Studies on Stem Tissue Culture and Organgenesis of Aloe vera

tem segments of Aloe vera were cultured on MS medium with different hormones and successfully regenerated into a large number of plantlets via callus-globoid-adventitious bud. In application of various cytokinins, the effect of zeatin on organgenesis was better than kinetm and the enhanced effect was apparent when NAA was combined with zeatin or kinetin on MS medium. The best results were obtained on the medium with zeatin 2ppm+NAA 0.5ppm. With scanning electron microscope and histocytological method the observation showed that the globoid which is a cell mass with hollow on the top originated from the surface of callus. After differentiation of scale primordium twice, the primordium of adventitious bud was formed between scale primordia. Finally, the adventitious bud was developed into plantlet. Therefore the globid can be considered as a proliferation unit producing adventitious bud.     

Light cytokinin interactions in shoot formation in epicotyl cuttings of Troyer citrange cultured in vitro

Ilumination did not affect the pathway of shoot regeneration at the cut edges of epicotyl explants of Troyer citrange (Moreira-Dias et al. 2000, 2001), but signigficantly affected the number of developed shoots and the response to exogenous cytokinins. Shoot regeneration at the apical end occurred through a direct organogenic pathway without callus formation. For explants incubated in the light, this regeneration did not require cytokinin addendum, but the number of shoots formed was significantly increased by benzyl adenine, but not by zeatin or kinetin. Incubation in the dark almost suppressed shoot formation at the apical end. The addition of benzyl adenine or kinetin, but not of zeatin, restored shoot formation in the dark to the value obtained in the light. At the basal end of the explants shoot regeneration occurred through an indirect organogenic pathway after the formation of a primary callus. In explants incubated in the light, callus formation and shoot growth was supported by a low (0.5–1 mg l⁻¹) benzyl adenine concentration and by zeatin. Kinetin did not support callus growth. Shoot formation was higher in the presence of benzyl adenine (0.5–1 mg l⁻¹) than of zeatin, but was inhibited by a high (5 mg l⁻¹) benzyl adenine concentration. Incubation in the dark increased callus growth and shoot formation at the basal cut as compared to explants incubated in the light. The three cytokinins tested supported callus growth and shoot formation in the dark, zeatin being the most effective and kinetin the least. In terms of number of shoots developed, the optimum cytokinin addendum depended on the pathway of organogenesis and the conditions of incubation. The maximum number of shoots developed at the apical end was obtained when the incubation was performed in the light in the presence of benzyl adenine. At the basal end, the optimal conditions were incubation in the dark in the presence of zeatin. It was not always possible to define an optimal cytokinin concentration as the curve concentration/response varied from experiment to experiment, which seemed unrelated to the endogenous cytokinin concentration in the explants.     

Alkaloid production in Catharanthus roseus cell cultures

Callus derived from hypocotyls of periwinkle, Catharanthus roseus, responded to culture on nutrient media supplementedwith IAA, BA, and zeatin with shoot formation at low frequencies. However, shoot regenerating callus could be very successfully propagated and subcultured. Alkaloid profiles of callus derived from the original explants (hypocotyls) as well as callus derived from regenerated shoots were almost identical. Subcultures of old callus (initiated in 1978) failed completely to grow shoots. In programs for long-term preservation of alkaloid producing cell lines by regeneration and storage of shoots, selection for ability to form shoots would have to precede selection for alkaloid production.Abbreviations IAA indolyl-3-acetic acid - IIAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzyladenine NRCC No. 20087     

Factors affecting in vitro shoot formation from vegetative shoot apices of apple and relationship between organogenic response and cytokinin localisation

ABSTRACT

The effects of macro- and micro-elements, benzyladenine (BA) concentration, and the period of auxin application on adventitious shoot formation from callus originating from vegetative shoot apices were tested on apple (Malus domestica Borkh) rootstock Jork 9. The putative relationship between organogenic response and cytokinin localisation was also studied by an immunolocalisation technique for in situ determination of free cytokinins. The use of MS (Murashige & Skoog, 1962) salts in the medium instead of those of LP (Quoirin & Lepoivre, 1977) had a strong positive effect both on shoot formation rate and on the number of shoots produced. The highest organogenic response from callus was induced using 17.8 μM BA in the presence of 2.7 μM NAA and by maintaining the explants for 20 days in darkness, then transferring them to fresh auxin-free medium and to the light. The in situ localisation studies, performed using antibodies with a marked specificity against zeatin and isopentenyladenine, revealed changes in the localisation of free zeatin in the tissues during the shoot-forming process, in particular during the active cell division phase leading to callus formation, and in the initial phase of bud formation. Changes in zeatin distribution in the tissues of the vegetative shoot apex during shoot formation may indicate a role for this cytokinin free base in cell differentiation and organogenesis.     

金塔柏扦插不定根形成与内源激素的调控研究

金塔柏（Platycladus orientalis ‘Beverleyensis’）是重要的观赏树种。生长素（IAA）、玉米素（ZT）、脱落酸（ABA）和茉莉酸（JA）在金塔柏扦插不定根再生过程中起着重要的调控作用,但不同发育阶段内源激素的动态变化及其对不定根发生的影响仍不清楚。以金塔柏半木质化枝条为材料,采用连续组织切片技术观察了不定根发生过程,利用高效液相色谱串联质谱法检测了4种内源激素含量的动态变化。结果表明,金塔柏不定根原基起源于愈伤组织、髓射线、木质部、维管形成层、次生韧皮部、皮层、髓射线与形成层交界处等部位,属于多位点发生模式和多类型生根方式。在不定根形成过程中,随着愈伤组织的形成,IAA和ZT含量下降,ABA和JA含量升高;随着根原基的分化,IAA和ZT含量缓慢升高,ABA和JA含量下降;随着不定根形成与伸长,IAA、ZT、JA逐渐升高,ABA维持在低水平。激素平衡分析发现,IAA/ABA比值和IAA/JA比值下降、IAA/ZT比值上升利于愈伤组织的形成,反之利于根原基的诱导分化,而IAA/ABA比值升高,IAA/ZT和IAA/JA维持在较低水平利于不定根形成与伸长。研究结果为揭示不同内源激素对金塔柏扦插不定根再生的调节作用提供了依据。     

Organogenesis from internode-derived nodules of Humulus lupulus var. Nugget (Cannabinaceae): histological studies and changes in the starch content

The sequence of histological and histochemical events occurring during organogenesis from Humulus lupulus var. Nugget internode-derived nodules was studied. Sections were made and studies were carried out from the start of culture treatment until the development of shoot buds. Cell division was observed in both cambial and cortical regions during the first week of culture establishment. Cell division in cortical cells led to the formation of an incipient callus tissue. From the calluses prenodular structures of cambial origin appeared and gave rise to nodules from which shoot buds formed. Nodules kept separating into "daughter nodules" from which arose an increasing number of shoot buds. Iodide staining showed a strong starch accumulation in callus tissue and in prenodular structures. During shoot-bud primordia formation starch content decreased in nodules. Some starch was also noted in control explants (cultured on basal medium), however at a lower level than that observed in explants cultured on media with growth regulators. Shoot-bud regeneration was not observed in control explants.     

Expression of a cytokinin synthesis gene from Agrobacterium tumefaciens T-DNA in basket willow (Salix viminalis)

Willow cells transformed with an ipt gene from Agrobacterium tumefaciens grow in tissue culture as undifferentiated callus without shoot induction. We show that the transformed calluses contained high levels of the cytokinins 9-β- d -ribofuranosyl zeatin and its monophosphate, demonstrating the presence of a functional isopentenyl transferase enzyme. The ipt gene was transcribed at different levels in different transformed callus lines. The absence of shoot differntiation is apparently not due to a lack of zeatin-type cytokinins in the transformed callus.     

Morpho-Histological Investigation of Plantlet Formation In Vitro in Taiwania flousiana Gaussen

Histological events during adventitious shoot formation in cultured shoot apex of 10–12-day-old seedlings and adventitious root formation in the elongated shoot of Taiwania floudana Gaussen were examined. Ceils of the peripheral subsurface layers of the shoot apex responded to cytokinin and divided into meristematic cells from which the shoot primordia were proliferated. A few bud primordia also originated from the epidermis and hypodermis of the adaxial surface of the cotyledon. The parenchyma of leaf gap of the shoots cultured in rooting medium dedifferentiated to regain the capacity of division and form adventitious root. Besides, cells that had relatively low potential of differentiation, such as the cortex parenchyma, pith ray, phloem parenchyma and cambium zone, albeit initiated to divide, but seldom formed root primordium. The origin of the adventitious roots in the leaf gap facilitated the establishment of the vascular connection between the shoot and root.     

Adventitious shoot production from strips of stem in the Dutch elm hybrid ‘Commelin’: plantlet regeneration and neomycin sensitivity

The regenerative ability of small strips of stem of the Dutch elm hybrid ‘Commelin’ was tested as well as its sensitivity to neomycins. Cambium explants (1 mm thick), were excised from woody stems collected in the field. Up to 20 buds/explant were induced within 2–3 weeks giving 2–5 rootable shoots/explant after 5–6 weeks. Shoot excision every week from week three improved the yield up to 7 shoots per explant. Fourteen and 2.9 μM GA3 promoted shoot growth. Cytokinins (1 μM zeatin or 5 μM BA or 0.05 μM TDZ) completely inhibited shoot production and promoted callus formation. Kanamycin and paromomycin at between 240 and 360 μM inhibited shoot formation as did geneticin at 80 μM. The shoot-forming ability of the explants was high from leaf fall in the autumn to the spring flush, but could be maintained up to September by using cold storage (5–7 °C). Ninety-six percent of the shoots rooted with 0.5 μM IBA and were successfully acclimatized despite having a large basal callus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Shoot histogenesis in tobacco callus cultures

Summary The earliest histological event observed in light-grown shoot-forming tobacco (Nicotiana tabacum L. cv. Wisconsin 38) callus was the deposition of safranin-stainable substances (probably suberin) on cut, exposed cell surfaces. This was followed by the initiation of cell files and the appearance of starch granules. Nodules with lignified tracheary elements also were observed in the upper part of the callus. Pronounced starch accumulation occurred in the lower part of the callus in which protrusions of tissue into the medium occurred. Meristemoids were found in these protrusions as well as elsewhere. In between meristemoids, parenchyma cells with starch granules of varying sizes were observed. Cell strands that connected with the meristemoids also were observed. These strands often terminated at the surface of the protrusion at which point shoot apices originated. The earliest shoots were formed in these protrusions. With time, additiional shoots were formed in other parts of the bottom of the callus and finally in the top part of the callus on prolonged culture. The determination of the loci at which shoot primordia were formed sequentially, was interpreted in relation to the physiological gradient concept. This work was carried out while E. M. was a Visiting Scientist at the University of Calgary under the Scientific Exchange Program between the National Research Council of Canada and the Japan Society for the Promotion of Science. Support for this study was provided by N.R.C. (Canada) Grant A-6467 to T. A. T.     

Sugar beet (Beta vulgaris L.) morphogenesis in vitro: Effects of phytohormone type and concentration in the culture medium, type of explants, and plant genotype on shoot regeneration frequency

In vitro regeneration techniques have been optimized for seven strains and cultivars of sugar beet (Beta vulgaris L.) bred in Russia. The frequency of shoot regeneration from somatic cells and tissues of sugar beet varies from 10 to 97% depending on the explant type, culture-medium composition, and genotype. The in vitro regeneration potential has been estimated in plants with different genotypes. The effect of medium composition (phytohormones and carbohydrates) on the frequency of the formation of a morphogenic callus competent for plant regeneration has been determined. The effect of the types and concentrations of various cytokines (zeatin, kinetin, and 6-benzylaminopurine) on direct shoot regeneration from cotyledon nodes has been estimated. The culture-medium composition has been optimized for direct shoot regeneration from petioles. The effects of different concentrations of abscisic acid on the frequency of shoot regeneration from a morphogenic callus has been studied. Micropropagation has been used to obtain petiole explants and reproduce the shoots obtained by direct regeneration from cotyledonnodes, petioles, and calluses. Improved shoot-regeneration methods can be used for both agrobacterial and bioballistic genetic transformation of the sugar beet genotypes studied.     

Sugar beet (Beta vulgaris L.) morphogenesis in vitro: effects of phytohormone type and concentration in the culture medium, type of explants, and plant genotype on shoot regeneration frequency

In vitro regeneration techniques have been optimized for seven strains and cultivars of sugar beet (Beta vulgaris L.) bred in Russia. The frequency of shoot regeneration from somatic cells and tissues of sugar beet varies from 10 to 97% depending on the explant type, culture-medium composition, and genotype. The in vitro regeneration potential has been estimated in plants with different genotypes. The effect of medium composition (phytohormones and carbohydrates) on the frequency of the formation of a morphogenic callus competent for plant regeneration has been determined. The effect of the types and concentrations of various cytokines (zeatin, kinetin, and 6-benzylaminopurine) on direct shoot regeneration from cotyledon nodes has been estimated. The culture-medium composition has been optimized for direct shoot regeneration from petioles. The effects of different concentrations of abscisic acid on the frequency of shoot regeneration from a morphogenic callus has been studied. Micropropagation has been used to obtain petiole explants and reproduce the shoots obtained by direct regeneration from cotyledon nodes, petioles, and calluses. Improved shoot-regeneration methods can be used for both agrobacterial and bioballistic genetic transformation of the sugar beet genotypes studied.     

Endogenous gibberellin levels influence in-vitro shoot regeneration in Arabidopsis thaliana (L.) Heynh.

The regeneration of shoot buds from callus cells in vitro is an important technique in modern plant genetic manipulation. Whilst it is clear that genetic factors play a major role in determining the ability of callus cells to become organized into regenerating shoot buds, the precise nature of these factors remains unknown. Here we show that callus derived from mutants of Arabidopsis thaliana which have reduced levels of endogenous bioactive gibberellins (GAs), or reduced responsivity to GAs, regenerates shoot buds more readily than does callus derived from wild-type controls. In addition, exogenous GA reduces, and exogenous paclobutrazol (a GA-biosynthesis inhibitor) increases, the frequency of shoot bud regeneration from wild-type callus. These results show that GA levels play a role in regulating shoot bud regeneration from callus, and suggest that variation in endogenous GA levels or responsivity may account for a major component of the genetic variation in shoot bud regeneration frequency described in other species.     

Interrelationship of Abscisic Acid and Gibberellic Acid in the Promotion of Callus Formation in the Abscission Zone of Citrus Bud Cultures

The mechanism of ABA-induced callus formation was studied in sterile bud cultures of Citrus [Citrus sinensis (L.) Osbeck] on defined media. ABA was found to promote callus formation in the abscission zone between the petiole and the branch while inhibiting bud growth. The promoting effect of ABA was dependent on the physiological state of the shoot from which buds were excised, and on the size of the explant. Callus formation was highest in autumn and summer (i.e. younger) buds, and lowest in older buds excised from previous summer flush. GA was only slightly active in promoting callus formation when applied separately, but showed a highly synergistic effect when applied with ABA: maximal callus formation was attained at a combination of 10^?5M ABA and 10^?6 MGA in the medium. Subcultures of ABA-induced callus revealed that ABA inhibited the growth of isolated subcultured callus, while IAA and kinetin, and especially GA, promoted its rapid proliferation. A general decrease in protein synthesis was found in the abscission zone during the first 5 days of induction, while total protein content changed only slightly. The results suggest that ABA-induced callus formation in Citrus bud explants is a multiphasic phenomenon involving, at least, two stages: (1) activation of certain cells in the abscission zone by ABA, resulting in the formation of callus layers, and (2) subsequent proliferation of the callus tissue, which is dependent on the hormonal balance in the explant. This growth-promoting effect of ABA seems to be a general phenomenon in explants exposing a cut-surface.     

Direct and efficient plant regeneration from leaf explants of Solanum tuberosum l. cv. Bintje

Summary A two-step procedure was used for plant regeneration from in vitro grown leaf strips (2–3 mm wide) of cv. Bintje. Step I medium was designed with 2,4-dichlorophenoxycetic acid (2,4-D) at 0.0 or 9.0 M, in combination with 2.28 M kinetin (K), benzyl adenine (BA), zeatin (Z) or zeatin riboside (ZR). Step II media were 2,4-D-free media containing 5.78 M gibberellic acid (GA3) and growth regulators similar to those of step I media. Leaf explants cultured in medium I containing zeatin riboside or zeatin for 6 days and then subcultured in medium II containing zeatin riboside produced numerous shoots without callus formation. Zeatin riboside containing step I and II media caused shoot regeneration in a high number (97.5±2.2) of explants. Approximately, 33.7±8.4 shoots were regenerated from each leaf explant.Abbreviations BA benzyladenine - Z zeatin - ZR zeatin riboside (trans isomer) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Callus induction and plant regeneration from immature embryos of rye and triticale

Callus cultures were established from the scutellum, scutellar node and radicle region of immature embryos of rye and octoploid triticale on modified Murashige-Skoog basal medium supplemented with various growth regulators. 2, 4-D, 2, 4, 5-T and 2, 4, 5-Cl, POP were found suitable for initiation and maintenance of callus cultures. Cytokinins had no or inhibitory effect on callus induction and growth. On basal medium containing 5 mg/l of 2,4,5-Cl₃ POP, 16% of triticale and 17% of rye primary cultures exhibited shoot bud regeneration after 3–4 weeks. Transfer of such cultures to basal medium supplemented with zeatin or zeatin in combination with IAA further promoted shoot elongation and plantlet formation. Plantlets were rooted on basal medium containing 1 mg/l NAA and were eventually transferred to soil. Chlorophyll variants were observed in about 6% of triticale cultures.     

Changes in the microtubular cytoskeleton precede in vitro tuber formation in potato

Summary An in vitro system for tuber formation was used to study early morphological and cytological changes occurring during tuber formation in potatoes, with special emphasis on the orientation of the microtubular cytoskeleton, visualized immunocytochemically. Axillary buds from potato plants were cultured in the presence or absence of gibberellin (GA), resulting in either tuber formation (without GA) or shoot formation (GA added). Tuber formation in the absence of GA was highly synchronous in individual buds, enabling the dissection of various aspects of tuberization. Under both conditions, starch started to accumulate. In the absence of GA, starch levels rapidly increased, concomitantly with tuber formation, whereas it slightly decreased in the presence of GA. Up to 4 days, the cortical MTs in the cells were oriented perpendicular to the longitudinal axis of the developing buds. Under tuber-inducing conditions this orientation changed into a longitudinal one at day 5. This change preceded a change in the direction of cell expansion. In the presence of GA no such reorientation was observed, cells continued to grow longitudinally, and a stoloniferous shoot was formed. The cytoskeletal changes preceded the visible swelling of the buds, observed after day 5, demonstrating that the reorientation of the microtubular cytoskeleton is one of the earliest steps observed so far in tuber formation in potatoes.Abbreviations GA gibberellin - MTs microtubules - PBS phosphate buffered saline - SD short-day     

Efficient shoot regeneration from internodal explants of Populus angustifolia, Populus balsamifera and Populus deltoids

In the present study, interactions between the duration of treatment with auxin and different cytokinins and their effect on shoot regeneration were evaluated with the aim to establish a rapid and efficient in vitro regeneration method applicable to a variety of Populus species. Three different species, Populus angustifolia, P. balsamifera, and P. deltoids, were chosen for that purpose. We were successful in regenerating plantlets from stem and petiole explants from all three chosen species using a four-step simple procedure. The first step was callus induction when the explants were exposed to an auxin-rich medium for 0-20 days. During the second step, they were transferred onto a cytokinin-rich medium for shoot bud induction. In the third step, the shoots regenerated were transferred onto a medium with reduced levels of cytokinins to promote shoot proliferation and elongation; finally, in the fourth step, the shoots were rooted and acclimated. A short period (6-10 days) of time of exposure to auxin was sufficient for shoot regeneration. A culture time longer than ten days in callus induction medium drastically reduced the efficiency of shoot regeneration. Besides, cytokinin type and concentration also affected the frequency of shoot induction. A 0.2 mg/l concentration of 2,4-D for callus induction followed by 0.02 mg/l of Thidiazuron for shoot formation proved to be the best treatment for adventitious shoot bud multiplication, generating a maximum of 10-13 shoots of P. balsamifera and P. angustifolia in ten weeks. In contrast, for P. deltoids, a combination of 1.1mg/l 2,4-D, 1.0mg/l NAA, 0.1mg/l zeatin for callus induction followed by a combination of 1mg/l zeatin plus 1.0mg/l BA for shoot bud induction was found to be the most effective, generating on average 15 shoots over a period of ten weeks.     

影响沙田柚叶片离体培养的因素研究

研究了离体培养条件下影响沙田柚叶片愈伤组织诱导和分化的一些因素。结果表明 ,2 ,4 D可以诱导愈伤组织的形成 ,高浓度的蔗糖 (6% )显著提高叶片愈伤诱导率与愈伤组织重量 ,且在 2 ,4 D和蔗糖之间存在着相互作用。外源GA3处理抑制愈伤组织的诱导和生长 ,而CCC与ABA处理显著提高叶片的愈伤诱导率和愈伤组织生长量。愈伤组织转移到附加 3 .0mg/LBA的MS分化培养基上可以分化出芽 ,0 .2 5mg/LGA3的加入可以进一步提高愈伤组织的分化率和每块愈伤组织的再生芽数。     

Plantlet regeneration from primary callus cultures of Lilium brownii F.E.Br. ex Miellez var. giganteum G. Y. Li & Z. H. Chen,a rare bulbous germplasm

Lilium brownii F.E.Br. ex Miellez var. giganteum G. Y. Li & Z. H. Chen, an endangered valuable genetic resource, was used to establish and optimize a callus propagation system and to investigate the effects of internal and external phytohormones for the purpose of germplasm conservation. Of the combinations and concentrations of auxins and cytokinins examined, Murashige and Skoog (MS) medium supplemented with 8 g L⁻¹ agar, 30 g L⁻¹ sucrose, 0.45 μM 2,4-dichlorophenoxyacetic acid, 2.69 to 5.37 μM α-naphthaleneacetic acid, and 0.44 μM 6-benzyladenine, 0.45 μM thidiazuron, and 0.28 μM zeatin riboside generated the best results, effectively promoting callus proliferation. Four callus types could be discriminated, of which type A (yellowish, granular) and type B (yellow, medium-granular) were dry, friable, and grew well. Periodic acid-Schiff staining revealed small and regular cells, with numerous starch granules surrounding each nucleus. In culture, callus clumps produced an average of 14.33 shoots under “MS + 7-d-dark–light” treatment with 100% regeneration frequency. Bulblets formed within 60 d after shoot transfer to bulblet formation medium. Type A and B callus was likely to be embryogenic, according to morphology, cytology, and high shoot regenerating capacity. Examination of endogenous phytohormone levels showed that the abscisic acid to indole-3-acetic acid (ABA/IAA) ratio gradually increased with increasing diameter of callus clumps treated with all exogenous phytohormones, except zeatin riboside, leading to the hypothesis that callus induction competence was closely associated with endogenous ABA/IAA ratio. This first report should assist further genetic studies of this rare Lilium and other bulbous plants.