The in vitro propagation of amaryllis (Hippeastrum spp. Hybrids)

Summary A new, rapid technique for the propagation of amaryllis (Hippeastrum spp. hybrids) by means of tissue culture is reported. Leaf bases, scapes, peduncles, inner bulb scales and ovaries were cultured successfully in vitro and plantlets were induced readily at various concentrations of growth regulators. Some plantlets also were produced in the absence of growth regulators. The most productive tissues for propagation were inverted scapes and peduncles, cultured in a modified Murashige and Skoog salt solution with added organic constituents and 1 mg per 1 (4.5μM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg per 1 (4.4μM) 6-benzylaminopurine (BAP). Plantlets induced axenically also grew roots on the generalized shoot-inducing medium so that no special rooting medium was required. Although friable callus was obtained from ovary tissue cultured on a medium containing 2 mg per 1 (11μM) naphthaleneacetic acid and 4 mg per 1 (18μM) BAP, it produced shoots after 8 weeks of further subculture on the same medium. An average of 10 rooted plantlets was obtained from each scape or peduncle explant on the shoot-propagating medium. Thus, if 45 explants are obtained from each bulb, 450 rudimentary plantlets could be obtained from each mother bulb in 8 weeks of culture. This is a substantial increase over present propagation methods. This work was supported by a grant-in-aid of research, to Bruce G. Cumming, from the National Research Council of Canada.     

Morphogenetic responses obtained from a variety of somatic explant tissues of data palm

Shoot tip, bud, leaf, stem and root explants from bearing trees, offshoots, seedlings, and asexual plantlets ofPhoenix dactylifera L. were cultured on modified Murashige and Skoog nutrient media containing 3 g/l activated charcoal, 100 mg/l 2,4-dichlorophenoxyacetic acid, 3 mg/l N ⁶-(Δ²-isopentyl)adenine to obtain callus. Differential morphogenetic responses were obtained from calli dependent on the explant type and parent source. Subcultured shoot tips and leafy lateral buds callus on nutrient media devoid of charcoal and supplemented with 0.1 mg/l α-naphthaleneacetic acid (NAA) produced adventitious plantlets. Subcultured leaf calli produced roots only. Root callus failed to exhibit any morphogenetic response upon subculturing. Undifferentiated non-leafy buds and stem tissues did not give rise to callus, regardless of the parent source. Generally, the best callus and embryogenetic responses from explants were obtained from seedling and plantlet parent sources. Similarly, organogenetic responses such as root formation and shoot development from shoot tips cultured on media containing 10 mg/l NAA were also related to the parent explant source. Mention of a trademark or proprietary product in the paper does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be available.     

A comparison of in vitro flowers to in vivo flowers of haploid and diploidNicotiana tabacum L.

Thin cell layers (TCLs) were cultured from inflorescences of diploid (2n=4x=48) and haploid (2n=2x=24)Nicotiana tabacum L. "Samsun" and the subsequent flowers formed in vitro were then compared to in vivo flowers. Plants derived from TCLs possessed flowers that were typical of their seed or androgenetically-derived counterparts, whereas de novo flowers from TCLs were abnormal when compared to their counterparts. The TCLs of haploid plants produced more flower buds than diploid TCLs, and did so in a shorter period of time. In vitro flowers and anthers at both ploidy levels were considerably smaller than the in vivo flowers; in vitro flowers also had variable numbers of anthers and pistils. The embryogenic capacity of anthers taken from in vivo diploid flowers was 5 times greater than that of in vitro diploid or haploid anthers. In vivo haploid anthers produced no embryoids, whereas in vitro haploid anthers did produce embryoids. Observations of mitotic cells in root tips of plants derived from anther cultures of in vitro haploid flowers revealed a mixoploid nature. Diploid meiosis was regular and haploid meiosis was irregular regardless of the origin (in vitro or in vivo) of the flowers.Supported by state Hatch funds.     

Transformation of pollen embryo-derived explants by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> in <Emphasis Type="Italic">Hyoscyamus niger</Emphasis>

Leaf, root, stem, petiole, hypocotyl, and zygotic embryo explants, as well as pollen embryoids, and redifferentiated tissues from pollen embryoid-derived plantlets of Hyoscyamus niger L. (black henbane) were inoculated with Agrobacterium tumefaciens, harboring binary vectors (pGS Gluc1) and then cultured on media containing kanamycin. Transient -glucuronidase activity and kanamycin resistant callus formation were influenced by explant origin. Transgenic calluses were obtained at a frequency of up to 30% from all the explants tested. However, transgenic shoots were obtained only from the hypocotyl of plantlets derived from pollen embryoids. Transformation was confirmed by the ability of leaf segments to produce kanamycin resistant calluses, -glucuronidase histochemical and flurometric assays, polymerase chain reaction and Southern blot analysis. The results show that pollen embryoid-derived explants may be an alternative source for both efficient transformation and regeneration of transgenic plants in recalcitrant species.     

Stimulation of somatic embryogenesis and plant regeneration from anther culture of Vitis vinifera cv. Cabernet-Sauvignon

Somatic embryogenesis and subsequent diploid plants have been obtained from anthers of Vitis vinifera Cabernet-Sauvignon, a cultivar so far considered as recalcitrant to in vitro regeneration. Anthers enclosing microspores near the first pollen mitosis were found to be the most responsive. However, from a practical point of view anther length proved to be an easier criterium for determining the optimal physiological anther stage. Calli derived from the anther somatic tissues produced embryoids only when cultured on a medium supplemented with casein hydrolysate. Glutamine and adenine were found to stimulate this embryoid production. Evidence is presented that early removal of cotyledons increases the frequency of normal development of embryoids into plantlets.Abbreviations MS Murashige and Skoog medium (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphtaleneacetic acid - BA 6-benzylaminopurine     

In vitro induction of multiple shoots and plant regeneration in cotton (Gossypium hirsutum L.)

Induction of multiple shoots in cotton (Gossypium hirsutum L. cv. Anjali-LRK 516) has been achieved with cotyledonary nodes devoid of cotyledons and apical meristems. Explants from 35-day-old seedlings yielded the maximum number of shoots (4.7 shoots/explant) using Murashige and Skoog (MS) basal medium supplemented with 6-benzylaminopurine and kinetin (2.5 mg/1 each). Explants from 35-day-old seedlings raised in glass bottles produced a higher number of multiple shoots (8.3 shoots/explant) than those grown in glass tubes and cultured on the same shoot induction medium. Elongation of multiple shoots was obtained on liquid or agar MS basal medium without phytohormones. In vitro shoots were rooted on half-strength agar-solidified MS basal medium or with 0.05 or 0.1 mg/1 naphthaleneacetic acid. Hardening and survival of tissue culture plantlets was 95% under greenhouse conditions.Abbreviations BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - MS Murashige and Skoog medium - NAA -Napthaleneacetic acid     

Thidiazuron promotes high frequency regeneration of peanut (Arachis hypogaea) plants in vitro

Multiple shoots were induced on Valenciatype peanut (Arachis hypogaea L.) explants cultured in vitro on a nutrient medium supplemented with thidiazuron. Zygotic embryos excised from mature seeds were germinated on Murashige-Skoog nutrient medium, and the resulting plantlets (8 days-old) were used as a source of explants. When cultured on a nutrient medium with increasing levels of thidiazuron (0.5 to 30 mg/l), expiants from various parts of the peanut plant (except the root) produced multiple shoot primordia which subsequently developed into individual shoots. Hypocotyl and cotyledon explants produced shoots in higher numbers than other explants (20 shoots per hypocotyl explant at all thidiazuron concentrations and 15 shoots per cotyledon explant at 30 mg/l). Shoots rooted normally on a basal Murashige-Skoog medium containing charcoal and developed into healthy and fertile plants when planted in soil.Abbreviations TDZ thidiazuron - MSO Murashige and Skoog (1962) basal medium - BA 6-benzylaminopurine     

Plant regeneration from seedling explants of green bean (Phaseolus vulgaris L) via organogenesis

Green bean (Phaseolus vulgaris L.) plants were regenerated from 3-day old seedling explants via organogenesis. The explants contained a cotyledon and a small portion (2–3 mm) of embryonic axis split in half. Explants were cultured on a defined medium containing glutamine as the sole nitrogen source. A ring of meristematic tissue was produced at the base of the axillary bud located at the cotyledonary node. The meristematic tissue was produced only if the axillary bud was present together with the cotyledon in the explant. Buds and shoots developed from the meristematic ring. Selected shoots produced roots when excised from the cluster of buds and transferred to root induction medium. Rooted shoots (plantlets) grew well and produced viable seeds when grown in the greenhouse. Histological studies revealed the origin of buds from the peripheral layers of the meristematic ring.Production of buds and shoots was a continuous process, so that new shoots could be removed from the explant for plantlet production every 10–14 days. With the cultivar Dark Red Kidney, an average of 49 buds and 8 shoots were regenerated per explant by 30 days after culture initiation. Sixty-seven percent of the shoots produced roots, and 90–95% of the plantlets survived greenhouse acclimatization to produce healthy plants.     

Embryogenesis and plantlet development in the bamboo Phyllostachys viridis (Young) McClure

Leaf explants of Phyllostachys viridis (Young) McClure were cultured on Murashige and Skoog medium supplemented with 9×10^-6 M 2,4-dichlorophenoxyacetic acid. Numerous embryoids were observed. On transfer to Murashige and Skoog medium lacking hormones, plantlets developed within two weeks and were later successfully transferred to the field.     

Somatic embryogenesis and plantlet regeneration in the genus Secale

Summary A tissue culture of five wild species of the Secale genus, i.e., S. africanum (Stapf.), S. ancestrale (Zhuk.), S. kuprianovii (Grossh), S. segetale (Rosher.), and S. vavilovii (Grossh), from immature embryos of sizes (stages) varying between 1.0 mm to 3.0mm, cultured on MS (1962) mineral nutrient medium supplemented with 0.62 mg/1–5.0 mg/1 of 2,4-D, was established. Initially various types of callus were observed and a correlation between genotype, size of explant and 2,4-D concentration was found. The best embryogenic response was observed when explants were smaller than 1.0 mm. Induction of somatic embryogenesis of 2.0 mm–3.0 mm explants required a higher concentration of 2,4-D. Most embryoids were formed in the presence of 5.0 mg/l of 2,4-D. Secale africanum and S. kuprianovii appeared to have the highest embryogenic capacity among the five investigated species. For embryoids germination to plantlets the MS medium supplemented with GA₃ and cytokinins was used. Ultimately, out of the 932 regenerants obtained 364 originated from somatic embryogenesis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA3 deGibberellic acid - BAP Benzylaminopurine     

甜橙成熟果实未发育胚珠的离体培养

路比血橙和锦橙成熟果实的未发育胚珠在含ME(1000mg/l)的MT培养基上培养,胚状体诱导率和每个胚珠平均产生胚状体数均较高。带胚状休的愈伤组织转入该培养基上继代培养,仍可保持其胚性特点。将子叶形胚状体转至含GA(1mg/l)和CH(500mg/l)的MT培养基上,成苗率较高。这为扩大柑桔组织培养的外植体来源和克服外植体的季节限制提供了新途径。     

In vitro culture of sugarcane infected with maize streak virus (MSV)

Young buds and leaf tissues of sugarcane cv. R 574 (a Saccharum hybrid), healthy or infected with Maize Streak Virus (MSV) were cultured in vitro. The success rate of the cultures from infected material is lower than that from healthy material. The symptoms of the disease were displayed on most of the plantlets produced from buds but only on 6% of the plantlets regenerated from calli. The indirect double-antibody sandwich ELISA technique using a monoclonal antibody makes it possible to identify virus-infected plantlets. In vitro, the infection results in the reduced growth of shoots.     

A comparative study of somatic embryogenesis in Secale vavilovii

Summary The ability of immature embryos, inflorescences and leaves of Secale vavilovii to form embryogenic callus was tested on Murashige and Skoog (1962) medium supplemented with different concentrations of 2,4-D. All cultured immature embryos formed calluses. The highest percentage of embryogenic callus production was from 1–2 mm embryos. Young leaves also formed calluses, mainly from the 10–15 mm basal segment, the percentages of embryogenic calluses being higher when cultures were maintained in darkness. Embryogenic calluses were obtained also from all the cultured immature inflorescences, in the three cases, rooted green plants were obtained and grown in soil. Comparison of the responses of the three explants used indicates that immature inflorescence is the most useful explant for obtaining regenerated plants in Secale vavilovii.     

Studies on Induction of Pollen Plantlets from the Anther Cultures of Lily

Anthers with the filament of lily (Lilium davidii var. Willmottiae (Wilson) Roffill) were cultured on modified MS medium. Supplemented with different concentrations and compatible ratios of growth hormones (Z 2 mg/L,or 2,4-D 2 mg/L + KT 2mg/L, or 2,4-D 4mg/L+ 6 BA 2 mg/L). At this time the pollen grains in the anthers were at the late uninueleate stage. Anther cultures were incubated at 25—27 ℃, and illuminated with daylight of about 800–1200 lx. After 30 days, the calli or embryoids were produced from anthers. The frequency of the calli or embryoids induction was 8.89%. After transfer eventually to the differentiation medium, these calli or embryoids developed into plantlets in 70 days. Among the root tips of regenerated plantlets haploid, diploid and aneuploid cells were found, but the haploid cells were produced in about 86.4% of the root tips. It is quite evident that haploid plantlets are derived from the pollen grains.     

Plantlet differentiation in leaf and root cultures of birch (Betula Pendula roth.)

The newly-formed leaves on plantlets differentiated from shoot bud cultures of Betula pendula, when excised and grown on a fresh medium produced callus from the margins or regenerated leafy shoots, roots and plantlets. After 4 weeks, upon transfer to murashige and Skoog (MS) medium supplemented with 3-indoleacetic acid (IAA) + 6-(4-hydroxy-3-methyl-trans-2-enyl)aminopurine (zeatin) + 6-aminopurine (adenine), 15–20 plantlets were produced from each explant. Likewise, the roots also showed meristematic activity at several sites, and produced nodulated callus on MS + α-naphthaleneacetic acid (NAA) + 6-(3-methyl-2-butenyl-amino)purine (2-iP) + adenine, and ultimately differentiated plantlets. Anatomical studies showed that initiation of callus takes place by meristematic activity in epidermal cells of leaves, and cortical cells of roots. Cytological investigations revealed no change in chromosomal complement.     

Thidiazuron-induced de novo shoot organogenesis on seedlings,etiolated hypocotyls and stem segments of Huang-qin

Development of an efficient in vitro propagation system for Huang-qin (Scutellaria baicalensis), a traditional Chinese medicinal plant used in the treatment of a wide range of human ailments, is described. Thidiazuron [TDZ: N-phenyl-N′- (1,2,3-thidiazol-5-ylurea)] effectively induced regeneration on cultured intact seedlings, etiolated hypocotyl explants and sterile stem segments of Huang-qin. Histological examinations of excised hypocotyl or nodal explants revealed that adventitious shoots formed through an intermediate callus. Comparison of TDZ-induced regeneration in the three tissue types indicated that isolation of explants was not essential for optimal regenerative efficiency. Significantly more regenerants formed along hypocotyls of intact seedlings (20 shoots/explant) than were observed on excised hypocotyls (9.7 shoots/explant) indicating that endogenous metabolites produced in adjacent tissues provided resources for the shoot initiation. More than 95% of de novo regenerants formed roots and then intact plantlets under either sterile culture or greenhouse conditions. Regeneration protocols developed in this study may provide the basis for improvement of this crop through the identification of medicinally active constituents and eventual development optimized pharmaceutical products. This revised version was published online in June 2006 with corrections to the Cover Date.     

Asexual embryogenesis and plant regeneration in Quercus

Red oaks (Quercus rubra L.) were regenerated via direct and indirect asexual embryogenesis from immature zygotic embryo tissues. Late heart and early cotyledonary explants cultured in light on modified MS medium proved to be most embryogenic. Embryoids arose from explants cultured on various combinations of 2,4-D and BA. However, the highest percentages of normal polar embryoids were produced by explants cultured on growth-regulator-free media. Epicotyl dormancy of embryoids was overcome by desiccation (air drying and use of an osmoticum) and rehydration treatments. Asexual plantlet development paralleled developmental changes associated with seed germination. White oak (Quercus alba L.) embryoids were also regenerated, but failed to germinate.     

魔芋属植物愈伤组织的诱导和再生植株的研究

用云南魔芋(Amorphophallus yunnanensis)、白魔芋(A.albus)、花魔芋(A.revieri)和勐海魔芋(A.bannaensis)等四个种的叶、鳞叶、花序、匍匐茎、块茎和茎尖为外植体,诱导产生小植物。可通过三种途径获得魔芋再生植株:(1)由外植体诱导愈伤组织,再分化小植株:在添加0.5—1.0mg/1 NAA和BA或KT、ZT的MS培养基上,高频率地诱导瘤状愈伤组织形成,发现NAA对愈伤组织的诱导是必不可少的,而细胞分裂素与之组合,促进瘤状愈伤组织的形成和发育,在无激素或低浓度激素的培养基上,瘤状愈伤组织分化出小植物;(2)茎尖和鳞叶、块茎切块培养,诱导形成多芽苗,产生再生植株;(3)块茎切块诱导产生微型小块茎,然后分化芽和根,长成小植物。本研究为魔芋的快速繁殖提供了新的手段。     

Micropropagation ofFagus sylvatica L.

Summary An in vitro shoot multiplication system was established from juvenileFagus sylvatica L. tissues, and plantlets were regenerated. Embryonic axes were excised from beech seeds and germinated in vitro on media supplemented with 6-benzyladenine (BA) to obtain plantlets with axillary shoots. Shoot multiplication was maintained by sequential subculture of axillary shoot tips and basal segments on Woody Plant Medium supplemented with 0.5 mg/liter BA+2 mg/liter zeatin+0.2 mg/liter naphthaleneacetic acid (NAA). The effeciency of shoot multiplication clearly depended on the kind of explant used. Transfer to fresh medium every 2 wk during the 6-wk multiplication cycle improved multiplication rates. In the rooting stage, an initial 7-day dark period significantly improved rooting capacity and accelerated the emergence of roots on auxin-treated shoots. Adventitious buds were induced on the intact hypocotyls of the whole plantlets derived from the initial embryonic axis explants, especially on those cultured on medium with 1 mg/liter BA. Cotyledon and hypocotyl segments isolated from seedlings grown in vitro from embryos also exhibited capacity for adventitious bud formation, especially when cultured on media supplemented with 0.5 mg/liter BA + 0.1 mg/liter NAA.     

Somatic embryogenesis and plant regeneration in different organs ofEuterpe edulis mart. (Palmae): Control and structural features

Somatic embryogenesis and further plant regeneration were observed using zygotic embryos, young inflorescences and young leaves ofEuterpe edulis (Palmae) as explants. Both for the cultures of zygotic embryos and inflorescences, activated charcoal in the medium was essential for the establishment of viable cultures. Embryogenesis was induced by using a gelled basal medium with MS or Euwens salts supplemented by high 2, 4-D levels (50–100 mg L⁻¹). The embryogenic process was direct without a callus stage. For further development, cultures with globular or post-globular embryos were transferred to the basal medium with 2-iP (2.5 mg L⁻¹) and NAA (0.1 mg L⁻¹). To convert embryos to plantlets, cultures were transferred to a third medium in which sucrose and salts were reduced to the half-strenght of the basal medium, without growth regulators. In the case of liquid medium, with either 2, 4-D or NAA (10–20 mg L⁻¹). The developmental stage of each explant was critical for the induction of embryogenesis. The histological study of embryogenic cultures revealed that in the case of zygotic embryos, somatic embryos arise directly from the surface of the cotyledonar node, or from subepidermal tissues. In the inflorescences, a pro-embryogenic tissue is formed at the floral primordium region; in the leaves, the first morphogenic event is cell proliferation in the vascular parenchyma.