Plant regeneration via direct embryo axis-like shoot and root formation from excised cotyledon explants of ginseng seedlings

Summary Cotyledon explants of Panax ginseng at various developmental stages were cultured on Murashige and Skoog (MS) medium with 0.5 μM indole butyric acid and 8.8 μM N⁶-benzyladenine. Upon culturing of cotyledon explants from mature zygotic embryos, 34% of the explants formed somatic embryos, and 46% formed adventitious shoots. In the cotyledon explants from 1-wk-old seedlings, embryo axis-like shoots and roots developed at a high frequency (79%) near the excised portion of the cotyledon base. The developmental pattern of embryo axis-like organ formation was structurally different from that of somatic embryos and adventitious shoots but similar to that of parts of the embryo axis of zygotic embryos. In the early stages of embryo axis-like organ formation, epicotyl-like shoot primordia were developed directly from the cotyledon base after 2 wk of culture; subsequently roots developed near the base of the epicotyl-like shoots and eventually regenerated into plantlets with both shoots and roots. The frequency of embryo axis-like organ formation declined as the growth of seedlings proceeded. In addition, the frequency of somatic embryo and adventitious bud formation rapidly declined with the age of the cotyledons. Plant regeneration via embryo axis-like organ formation might be a new pattern of morphogenesis in P. ginseng cotyledon culture.     

Effect of Auxin and Cytokinin on Newly Synthesized Proteins of Cultured Douglas Fir Cytoledons

Newly synthetized cyloplasmic proteins of Douglas fir cotyledon culture capable of initiating adventitious bud formation (bud culture) were compared with those of morphologically distinct types of cultures, bud-callus culture (some adventitious buds with callus), and callus culture (no adventitious buds; only callus). SDS polyacrylamide gel electrophoretic profiles of double labeled (¹⁴C-and ³H-) proteins showed that a persistent increase of low molecular weight proteins (16,000 to 20,000 daltons) was associated with bud culture. Similar electrophoretic profiles of proteins extracted in the absence and in the presence of protease inhibitor (4-mcthylbenzene sulfonyl fluoride) or phenolic compounds absorbents (Dowex I-X8 and polyvinylpolypyrolidine) were obtained indicating that the detected low molecular weights of these proteins represented the real monomeric state of these molecules.     

Optimization of in vitro bud induction and plantlet formation from mature embryos of Aleppo pine (Pinus halepensis Mill.)

Summary Studies were undertaken to optimize tissue culture conditions for micropropagation of Aleppo pine (Pinus halepensis Mill.) from mature embryos and various explants of the embryo. Over 90% of the embryo explants gave rise to adventitious buds within 4 wk. Intact embryos were the most suitable explants for shoot bud induction. Both isolated cotyledons and hypocotyls produced adventitious buds, but these developed slowly and failed to elongate. N⁶-Benzyladenine (BA) alone at 5.0μM was the most effective cytokinin when added to gelled to gelled von Arnold and Eriksson’s (AE) medium containing 3% sucrose. Adventitious bud development was achieved on hormone-free AE medium, and shoot elongation was optimum on three quarter-strength Bornman’s MCM medium, with 0.1% conifer-derived activated charcoal. Shoots were multiplied on three-quarter strength MCM medium, containing 5μM BA. To induce adventitious roots on the elongated shoots, pulse treatment with 1 mM IBA for 6 h, followed by the transfer of the shoots to sterile peat:vermiculite (1:1) mixture, was beneficial. After acclimatization for 3 to 4 wk under mist, almost all the rooted shoots could be transplanted successfully to the greenhouse, where the plants exhibited normal growth habit. Histologic studies on the ontogeny of adventitious shoot formation from mature embryo explants revealed temporal structural changes in different parts of the explant. Induction of mitotic divisions on the shoot-forming medium resulted in the formation of meristemoids in the epidermal and subepidermal layers of the explant, located initially at both the tips of the cotyledons and the axils of adjacent cotyledons. Shoot buds arising in the axils of adjacent cotyledons were due to new cell division and not to any preexisting meristem.     

Plant regeneration via organogenesis from adventitious bud explants of a medicinal herb species, <Emphasis Type="Italic">Polygonatum cyrtonema</Emphasis>

Summary Explants derived from adventitious buds, rhizomes, stems, and leaves of a medicinal plant, Polygonatum cyrtonema, were studied for plantlet regeneration, and only adventitious bud explants were able to be regenerated into plantlets. Regeneration was also accompanied by the formation of rhizome-like tissue, the medicinal portion of the plant. The optimum hormone combination for plantlet regenertion was 4.44 μM benzyladenine plus 2.26 μM 2,4-dichlorophenoxyacetic acid, at which new adventitious buds were obtained from 96.6% of the adventitious bud explants, with an average of 5.2 buds per explant. The best medium for root induction was half-strength Murashige and Skoog medium with 4.57 μM α-naphthaleneacetic acid, as 92% of regenerated buds rooted. Regenerated plantlets were successfully transferred to a greenhouse with 86% survival. Histological observation indicated that new adventitious buds originated from the superficial meristematic cell cluster of the granular callus induced from adventitious bud explants via organogenesis.     

Secondary somatic embryogenesis and plant regeneration in cassava

Somatic embryos isolated from mature seed-derived cotyledon cultures of cassava (Mannihot esculenta Crantz) underwent direct secondary somatic embryogenesis or plant development under appropriate incubation conditions. Isolated somatic embryos were subjected to a two-stage culture procedure similar to that which induced their development on cotyledon explants. This involved incubation for 24–30 days on Murashige and Skoog basal medium supplemented with 2–8 mgl^-1 2,4-dichlorophenoxyacetic acid (2,4-D) (Stage I medium) before transfer to medium supplemented with 0.01 mgl^-1 2,4-D and 0.1 mgl^-1 6-benzylamino purine (BAP) (Stage II medium). Under these conditions, secondary somatic embryos developed directly from the cotyledons and shoot-tip region of primary somatic embryos by a developmental process morphologically very similar to that occurring on zygotic cotyledon explants. Apical shoot extension and adventitious root formation occurred when somatic embryos were isolated from parental cultures and incubated on Stage II medium. Somatic embryo-derived plants growing in greenhouse conditions appeared morphologically normal when compared with non-regenerated plants.     

Micropropagation of Guazuma crinita mart. by root and petiole culture

Summary Shoot organogenesis of Guazuma crinita Mart. from root and petiole explants was obtained via adventitious bud formation. Root segments and petiole explants excised from in vitro generated plantlets were cultured on woody plant medium (WPM) supplemented with [trans-6-(4-hydroxy-3-methylbut-2- enyl)aminopurine] (zeatin) or with [6-benzyladenine] (BA). After 45 d of culturing, clumps of green bulbous structures containing small adventitious buds (clusters) were generated in all explants cultured with 10 μM zeatin under a photon flux density of 65 μmol m⁻² s⁻¹. For subsequent shoot differentiation, clusters were transferred onto medium containing 1 μM zeatin. After 60 d of culturing, 30% of clusters generated from petiole explants developed into plants. The regenerated plantlets were successfully acclimatized and all survived and grew well. No morphological abnormalities were observed.     

Differential Reactivity in Epidermal Cells of Begonia rex Excised and Grown in vitro

Thin explants composed of the epidermis and underlying collenchyma excised from leaf veins of Begonia rex and cultured in vitro are capable of neoformation of unicellular hairs, roots and buds. Unicellular hairs were formed over the entire surface of the explant when 10⁻⁶M indole acetic acid or 10⁻⁷M naphthaleneacetic acid (NAA) was added to the basal medium; each epidermal cell was potentially involved. The epidermis was most sensitive to a NAA treatment during the first few days of culture but 30% of the explants could still react after 4 days of culture without NAA. When NAA (5 × 10⁻⁷M) and a cytokinin, zeatin (10⁻⁷M), were added together, roots were formed from epidermal tissue after numerous divisions in the original cells. Their initiation was not related to particular cells. Buds were formed when a cytokinin (10⁻⁶M) was added to the basal medium; bud meristems were formed from small groups of cells surrounding basal cells of glandular hairs. Hair formation was inhibited by either high (32–27°C) or low (12°C) temperatures applied continuously. 32–27°C seemed to inhibit elongation of the hairs specifically, whereas 12°C inhibited earlier phases in hair formation. This hypothesis was supported by short temperature treatments applied at different times during hair formation.     

In Vitro Plantlet Formation in Mangosteen (Garcinia mangostana L.)

Optimum conditions were determined for in vivo growth and multiplicationof Garcinia mangostana L. using explants from aseptically germinatedseedlings and field-grown plants. Proliferating shoots wereobtained from cotyledon segments cultured on modified Murashigeand Skoog's (1962) medium with 6-benzylaminopurine. Juvenileleaf segments produced adventitious buds on Woody Plant Medium(Lloyd and McCown, 1981). Root segments gave few buds. Shoottip, nodal, and internodal explants gave multiple axillary andadventitious buds. Shoots were multiplied by enhanced axillaryand adventitious bud formation. The shoots were rooted withindolebutyric acid treatment. Rooted shoots were readily establishedin vermiculite: sand (1:1) mixture. Garcinia mangostana L., Mangosteen, tissue culture, shoot regeneration, bud development     

Adventitious shoot bud formation and plant regeneration from <Emphasis Type="Italic">In vitro</Emphasis>-cultured stem segments of reed (<Emphasis Type="Italic">Phragmites communis</Emphasis> Trin.)

Summary A protocol for large-scale propagation of Phragmites communis Trin. by adventitious bud formation and plant regeneration was established. Adventitious buds were induced through either the indirect pathway or the direct pathway from stem explants of Phragmites communis. In the indirect pathway, it was essential to decrease the level of 2,4-dichlorophenoxyacetic acid from 9.1 to 0.5 μM to induce adventitious buds and achieve plant regeneration. In the direct pathway, the effects of different benzylaminopurine (BA) concentrations in the medium, and different positions of the explants, on adventitious bud formation were determined. Murashige and Skoog (MS) medium supplemented with 5.4μM α-naphthaleneacetic acid (NAA) and 53.4 μM BA, and the bottom part of stem explants were most responsive for the differentiation of adventitious shoot buds. The highest differentiation frequency was 20–30 adventitious shoot buds per stem node tissue. Elongation and proliferation of adventitious buds were achieved on MS medium supplemented with 13.3 μM BA and 5.4 μM NAA. Shoots were rooted in liquid half-strength MS medium with 5.4 μM NAA+4.9 μM indole-3-butyric acid. Rooted plants survived (87.5%) and grew well after transfer into soil for 4 wk. More than 20 000 regenerated plants of a salt-tolerant variant line of Phragmites communis have been produced. This protocol is useful for clonal micropropagation and possibly for Agrobacterium- mediated gene transfer in P. communis.     

Shoot regeneration from seedling explants of Acacia mangium Willd

Summary Regeneration of adventitious shoots was obtained in over 80% of explants, consisting of wounded cotyledonary nodes of Acacia mangium, by culturing germinated seedlings on DKW medium with combinations of N⁶-benzyladenine and either thidiazuron or N-(2-chloro-4-pyridyl)-N-phenylurea. Electron microscopy showed the presence of adventitious buds arising from wound tissue of the cotyledons and cotyledonary nodes. Shoot regeneration was also obtained at lower frequency in isolated cotyledon explants cultured with 6% sucrose alone (10%), or with 3% sucrose and 30.0 mg l⁻¹ (0.1 μM) 2–4-dichlorophenoxyacetic acid (2,4-D; 16%). With 2,4-D,>60% of explants produced organized structures but these did not develop into shoots or somatic embryos. Shoot formation was not induced in either hypocotyl or root explants.     

Adventitious bud induction in Eucalyptus globulus Labill

Summary Adventitious buds and shoots of Eucalyptus globulus Labill. (Tasmanian Bluegum) have been regenerated from cotyledons and hypocotyls from mature embryos and seedlings. Adventitious buds, were induced at high frequency with 0.05 μM thidiazuron in combination with 0.2 μM 2,4-dichlorophenoxyacetic acid or 5 μM α-naphthaleneacetic acid. Culture of explants in the dark inhibited bud induction, but up to 86% of cotyledons, longitudinally split just prior to culture, produced adventitious buds, in the light. Development of buds into shoots occurred only at low frequency, after transfer to media containing N⁶-benzylaminopurine.     

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.     

Possibilities and constraints in the regeneration of trees from cotyledonary needles of Picea abies in vitro

Although use of embryonic or seedling tissues for mass clonal micropropagation in vitro in conifer reforestation programmes is questionable, there is a potential application in the regeneration of plants from scarce and costly seed derived from controlled pollination. In addition, in vitro culture shortens considerably the lag phase in numbers during the initial stages of vegetative propagation via rooted cuttings. Successive steps of the present technique are described whereby cotyledonary needles (secondary explants) were subcultured on a hormone-free medium after administration of cytokinin or auxin to 14-day-old seedling (primary) explants of Picea abies. For bud induction, N⁶-benzyladenine (BA) was applied either as a short-duration (3 h), high-concentration (125 μM) pulse or by vacuum infiltration and incubation in a BA-containmg (5 μM) infusion medium. Induced adventitious shoots were elongated with the aid of far-red light and rooted in vivo after a long-duration (12 h), high-concentration (625 μM) application of indolebutyric acid. Pulse and infusion treatments resulted in the induction of greater numbers of adventitious buds (average of 12 per needle) over a three to four week shorter culture period than was the case with the conventional inclusion of growth regulators in the agarified medium. No exogenous auxin was required in the bud-induction programme; its inclusion even at nanomolar levels promoted histo- rather than morphogenesis. In cotyledonary needles, to the primary explants of which BA was applied as a pulse or by infusion, the cell divisions which gave rise to the meristemoids from which adventitious buds were produced, appeared to commence mainly in undifferentiated hypodermal layer cells but also in the mesophyll immediately below. By contrast, where BA was incorporated in the agarified medium the first divisions occurred mainly in cells of the epidermal layer. A number of factors affected plantlet regeneration, for instance seed variability, age of seedlings, and mode of application of growth substances. It should also be accepted that the xeromorphic nature of the conifer leaf might impose physiological and morphological constraints on its culture in vitro that could militate against easy morphogenic manipulation. It is deemed essential that the current mean ratio of regenerated plants to cotyledonary needles of 1:1 be increased 10 to 20 fold in order to approach commercial feasibility.     

Adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (<Emphasis Type="Italic">Crataegus pinnatifida</Emphasis> Bge. var. <Emphasis Type="Italic">major</Emphasis> N.E.Br.)

Hawthorn (Crataegus spp.) is an important plant with a long history as an ornamental and a source of medicine. A protocol is outlined for adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (C. pinnatifida Bge. var. major N.E.Br.). Adventitious buds were induced on both the leaves of sprouting winter buds and the leaves of in vitro plants, but the percentage of bud regeneration from leaves of in vitro plants was very low—less than 6%. On N6 medium supplemented with 31.08 μM BA and 9.67 μM NAA, the percentages of bud regeneration from leaves of sprouting winter buds of cultivars “Liaohong” and “Qiujinxing” were 31.4% and 17.6%, respectively. The regeneration abilities of three kinds of cotyledon explants, immature cotyledon, mature cotyledon, and cotyledon leaf, were compared. The percentage of bud regeneration from cotyledon leaves was higher. On MS media supplemented with 4.44 μM BA and 4.54–9.08 μM TDZ, the percentages of bud regeneration from cotyledon leaves of cultivars “Qiujinxing” and “Xiajinxing” were 27.7 ± 7.8% and 20.1 ± 4.7%, respectively, and the numbers of buds per explant were 5.9 ± 1.6 and 3.2 ± 0.7, respectively. On B5 medium supplemented with 2.22 μM BA, 2.32 μM Kn, and 0.57 μM IAA, adventitious buds grew quickly and 80–100% of buds developed into shoots. The shoots rooted successfully with the two-step rooting method. Ninety days after transplantation, more than 80% plants were survived. This system of adventitious bud regeneration from leaf and cotyledon explants could be useful for the genetic transformation and polyploidization of Chinese hawthorn.     

adventitious shoot regeneration from petiole explants of Heracleum candicans wall

Summary A method for adventitious shoot induction from petiole explants of Heracleum candicans is reported. Shoot buds were induced on Murashige and Skoog (MS) medium with 4.4μM 6-benzylaminopurine (BA) and 1.1 μM 2,4-dichlorophen-oxyacetic acid (2,4-D). A wound response in the presence of BA and 2,4-D at the time of culture was necessary for inducing shoot buds. The shoot bud regeneration was significantly influenced by size, type and orientation of explants on the culture medium. These shoot buds developed into 4–5 cm shoots upon transfer to a medium containing 1.1μM BA and 0.5 μM α-naphthaleneacetic acid (NAA). The regenerated shoots formed rooted plantlets on MS medium supplemented with 4.9 μM indole-3-butyric acid (IBA). About 15 plants were established in the field for further evaluation.     

An in vitro technique for the production de novo of multiple shoots in cotyledon explants of cucumber (Cucumis sativus L.)

A technique is described for the production de novo of cucumber (Cucumis sativus L.) shoots in the presence of cytokinin using cotyledon explants. The shoots, which arose from adventitious buds and not from enhanced axillary branching, are confined to a specific region at the base of the cotyledon. Concentrations (4 mgl^–1 or less) of the cytokinins 6-benzylaminopurine, kinetin and N⁶-(2-isopentenyl)adenine, are all effective in producing adventitious buds. It is possible to achieve a yield of 23 shoots per cotyledon by removal of the axillary bud. The yield is increased to 50 shoots per cotyledon by cutting the basal region of the cotyledon into small pieces prior to culturing. These techniques may be useful for transformation studies in cucumber.     

Adventitious shoot regeneration fromFagus sylvatica leaf explantsin vitro

Summary Adventitious shoots were induced on transversally divided expanding leaves fromFagus sylvatica shoot cultures of juvenile origin. Adventitious shoot buds formed mainly on callus that developed on the petiole stump or on the cut across the midrib of distal leaf halves. However, sometimes they arose directly from leaf tissue. An anatomical study confirmed both the direct and indirect origin of the adventitious buds. The best results were obtained by culturing proximal leaf sections on woody plant medium supplemented with 2.9 μM indole-3 acetic acid in combination with 8.9 μM benzyladenine or 2.3 μM thidiazuron (TDZ). Proximal explants were more responsive than distal explants in terms of both callus formation and bud regeneration, regardless of the induction medium or clone tested. Bud formation capacity was influenced by the genotype of the stock shoot culture and was enhanced by an initial 10 d darkness, but was inhibited by longer periods of darkness. Caulogenic competence was significantly affected by the duration of exposure to TDZ; in particular, adventitious shoot length was depressed by increasing the exposure period. Three weeks culture with TDZ was the most efficient treatment for shoot production and elongation. Further shoot development was promoted by subculturing the explants to the same medium used for the maintenance of the stock shoot cultures. Shoots so obtained were multiplied and rooted producing plantlets of adventitious origin.     

Adventitious bud development and regeneration in Tillandsia eizii

Summary The bromeliad Tillandsia eizii is a stricking species with large, colorful, and persistent inflorescences that can reach 1 m in length. The value of this plant as an ornamental and its importance in cultural and religious activities has led to its overcollection in the wild. Clonal propagation via tissue culture may be a means to repopulate native stands while meeting the demands for this species as an ornamental and ceremonial plant. Adventitious bud proliferation was induced from axenically germinated scedling material. Parameters evaluated were the age of explant material at the time of transfer onto bud-induction medium, the concentration of plant growth regulators, and the period of exposure to induction medium. Light and seanning electron microscopy (SEM) established the origin and development of buds. Twelve-week-old seedling explants rapidly initiated adventitious buds after a 30-d induction period on shoot-initiation medium. Adventitious buds were induced in 40% of the explants placed on media with 2 mg l⁻¹ 6-benzylaminopurine (BA) (8.88 μM) plus 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA) (0.54 μM) with some cultures becoming highly prolific after repeated subeulture. Shoots elongated in proliferating cultures, and plants were successfully acclimatized and planted into the greenhouse. The results indicate that tissue culture may be used as a means to propagate this epiphytic bromeliad species, which is being seriously affected by deforestation and habitat destruction. In addition, adventitious bud proliferation can provide a means to propagate superior genotypes.     

An efficient in vitro method for mass propagation of Potentilia potanii wolf

Summary This study reports a method for high-frequency shoot organogenesis and plant establishment of Potentilla potaninii Wolf. Hypocotyl and cotyledon explants of P. potaninii were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of benzyladenine (BA) and &#x03B1;-naphthaleneacetic acid (NAA) to induce adventitious shoot formation for micropropagation. The highest frequency of adventitious shoot regeneration was achieved from hypocotyl and cotyledon explants grown on MS medium supplemented with 5.0 mgl^&#x2212;1 BA and 1.0 mgl^&#x2212;1 NAA. The regenerated shoots rooted most efficiently on half-strength MS medium supplemented with 1.0 mgl^&#x2212;1 NAA and 0.5 mgl^&#x2212;1 indole-3-acetic acid or indole-3-butyric acid. The acclimatized plants with normal morphology and growth characters flowered and set seeds in the following year.     

Direct and indirect somatic embryogenesis on cotyledon explants of <Emphasis Type="Italic">Quassia amara</Emphasis> L., an antileukaemic drug plant

Summary In vitro propagation of Quassia amara L. (Simaroubaceae) was attempted using mature and juvenile explants. Attempts to establish in vitro culture using leaf and internode explants from a plant more than 15yr old were unsuccessful due to severe phenolic exudation. Plant regeneration through direct and indirect somatic embryogenesis was established from cotyledon explants. Murashige and Skoog (MS) medium with 8.9 μM N⁶-benzyladenine (BA) and 11.7 μM silver nitrate induced the highest number (mean of 32.4 embryos per cotyledon) of somatic embryos. Direct somatic embryogenesis as well as callus formation was observed on medium with BA (8.9–13.3 μM). Semi-mature pale green cotyledons were superior for the induction of somatic embryos. Embryos developed from the adaxial side as well as from the point of excision of the embryonic axis. More embryos were developed on the proximal end compared to mid and distal regions of the cotyledons. Subculture of callus (developed along with the somatic embryos on medium with BA alone) onto medium containing 8.9 μM BA and 11.7 μM silver nitrate produced a mean of 17.1 somatic embryos. Primary somatic embryos cultured on MS medium with 8.9 μM BA and 11.7μM silver nitrate produced a mean of 9.4 secondary somatic embryos. Most of the embryos developed up to early cotyledonary stage. Reduced concentration of BA (2.2 or 4.4 μM) improved maturation and conversion of embryos to plantlets. Ninety percent of the embryos converted to plantlets. The optimized protocol facilitated recovery of 30 plantlets per cotyledon explant within 80d. Plantlets transferred to small cups were subsequently transferred to field conditions with a survival rate of 90%.