Vegetative propagation of adult Eucalyptus grandis X urophylla and comparison of growth between micropropagated plantlets and rooted cuttings

Methods for the production of micropropagated plantlets and rooted cuttings were developed and used to vegetatively multiply adult Eucalyptus grandis X urophylla. Rooting success was less than 5% when cuttings excised from twigs of 3-year-old trees were used. The rooted cuttings were grown in the greenhouse as explant- or cutting-donors and maintained at a height of 30 to 100 cm by trimming back periodically. Good rooting success (95%) of cuttings was obtained for epicormic shoots produced from donor plants after trimming 5 times. Explants of both apical and axillary buds taken from the donor plants produced multiple shoots when cultured in vitro. In vitro multiple shoot production was optimal on MS medium containing 0.1 mg/l BA and 0.01 mg/l NAA averaging 13.7 shoots per explant in a 40-day culture period. Shoot elongation was accelerated on a modified MS medium containing half strength potassium nitrate and sucrose. Elongated shoots excised at approximately 1.5 cm in length were successfully rooted on media with NAA or IBA concentrations ranging from 0.1 to 10 mg/l. Root formation was optimal on medium consisting of full strength MS basal macro elements and vitamins, half strength micro elements, 1% sucrose and supplemented with 0.3 mg/l IBA. In the field test, no significant differences were found in tree height and DBH between micropropagated plantlets and rooted cuttings at 1 and 3 years old, with the exception at 2 years old. A considerable difference arose between the 2 types of vegetative propagules in physiological response to flowering, caused by dissimilar degrees of rejuvenation.Abbreviations BA Benzyl-Aminopurine - NAA Naphthalene Acetic Acid - IBA Indole-3-Butyric Acid - MS medium Murashige and Skoog's medium - DBH Diameter at Breast Height     

The effect of etiolation and shading of stock plants on rhizogenesis in stem cuttings of <Emphasis Type="Italic">Cotinus coggygria</Emphasis>

In order to improve vegetative propagation of a difficult to root Cotinus coggygria the stock plants were subjected to: etiolation, shading and spraying with IBA, combined with the application of two commercially available rooting powders. The IBA treatment was more suitable for rooting of C. coggygria cuttings than the NAA application and it enhanced rhizogenesis regardless of the form of auxin application (foliar application to a stock plant or a rooting powder used directly on cuttings) and the amount of light provided to stock plants. Etiolation did not improve rhizogenesis in stem cuttings, however, reduction of light intensity by 50% and 96% of the ambient prior to harvest of cuttings affected rooting positively. Positive effects of shading can be ascribed to changes in shoot anatomy, i.e. a weaker sclerenchyma development. Synergistic effect of shading and foliar auxin application can result from the increase in leaf blade area and/or thinner lower epiderm. Enhanced rooting in cuttings from shoots grown out under reduced light intensity was accompanied by decrease in the contents of total soluble sugars, soluble proteins and free ABA and by increase in total chlorophyll, free amino acids, polyphenolic acids and free IAA contents.     

The effect of shading of stock plants on rhizogenesis in stem cuttings of Berberis thunbergii ‘Red Rocket’

In order to improve vegetative propagation of Berberis thunbergii ‘Red Rocket’, stock plants were subjected to shading and IBA treatment, as well as to the application of two commercially available rooting powders. Spraying stock plants with IBA 24 hours prior to cutting harvest gave the effect comparable to the use of rooting powders. Reduction of light intensity by 50% and 96% of ambient prior to harvest of cuttings improved rooting. Positive effect of shading results in changes in shoot anatomy, i.e. a weaker sclerenchyma development. Enhanced rooting in cuttings from shoots grown under reduced light intensity was accompanied by a decrease in the concentration of soluble proteins and by an increase in total chlorophyll content while the concentration of total soluble sugars remained unaffected.     

Metabolic changes during rooting in stem cuttings of Casuarina equisetifolia L.: effects of auxin, the sex and the type of cutting on rooting

Rooting in terminal shoot and lateral shoot cuttings from 10-year-old elite trees of Casuarina equisetifolia L. in different sex groups was achieved after 20 days when the basal ends of the cuttings were dipped for 3 h in 20 ppm indole-3-butyric acid (IBA). Shoots derived from male plants rooted better than their female and monoecious counterparts, and the lateral shoots were more responsive to rooting than the terminal shoots. During rooting, the metabolic activities varied in both lateral shoot and terminal shoot cuttings derived from plants under different sex groups. Peroxidase and polyphenoloxidase activities were high during root initiation and showed a sharp decline thereafter. The polyphenoloxidase activity was higher in the lateral shoot than the terminal shoot cuttings. The rooted plantlets survived and established well in the field.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - PVP polyvinylpyrrolidone     

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.     

Quantification of IAA Metabolites in the Early Stages of Adventitious Rooting Might Be Predictive for Subsequent Differences in Rooting Response

Stock mother plants have gained importance in the process of adventitious rooting of woody plants in recent years. The present study reveals the role of the cutting position from a stock mother plant for subsequent rooting. Cuttings of Prunus subhirtella Miq. var. Autumnalis which originated from suckers at the bottom of a tree developed a rooting system of better quality (rooted cutting without callus formation, more main roots) compared to cuttings which originated from shoots at the top of a tree. The latter accumulated significantly more indole-3-acetic acid (IAA) with the highest value of 25.37 μg g^?1 FW on the severance date. These cuttings also contained more indole-3-acetyl-aspartate (IAA-Asp) on the second day after severance with the highest value of 4875.95 μg g^?1 FW compared to cuttings from the bottom-deriving suckers. The latter metabolised IAA primarily via 2-oxindole-3-acetic acid (oxIAA) and indole-3-methanol because the concentrations of these compounds increased at the base of these cuttings. The highest concentration of oxIAA, 8.3 mg ekv. IAA g^?1 FW, was measured 1 h after severance in cuttings from the bottom-derived suckers. With 590.5 ng ekv IAAg^?1 FW, the indole-3-methanol values were also significantly higher in the cuttings from the bottom shoots compared with cuttings from the top of the tree.     

Adventitious root formation in cuttings of loblolly pine (Pinus taeda L .): developmental sequence and effects of maturation

 Adventitious root formation in cuttings from fascicular shoots in loblolly pine (Pinus taeda L.) consists of four more or less discontinuous stages: (1) proliferation of cells at the base of the cutting, (2) differentiation of wound vascular tissue and periderm, (3) dedifferentiation of a zone near the wound cambium and wound phloem to form a root initial, and (4) formation of a root meristem. Anatomical changes during adventitious root initiation are described in cuttings from donors of different types and ages. Cuttings from seedlings and 3- to 7-year-old hedged stock plants rooted better than cuttings from 3-year-old tree form donors. It is concluded that the loss of rooting capacity in loblolly pine can be arrested by shearing loblolly pine stock plants to low hedges. The process of root initiation, however, was similar in cuttings from all sources and is apparently not the cause for the rapid decline of rooting potential with increasing age of the donor plant. Received: 3 June 1997 / Accepted: 15 August 1997     

Stock-plant etiolation causes drifts in total soluble sugars and anthraquinones, and promotes adventitious root formation in teak (Tectona grandis L. f.) coppice shoots

The effects of stock-plant etiolation on coppice-shoot growth, drifts in total soluble sugars and anthraquinones (AQs; C₁₄H₈O₂), and rooting potentiality of shoot cuttings were examined in Tectona grandis L. f. (clone FG1). When seedlings were one-year-old, they were coppiced and maintained in the dark for etiolation, with a parallel set kept under natural light in an open environment. Coppice shoots were made into single-node leafy cuttings (SNCs), which were cultured under intermittent mist for rooting. These SNCs were treated with different concentrations of NAA (0, 2000 and 3000 mg l⁻¹). Etiolation significantly increased the coppice-shoot length, internode length, number of coppice shoots, number of leaves, number of nodes and total soluble sugars. The HPTLC analysis showed qualitative and quantitative differences in AQs in coppice shoots obtained from etiolated and non-etiolated stock plants. The study showed that AQs could be used as a marker for maturity and juvenility in teak. Stock-plant etiolation caused a significant increase in percent rooting and sprouting, shoot length, number of shoots and number of leaves per SNC, but a decrease in callusing at the base of the SNC. NAA at 2000 and 3000 mg l⁻¹ had inhibitory effects on rooting and sprouting of SNCs. The result showed that stock-plant etiolation fostered rooting by rejuvenating the coppice shoots.     

Rooting and vitality of poinsettia cuttings was increased by arbuscular mycorrhiza in the donor plants

In this paper, we provide evidence that the rooting performance of cuttings can be improved by the arbuscular mycorrhizal (AM) symbiosis of donor plants. Poinsettia stock plants were inoculated with the Glomus intraradices isolate H510 and grown in three different cultivation systems (two organic and one conventional). AM colonization was not related to P availability in the substrate. Decay of the excised cuttings in response to unfavorable postharvest storage conditions was significantly reduced by AM colonization of the stock plants. In most cases, AM significantly promoted the formation of adventitious roots in the stored cuttings. The strongest effect of AM was found when donor plants were grown in a modified organic substrate; then AM-conditioned cuttings showed higher leaf sugar levels and a changed kinetic of carbohydrates during storage. Analyses of N, P, and K in cuttings did not indicate a nutritional effect. The results support the idea that an altered carbohydrate metabolism and plant hormones can contribute to improved rooting performance of cuttings excised from mycorrhizal donor plants.     

The effect of indole-3-acetylaspartic acid on adventitious root formation on bean cuttings

The influence of indole-3-acetylaspartic acid (IAAsp) on rooting of stem cuttings from bean plants (Phaseolus vulgaris L.) of different ages, cultivated at different temperatures (17°, 21° and 25°C) was studied and compared to that of indole-3-acetic acid (IAA). At a concentration of 10^–4 M, IAAsp only nonsignificantly stimulated adventitious root formation, approximately to the same level as IAA in all treatments. IAAsp at 5×10^–4 M further enhanced rooting, by up 200% of control values, with little influence of temperature conditions and stock plant age. This concentration of IAA usually stimulated rooting more than the conjugate. The largest differences between the effects of IAAsp and IAA occured at the highest cultivation temperature of 25°C where stock plant age also influenced the response. The number of roots produced in comparison with the control, was enhanced from 350% on cuttings from the youngest plants to more than 600% on cuttings from the oldest. In contrast to the conjugate, 5×10^–4 M IAA induced hypocotyl swelling and injury of the epidermis at the base of cuttings, in all treatments.     

In vitro propagation of Halesia carolina L. and the influence of explantation timing on initial shoot proliferation

Halesia carolina L., a small, ornamentally valuable tree, is difficult to propagate due to the complexity of seed propagation and the unavailability of propagules for conventional vegetative propagation. A micropropagation system was developed to facilitate easy propagation of this species. Actively growing shoot tips achieved optimum shoot proliferation from axillary buds when placed on Woody Plant Medium supplemented with 1.0 to 2.5 mg/l benzyladenine. The addition of 0.1 mg/l naphthaleneacetic acid had little effect on culture performance. Murashige and Skoog medium was incapable of supporting vigorous shoot proliferation. Non-sterile rooting conditions provided better rooting and subsequent plantlet growth, when compared to an in vitro rooting method. The seasonal fluctuations in the stock plant dramatically affected the shoot proliferating potential of the explants in vitro. Rapidly elongating shoots formed shoot proliferating cultures more slowly than explants taken either before or after the rapid elongation phase.     

Adventitious Root Formation Induced by Gibberellic Acid and Regulated by the Irradiance to the Stock Plants

Pea plants (Pisum sativum L. cv. Alaska) were grown from seeds for eleven days at different irradiances. Cuttings were then excised and rooted at 16 W × m^?2. Gibberellic acid (GA₃, 10^?11 to 10^?3M) was applied to the cuttings immediately after excision. Cuttings from stock plants grown at the highest level of irradiance (38 W × m^?2) formed the lowest number of roots. An increasing number of roots per cutting was obtained by decreasing the irradiance to the stock plants. In cuttings from stock plants grown at low irradiances, low concentrations of GA₃ (10^?8 and 10^?7M) promoted root formation further. No effect on rooting by these GA₃ concentrations was observed when applied to cuttings originating from stock plants grown at the high irradiances. Root formation in all cuttings was inhibited by GA₃ at concentrations higher than 10^?6M. The degree of inhibition by GA₃ was influenced by the irradiance pretreatment and was increased with an increase in the irradiance during the stock plant growth. Seeds from different years produced cuttings with different response patterns regarding the irradiance and GA₃ effects on rooting.     

Stock plant etiolation and stem banding effect on the auxin dose-response of rooting in stem cuttings of Carpinus betulus L. ‘Fastigiata’

Experiments were undertaken to determine the effect of stock plant etiolation and stem banding, prior to cutting propagation, on the auxin dose-response of rooting in Carpinus betulus L. Fastigiata stem cuttings. Stock plants were forced in a greenhouse, etiolated for 10 days and banded with black, light-tight Velcro for 8 weeks. Indole-3-butyric acid was applied to cuttings at concentrations ranging from 0 to 79 mM. Rooting percentages and numbers increased to a peak reponse at 20 mM in light-grown and 40 mM in etiolated shoots, followed by an inhibition at higher concentrations for all except etiolated and banded shoots. Cuttings prepared from shoots which had been etiolated or banded rooted better than controls at low and optimal IBA concentrations. Cuttings from shoots receiving both etiolation and banding also yielded higher rooting percentages and more roots per rooted cutting. Furthermore, etiolation and banding reduced the sensitivity of cuttings to supra-optimal auxin-induced inhibition of adventitious root initiation.     

Influence of Cotyledon Excision and Sucrose on Root Formation in Pea Cuttings

Sucrose was supplied to stock plants of Pisum sativum L. cv. Alaska grown at different levels of irradiance. There was no significant effect on the rooting of the cuttings by sucrose supply to intact plants regardless of the irradiance. However, an increase in the number of roots per cutting was obtained at increasing concentrations of sucrose when the stock plants had been grown at 4 W m^?2 and their cotyledons had been removed two days before the cuttings were excised. Cotyledons were removed from stock plants at different times before the excision of cuttings with the intent to regulate the endogenous supply of carbohydrate. The number of roots per cutting was reduced by removal of the cotyledons and this reduction was correlated to the number of days the stock plants had grown without cotyledons as well as to the irradiance pre-treatment. A greater reduction occurred in cuttings from plants grown under 4 W m^?2 than from those grown under 38 W m^?2. The growth of the stock plants and the subsequent stem growth of the cuttings was determined by the irradiance to the stock plants and by the time of removal of the cotyledons. Exogenous supply of sucrose had no effect on the stem growth of the cuttings.     

Callus and shoot formation in organ and tissue cultures of Hedera helix L., English ivy

When shoots of young plants of hemp (Cannabis sativa L.) and spinach (Spinacea oleracea L.) were cultured as cuttings and allowed to regenerate advenitious roots, ca. 80–85% became female (formed pistillate flowers) regardless of whether the leaves were left on the plants or were cut off (except for the 2–3 uppermost ones) after the beginning of adventitious-root formation. But when the leaves were cut off and the cuttings treated with gibberellic acid (GA₃, 25 mg/l) ca. 77–80% of the plants became male (formed staminate flowers). The result was quite similar when roots and leaves of young hemp plants were removed at the same time and the cuttings treated with GA₃. It is suggested that the leaves play an essential role in sex expression in hemp and spinach and that this role is related to gibberellin synthesis in the leaves.     

Light effects on root formation in aspen and willow cuttings

The effect of light on rooting of leafy cuttings of aspen (Populus tremula × tremuloides) and a willow hybrid (Salix caprea × viminalis) was investigated under controlled conditions in water culture. Two levels of irradiance were used, 40 and 8 W m^?2. The lower level gave the best rooting of aspen cuttings, both when applied to the stock plants before the cuttings were taken and when given to the cuttings during the rooting period. Irradiation of the cutting base during the rooting period inhibited rooting almost completely in aspen and decreased the number of roots formed in the Salix hybrid. Net photosynthesis in the cuttings of Salix decreased considerably after excision and increased again after formation of roots. Indirect evidence indicated that photosynthesis was even more affected in aspen cuttings. The possible roles of carbohydrates and inhibitors in the light effects are discussed.     

Stimulation of adventitious rooting of Taxus species by thiamine

Summary Results obtained from using root inducing compounds on Taxus species cuttings suggested that rooting could be significantly enhanced by the presence of thiamine. This observation was verified using a root inducing solution containing a set concentration of IBA (0.2%), NAA (0.1%), and supplemented with various concentrations of thiamine. The best rooting response for Taxus cuspidata stem cuttings was found using this solution supplemented with 0.08% thiamine. Rooted cuttings were easily established and developed into vigorous plants. In addition, Taxus brevifolia shoots obtained from tissue cultures via in vitro organogenesis also responded favorably to this 0.08% thiamine supplemented rooting solution.     

VEGETATIVE PROPAGATION OF CHENOPODIUM QUINOA BY SHOOT TIP CULTURE

An in vitro procedure is described for vegetative propagation of two varieties of Chenopodium quìnoa. Shoot tips of seedlings and adult plants were stimulated to produce multiple shoots by axillary branching in a B5 medium modified by decreasing sucrose (10 g/l), increasing nitrate (2,700 mg/l) and phosphate (315 mg/l) salts, and to which glycine was added (4 mg/l). This modified medium was supplemented with benzyladenine (0.22 mg/l) and naphthaleneacetic acid (0.018 mg/l). These conditions allowed development of more than twice the average number of shoots per culture as did the non-modified B5 medium. Absence of exogenous growth substances stimulated shoot elongation and rooting more effectively than auxins or gibberellic acid added to the medium. Rooted plants, transferred to a greenhouse, grew to 2 m at the flowering stage.     

Seasonal changes in adventitious root formation in hypocotyl cuttings of Pinus sylvestris: Influence of Photoperiod during stock plant growth and of indolebutyric acid treatment of cuttings

Seedlings of Pinus sylvestris were grown for 6 weeks under natural light conditions in a temperature controlled environment room. Cuttings from these plants were rooted in tap water or in indolebutyric acid (IBA) solutions for 60 days at an irradiance of 16 W m^-2. Experiments were performed at 3-week intervals during two growth seasons. — Seasonal changes in root formation were found in control cuttings as well as in IBA treated cuttings. The number of roots and the percentage of cuttings that rooted were high during early spring and autumn. During the summer period hardly any roots were formed. Stimulation of root formation by IBA occurred manily during spring and autumn when cuttings already possessed the ability to form roots. — The influence of photoperiod during stock plant growth was also investigated. Shorter photoperiod resulted in an increase in the number of roots and rooting percentage. The period during summer where rooting was inhibited under natural light conditions was considerably shortened when stock plants were grown at a photoperiod of only 4 h. The results demonstrate the importance of the growing conditions for stock plants for subsequent root formation. The results are discussed with special reference to the role of irradiance.     

An improved protocol for in vitro multiplication of Bacopa monnieri (L.)

A cost-effective and efficient protocol has been described in the present work for large scale and rapid in vitro propagation of a valuable medicinal herb Bacopa monnieri (L.) by shoot bud proliferation on internodal segments isolated from field grown mature plants. This shooting was achieved on low concentration of cytokinin with BAP 1.0 mg/l and Kn 0.5 mg/l within 3 weeks of inoculation contrary to earlier reports where auxin is also used in combination with cytokinin. Normally internodes gives rise to a clump of shoots (18 shoots). On an average within a period of three subcultures the internode explant generated 324 shoots thereby improving the economics of the cost of the plants produced and time required. For rooting this is a first report of its growth in a liquid culture. The regenerated plants resemble the mother plants in the general habitat without any morphological variation. This paper reports a very simple, reproducible procedure for in vitro propagation of Bacopa monnieri (L.) that can be used to form a foundation stock of elite plant material for large-scale cultivation.