Floral induction (FI) in longan (Dimocarpus longan, Lour.) trees: Part I. Low temperature and potassium chlorate effects on FI and hormonal changes exerted in terminal buds and sub-apical tissue

Floral induction (FI) in longan (Dimocarpus longan, Lour.) trees was achieved in controlled greenhouse experiments by low temperature (LT) and by the application of potassium chlorate at high temperature (HT + KClO₃) during two consecutive seasons. The latter treatment was successful also at temperatures above 20 °C. The present experiment was conducted to investigate possible alterations in the concentrations of plant hormones exerted by these two methods and to relate them to the floral induction process. The following hormones were examined by radioimmunoassay in the shoot apical buds (SAB), and in sub-apical bark and wood: the auxin indole-3-acetic acid (IAA); gibberellins (GAs); and the cytokinins zeatin/zeatin riboside (Z/ZR) and isopentenyl-adenine/isopentenyl-adenosine (iP/iPA).     

Floral induction (FI) in longan (Dimocarpus longan, Lour.) trees. III: Effect of shading the trees on potassium chlorate induced FI and resulting hormonal changes in leaves and shoots

Previous experiments demonstrated that treatment of longan trees with potassium chlorate (KClO₃) induces “off season floral induction” (FI) even in the absence of the naturally required cool temperature [Manochai, P., Sruamsiri, P., Wiriya-alongkorn, W., Naphrom, D., Hegele, M., Bangerth, F., 2005. Year around off season flower induction in longan (Dimocarpus longan, Lour.) trees by KClO₃ applications: potentials and problems. Sci. Hortic. 104, 379–390]. Potassium chlorate, however, cannot replace the presence of functional mature leaves and sufficient light intensity. Here we examined in more detail the effect of shade (about 10% of natural sunlight) on KClO₃ affected hormone concentrations/transport of leaves and shoot apical buds (SAB) and their interactions with FI.     

Floral induction (FI) in longan (Dimocarpus longan, Lour.) trees—The possible participation of endogenous hormones: II. Low temperature and potassium chlorate effects on hormone concentrations in and their export out of leaves

Floral induction in longan (Dimocarpus longan Lour.) trees by low temperature (LT) or potassium chlorate (KClO₃) treatment, is supposed to act, at least partially, by their interference with the hormonal concentration and/or export out of leaves. Therefore the effect of LT and KClO₃ on hormone concentration and transport rate of leaves was investigated in these experiments. Results of experiments below show that LT-, but not KClO₃, treatment considerably increased the leaf export of N⁶ (Δ²-isopentenyl) adenine/N⁶ (Δ²-isopentenyl) adenosine (iP/iPA) together with indole acetic acid (IAA). Export rates of Zeatin/Zeatin riboside (Z/ZR) and gibberellins (GA) were very low and often below the detection limit (<10 ng). Practically no correlation between the concentrations of IAA, and iP/iPA-type CK of leaves and their export rates out of leaves after LT- or KClO₃ treatment were observed. This suggests a specific and independent export rate regulation by these two treatments. Potassium chlorate also strongly increased the production of ethylene in leaves. Whether this burst in ethylene is involved in the FI process, or is merely a stress response of the plant against the herbicide KClO₃, is unknown but could easily be tested by using inhibitors of ethylene biosynthesis/action. The results of these experiments and their effect on hormonal change and FI in the shoot (results of Part I experiments) are discussed.     

氮素形态对平邑甜茶细胞分裂素水平和叶片生长的影响

以平邑甜茶(Malus hupenensis Rehd. ) 实生苗为试材, 在水培条件下研究NO₃^- 和NH₄⁺对植株不同器官细胞分裂素水平及对叶片生长的影响。结果表明: NO₃^- 处理0.5 h后, 根和叶片玉米素及其核苷(Z+ZR) 含量迅速升高, 并且始终高于NH₄⁺处理和对照。茎部玉米素及其核苷含量在NO₃^- 处理1 h后迅速升高, 但之后与NH₄⁺处理及对照差异不显著。就异戊烯腺嘌呤及其核苷(iP+iPA) 而言, 总体上看根中以NO₃^- 处理较高, 茎中各处理差异不大, 叶片中则以NH4+处理较高。NO₃^- 处理与NH₄⁺处理相比, 更能促进叶片生长, 这可能与叶片中较高的玉米素及其核苷含量和较低的ABA含量有关。     

胡萝卜雄性不育系和保持系生理生化特性的比较分析

采用ELISA和分光光度法,测定了新黑田五寸胡萝卜雄性不育系及其保持系不同发育时期花蕾和叶片中IAA、GA3、ABA、Z+ZR、蛋白质、游离脯氨酸、MDA含量以及POD、CAT的活性,讨论分析了生理生化指标与胡萝卜雄性不育的关系。结果表明:花蕾中蛋白质含量不育系明显亏缺,中花蕾、大花蕾和叶片中游离脯氨酸含量不育系显著低于保持系;花蕾中POD、CAT活性和MDA含量不育系高于保持系;花蕾中IAA和ABA积累,Z+ZR和GA3亏缺是胡萝卜雄性不育的特征。中花蕾、大花蕾中IAA/GA3、IAA/ABA、IAA/(Z+ZR)、GA3/ABA、(Z+ZR)/ABA、(Z+ZR)/GA36个内源激素比值均表现为不育系高于保持系。     

KClO3 applications affect Phalaenopsis orchid flowering

Potassium chlorate (KClO₃) treatments are known to promote flowering in longan plants. Potential effects of KClO₃ on Phalaenopsis orchid flowering were investigated in the present study. However, increasing application concentrations of 2, 4, 8 and 16 mmol/L KClO₃ delayed spike emergence by 5, 6, 18 and 26 days, respectively. Moreover, they reduced final spike length by 2.1%, 4.0%, 16.2% and 46.1%, respectively. Nonetheless, application of KClO₃ at 4 and 8 mmol/L advanced the time to appearance of the first open flower by 13 and 24 days, respectively. Use of 8 mmol/L KClO₃ also increased the number of floral buds by 16%. Treatments with KClO₃ tended to reduce flower size. Overall, the data suggest that application of KClO₃ at an appropriate concentration (e.g. 8 mmol/L) can increase the number of floral buds and advance the time to Phalaenopsis orchid flowering, but may reduce flower size.     

荔枝环剥时期对新梢生长及碳素储备的影响

 枝梢环剥显著抑制了‘怀枝’荔枝新梢的生长，并导致射线细胞和髓部大量积累淀粉。梢芽萌发或伸长前环剥可完全抑制新梢生长，而新梢快速伸长期环剥对其抑制效应最弱。秋季环剥对新梢的抑制和对淀粉积累的促进效应明显高于春季环剥。环剥口以上的老叶面积和环剥位置并不影响环剥对新梢生长的抑制效应和促进淀粉的积累效应，但环剥口以上单位枝梢长度的叶面积与淀粉含量呈显著正相关。     

Effects of temperature and daylength on growth and flowering of roselle, Hibiscus sabdariffa L.

Hibiscus sabdariffa L. was found to show an ambiphotoperiodic reaction, flowering both in short days and in extremely long days, but remaining vegetative (at least at 21–25° C) in a 16 h day. Flowering had a dual effect on growth. When floral induction was strong, lateral buds developed into flowers and the number of branches was proportionally reduced. The first stage of floral induction, however, was accompanied by a decrease of apical dominance. Plants shifted to long days after only a short stay at an inductive daylength, and plants moved to short days after a prolonged stay in long days, formed many more branches and a much greater vegetative mass than plants grown in continuous short days or long days.Plants were already sensitive to daylength in the cotyledon stage. Floral induction was not carried over from short days into long days. After marginal induction, floral buds reverted to vegetative shoots, or aborted and were replaced by a shoot from the axil of a bract.In short days, seedlings form shallowly three-lobed leaves until the generative stage, when only entire leaves are formed. In long days, the leaves are deeply five-lobed, also when the plant flowers.     

In vitro morphogenesis and adventitious shoot mass regeneration of Vriesea reitzii from nodular cultures

Micropropagation systems based on nodular cultures (NCs), are considered as an intermediary in vitro morphogenetic route, diverging from regenerative systems based on organogenesis and somatic embryogenesis. The aim of this study was to establish a regenerative protocol based on the induction and development of NCs in Vriesea reitzii, an endangered bromeliad from the Atlantic forest which also has ornamental value. Additionally structural analyses were performed in order to better understand this in vitro morphogenetic route. NCs were regenerated in MSB culture medium free of PGR or supplemented with different levels of NAA alone or in combination with in combination with 2-iP. The subculture of these NCs on MSB medium supplemented with 10 μM of GA₃ promoted the synchronized shoot elongation. A regenerative efficiency of 12.4 g g⁻¹ of NCs was obtained, and this results in 5300 microshoots after 10 weeks in culture. The structural analyses of the NCs revealed that the regenerative process occurs from the proliferation of meristematic cell groups resulting in the development of multiple shoot meristems and buds. The development of NCs leads to the formation of monopolar structures called microshoots, which evolve to elongated shoots. Intermediary features shown in NCs are consistent with their classification as an intermediary system among organogenesis and somatic embryogenesis.     

Auxin-dependent development and habituation of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures

The influence of IAA (1.0 mg dm⁻³), and IBA (1.16 mg dm⁻³), on the development of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures was examined. Depending on the kind of auxin and 2iP concentration in vitro cultures consisted of various number of axillary (AX) and adventitious (AD) shoots. Three different categories of AD shoots were found: leaf shoots (AD-L), node-adjoin shoots (AD-P), and base-adjoin shoots (AD-M, madshoots). The AX shoots were the least habituated (towards auxins, cytokinins and vitamins) whereas the AD-M shoots (madshoots) the most. In comparison to IAA, IBA caused dying or callusing higher number of initial explants. However, IBA generally suppressed development of AD shoots, especially madshoots whereas slightly weakened multiplication of AX shoots. IBA significantly enhanced elongation of AX shoots also. Axillary shoots obtained on IBA-media had relative long internodes and rigid, well-developed leaves. The adventitious shoots, especially base-adjoin (AD-M) ones, were easily distinguishable as were more thin and fragile, more or less vitrified, and had short internodes and smaller, sometimes unfolded leaves. Development of blueberry in vitro cultures on auxin-free and IAA-supplemented media was similar. AX shoots grown on such media resembled AD shoots. 2iP applied in higher doses along with IAA promoted much proliferation of AD than AX shoots. In contrast, 2iP applied in higher doses together with IBA stimulated significantly only growth of AX shoots whereas in general, development of adventitious shoots was not affected. Micropropagation carried out through routine method based on subculturing of shoot explants or shoot clumps on the medium supplemented with IAA (4 mg dm⁻³) and 2iP (10–15 mg dm⁻³) as well as stimulation of shoot elongation on the blank medium causes in fact the propagation of highbush blueberry through highly habituated adventitious madshoots. Replacement of IAA by IBA facilitates micropropagation of highbush blueberry cv. Herbert through axillary shoots.     

In vitro shoot regeneration from stored mature cotyledons of sweet cherry (Prunus avium L.) cultivars

Shoot regeneration was achieved from stored mature cotyledons of sweet cherry (Prunus avium L.) in vitro. The influences of different cytokinins (thidiazuron (TDZ) and benzylaminopurine (BA)) and their levels, the dark incubation for the first 10 days of the culture and TDZ pretreatments on shoot regeneration were determined. All varieties regenerated in the presence of TDZ if cotyledons were maintained in darkness for the first 10 days of the culture; however, only three varieties regenerated with low frequencies in the absence of dark incubation. Dark incubation at the early stage of culture was critical for obtaining higher regeneration efficiencies from stored cherry cotyledons. TDZ was more effective than BA in inducing shoot regeneration. The highest regeneration efficiencies were obtained with intermediate concentrations of TDZ (3.6 and 7.2 μM) in combination with dark incubation and the best regeneration frequencies for ‘Vista’, ‘Sunburst’, ‘Tehranivee’, ‘Vouge’ and ‘Heidelfingen’ cotyledons were 70.0%, 53.3%, 23.3%, 30% and 26.6%, respectively.     

Improvement of caper (Capparis spinosa L.) propagation using in vitro culture and gamma irradiation

Some of the factors influencing the propagation of caper (Capparis spinosa L.) plants in vitro and germination of the seed were studied. The number of adventitious shoots emerging from caper stems cultured in vitro increased from 2.2 shoots per explant when the growth medium contained 2 mg/L of gibberellic acid (GA3) to 5.5 when the growth medium contained 2 mg/L zeatin riboside (ZR) and 1 mg/L naphthalene acetic acid (NAA). The best medium for callus formation from leaf and stem parts contained the growth regulators 1 mg/L 6-benzylaminopurine (BAP) and 0.1 mg/L NAA and the best medium for plant regeneration contained 1 mg/L kinetin and 0.1 mg/L indole-3-acetic acid (IAA). The effect of gamma irradiation on the growth of caper shoots in vitro was also studied. A 10 Gy dose of gamma irradiation stimulated growth of shoots up to 200% and increased shoot rooting percentage from 75 to 100%.     

Nitrate reduces the detrimental effect of potassium chlorate on longan (Dimocarpus Longan Lour.) trees

A recent finding that potassium chlorate induces longan (Dimocarpus Longan Lour.) flowering at any seasons has led to wide use of this chemical in longan industry for off-season harvest, despite the risk of injury to the tree. In this paper, we examined the influences of pretreatment of nitrate by foliar spray with 3% potassium nitrate on the responses of potted longan (Dimocarpus longan Lour.) cv. Shixia (a subtropical ecotype) trees to soil applications of potassium chlorate at 10 and 20 g plant⁻¹. Chlorate treatments caused severe leaf drop and chlorophyll breakdown and suppressed budbreak, flowering and shoot growth. Pretreatment of potassium nitrate reduced the severity of leaf drop and chlorophyll breakdown caused by chlorate but the extend was less significant in high dosage chlorate (20 g plant⁻¹) treatment than low dosage (10 g plant⁻¹) chlorate treatment. Potassium nitrate did not alleviate the suppression effect of chlorate on budbreak, flowering and shoot growth, although it promoted these processes per se. Chlorate treatment resulted in only a transient accumulation of chlorate in the leaves, which peaked around 14 days after chlorate treatment (DACT), while chloride accumulated constantly within 28 DACT. Pretreatment of potassium nitrate tended to promote chlorate accumulation but did not cause a significant change in chloride accumulation. Severity of leaf drop was not significantly correlated to chlorate accumulation but significantly to chloride accumulation, suggesting that toxicity of chlorate to longan tree was realized during the chloride-producing process of chlorate reduction. Nitrate influenced the correlations between severity of leaf drop and chloride accumulation by reducing the slopes of the regressed lines of leaf drop versus chloride, indicating that nitrate reduced the sensitivity of longan trees to the toxic intermediates produced during chlorate reduction. The results indicate that the application rates were excessive for potted trees. The potential benefit of potassium nitrate in a longan production system based on application of chlorate needs to be evaluated in field trees.     

In vitro propagation of wavy-leaved Indian paintbrush (Castilleja applegatei Fern.)

Castilleja spp. (Indian paintbrush, Orobanchaceae) are desirable ornamental plants with showy floral bracts that are native throughout much of the western U.S. Propagation of these hemiparasites by seed is usually successful only when a host species is present, and asexual propagation through traditional methods has proven to be extremely difficult. In this study we present an effective shoot culture micropropagation system for Castilleja applegatei Fern., the wavy-leaved Indian paintbrush. In vitro shoot tips of three C. applegatei clones were cultured for 28 days on woody plant medium (WPM) supplemented with four concentrations of each of three cytokinins: 6-benzylaminopurine (BA), 6-(y,y-dimethylallylamino)-purine (2iP) and zeatin. Responding explants, shoot number per responding explant and shoot length were determined. In vitro rooting of microcuttings cultured for 42 days on media containing three concentrations of indole-3-butyric acid (IBA) were also evaluated. A high percent response to shoot induction was observed across all treatments, ranging from 94.5 to 100%. C. applegatei responded best to zeatin, which resulted in both the highest mean shoot number and mean shoot length among the cytokinins tested. The best overall shoot multiplication occurred on media with 4.0 μM zeatin, yielding a mean shoot number of 4.11 and mean shoot length of 3.95 cm. The mean highest rooting response (66.7%), root number (13.21) and root length (2.73 cm) were obtained on WPM supplemented with 10 μM IBA. Significant clone × treatment interactions existed for all variables except mean root number, thus the optimum treatments for both shoot multiplication and root induction were clone-dependent. Rooted microcuttings were acclimated ex vitro with an average success rate of 81.2%. This study demonstrates the considerable potential of an in vitro shoot culture system for the asexual propagation of Castilleja spp.     

Plant regeneration of pansy (Viola wittrockiana) ‘Caidie’ via petiole-derived callus

An in vitro plant regeneration protocol for pansy (Viola wittrockiana) cultivar ‘Caidie’ from petioles was established as following: callus induction on a half-strength MS medium supplemented with 0.45 μmol l⁻¹ 2,4-d plus 8.9 μmol l⁻¹ BA, callus subculture on medium F (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 0.44 μmol l⁻¹ BA) and then on medium T (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 2.2 μmol l⁻¹ BA), shoot regeneration on medium D3 (MS media supplemented with 2.9 μmol l⁻¹GA₃, 23.6 μmol l⁻¹ AgNO₃, 0.02% active charcoal and 4.5 μmol l⁻¹ TDZ), shoot multiplication on medium M (half-strength MS medium containing NAA 1.1 μmol l⁻¹, TDZ 9.1 μmol l⁻¹ and GA₃ 8.7 μmol l⁻¹), and then shoot elongation and rooting on medium R (MS medium supplemented with 1.1 μmol l⁻¹ NAA and 1.1 μmol l⁻¹ BA). Subculture on appropriate medium was found to be important for successful shoot regeneration.     

High-frequency embryogenesis,regeneration of broccoli (Brassica oleracea var. italica) and analysis of genetic stability by RAPD

High-frequency somatic embryogenesis and shoot regeneration of broccoli (Brassica oleracea var. italica) were achieved. Cotyledon and hypocotyl explants from four varieties of broccoli were cultured on MS and modified MS media (mMS, supplemented with PG-96 organic components) with different combinations of growth regulator. The effects of genotypes, different explants, growth regulator combinations, organic components and AgNO₃ on induction of calli and shoots were evaluated. The optimal media for inducting calli/shoots and roots were mMS medium containing 3% (w/v) sucrose and 0.8% (w/v) agar supplemented with NAA at 0.5 mg l⁻¹, 6-BA at 3.0 mg l⁻¹, AgNO₃ at 4.0 mg l⁻¹ and MS medium containing 3% sucrose and 0.8% (w/v) agar supplemented with NAA at 0.2 mg l⁻¹, respectively. The callus induction percentages were over 90% in all four varieties; shoot induction percentage was 92.5% and the average number of shoot per explant was 4.1 from cotyledon explant in variety Bishan. In this study, we established high-efficient embryogenesis and shoot regeneration system of broccoli and analyzed genetic stability of regenerants at DNA level using RAPD molecular marker. Out of 62 arbitrary primers screened using PCR amplification, 79 polymorphic bands were amplified from 20 primers. The results demonstrated the genetic stability of regenerants from the same variety.     

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

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

Regeneration from leaf explants and protoplasts of Brassica oleracea var. botrytis (cauliflower)

Adventitious shoot regeneration and protoplast isolation and culture were examined from leaf explants of in vitro shoot cultures of several cauliflower (Brassica oleracea var. botrytis) cultivars, sourced from Europe and Australia, was investigated with the aim to develop improved nuclear and plastid transformation protocols for this vegetable crop. Eight out of 10 cultivars regenerated shoots from at least 79% of leaf explants. Mesophyll protoplasts from leaves gave high yields and division frequencies. Growth of shoot cultures in large glass vessels with vented lids was the key factor in obtaining high protoplast division frequencies of up to 71% and at least 70% of protoplast calluses regenerating shoots.     

The number of leaves required for floral induction and translocation of the florigenic promoter in mango (Mangifera indica L.) in a tropical climate

Mango flowering appears to be determined by a temperature-regulated florigenic promoter (FP) synthesized in leaves and translocated to buds in phloem. The number of leaves required for flowering was investigated in ‘Keitt’ and ‘Tommy Atkins’ mango trees exposed to tropical conditions in Colombia. Data were compared with a previous study conducted under cool, floral-inductive conditions in Florida (Davenport et al., 2006). Leaf-number treatments consisted of 0, 1/8, 1/4, 1/2, 1, 2, 3, or 4 leaves on each of 20–40 stems per branch. The long distance translocation experiment consisted of a terminal donor stem and five defoliated receiver stems on each treatment branch. Treatments were 0, 1, 3, or 5 leaves on the donor stem. Every treatment branch in both experiments was girdled to isolate it from the rest of the tree, and developing panicles were removed to stimulate lateral shoot initiation at a time when conditions were right for floral induction in those buds. Treatment stems bearing no leaves produced only vegetative shoots in both cultivars in both experiments. The minimum number of leaves per stem (1/8 of a leaf and 1/4 of a leaf) was sufficient to induce 6% and 1% reproductive shoots with 94% and 99% vegetative shoots in Tommy Atkins’ and ‘Keitt’ stems, respectively. Branches bearing 4 leaves per stem in ‘Tommy Atkins’ trees produced the maximum mean flowering response with 45% reproductive shoots and 55% vegetative shoots. The maximum ‘Keitt’ response was 22% reproductive and 78% vegetative shoots with 4 leaves per stem. The donor stem of the 1-leaf treatment and the donor and first receiver stem of the 3-leaf treatment in the translocation experiment were induced to flower in ‘Tommy Atkins’ trees. The more distal receiver stems from the donor were vegetative. The 5-leaf donor treatment-induced reproductive shoots as far as the third leafless receiver stem located 52 cm from the donor. Proportions of flowering shoots decreased with distance from the donor, and the level of the FP was apparently insufficient to reach the fourth and fifth receivers since their shoot responses were vegetative. Only vegetative shoot responses were obtained in the translocation experiment conducted in ‘Keitt’ trees. The leaf number and translocation experiment results support the hypothesis that far less FP is synthesized in both ‘Tommy Atkins’ and ‘Keitt’ leaves during warm, tropical conditions than is synthesized in ‘Keitt’ leaves exposed to cool, subtropical conditions.     

Clonal propagation of Rosa clinophylla Thory. through axillary bud culture

A protocol for clonal propagation of Rosa clinophylla, a rare and endangered species but very important for breeding purposes had been standardized through in vitro axillary bud culture. Although cytokinins alone were able to induce shoot buds, but their proper growth and number could be increased only when they were used in combination with GA₃. However, there was shoot tip necrosis and leaf fall in the proliferated shoots. AgNO₃ at 58.85 μM proved effective to avoid shoot necrosis and yellowing of leaves. Activated charcoal (AC) at 250 mg l⁻¹ was found necessary at all the stages of shoot multiplication as well as rooting. Ninety percent rooting could be achieved in 1/2 MS medium supplemented with 4.92 μM IBA and 250 mg l⁻¹ AC. Rooted plantlets were hardened and transferred to the field successfully with 80% survival rate.