Sequential decline in fruit resource allocation within inflorescences of Sagittaria trifolia: a test of non‐uniform pollination hypothesis

Flowering plants often exhibit declining resource investment to floral organs, fruits and seeds temporally or spatially in an inflorescence. To account for such variances, non‐uniform pollination hypothesis, which highlights various mating environments each flower experiences, provides adaptive significance for allocation patterns but with controversial supports. Sagittaria trifolia (Alismataceae) was used to examine differences in seed number, seed weight and germination rate among sequential fruits within inflorescences. Ovule number was also investigated to evaluate allocation patterns in the floral stage. To test the non‐uniform pollination hypothesis, we used three polymorphic microsatellite loci of S. trifolia to estimate the seed outcrossing rate in proximal and distal fruits. The results showed that the seed number, average seed weight and seed germination rate of S. trifolia gradually decreased from proximal to distal fruits within inflorescences. The percent of decrease in seed number between two contiguous fruits was 14.68 ± 3.22%, which was much stronger than the percent of decrease in ovule number at 6.95 ± 1.60%. Both proximal and distal fruits within inflorescences had high outcrossing rates (81.5 ± 5.0%, proximal; 82.3 ± 6.9%, distal) and they did not differ significantly. Overall, there was an acropetal decline of resource allocation to fruits within inflorescences of S. trifolia. Allocation pattern to ovules was not a limiting factor for seed production. The lack of difference in outcrossing rate between proximal and distal fruits indicated that the allocation strategy was probably not caused by non‐uniform pollination, but more likely position effects.     

Shoot development ofAucuba japonica I. Morphological study

The shoot development ofAucuba japonica was studied morphologically. The shoot shows dichasial branching in connection with the formation of a terminal inflorescence and shows a decussate phyllotaxis even in the reproductive phase. The sequence of initiation of successive foliar appendages is very precise, hence the foliage leaf, scale leaf and bract can be compared with each other even at their stages of initiation. In the stage of proximal foliage leaf formation the shoot apex is flat, while in the stage of formation of distal foliage leaves, bud scales and proximal bracts, it becomes concave. In the stage of formation of distal bracts the apex becomes domed. Plastochron durations are relatively long in the vegetative phase in comparison with other plants, and the duration from initiation of the first pair of appendages to that of the second is about one and a half months. Both male and female inflorescences exhibit basically a thyrsoid type of monotelic synflorescence.     

Regulation of reproduction in wild and cultivatedLycopersicon esculentum Mill. by vesicular-arbuscular mycorrhizal infection

Summary We examined how mycorrhizal infection byGlomus etunicatum Becker and Gerd. affected flowering phenology and components of reproduction in eight wild accessions and two cultivars ofLycopersicon esculentum Mill. We did this by performing a detailed demographic study of flower, fruit and seed production. Mycorrhizal infection had variable effects on the ten accessions. Infection significantly decreased the time taken to initiate flowering in some accessions. In addition, infection increased flowering duration in some accessions. In many accessions, infection significantly increased seed production, primarily by increasing the number of inflorescences and infructescences. In some accessions, mycorrhizal infection also increased the proportion of flowers that produced mature fruits or the number of seeds per fruit. Among accessions, shoot phosphorus content was correlated with seed productivity for both mycorrhizal and non-mycorrhizal plants. However, non-mycorrhizal plants produced more seed biomass per mg of shoot phosphorus than mycorrhizal plants.     

Shoot regeneration,re-flowering and post flowering survival in bamboo inflorescence culture

In vitro grown inflorescences of Bambusa edulis were used to investigate the process of vegetative shoot growth in detail. The findings revealed that auxins and ACC could be significant growth regulators in this process. Overall, auxins [NAA, indolebutyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4-D)] induced inflorescences to grow vegetative shoots. However, the efficiency of shoot regeneration varied. A greater percentage (27.3–34.5) of inflorescences in the 5 mg l⁻¹ NAA, 10 mg l⁻¹ NAA, and 1 mg l⁻¹ 2,4-D treatments formed more vegetative shoots than those exposed to other treatments. IBA promoted shoot regeneration less effectively than NAA and 2,4-D. Fifty percent of regenerated vegetative shoots flowered after 2 months when the medium was supplemented with 5 mg l⁻¹ NAA. All shoots that received 1 mg l⁻¹ 1-amino-cyclopropane-1-carboxylic acid (ACC) flowered in 5 mg l⁻¹ NAA medium. Rooted plantlets were used to examine their survival following in vitro flowering. All plantlets with vegetative shoots, even those with inflorescences, survived and grew.     

Hydrophilous pollination and reproductive morphology in the seagrassPhyllospadix scouleri (Zosteraceae)

In the seagrassPhyllospadix scouleri (Zosteraceae) floral aggregates (spadices) occur on short lateral axes produced by subordinate vegetative shoots. The filamentous pollen is both dispersed on the surface of the sea as well as below the surface. In surface pollination, snowflake-like search vehicles (pollen rafts) float and collide with any rigid female inflorescences that emerge from the water surface. In submarine pollination, collinear bundles of pollen grains are dispersed. Analysis of seed set indicates ovule position within the inflorescence to affect likelihood of fertilization in submarine, but not surface-pollinated inflorescences. Agamospermy appears to be unlikely, but the strongly female-biased shoot sex ratio remains to be explained.     

The use of phenotypic markers to identify Brassica oleracea genotypes for routine high-throughput Agrobacterium-mediated transformation

Doubled haploid (DH) genotypes from a genetic mapping population of Brassica oleracea were screened for ease of transformation. Candidate genotypes were selected based on prior knowledge of three phenotypic markers: susceptibility to Agrobacterium tumefaciens, shoot regeneration potential and mode of shoot regeneration. Mode of regeneration was found to be the most significant of the three factors. Transgenic plants were successfully obtained from genotypes that regenerated multiple shoots via a distinct swelling or callus phase. The absence of tissue culture blackening (associated with genotypes that formed callus) was found to be critical for transformation success. Transgenic shoots were obtained from genotypes that regenerated via an indirect callus mode, even when susceptibility to Agrobacterium was low. The most efficient genotype (DH AG1012) produced transgenic shoots at an average rate of 15% (percentage of inoculated explants giving rise to transgenic plants). The speed and efficiency of regeneration enabled the isolation of transgenic shoots 5–6 weeks after inoculation with A. tumefaciens. This line was also self-compatible, enabling the production of seed without the need for hand-pollination. A genetically uniform DH genotype, with an associated genetic map, make DH AG1012 highly desirable as a potential model B. oleracea genotype for studying gene function. The possibility of applying the same phenotypic tissue culture markers to other Brassica species is discussed.     

Dry matter accumulation at specific pod positions on field bean (Vicia faba L.) plants and possible relations with indoleacetic acid and abscisic acid levels

Seed weights at specific positions within inflorescences of field bean plants (Vicia faba L.) were varied by removal of flowers. The inflorescences of two regions (nodes 3+4 and nodes 5+6, counted from the bottom of the plant) were used for manipulations and investigations. The two proximal flowers of the manipulated inflorescence were removed in order to vary the development and seed weights of distal pods (see Fig. 1). Dry matter accumulation and IAA- and ABA-concentrations in seeds were investigated during the filling period. Treatment effects in both regions were similar during two seasons. The removal of proximal flowers prevented the usually observed drop of distal pods and favoured the accumulation of dry matter and IAA in seeds, whereas the variation of ABA-concentrations partly depended on interaction with season. Whether these effects contribute to a signal leading to the establishment of processes preceeding dry matter accumulation or are a consequence of such processes is questionable.     

Shoot organogenesis from cultured seed explants of peanut (Arachis hypogaea L.) using thidiazuron

Summary Thidiazuron (TDZ) was utilized to induce adventitious shoot formation from the hypocotyl region of cultured seed explants of peanut (Arachis hypogaea L.). Excision of the radicle from seed explants was more stimulatory to shoot initiation than removal of the epicotyl alone. Removal of both the radicle and the epicotyl from seeds resulted in a 37-fold increase in the frequency of shoot production when compared to intact seeds. Half seed explants with epicotyl and radicle removed produced the greatest number of shoots per explant. Explants from mature seeds were more responsive to TDZ than immature seed-derived explants. A 1-wk exposure to 10 μM TDZ was sufficient to stimulate the initiation of adventitious shoots that subsequently developed into plants. High frequency of shoot initiation was readily induced in a variety of genotypes ofA. hypogaea and a wild peanut (A. glabrata). Plants regenerated from shoots induced by TDZ were phenotypically normal and fertile.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration through somatic embryogenesis and direct shoot organogenesis in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.

An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.     

Factors affectingin vitro establishment and shoot proliferation ofRosa hybrida L. andRosa Chinensis minima

Summary The effects of nodal explants collected at different plastochrones, use of various benzyladenine (BA) concentrations, sources of carbohydrates, and phases of the culture medium on shoot establishment and proliferation ofRosa hybrida L. andR. chinensis minima were evaluated. Higher numbers of shoots per explant were obtained fromR. hybrida cv. Carefree Beauty explants proximal to the apical meristem than those from distal nodes. However, proliferating shoots derived from plastochrones proximal to the apical meristem had a lower number of leaves/explant and were shorter than those derived from other distal plastochrones. Although shoot proliferation increased with higher BA concentration in the medium, a concentration of 4.4 μM BA was found optimum for axillary bud-break and shoot development forR. hybrida cvs. Adelaide Hoodless and Cuthert Grant. A higher shoot proliferation rate was observed forR. hybrida cv. Carefree Beauty explants grown on a medium containing 55.5 mM fructose than 58.4 mM sucrose. However, no differences were observed forR. hybrida cv. Cuthert Grant grown on a medium containing either fructose or sucrose. The mean number of shoots/explant was higher forR. chinensis minima cv. Red Sunblaze explants grown on a liquid (4.5) than on a solid medium (1.7) for the first reculture; while no significant differences between the two phases of the medium were observed for the second reculture. However, a higher mean number of shoots/explant was observed on solid-phase (4.0) than liquid-phase medium (3.4) for the third reculture. A higher mean number of leaves/shoot was obtained on a solidified medium rather than liquid medium in the first two consecutive recultures, while no differences were observed for the third reculture. Although a significant effect of BA concentration on mean number of shoots/explant was observed for Red Sunblaze nodal explants, the influence of BA concentration decreased in the two consecutive cultures for both phases of the medium. Hyperhydricity was observed on Red Sunblaze shoots grown on the liquid-phase medium.     

Multiple shoot induction by benzyladenine and complete plant regeneration from seed explants of chickpea (Cicer arietinum L.)

The efficacy of benzyladenine (BA) to induce multiple shoots from seed explants of chickpea (Cicer arietinum L.) was assessed. Shoot differentiation was influenced by the type of seed explant, genotype and concentration of BA. Orientation of the explant also strongly influenced the shoot regeneration response. The optimum BA concentration for shoot/shoot bud regeneration was genotype dependent. Two types of BA-induced response were observed: (1) at less than 7.5 gm BA, direct shoot differentiation (2 to 4-cm-long shoots) was observed within 30 days; (2) at higher BA concentrations (75–100 m), shoot/shoot bud differentiation was achieved in 45–90 days. A high BA concentration inhibited subsequent rooting of shoots. Roots, however, could be easily induced on shoots derived from <12.5 m BA. Following transfer to soil, 80% of the regenerants developed into morphologically normal and fertile plants.Abbreviations BA Benzyladenine     

The genetics and ecology of seed size variation in a biennial plant,Hydrophyllum appendiculatum (Hydrophyllaceae)

The goal of this study was to elucidate the sources of seed size variation in Hydrophyllum appendiculatum, an outcrossing, biennial plant. The genetic basis of seed size variation was examined with a diallel breeding design. The analysis did not reveal any evidence for additive genetic variance, suggesting that seed size could not evolve in response to natural selection. A series of greenhouse experiments was conducted to determine the sensitivity of seed weight to a number of ecological variables. Seed weight was affected by inbreeding depression: seeds produced by self-pollinations were significantly lighter that outcrossed seeds. Maternal plants did not differentially provision seeds that were the result of crosses between subpopulations (separated by 300 m) or between populations (separated by 1.7 km). Mean seed size was independent of the number of outcrossed pollen donors (one vs. many) that sired seeds on an inflorescence; however, the variance was greater on inflorescences pollinated by multiple donors. Direct manipulations of the abiotic environment showed that seed size was greater on plants growing under full sunlight compared to shaded plants. Seed size was unaffected by soil type, fertilizer addition, or defoliation. Finally, I determined the effect of varying pollination intensity at the level of a single inflorescence, and at the whole plant level. Seed weight was greatest on plants that had only 1 and 5 inflorescences pollinated, and least on those that had 10 and 20 pollinated. At the inflorescence level, seed weights were greatest on those where all flowers were pollinated, compared to inflorescences where only half of the flowers were pollinated. Perhaps the greatest contributor to variance in seed size in this species was the temporal decline within plants through the flowering season. These results indicate that maternal plants are not capable of producing uniform seed crops. Rather, the final distribution of seed size produced by plants within a population will necessarily vary and be the result of pollination effects, heterogeneity in the abiotic environment, and developmental constraints.     

A Developmental Pattern of Flowering in Colored <Emphasis Type="Italic">Zantedeschia</Emphasis> spp.: Effects of Bud Position and Gibberellin

The restricted flowering of colored cultivars ofZantedeschia is a consequence of developmental constraints imposed by apical dominance of the primary bud on secondary buds in the tuber, and by the sympodial growth of individual shoots. GA₃ enhances flowering inZantedeschia by increasing the number of flowering shoots per tuber and inflorescences per shoot. The effects of gibberellin on the pattern of flowering and on the developmental fate of differentiated inflorescences along the tuber axis and individual shoot axes were studied in GA₃ and Uniconazole-treated tubers. Inflorescence primordia and fully developed (emerged) floral stems produced during tuber storage and the plant growth period were recorded. Days to flowering, percent of flowering shoots and floral stem length decreased basipetally along the shoot and tuber axes. GA₃ prolonged the flowering period and increased both the number of flowering shoots per tuber and the differentiated inflorescences per shoot. Activated buds were GA₃ responsive regardless of meristem size or age. Uniconazole did not inhibit inflorescence differentiation but inhibited floral stem elongation. The results suggest that GA₃ has a dual action in the flowering process: induction of inflorescence differentiation and promotion of floral stem elongation. The flowering pattern could be a result of a gradient in the distribution of endogenous factors involved in inflorescence differentialtion (possibly GAs) and in floral stem growth. This gradient along the tuber and shoot axes is probably controlled by apical dominance of the primary bud. Online publication: 7 April 2005     

Performance and impact of the biological control agent Xubida infusella (Lepidoptera; Pyralidae) on the target weed Eichhornia crassipes (waterhyacinth) and on a non-target plant, Pontederia cordata (pickerelweed) in two nutrient regimes

Xubida infusella (Walker) (Lepidoptera: Pyralidae) is potentially a useful biological control agent targeting Eichhornia crassipes (waterhyacinth) in the USA but many regions infested with waterhyacinth are also inhabited by an alternative native host, Pontederia cordata (pickerelweed). Experiments were conducted in Australia to assess the impact of X. infusella on pickerelweed compared to waterhyacinth where both these plants were available and X. infusella had already been released. Overall X. infusella had a greater impact on pickerelweed than on waterhyacinth. More than one larva per plant was required to reduce the total shoot dry weight of waterhyacinth but only one larva per plant reduced the total shoot dry weight of pickerelweed. Insect feeding caused the number of secondary shoots (daughter plants) of pickerelweed to double whereas the number of daughter plants produced by waterhyacinth remained unchanged. We suggest this indicates a considerable impact on pickerelweed rather than effective compensation for insect damage because the shoots produced were very small. Waterhyacinth produced a constant number of daughter plants when fed on by up to three larvae per plant. Higher nitrogen status of both species of host plant increased the rate of larval development and pupal weight of X. infusella. The weight and fecundity of X. infusella reared on pickerelweed were lower than those reared on waterhyacinth but large numbers of progeny were produced on both plant species. This experiment demonstrates a considerable impact of X. infusella on pickerelweed suggesting this plant is at risk from this agent if released in the USA where pickerelweed is present. The considerable impact on waterhyacinth demonstrates the potential for this insect to contribute to waterhyacinth control in countries where risk assessment favours release.     

A rapid in vitro propagation protocol for Piper barberi Gamble, a critically endangered plant

Summary A protocol is described for rapid multiplication of Piper barberi Gamble (Piperaceae) through shoot tip and nodal explant cultures. Nodal explants with a single axillary meristem showed three times better response with respect to shoot proliferation when compared to shoot tip explants. The best shoot proliferation response of nodal explants was observed with a cytokinin combination of N₆-benzyladenine (4.43 μM) and kinetin (2.32 μM), with 88% bud break. The number of shoot initials (2.4) produced per nodal explant was twice the number of shoot initials (1.2) per shoot tip. An average of 6.9±0.58 adventitious shoots were observed from the proximal end of the internodal explants on Mursashige and Skoog (1962) (Ms) basal medium supplemented with N₆-benzyladenine (2.22 μM) and kinetin (0.46 μM). A multiplication rate of 82 shoots per explant could be achieved after 9 wk of subculturing. The in vitro shoots were rooted on one-half and one-quarter MS basal medium. The shoots rooted on one-quarter MS in the dark produced eight roots with an average root length of 3.36 cm and 98% survival. These plants were transferred to the field with a survival rate of 75%.     

Assessment of somaclonal variation in <Emphasis Type="Italic">Dieffenbachia</Emphasis> plants regenerated through indirect shoot organogenesis

The occurrence of somaclonal variation among regenerants derived through indirect shoot organogenesis from leaf explants of three Dieffenbachia cultivars Camouflage, Camille and Star Bright was evaluated. Three types of somaclonal variants (SV1, SV2, and SV3) were identified from regenerated plants of cv. Camouflage, one type from cv. Camille, but none from cv. Star Bright. The three variants had novel and distinct foliar variegation patterns compared to cv. Camouflage parental plants. Additionally, SV1 was taller with a larger canopy and longer leaves than parental plants and SV2. SV2 and SV3 did not produce basal shoots (single stem) but basal shoot numbers between SV1 and parental plants were similar ranging from three to four. The variant type identified from regenerated cv. Camille had lanceolate leaves compared to the oblong leaves of the parent. This variant type also grew taller and had a larger canopy than parental plants. The rates of somaclonal variation were up to 40.4% among regenerated cv. Camouflage plants and 2.6% for regenerated cv. Camille. The duration of callus culture had no effect on somaclonal variation rates of cv. Camouflage as the rates between plants regenerated from 8 months to 16 months of callus culture were similar. The phenotypes of the identified variants were stable as verified by their progenies after cutting propagation. This study demonstrated the potential for new cultivar development by selecting callus-derived somaclonal variants of Dieffenbachia.     

Effects of seed phosphorus concentration on the emergence and growth of subterranean clover (Trifolium subterraneum)

Subterranean clover seed (Trifolium subterraneum cv. Dalkeith) with phosphorus concentrations of 0.75% (high P seed) and 0.48% (low P seed) and of uniform size (2.0–2.4 mm diameter) was used to measure the effect of seed P concentrations on seedling emergence and growth.Seedling emergence numbers were 35% greater for the high P seed, and this effect was independent of external P supply. High P seed also emerged more quickly than low P seed.Leaf emergence was faster and shoot dry weight was greater for seedlings grown from high compared with low P seed, but only when external P supply was deficient for plant growth. Phosphorus concentrations in the shoots of two-week old seedlings were 32–51% higher for high P seed, although by four weeks plants grown from high and low P seed had similar concentrations of P in their shoots. We suggest that establishing pastures using high P seed would improve both early and late season pasture production.     

In vitro plantlet regeneration from cotyledon, hypocotyl and root explants of hybrid seed geranium

In vitro plantlet regeneration systems for the seed geranium (Pelargonium x hortorum Bailey) using cotyledon, hypocotyl and root explants were optimized by studying the influence of seedling age, growth regulators and excision orientation on organogenesis. Indole-3-acetic acid combined with zeatin yielded the highest rate of shoot production on cotyledon explants (0.2–2 shoots per explant). More shoots were produced on explants cut from the most basal region of cotyledons from 2 to 4-day-old seedlings than from older seedlings or more distal cut sites. Hypocotyl explants produced the highest number of shoots, up to 40 shoots per explant, on indole-3-acetic acid (2.8–5.6 mM) + zeatin (4.6 mM) or thidiazuron (4.5 mM). Maximum shoot formation (0.3–1.4 shoots per explant) on root explants occurred when they were cultured on medium containing zeatin. Regenerated shoots rooted best on a basal medium containing no growth regulators. There were substantial differences among cultivars in shoot formation from each of the explant systems.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - TDZ thidiazuron     

A study of some factors affectingAgrobacterium transformation and plant regeneration ofDendranthema grandiflora Tzvelev (syn.Chrysanthemum morifolium Ramat.)

In an attempt to develop a system for producing transformed plants from explants ofDendranthema grandiflora, the susceptibility of the cultivar Super White to various wild-type strains ofAgrobacterium tumefaciens andA. rhizogenes was investigated. Tumour formation was not a reliable indicator of the ability of a related disarmed strain to mediate transformation. Following inoculation of explants with disarmedAgrobacterium strains, a number of shoots developed on selective media. However, none of these shoots were transformed. By co-cultivating stem internode explants with a mixed inoculum of wild-type and disarmed strains, it was possible to obtain a callus stably transformed withAgrobacterium carrying a disarmed T-DNA. Histological analysis of explants revealed that shoot regeneration initially occurred from the cells of the epidermis and subsequently from the cortex. However, the cells which were susceptible to T-DNA transfer were confined to the vascular tissue.     

Somatic embryogenesis inAesculus

Summary Somatic embryogenesis was observed with explants taken from four types ofAesculus tissue: (a) shoots of 4-wk-oldin vitro germinated excised embryos (seed fromA.×arnoldiana), (b) roots of 4-wk-oldin vitro germinated excised embryos (seed fromA.×arnoldiana), (c) shoots from newly forced 3-yr-old seedlings (A. glabra), and (d) newly forced shoots from a 30-yr-old tree (A.×arnoldiana “Autumn Splendor”). Shoots provided three types of explants, single node, shoot apex, and internodal section, and all exhibited embryogenesis. Proembryogenic masses developed in a few cases after 6 wk in culture but were more commonly seen after 3 mo. The yellow, friable proembryogenic masses emerged from proximal cut ends of explants. Almost all cultures that formed embryos possessed leaves, either from developing apical or axillary buds or from adventitious buds, prior to the emergence of proembryogenic masses. Only tissues that had begun to senesce and had been exposed to cytokinin (benzyladenine at 5 or 25 μM) formed somatic embryos. Embryos with distinct cotyledonlike structures and root/shoot axes developed during the 10 to 16 wk following the inital emergence of proembryogenic masses. Enhanced frequency of embryogenesis was obtained by dark culture of root and shoot explants from 4-wk-old germinated embryos (A.×arnoldiana) and by dark and cold (5°C) treatment of shoot tissue cultures derived from 3-yr-old seedlings (A. glabra). Embryogenic potential was greatest in the most juvenile tissue and least in the mature tissue. Five percent of shoot explants taken from the 30-yr-old select treeA.×arnoldiana “Autumn Splendor” produced somatic embryos.