Softwood Shoots Research Articles

An Efficient Propagation Approach to Forcing Softwood Shoots from Epicormic Buds and Subsequent Rooting of Paulownia elongata S. Y. Hu.

The current research describes the multiplication of Paulownia elongata S. Y. Hu, a timber plant, through the forcing of softwood shoots from epicormic buds under glasshouse conditions in spring and fall seasons. Different growth media were used to compare their effect on the forcing potential of epicormic buds. For this, 25-30-cm-long and 1.2-2-cm-diameter stem segments taken from the lower juvenile portion of a mother plant were placed horizontally in flat trays containing media, i.e., sterilized well-moistened sand, peat moss, perlite, and vermiculite individually. Furthermore, 4-6-cm-long forced softwood shoots were detached and treated with various concentrations of IBA (indole-3-butyric acid) and NAA (α-naphthyl acetic acid) either individually or in combinations for subsequent rooting. The response of shoot forcing was better in spring as compared to fall in terms of shoot length (cm), and number of shoots or leaves; however, an earlier bud break was observed during fall after 30 days of the initial experiment. The use of peat moss and vermiculite proved to be equally suitable for early bud break in both seasons, whereas in terms of shoot and leaf number as well as the shoot length (cm), the best outcome was observed in sand. Best rooting was observed at 3 gL-1 IBA + 3 gL-1 NAA in terms of root number per shoot, root length (cm), and days to root initiation while using sand as the growth medium after 50 days of the rooting experiment. The successfully established plantlets were further shifted to soil at Botanical Garden, University of the Punjab, Lahore, Pakistan, exhibiting an 87.5% survival rate. On the basis of the results obtained, it may be concluded that reasonable softwood shoot forcing in P. elongata may further be exploited for its mass scale nursery propagation as well as use in future in vitro studies.

EFFICIENT CLONAL PROPAGATION OF GUAVA CULTIVARS BY MINI SOFTWOOD CUTTINGS

Guava (Psidium guajava L.) is commercially propagated by seeds in Punjab and vegetative propagation systems are lacking. Propagation by stem cuttings is handicapped by the non-availability of plant material in bulk for cuttings and season dependency. Hence, juvenile mini softwood (MSW) shoot cuttings (3"-4") of Gola (Round) cultivar having two to three nodes were treated with plant growth regulators (PGRs) including indole butyric acid ‘IBA’ or naphthalene acetic acid ‘NAA’ in combinations with benzyl aminopurine ‘BAP’ and planted in sand, silt and coco peat (40:40:20 v/v) under CRD layout for PGR optimization. Cuttings treated with BAP and IBA (2 mgL-1 + 3 mgL-1) started early sprouting in 8 days and induced a greater number of branches (5-6), number of leaves (11), number of roots (6-7) and long roots (14 cm). Whereas, sprout length was higher up to 14 cm in cuttings treated with BAP and IBA (3 mgL-1 + 1 mgL-1). In the second part of the study, application of the optimized PGR treatment of BAP and IBA (2 mgL-1 + 3 mgL-1) to MSW cuttings of different commercial strains of cultivars Gola (Round) and Surahi (Pyriform) showed a better number of roots (5, 4), root length (13 cm, 11 cm), bud sprouting (100% and 80%) and number of nodes (5 each) in Gola (Sadabahar ‘G1’) and Surahi (Sindhi Bao ‘S2’), respectively. The rooted cuttings were maintained under standard growth room conditions and higher humidity till proper plant growth and the established plantlets were transferred to green house. This study established an efficient clonal propagation system using MSW cuttings thus reducing the plant material and PGRs required for mass multiplication of elite guava cultivars. Keywords: Auxin, cytokinin, softwood cuttings, shoot, rooting.

First Report of Krymsk® 5 (cv. VSL 2) Cherry Rootstock In Vitro Propagation: Studying the Effect of Cytokinins, Auxins and Endogenous Sugars

Krymsk® 5 (VSL-2) is a semi-dwarf cherry rootstock adaptable to a range of climates. The present study aimed to establish the first efficient in vitro propagation protocol for this rootstock. Therefore, six cytokinines, four adenine type (6-benzyladenine, 2-isopentenyladenine, kinetin and meta-topolin) and two phenylureas (thidiazuron and forchlorfenuron) at three (2.4 μΜ, 4.8 μΜ and 9.6 μΜ) concentrations plus three (0.24 μΜ, 0.48 μΜ, 0.96 μΜ) for thidiazuron only were tested during the multiplication stage. 6-Benzyladenine was the most efficient cytokinin, based on the number of shoots produced (3.5 shoots at 9.6 μΜ) and the number of nodes per explant (10 nodes at 9.6 μΜ) whereas the other aromatic adenine tested, i.e. meta-topolin, presented the highest number of nodes per cm and node per shoot. During the rooting stage the synthetic auxins 1-naphthaleneacetic acid (1-NAA) and indolebutyric acid (IBA) were tested at concentrations of 0, 2.5, 5, 10 and 20 μΜ both separately and in all possible combinations. The percentage of successfully rooted explants reached 95% under the combination of 20 μΜ IBA plus 5 μΜ 1-NAA, whereas the highest number of roots recorded was 8.5 roots for the treatment 20 μΜ ΙΒΑ plus 2.5 μΜ 1-NAA. Furthermore, two different carbon sources were compared, the widely used sucrose and the endogenous sugar ratio of mother plant softwood shoot, sampled during late of May. Endogenous sugar ratio proved to be the preferable carbon source, since it increased the number of shoots produced and almost doubled the number of produced nodes per explant.

Establishment of Embryogenic Cultures and Efficient Plant Regeneration System from Explants of Forced Softwood Shoots of Teak (Tectona grandis L.)

The present study highlights an efficient plant regeneration system in teak (Tectona grandis L.) using forced softwood shoots as an initial plant material. Forced softwood shoots of teak were cut to prepare shoot tip, nodal and internodal explants and cultured on Murashige and Skoog (MS) medium + NAA (1, 3, 6, 10, and 15 µmol ⋅ L−1) or TDZ (0.001, 0.01, 0.1, 1, 4, 8, 10, 12 µmol ⋅ L−1) for callus induction. Such calluses were further grown on the same levels of TDZ or 0.4, 1, 4, 8, 10 µmol ⋅ L−1 BA + 1 µmol ⋅ L−1 IBA or GA3. Callus induction was the highest with 4.55 cm3 callus volume and 5.75 g dry weight at 0.1 µmol ⋅ L−1 TDZ from shoot tips after 35 days. Embryogenic calluses were then shifted to 6, 8 or 12 µmol ⋅ L−1 TDZ + 2 µmol ⋅ L−1 BA or IBA along with 5 mmol ⋅ L−1 ascorbic acid (AA) for shoot regeneration from embryogenic cultures. The highest embryogenesis (100%) with 36.4 globular and 5.5 heart-shaped embryo-like structures was obtained at 8 µmol ⋅ L−1 TDZ + 2 µmol ⋅ L−1 BA after 63 days. Such cultures when further maintained on the same medium up to 150 days resulted in 100% shoot regeneration with 16.4 mean shoots. Shoots were elongated up to 50 mm on agar medium + 8 µmol ⋅ L−1 BA + 1 µmol ⋅ L−1 GA3. An efficient rooting response (70%) was achieved having 4.50 mean number and 49.10 mm root length at 8 µmol ⋅ L−1 IBA + 8 µmol ⋅ L−1 NAA + 0.1% activated charcoal after 36 days. Rooted shoots were acclimatized in glasshouse, achieving 56.6% plantlet survival.

Improvement of grafting efficiency in hard grafting grape Berlandieri hybrid rootstocks by plant growth-promoting rhizobacteria (PGPR)

Certain grapevine rootstocks in Vitis berlandieri hybrids (such as 41 B, one of the most important rootstocks used in worldwide viticulture) have great potential to use owing to its great resistance to calcareous soils and phylloxera. But their use is limited due to the difficulties in propagation and grafting. PGPR is reported to stimulate division and enlargement of plant cells that might result in better fusion at graft union point. This investigation was thus conducted to reveal the effects of plant growth promoting rhizobacteria (Pseudomonas putida strain BA-8, Bacillus simplex strain T7) on callus formation, graft success, vegetative development and subsequent bud fruitfullness of Alphonse Lavallée grape cultivar.The green softwood shoots of Alphonse Lavallée (AL) grape were grafted on the rootstocks 41 B or 1103 P by modified cleft green-grafting technique in controlled glasshouse. Before grafting, the single scion nodes were divided into four treatment groups. Three of them were separately dipped into bacterial suspension (109CFUml−1) of either of two strains or their combination for an hour. One of the treatment groups was dipped into the tap water (as control). Each treatment has contained thirty grafts, divided into three replicates.In both graft combinations, combined application of BA-8 and T7 strains resulted in 100% callusing compared with the uninoculated control where the percentages were 93.3 and 90.0% for AL/41 B and AL/1103 P combinations, respectively. Individual or combined applications of the strains also had remarkably positive influences on full callusing rates. The highest shoot length values were obtained from combined application (113.5cm) and BA-8 strains (100.9cm) in AL/41 B and AL/1103 P graft combinations, respectively. Inoculation of the graft scion parts with BA-8 strain resulted in the highest shoot lignification lengths in both AL/41 B and AL/1103 P combinations with the values of 75.4 and 75.6cm, respectively while uninoculated grafts of the representing combinations were as low as 59.0 and 55.3cm. In AL/41B, the highest (100%) graft survival rate in nursery was recorded in plants treated with bacterial combination. Combined application as well as BA-8 performed better in AL/1103P. The findings imply that PGPR can be employed in ecological and sustainable production of nursery material and point to the feasibility of commercially synthetic auxin replacement by PGPR-based ecological treatment.

The influence of plant growth regulators on explant performance, bud break, and shoot growth from large stem segments of Acer saccharinum L

Benzyladenine (BA) and/or gibberellic acid (GA3) were applied in 20% white exterior latex paint separately at 0, 0.3, 1, 3, 10, or 30 mM; and at 1, 10, or 30 mM of each plant growth regulator (PGR) in a 3 × 3 factorial to 40 cm long stem segments of Acer saccharinum L. Softwood shoots were forced from these stem segments at various times of the year in a greenhouse and in a laboratory, these resulting shoots were surface disinfested and used as explants in vitro on Driver and Kuniyuki Walnut medium with 0 or 0.01 μM thidiazuron (TDZ). There was some response to the plant growth regulators applied in paint for shoot production from the stem segments and in vitro. Explants from softwood shoots forced from stems painted with 3 mM BA and cultured on medium with 0.01 μM TDZ produced more shoots than explants taken from softwood shoots forced with other BA concentrations or controls. Callus also grew significantly more on explants from stems treated with 3 mM BA cultured on 0.01 μM TDZ than explants harvested from stems painted with other concentrations of BA excluding 10 mM BA. When stem segments treated with BA plus GA3 were compared as a group to controls, more and longer softwood shoots grew on the stems painted with PGRs when all four runs were pooled (Sept. 2005 through Feb. 2006). Application of PGRs in paint extends the season of production of softwood shoots that may be used as explant materials and their subsequent performance in vitro.

An efficient method for clonal propagation and in vitro establishment of softwood shoots from epicormic buds of teak (Tectona grandis L.)

Softwood shoots were produced from 40 cm long stem segments placed horizontally in flat trays containing sterilized sand under natural light or shade conditions for subsequent rooting and micropropagation studies in teak (Tectona grandis L.). Higher number of shoots (6.17) per log was produced under natural light as compared to shade conditions. Forcing was also better in natural light as compared to shade in terms of shoot length, number of nodes or leaves. For rooting, 2–4 cm long softwood shoots were excised and treated with either indole-3-butyric acid (IBA) or α-naphthyl acetic acid (NAA) at 0, 1000, 2000 or 3000 μmol·L−1 each or with combinations (1000 + 1000, 2000 + 2000 or 3000 + 3000 μmol·L−1) and then placed in flat trays containing autoclaved sand at 25 ± 2°C in 16 h photoperiod at 35 μmol·m−2·s−1. After 28 days, softwood cuttings treated with IBA + NAA (3000 + 3000 μmol·L−1) had highest rooting percentage (89.3%) with 5.5 mean roots. Shoot apex and nodal explants of softwood cuttings were pretreated with 0.1% (w/v) ascorbic acid, boric acid, activated charcoal, citric acid, glutamine or polyvinylpolypyrollidone (PVP) for 24 h to remove phenolic compounds before surface disinfestation. Glutamine (Gl) and PVP were equally effective resulting in 60% establishment of shoot apices on MS medium supplemented with 10 μmol·L−1 6-benzylaminopurine (BAP) + 5 μmol·L−1 NAA. Using shoot apices, highest (42.80) number of multiple shoots with 54.33 mm shoot length were obtained on MS + BAP (8.8 μmol·L−1) + IBA (2 μmol·L−1) after 45 days. Shoots were successfully rooted and acclimatized to greenhouse conditions.

Effect of branch position and auxin treatment on clonal propagation of Tectona grandis Linn. f.

The main objective of our clonal propagation program is the production of high quality timber and faster tree growth. Teak (Tectona grandis Linn. f.) is an important plantation species in the tropics but the demand of teak timber is increasing globally. Teak planting stock is still produced from seeds of unselected elite genotypes which may lead to poor stand establishment. The number of seeds per fruit and their germination ability are also variable and limited by several factors. Therefore an alternative method i.e., clonal propagation is required for mass multiplication of elite trees. It is an established fact that the clonal propagation or adventitious root formation in cuttings is markedly affected by several external and internal factors. Considering these, an experiment was conducted on leafy soft wood shoot cuttings of 3-year-old hedged teak stock plants grown in a hedge garden to study the effects of branch position and auxin treatment on adventitious root formation. A factorial completely randomized design was used for experimentation. Coppice shoots were collected from basal, middle and upper positions of the hedge garden; and mono-nodal leafy cuttings were prepared. These cuttings were treated with different concentrations of IBA and NAA; and were cultured in a mist chamber for rooting where the relative humidity was maintained at 85 ± 2% with maximum and minimum day-night temperature at 32 ± 1°C and 26 ± 1°C respectively. After 45 days the cuttings were removed from the rooting medium and observations were made. It was observed that the rooting parameters viz., percent rooting and percent sprouting, mean number of leaves, mean number of shoots and length, and mean number of roots and length per cutting were significantly influenced by the branch position and auxin treatment. Treatment with 4000 ppm IBA increased percent rooting and percent sprouting whereas NAA suppressed it. Treatment with 4000 ppm IBA also increased the mean number of leaves and shoots, and the length, and the mean number of roots per cutting. The highest mean number of roots and length were observed in cuttings taken from the branch emerging at the upper position followed by branches of middle and lower position. Cuttings of branches at a middle position showed the highest values of percent rooting, percent sprouting, mean number of leaves and shoots and mean shoot length per cutting. Furthermore, the interactive effect of branch position and auxin treatment on rooting and sprouting response was also found to be significant. Overall, the findings of this investigation suggest that middle branch position in hedge plants and 4000 ppm IBA can be made to induce maximum rooting and to produce high quality planting stock material for clonal forestry program of teak.

The Influence of Plant Growth Regulators on Budbreak and Shoot Growth from Large Stem Segments of Acer saccharinum L.

A factorial combination of gibberellic acid (GA3) and benzyladenine (BA) was applied in 20% white exterior latex paint to large (40 cm long, >2.5 cm diameter) stem segments of Acer saccharinum L. (silver maple) to determine the effects on forcing new softwood shoots in the greenhouse or laboratory and the subsequent growth of these new shoots in vitro. Stem segments were harvested from 10-year-old field-grown coppice shoots. The GA3/BA-paint mixes were applied to the entire stem segments that were forced in plastic flats filled with 1 perlite: 1 vermiculite (by volume) and watered with care so as not to wet the new softwood shoots. The flats and stem segments were drenched weekly with Zerotol (0.18% H2O2). The softwood shoots were harvested when they were at least 3 cm long. After disinfesting and rinsing, the nodal and shoot tip explants were established aseptically in vitro on DKW medium with no cytokinin or with 10-8M thidiazuron. Coppice shoots were harvested, cut, and painted on 9 Sept., 28 Oct., and 12 Dec. 2005. Although there were no significant differences in shoot production among stem segments painted with various combinations of GA3/BA, stems treated with plant growth regulators produced a mean of 2.7, 1.8, or 0.5 shoots for the three harvest dates compared to 0.5, 0.0, or 0.25 shoots on control stem segments. It is well-known that shoot forcing is poor from September through January; however, use of GA3/BA resulted in growth of dormant epicormic shoots. Shoot tip explants produced the most shoots in vitro after 8 weeks if they were harvested from stem segments treated with 0.03 mM GA3, whereas nodal explants produced the most shoots if harvested from segments that had been treated with 0.01 mM GA3.

Differentiation of the dynamic variables affecting rooting ability in juvenile and mature cuttings of cherry (Prunus avium)

SummaryThe rooting potential of four types or origins of Prunus avium cuttings from the same mature trees (over 20 years-old) was compared using a mist propagation bed during early Summer (June). The cuttings originated from juvenile sucker shoots of the current and previous year, and mature crown shoots (current year’s lateral ‘long-shoots’ and multi-year terminal ‘short shoots’). The morphological differences in inter-node length, stem diameter and leaf area between the four cutting types were highly significant (P = 0.05), leading to large differences in cutting volume and, so it is argued, to assimilate reserves. Juvenile cuttings rooted well (65% and 77% rooting for hardwood and softwood shoots, respectively), while mature cuttings rooted poorly (4% and 7% for mature hardwood and softwood cuttings, respectively). Leaf abscission was significantly more frequent in mature hardwood cuttings (16 – 78%) than in the other cutting types (1.6 – 9%) at the end of the propagation period. Leaf loss resulted from two processes: abscission and leaf rotting. Physiologically (i.e., in carbon assimilation, leaf transpiration and stomatal conductance), the four cutting types were not significantly different early in the post-severance period (day-4); but, by day-22, stomatal conductance was lowest in mature hardwood cuttings that still had leaves. At this time, the most physiologically-active unrooted cuttings were from juvenile hardwood and mature softwood shoots. The extent of physiological and morphological variability between cutting types and their probable impact on processes affecting rooting ability is complex and highly interactive. Consequently, it is not possible to explain the causes of the variation in rooting ability between juvenile and mature cuttings, although this study suggests that the constraints to rooting are likely to reflect physiological differences between the different cutting types. It is concluded that, to resolve the debate about factors that affect the rooting ability of juvenile and mature cuttings (i.e., ontogenetic vs. physiological ageing), there is a need to achieve morphological and physiological comparability in the tissues.

(172) The Influence of Environment, Media, and Zerotol on Forcing and In Vitro Establishment of Softwood Shoots from Large Stem Segments of Acer saccharinum and Fraxinus pennsylvanica

The objectives of this research were to study the effects of three environments (lab, mist, or fog), four media treatments [perlite, vermiculte, 1 perlite: 1 vermiculite (by volume), or a control (empty flats)] and zerotol treatments on shoot forcing and subsequent transfer of explants to in vitro conditions. Stem segments from field-grown trees were cut to 40-cm lengths before being placed in flats with the media treatments. Half of the flats under mist and fog were drenched weekly with zerotol (0.18% H2O2). In a separate study, silver maple was forced under mist and drenched weekly with zerotol at 0%, 0.09%, 0.108%, 0.135%, 0.18%, 0.27%, or 0.54% H2O2. Shoots (≥5 cm) were harvested and nodal and shoot tip explants were surface disinfested and placed in vitro on DKW medium with 10-8 M thidiazuron plus 1.0 μM indolebutyric acid. Species did not interact with environment, media, or zerotol treatment, and silver maple produced a mean of 6 shoots per stem segment, while green ash produced a mean of 1.2 shoots. There was a significant interaction among perlite, vermiculite and environment, with the most shoots (6.7/stem segment) produced under mist in the perlite: vermiculite mix. Silver maple explants from the lab had only 4% microbial contamination, whereas 68% of explants from fog and 92.2% of explants from mist were contaminated. When forcing was under fog, in perlite, and drenched with zerotol, explants had a 43% rate of contamination. In a separate study, when silver maple stems were placed under mist and drenched weekly with 0.18% H2O2, 46% (18 of 39 explants) established cleanly in vitro. Contamination was higher with misted explants that were drenched with higher or lower concentrations of zerotol.

The Influence of Location of Main Stem Segment Origin on Forcing and Rooting of Softwood Shoots of Betula nigra L.

The objective of this study was to investigate the effects of the position on the main stem that large stem segments were harvested from on forcing and subsequent rooting of Betula nigra L. (river birch) softwood shoots. The main trunks of eight adult-phase native trees (four trees per run of the experiment) were cut into 50-cm long segments from the ground up. The segments placed in horizontally in 52 × 25 × 6.5 cm (l × w × h) flats containing perlite and were positioned so the bottom one-third of the stem was within the medium. Shoots were forced under natural photoperiod and intermittent mist. This experiment was conducted twice. Data were collected weekly for fourteen weeks on the number of softwood shoots each segment produced, shoot length, number of rootable shoots (>6 cm long), the length of time that the stem segments produced rootable shoots, and the rootability of these shoots treated with 3000 ppm IBA in talc. The number of harvested shoots was greater in Run 1, with the basal segments producing the most harvestable shoots. However, the upper segments in Run 2 produced the most harvestable shoots. Softwood shoots that rooted were placed under intermittent mist. Out of the 540 harvested shoots for both runs, 82.4% rooted, with the majority of those from Run 1. Shoots harvested from this run began producing roots about 6 weeks after harvest, and continued until the end of the experiment. Run 2 shoots began root initiation about 3 weeks after harvest and ended about 2 weeks before the end of the experiment. Run 1 had an mean of 8.3 roots per shoot and Run 2 had an mean of 6.2 roots. The relationship between juvenility and shoot forcing and subsequent rooting will be discussed.

NOVEL METHODS IN MICROPROPAGATION

The focus of this paper is on two novel methods for micropropagation, forcing softwood shoots from large stem sections and encapsulation of buds and shoot tips for storage and facilitation of Stage IV, acclimatization. Neither method is being used widely by commercial micropropagation laboratories. Branches are excised from within the cone of juvenility of woody plants that were originally propagated from seed. The branches are cut into sections 30-50 cm long and placed horizontally in flats or benches filled with perlite. Irrigation must be done with care to avoid water contacting the stem segments or new softwood shoots. The softwood shoots are excised, surface disinfested and used as explants. If the softwood shoots are to be used as macrocuttings, forcing is best under intermittent mist. However, forcing in mist will result in microbial contamination if micropropagation is the goal. Alginate gel is used to encapsulate somatic embryos to produce synthetic seeds. The same gel matrix can be used to encapsulate buds and shoot tips from Stage II proliferating cultures. The encapsulated buds can be stored under refrigerated conditions or dehydrated and placed in liquid nitrogen. Additionally, shoots and roots can grow from the encapsulated shoot tips and buds and these can be transplanted to a greenhouse medium for acclimatization. Because of the nutrient salts and sucrose in the gel matrix, the incorporation of fungicides into the gel has reduced fungal growth during Stage IV and has increased the percent survival.

691 Obtaining Shoot Explants From Softwood Shoots Forced From Large Stem Segments of Woody Plants

Softwood shoots can be forced from sections of branches of trees or from basal stems of shrubs by cutting into ≈40-cm lengths and placing these segments horizontally in flats filled with perlite. We have had our best success using stems that are >1.5 cm in diameter. Although the best environment that we have found for producing the most and longest softwood shoots is under intermittent mist, this is unacceptable for producing explants because of microbial contamination. Rather, for micropropagation, watering must be done two to three times a day and care must be taken to avoid water spray onto the stem segments or the subsequent softwood growth. Irrigation can be by hand or by using drip irrigation. For trees, using the basal portions of large branches allows for selection of shoots from within the “cone of juvenility.” Theoretically, these should propagate better than shoots taken from the outer, more adult portions. Although late winter through spring are the best times for forcing, some shoots will grow if the stem sections are harvested nearly any time of the year, except for October through December in southern Illinois.

Axillary Shoot Proliferation of Adult Eastern Black Walnut

Softwood shoots of adult Juglans nigra (eastern black walnut) were forced from latent (epicormic) buds below the bark of large stem sections in a greenhouse. Once sufficiently long, the shoots were excised, surface-disinfested, and cut into 1-cm-long nodal or apical segments for establishment in culture vessels. Two experiments were conducted, each using explants of three different clones. The first experiment compared the effect of cytokinins: 0.3, 1.0, or 3.0 μM benzyladenine (BA) and 0.1, 0.3, or 1.0 μM thidiazuron (TDZ) arranged factorially. The basal medium was agar-solidified Long and Preece (LP) with 0.05 μM indole-3-butyric acid (IBA) and 30 g/L sucrose. The second experiment compared the agar-solidified basal media: Driver-Kuniyuki-Walnut (DKW), Woody Plant Medium (WPM) and LP, all with 1.0 μM BA, 0.3 μM TDZ, 0.05 μM IBA, and 30 g/L sucrose. Regardless of the BA concentration, explants on media containing 0.1 μM TDZ produced few, if any, axillary shoots while explants on media containing 1.0 μM TDZ excessive amounts of callus. Explants in media containing 0.3 μM TDZ, at all levels of BA, produced the greatest number of shoots and minimal callus. Male catkins were produced by 17 explants on various media. Fifteen of the catkin-producing explants were from one walnut clone. Axillary shoot number and callus production were not significantly affected by basal medium for any of the three clones.

Epicormic Shoot Forcing of Adult Red Maple, Sugar Maple, and Eastern Black Walnut

Dormant stem sections from three individuals each of Acer rubrum (red maple) and A. saccharium (sugar maple), and three clones of Juglans nigra (eastern black walnut) were collected from lower lateral branches of trees at least 30 years old. The stem sections were cut into 50-cm-long segments and placed horizontally in soilless media in a greenhouse. Two experiments were conducted to determine which treatments were most efficient for the production and elongation of epicormic shoots from latent buds below the bark. The first experiment consisted of a factorial arrangement of two levels of shading (not shaded or covered with 48% Saran shade cloth) and two levels of flooding (not flooded or flooded to a depth of 1/3 to 1/2 the diameter of the stem sections). Flooded treatments contained 0.025% NaClO to control microbial growth. The second experiment consisted of three media treatments: perlite, vermiculite, or a 1 perlite: 1 vermiculite (by volume) medium. No stem segments of any species in flooded treatments produced softwood shoots. Neither shading nor media treatments affected shoot number or length for any of the species. Additional sugar maple segments collected after budbreak produced more and longer shoots than those collected while dormant.

In vitro shoot proliferation of 5-leaf aralia explants from field grown plants and forced dormant stems

A. Sieboldianus (5-leaf aralia) is recalcitrant for micropropagation, but has very good landscaping potential. This research was conducted with the following objectives: (1) to study effects of BA, TDZ, CPPU, 2iP, kinetin and zeatin in woody plant medium on the performance of softwood shoot nodal explants produced by field grown 5-leaf aralia plants; (2) to investigate influences of BA or TDZ in the forcing solution on subsequentin vitro shoot initiation of nodal explants taken from forced softwood growth. Shoot initiation of softwood nodal explants from field-grown plants was promoted by adding BA, TDZ or CPPU to the culture medium. Kinetin, zeatin and 2iP were ineffective for micropropagation ofA. Sieboldianus. The forced softwood growth for use as explants was “primed” by forcing dormant stems in solution containing 200 mg 8-HQC per liter plus 2% sucrose, 44.4, 222, or 444 µM BA, or 45.4, 227, or 454 µM TDZ. BA and TDZ in the forcing solution enhanced subsequentin vitro axillary shoot initiation of nodal explants taken from forced stems by doubling the number of shoots produced per explant to 3.3 from 1.65 shoots per explant taken from field grown plants. This forcing solution technique also reduced the time needed from culture initiation to potted plants to half of the time needed for the conventional micropropagation method (12 to 14 vs. 25 to 27 weeks), thus expediting the micropropagation ofA. Sieboldianus.

Production of Shoots from Dormant Species of Acer as Influenced by Length and Caliper of Stem Sections

Abstract Stem sections removed from selected, dormant species of Acer L. (maples) were forced to produce softwood shoots under greenhouse conditions. Species and clones varied widely in the percentage of stem sections forming shoots, and in the number of shoots that were generated per section. Both length and diameter of sections influenced the number of shoots that were generated. With respect to length, it was best to use stem sections of 30 to 40 cm (11.8 to 15.7 in) since longer sections failed to produce additional shoots and occupied considerably more production space. Effect of increased stem diameter varied across species. For A. rubrum, the number of shoots produced was greatest on ‘large’ diameter (5.2 to 7.6 cm; 2.0 to 3.0 in) stem sections; shoot production was greatest on ‘medium’ diameter (3.3 to 5.1 cm; 1.3 to 2.0 in) stem sections of A. palmatum. Data indicate that an optimum diameter will have to be determined for each species that is propagated when using this method.

Epicormic Sprouts from Branch Segments of Mature Juglans nigra L. as an Explant Source for in Vitro Culture

At monthly intervals for 1 year, one branch was removed from the lower crown of three 30-year-old trees of black walnut (Juglans nigra L.). The basal 1.3 m of each branch was cut into four 32-cm-long segments that were placed horizontally in shallow plastic trays filled with perlite and watered daily with tap water. Branch segments cut early in the dormant season (29 Sept., 31 Oct., or 1 Dec.) or shortly after flushing (6 June) produced few, if any, epicormic sprouts. Approximately half the branch segments cut on 3 Jan. or 3 Feb. produced one sprout that elongated slowly. Most branch segments cut in the late dormant season (2 Mar., 30 Mar., 3 May) or growing season (5 July, 4 Aug., 6 Sept.) produced one or two sprouts >20 mm long. To prepare explants for in vitro culture, the terminal 2.5 cm was harvested when sprouts exceeded 3.0 cm, trimmed of all leaves, and disinfested. Explants were placed vertically in liquid Long & Preece (LP) medium supplemented with 3% sucrose, 0.3 μM TDZ, 0.05 μM IBA, and 1 μM BA. When shoots began to elongate (4 to 6 weeks), they were then placed horizontally on agar-solidified LP medium with liquid LP overlays to induce axillary shoot proliferation. Advantages of forcing epicormic sprouts on large branch segments are: 1) they can be a source of in vitro explant material for 6 to 7 months a year, 2) aseptic cultures can be easily obtained, 3) shoots from the base of branches may show more juvenility than shoots forced from branch tips, 4) softwood shoot wilting is not a problem as with forcing shoots from branch tips, 5) the procedure does not require preparing and changing forcing solutions, and 6) branch segments should have more stored food than dormant branch tips for forcing softwood growth.

Forcing Large-diameter Stems of Adult White Ash in a Greenhouse to Obtain Softwood Shoot Explants

Branches were collected from trees in July. Lengths with a 2.5-cm caliper were cut to 30 cm and placed horizontally into flats of perlite with half of the diameter of each stem above the perlite surface. Flats were watered daily with tap water and stems were kept moist. Buds swelled quickly, and after 6 days, small epicormic shoots were visible. These softwood shoots continued to elongate and retain good turgor for 2 weeks, when they were excised and placed in vitro. Shoottip and nodal explants were placed onto MS medium with 1 μM BA; 1 M IBA; and 0, 3, or 10 μM TDZ where shoots slowly elongated. Forcing large stems in the greenhouse has been superior to forcing smaller stem tips in the laboratory because of lower contamination (40% on shoots from large stems vs. up to 90% on shoots from small stems), longer life of the softwood shoots (less wilting from the larger stems), a longer time during the year for forcing, and the possibility of forcing shoots from more juvenile wood than stem tips.