thunbergii</Emphasis> cuttings

Adventitious rooting is essential for cutting propagation of pine wilt-resistant Pinus thunbergii. To examine a variety of adventitious rooting potentials among donor plants, cuttings were taken from 31 seedlings within a half-sib family. Rooting abilities of cuttings from each seedling ranged from 0 to 100%. When 11 ortets and 11 ramets (clonally propagated from each ortet) were used as donor plants, there was a positive correlation between rooting abilities of cuttings from ortets and ramets, suggesting that adventitious rooting is dependent on genetic factors in the donor plants. To promote adventitious rooting of cuttings by hormonal treatment, we examined the effect of soaking time in Oxyberon (19.7 mM indole-3-butyric acid (IBA) solution) on rooting. Ten minutes was the best soaking time for rooted cuttings to produce more adventitious roots without impairing normal growth. When cuttings were soaked in Ethrel diluent (69.2 μM ethephon) for 24 h before soaking in Oxyberon for 10 min, a significantly higher rooting ability was observed than those soaked in Oxyberon alone. Ethrel on its own barely affected rooting ability. The positive effect of the combinational treatment was confirmed in a similar experiment using authentic ethephon and IBA instead of Ethrel and Oxyberon. When cuttings were soaked in a mixture of ethephon and silver thiosulfate (STS), an ethylene action inhibitor, before IBA-soaking, the effect was partially diminished compared with combinational treatment without STS. These findings suggest that ethylene action caused by ethephon treatment promotes IBA-mediated adventitious rooting of P. thunbergii cuttings.