Cytofluorometric measurements of nuclear DNA in adventitious buds and shoots of Picea abies regenerated in vitro

Nuclear DNA content of adventitious buds and shoots of Picea abies (L.) Karst. developed in vitro was compared to that of buds collected from field-grown trees. Protoplasts were isolated from the different tissues and after fixation and staining with ethidium bromide the DNA content of their interphase nuclei was determined cytofluorometrically. The DNA pattern of the different tissues was within the same range and had the same distribution with one main peak.     

美国黄松成熟胚的离体培养与不定芽的形成

以美国黄松成熟胚为外植体进行离体培养诱导不定芽。正交试验和统计分析结果表明基本培养基的种类对外植体不定芽的诱导起主要作用。在GD培养基上附加0.5mg/L的6－BA,外植体不定芽的诱导率达55％,平均增值率为6,最大增值率达10。NAA不利于外植体不定芽的诱导,培养基中加入适量的添生炭有利于不定芽的形成和生长。     

In vitro adventitious root induction in Antirrhinum majus L.

A broadly applicable method for the successful induction of root systems in a number of cultivars of A. majus has been determined. This involves a double filter-paper bridge with a liquid medium for root induction and allows the transfer of culture-grown plantlets to a glasshouse environment with minimal disturbance to the plant as a whole. 100% survival of transferred plantlets has been achieved with the inclusion of a few simple precautions upon shoot transfer and during the initial stages of plant establishment in vivo.     

Uptake of benzyladenine in explants of Picea abies and Pinus sylvestris

Primary explants of Picea abies (L.) Karst. and Pinus sylvestris L. were incubated for various periods in solutions containing different concentrations of [¹⁴C]-benzyladenine. Time course analysis showed that uptake was linear for the initial 60 min; after this time linear uptake continued but at a much reduced rate. The amount of benzyladenine taken up by the explants saturated at a concentration about one third of that of the medium, Concentration dependence experiments showed that BA uptake was directly proportional to the external concentration. These results and those from experiments in which uptake was examined at different temperatures are consistent with a passive mode of BA uptake. The results are discussed with respect to in vitro micropropagation.     

Histological study of in vitro development of adventitious buds on leaf explants of oriental beech (Fagus orientalis lipsky)

Summary Adventitious shoots were induced on the proximal portion of leaves excised from Fagus orientalis shoot cultures derived from a 2-mo.-old or a 4-yr-old seedling. Up to 90% of the explants formed between 13 and 19 buds after culture on Woody Plant Medium containing 2.9 μM indole-3-acetic acid and 4.5 μM thidiazuron. Adventitious buds developed mostly on petiole stub callus, but also on the midvein. The histological events leading to shoot organogenesis were examined. Some shoots developed directly from subepidermis or epidermis, but most originated indirectly from cell file proliferation produced by periclinally dividing cells subadjacent to the epidermis. Some cells in the outermost layers of these files became meristematic and divided extensively, resulting in the formation of meristemoids after 16 d of culture. Dedifferentiation into meristematic cells, which exhibited a large, prominent nucleus, densely-stained cytoplasm, and a high nucleus-to-cell area ratio, was generally associated with anticlinal divisions in cells previously originated by periclinal division. Subepidermal cell proliferation occurred mainly in the abaxial surface of the explant, which initially formed a diffuse cambium and later evolved to a phellogenic cambium. Some meristemoids were also differentiated by lenticel phellogen. Organized cell divisions in meristemoids gave rise to bud primordia that emerged from the explant surface and differentiated a protoderm. The progressive structural differentiation of the apical meristem, leaf primordia, and procambial strands led, after about 28 d of culture, to shoots with vascular connections with treachery elements previously differentiated in adjacent tissues.     

In vitro phyllomorphic regeneration of shoot buds and shoots in Picea abies

Needles (10–15 mm) of frost-hardened 20–22-week-old (physiological age equivalent to 1 year) plants of Picea abies L. excised just after flushing, were induced to form adventitious shoot buds and shoots on media supplemented with BAP (6-benzylaminopurine) and NAA (1-napthaleneacetic acid). The addition of nanomolar concentrations (0.5–50) of NAA combined with 1–10 μM BAP considerably stimulated formation of pseudobulbils on the basal to mid-part of the needle axis, as well as their subsequent development into shoot buds and shoots. On a medium containing 10 μM BAP, pseudobulbils that formed at the needle base did not develop further, but became necrotic and died with the omission of NAA. With 5 μM BAP + 50 nM NAA the initial phase of development was slow, but later showed good response and up to 22% of the needles produced shoot buds. Two to three shoots per needle could be excised and subcultured individually onto fresh media. It is concluded that the level of endogenous auxin decreases progressively from the needle's base to its tip, so that that concentration of exogenous auxin (50 nM NAA) which promotes pseudobulbil and shoot-bud formation part-way along the needle axis, simultaneously inhibits their induction at the needle base.     

Effect of rigidity of gel medium on benzyladenine-induced adventitious bud formation and vitrification in vitro in Picea abies

Observations on the effects of different degrees of rigidity of both an agar (Tayio) and a non-agar (Gelrite) gel on the uptake of radiolabelled N⁶-benzyladenine (¹⁴C-BA) were also extended to mode of application and positioning of the explant. Regression analysis showed a highly significant inverse correlation between ¹⁴C-BA accumulation and degree of gel stiffness. Significantly greater numbers of adventitious buds per explant were induced at low to medium levels of rigidity (2.5–10 g Tayio 1⁻¹, 1–5 g Gelrite 1⁻¹); this advantage was almost completely nullified at the lower levels (2.5 and 5.0 g Tayio 1⁻¹, 1 and 1.5 g Gelrite 1⁻¹) as a result of the high incidence of vitrification. In addition to turgor distension, vitrified buds displayed cellular damage. Explants with their cotyledons flattened onto the agar surface accumulated less ¹⁴C-BA after 96 h than upright explants, but produced greater numbers of adventitious buds, pseudobuds and phylloids. It was suggested that BA was taken up only by "target" cells, presumably the differentiating subsidiary cells of those stomatal complexes in surface contact with the medium. Pulse treatments of relatively short durations (2 h) with optimal concentrations of BA (ca 125 μ M ), followed by subculturing on hormone-free media gelled with 10 g agar 1⁻¹, produced a satisfactory balance between yield and competence of adventitiously-induced buds.     

In vitro control of adventitious bud differentiation by inorganic medium components and silver thiosulfate in explants of Passiflora edulis F. flavicarpa

Summary Hypocotyl and leaf explants from Passiflora edulis F. flavicarpa were evaluated for morphogenesis when cultured on several nutrient media supplemented with benzyladenine and indoleacetic acid. The effect of silver thiosulfate on growth-regulator-induced morphogenesis was also investigated. Murashige and Skoog medium was more effective than woody plant medium in promoting adventitious bud differentiation. The omission of ammonium or nitrate from the Murashige and Skoog medium and a disequilibrium from the Murashige and Skoog nitrate: ammonium ratio drastically reduced the bud-forming capacity of the explants. The inclusion of silver thiosulfate in the culture medium significantly increased the differentiation and development of adventitious shoots. Regenerated shoots were excised and induced to root on basal Murashige and Skoog medium. Plants were transplanted to pots and grown ex vitro.     

Initial stages in the course of adventitious bud formation on embryos of Picea abies

Adventitious buds on embryos of Picea abies (L.) Karst. developed after a pulse treatment with 250 μ M benzyladenine (BA) of pH 5.5 for 2 h. Light and temperature regimes were not critical during the initial stages. Adventitious buds developed faster after a pulse treatment and the variation among different experiments was lower compared to when the embryos were cultured on media supplemented with BA. Various stages of the differentiation of adventitious buds were identified: stage 1 - appearance of meristematic centres (approximately the first two weeks); stage 2 - development of adventitious bud primordia (approximately the third week); stage 3 - adventitious bud development (from approximately the 4th to the 8th week). This system may be used for further studies on bud differentiation.     

In vitro adventitious bud regeneration from internode segments of beech

Internode explants collected from in vitro grown shoots of two clones of Fagus sylvatica L. (European beech) and five clones of F. orientalis Lipski (Oriental beech) were used to evaluate their bud regeneration capacity. Adventitious shoot-buds formed on callus, which developed from internode segments cultured in a Woody Plant Medium supplemented with different concentrations of either thidiazuron (TDZ) or benzyladenine (BA). After 4 weeks of culture on induction media, the explants were transferred to a proliferation medium supplemented with 2.2 μM BA, 9.1 μM zeatin and 2.9 μM indole-3-acetic acid (IAA) for another 8 weeks. Medium containing TDZ was much more efficient than medium containing BA in inducing adventitious buds, the optimal TDZ concentration being 4.5 μM and the optimal BA concentration 17.8 μM. Genotypic variation in shoot regeneration capacity was observed among the two Fagus species and between clones within each species, with a significant interaction between TDZ concentration and genotype regarding mean bud number. Thidiazuron induction medium supplemented with a range of individual auxins was investigated, and it was found that IAA or indole-3-butyric acid at 2.9 μM enhanced the bud forming capacity of explants. Morphogenic response varied significantly with the position of the internode along the stem. The highest regeneration potential was obtained from apical internodes, while those distal to the apex were the least productive. Elongated shoots of adventitious origin can be readily proliferated by axillary branching. This revised version was published online in June 2006 with corrections to the Cover Date.     

空心莲子草根中异常结构及不定芽的发育解剖学研究

运用光镜和扫描电镜的方法 ,观察了空心莲子草根的结构及不定芽的结构 ,结果表明 :根的初生木质部为二原型、三原型和四原型 ;初生结构和早期次生结构正常 ,异常的次生生长是由于次生韧皮部外侧发生的额外形成层所致 ,后一轮额外形成层起源于前一轮向外产生的薄壁细胞 ,结合组织为发达的薄壁细胞 ,二至四轮三生维管束排成整齐的同心环类型 ,不定芽主要起源于异常根的额外形成层 ,芽外有二至三轮鳞片 ,其内着生许多毛状物。     

In vitro adventitious shoot formation from embryonic and cotyledonary tissues of Pinus brutia Ten.

Adventitious buds were induced when isolated whole embryos, and excised cotyledons from treated seeds of Calabrian pine (Pinus brutia Ten.) were cultured on a cytokinin-supplemented medium. The adventitious buds formed directly from the cotyledons. The highest number of bud primordia were formed, with both embryos and excised cotyledons, after 6 weeks on a BAP — supplemented Schenk and Hildebrandt medium under a 16 h photoperiod. The buds, when separated and maintained individually on a full-strength medium without growth regulators, developed into well-formed shoots within 4 weeks. The average number of harvested shoots obtained (>1 cm in height) per seed over 24 weeks was 55; however, a maximum number of 152 shoots was obtained from one individual over the same period. The shoot forming capacity of the meristematic tissue was not lost after seven harvests.     

广藿香扦插繁殖不定根形成的解剖学研究

利用常规石蜡切片法,系统研究了广藿香不定根的起源和发育。观察结果表明：广藿香茎中没有先成根原始体,其不定根属于诱生根类型,起源于靠近维管形成层的初生韧皮部薄壁细胞中,愈伤组织内无不定根生成。本研究揭示了广藿香扦插生根的解剖学特征,从而为该药材在生产中提高扦插育苗成活率提供理论依据和实践指导。     

In vitro adventitious shoot regeneration from leaves of Prunus spp.

A high level of adventitious shoot regeneration was obtained from proliferating shoots in vitro for a range of Prunus spp. There was a significant variability in clone response to a range of adventitious shoot regeneration treatments. Treatment of apricot clone H.152 with Quoirin macroelements (C.R. Rech., Stu. Cult. Fruit. Maraîchères Gemblaux (1977) 93–117), and both apricot clone H.146 and hybrid plum clone P.1869 with half-strength Murashige and Skoog medium, consistently induced regeneration. Thidiazuron (TDZ) alone, or in combination with naptthaleneacetic acid (NAA), was most effective in stimulating adventitious shoot production, the optimum concentration being clone-dependent. Addition of silver nitrate (AgNO₃) to regeneration media enhanced regeneration by 10–40% and reduced the variability between experiments. Regeneration with AgNO₃ was obtained also for three other plum clones belonging to the P. marianna, P. domestica and P. insititia species.     

In vitro rooting of micropropagated shoots from juvenile and mature Pinus pinaster explants: influence of activated charcoal

The ability for adventitious rooting of micropropagated shoots from juvenile and mature Pinus pinaster Ait. explants was assessed in vitro on a rooting expression medium. The different rooting traits observed, namely the rooting rate, the number and the length of the adventitious roots, and the root score, were greatly influenced by the age of the donor plant: 98% of juvenile explants rooted, while only 49% of mature explants did. Addition of activated charcoal in the rooting expression medium improved the overall rooting capacity of the mature explants to an average of 78%. Whatever the plant material, the number and the length of the adventitious roots, as well as the root score, fluctuated according to the sampling date.Abbreviations BA 6-benzyladenine - NAA naphthaleneacetic acid - REM rooting expression medium - RIM rooting induction medium     

Activity of pectin esterase and cellulase in the abscission zone of citrus leaf explants

The activity of pectin esterase and cellulase in abscission of citrus explants was studied. No relation was established between pectin esterase and abscission, while cellulase activity was markedly increased before abscission and for a certain period after excision. IAA and cycloheximide delay abscission and cellulase activity, while ethylene and, to a lesser extent, GA₃ accelerate them. Application of cycloheximide during the lag period and before cellulase activity can be measured, inhibits to a certain extent the formation of cellulase. An escape from the inhibitory effect of cycloheximide is detected when inhibitor is supplied at the end of the lag period.     

In vitro regeneration of sesame (Sesamum indicum L.) from seedling cotyledon and hypocotyl explants

The goal of this study was to develop an efficient regeneration protocol to be used for genetic transformation of sesame. Published regeneration methods using benzyladenine (BA) and 1-naphthalene acetic acid (NAA) were unsuccessful for the cultivars used herein. Experiments were carried out using cotyledon and hypocotyl explants from the cultivar Mtwara-2. Later the optimised culture conditions were used to investigate the regeneration response of different genotypes. There was significant interaction between hormone treatments and macronutrients for shoot and root regeneration. Results also showed that shoot regeneration was significantly influenced by explant type. Shoots were only obtained from cotyledons whereas both cotyledons and hypocotyls could produce roots. Modified Murashige and Skoog (MS) medium with N6 macronutrients resulted in twice the shoot regeneration frequency obtained with ½MS macronutrients in the presence of thidiazuron (TDZ). The shoot regeneration frequency was significantly reduced when BA was used in place of TDZ. On shoot regeneration medium containing BA and NAA, only roots were formed. Replacing NAA with indole-3-acetic acid (IAA) greatly improved the regeneration of shoots. The optimum growth regulator combination for shoot regeneration was 20 μM TDZ together with 2.5 μM IAA, which gave a frequency of 63% and 4.4 shoots per regenerating explant for the best cultivar Ex-El. Genotypic differences were significant both for the number of explants regenerating shoots and the number of shoots produced per regenerating explant.     

Abscission of citrus leaf explants: no correlation with naphthaleneacetic Acid conjugation in the abscission zone

The role of α-naphthaleneacetic acid (NAA) in the control of abscission in Citrus (Citrus sinensis L. Osbeck) leaf explants and its conjugation were studied in non-aged and 24-hour-aged explants. Dipping non-aged explants in 1.5 micromolar NAA for 15 minutes immediately after excision did not delay abscission whereas 150 micromolar NAA effectively delayed it. As incubation time was prolonged up to 24 hours after excision, the delaying effect of both concentrations gradually increased. In general, both concentrations did not delay abscission when applied to 24-hour-aged explants held for an additional period of up to 24 hours. The uptake and conjugation of ¹⁴C-NAA to glucose and aspartic acid were similar in petiole, abscission zone, and leaf blade of non-aged and aged tissues, for all NAA concentrations. No correlation was established between the kinetics of abscission and the rate of conjugation in the abscission zone.     

In vitro regeneration of adventitious buds from leaf explants and their subsequent cryopreservation in highbush blueberry

Agarwood is the most valuable resinous wood used in the production of products such as incenses, perfumes, and traditional medicines. However, such production has led to the destruction of wild Aquilaria forests. In the current study, an in vitro culture system was established by combining Aquilaria malaccensis with a quasi-symbiotic bacteria strain, A329, cultured on a medium designed for agarwood production. The quasi-symbiotic bacteria A329 derived from A. malaccensis agarwood has Bacillus-like characteristics, is low in phytopathogens, and can coculture with A. malaccensis with the current designed culture medium. The prolonged culture induced agarwood production at the base of A. malaccensis in vitro. Using high solid-phase microextraction gas chromatography-mass spectrometry, 23 compounds were tentatively identified, including several fingerprinting compounds, agarospirol, eudesmanes, guaianes, valencanes, selinenes, aristolone and 2-(2-phenylethyl) chromone. Using the in vitro system established in this study, the duration of agarwood resin formation can be shortened to 3 months in a well-controlled environment with 100% yield, constituting a promising platform with high commercial value for the selection of Aquilaria spp., the quasi-symbiotic microbes, and the combination of the two.     

离体黄瓜子叶不定根发生的研究

离体黄瓜子叶在仅加有双蒸水的培养皿中能高频率发生不定根的现象,并对不定根发生过程中过氧化物酶（ＰＯＤ）活性、可溶性蛋白和核酸含量的变化进行了测定。：６ｄ苗子叶不定根发生率最高,不定根发生高峰是在培养后３ｄ～５ｄ,在不定根发生过程中,过氧化物酶生呈上升趋势,可溶性蛋白和核酸含蜈则降低。