Enhanced somatic embryogenesis in Selinum candolii DC. under a mineral oil overlay

Cell suspension cultures of Selinum candolii DC. obtained on liquid Murashige & Skoog's medium supplemented with 4.52 M 2,4-D and 1.16 M kinetin when plated on solid medium devoid of 2,4-D proliferated into a callus and subsequently produced 15–20 somatic embryos within 60 days. However, when the plated cells were overlaid with mineral oil, a decrease in callus formation coupled with a four-fold increase in the number of somatic embryos per gram fresh weight of the cells were observed after 30–45 days. Though no significant correlation could be found between the depth of mineral oil overlay and the number of somatic embryos produced, the embryoids that developed under mineral oil showed a lesser degree of secondary embryogenesis than the controls. The somatic embryos could be readily regenerated into plantlets.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - MS Murashige & Skoog's medium     

碳氮源对花榈木胚性愈伤组织诱导、发育及有机物积累的影响

为探讨花榈木体胚发生过程中不同碳氮源处理对胚性愈伤组织诱导、发育和有机物积累的影响,并筛选出有利于花榈木体胚发生的碳氮源,优化体胚发生体系,该研究以成熟胚为外植体,通过单因素试验分析3种碳源、4种蔗糖浓度和6种氮源处理下胚性愈伤组织诱导、发育和有机物积累的差异。结果表明：(1)蔗糖中胚性愈伤组织诱导率显著高于葡萄糖和麦芽糖,但其体胚诱导率、体胚分化率、胚性愈伤组织可溶性糖、淀粉和可溶性蛋白含量差异不显著。(2)随着蔗糖浓度的升高,胚性愈伤组织、体细胞胚(体胚)诱导率、体胚分化率、胚性愈伤组织重量和可溶性蛋白含量呈先升高后降低的趋势,均以添加30 g·L^-1蔗糖最高,而胚性愈伤组织可溶性糖和淀粉含量呈增加的趋势。(3)在6种氮源处理中,胚性愈伤组织诱导率以添加500 mg·L^-1谷氨酰胺的处理最高,体胚诱导率则以添加谷氨酰胺和水解酪蛋白的处理较高,但不同氮源处理间体胚分化率无差异;添加有机氮源的处理其胚性愈伤组织可溶性蛋白含量显著高于无氮源处理。总之,不同的碳氮源通过影响花榈木胚性愈伤组织的诱导、发育和有机物的积累,从而影响其体胚诱导率,但对体...     

Direct somatic embryogenesis and plant regeneration from single cell suspension cultures of Elymus giganteus Vahl

Summary A yellowish, nodular callus was induced from mature embryos of Elymus giganteus Vahl on MS medium containing 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l kinetin, from which a cell suspension culture was initiated in liquid MS medium supplemented with 0.5 mg/l 2,4-D, 1.0 mg/l kinetin and 0.2 mg/1 naphthaleneacetic acid (NAA). By filtering through a series of sieves with decreasing mesh sizes and collecting the resultant filtrate, a suspension culture composed mainly of single embryogenic cells was established. In a medium containing 0.3 mg/l 2,4-D, 1.0 mg/l 6-benzylaminopurine (6-BAP) and 500 mg/l casein hydrolysate (CH), the single cells underwent direct somatic embryogenesis resulting in the formation of proembryos. These proembryos developed into mature embryos when placed in a double-layer liquid overlay culture. Intact plants were developed from somatic embryos when they were transferred onto solidified MS medium without added growth regulators.     

Influence of dicamba and casein hydrolysate on somatic embryo number and culture quality in cell suspensions of Dactylis glomerata (Gramineae)

The effects of various concentrations and combinations of dicamba (3,6-dichloro-o-anisic acid) and casein hydrolysate on growth, mucilage accumulation, somatic embryo and root development in suspension cultures of Dactylis glomerata L. (orchardgrass) were examined. Fresh weight of culture tissue was increased with 20 M but not with 80 or 160 M dicamba in treatments with 1–4 g/l casein hydrolysate. Different casein hydrolysate concentrations did not alter the amount of mucilage (measured by viscosity) in the supernatant in the absence of dicamba. However, the addition of dicamba increased viscosity with 80 M giving the maximum response. Casein hydrolysate produced the greatest viscosity at 1–3 g/l in treatments where dicamba was present. Both dicamba and casein hydrolysate were required for development of somatic embryos. Dicamba at 40 M with 3–4 g/l casein hydrolysate produced approximately 2000 embryos/35 ml of suspension. Root development was inhibited by dicamba and stimulated by the presence of casein hydrolysate. The usefulness of medium component manipulations for influencing somatic embryogenesis and culture quality is discussed.     

Improvement of somatic embryogenesis in Medicago arborea

A number of medium constituents were evaluated in an attempt to improve somatic embryo production in Medicago arborea ssp. arborea, using cotyledons, petioles and leaves as explants. Two culture steps were applied: in the first stage (2 months), Murashige–Skoog (MS) medium was used, containing 2,4 dichlorophenoxyacetic acid (9M 2,4-D) and kinetin (9 M KIN) together with different nitrogen sources (alanine, glutamine, proline or tryptophan (2.5 and 5 mM); casein hydrolysate (100, 500 and 1000 mg l^–1; nitrate (4.69 and 9.39 mM) or casein hydrolysate (100 mg l^–1) and nitrate (4.69 mM)), polyalcohols (mannitol at 164 and 328 mM or sorbitol at 219 and 438 mM), sucrose (43.8 and 175.4 mM) or calcium (1.5 and 6 mM). In the second stage (3 months of cultivation), calli were transferred to a kinetin-free MS medium with 2,4-D (2.25 M) only. The inclusion of proline (2.5 mM) was the most effective treatment for the induction of somatic embryos, with the petiole being the best explant. Treatment with casein hydrolysate (100 mg l^–1) also improved the embryonic efficiency. The rest of the treatments neither affect nor inhibit the embryonic response.A special treatment with sorbitol (219 mM) in the second stage of cultivation produced a slight increase in embryogenesis, but less than that obtained with proline.     

Somatic embryogenesis in Encephalartos cycadifolius

Callus cultures of Encephalartos cycadifolius were established from zygotic embryo explants on a modified B5 medium containing 1 mg l^–1 2,4-D and 1 mg l^–1 kinetin. Callus was transferred to media containing various combinations of 2,4-D and kinetin for improvement of somatic embryogenesis. Somatic embryos were produced on media with several growth regulator combinations. The somatic embryos developed from proembryos, which developed long suspensors. A dicotyledonary embryo formed at the distal end of the suspensor. The embryos turned green in light. When transferred to a medium containing 1 mg l^–1 ABA the somatic embryos matured. The suspensors desiccated and these embryos rooted when transferred to a medium without phytohormones.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Enhanced development of somatic embryos of Plantago ovata Forsk. By additives

Summary Plantago ovata Forsk (commonly known as Isabgul) is an economically important medicinal plant. In the present investigation, in vitro plant regeneration of P. ovata was attempted through somatic embryogenesis. Casein hydrolysate and coconut water were used in different concentrations in Murashige and Skoog medium along with 1-naphthaleneacetic acid and N⁶-benzyladenine to increase the amount of callus and number of somatic embryos. Light and scanning electron microscopic studies followed the developmental stages of embryo formation. Results indicated that optimum concentrations of casein hydrolysate and coconut water are useful for promoting the growth of embryogenic cultures. However, a supra-optimal dose of casein hydrolysate and coconut water induced polyphenol synthesis and caused browning of callus and also eventual death of embryos. The use of additives such as coconut water and casein hydrolysate promotes large-scale production of P. ovata through in vitro somatic embryogenesis.     

High-frequency plant regeneration via somatic embryogenesis and organogenesis and in vitro flowering of regenerated plantlets in Panax ginseng

 The morphogenesis ability of light yellowish globular callus derived from cotyledons of mature zygotic embryos of Panax ginseng was investigated. The optimal media for somatic embryogenesis and shoot organogenesis were MS medium containing 0.5 mg l^–1 2,4-dichlorophenoxyacetic acid, 0.1 mg l^–1 6-benzyladenine (BA), and 500 mg l^–1 lactoalbumin hydrolysate, and SH medium supplemented with 0.5 mg l^–1 α-naphthaleneacetic acid, 0.1 mg l^–1 BA, and 500 mg l^–1casein hydrolysate. The influences of glucose, mannose, fructose, and sorbose in the media on somatic embryogenesis and shoot organogenesis were revealed as differences in the numbers of somatic embryos and adventitious shoots per gram of morphogenic callus. The best regeneration of somatic embryos was obtained on medium containing glucose, with a mean of 8.7 somatic embryos per gram of callus. The best regeneration of shoots was observed on medium containing fructose, with an average of 12.2 adventitious shoots per gram of callus. Of the somatic embryos 95% were converted into regenerated plantlets, and 100% of adventitious shoots rooted to form regenerated plantlets. Regenerated plants were successfully established in soil. Flowering was observed in 5.7% of the regenerated plants derived from shoot organogenesis and in 1.4% of the regenerated plants derived from somatic embryogenesis. Received: 1 December 1998 / Revision received: 13 September 1999 / Accepted: 20 September 1999     

Somatic embryogenesis in liquid cultures of a tetraploidAlstroemeria

A simple and efficient procedure was developed for regeneration of a tetraploid cultivar ofAlstroemeria (A. pelegrina x A. psittacina) via somatic embryogenesis in liquid cultures. Embryogenic callus induced from mature zygotic embryos, cultured on MS medium supplemented with 40 μM NAA and 20 μM kinetin, was used as inoculum for liquid cultures. Pre-culture of the callus on MS medium supplemented with 80 μM NAA for two days was essential for cell proliferation in the liquid medium. Embryogenic cell aggregates, obtained by sieving through a 750 μm nylon mesh, continued to proliferate in media containing 10 or 20 μM NAA and 10 or 20 μM kinetin. When transferred to a semi-solid half strength MS medium supplemented with casein hydrolysate, cell aggregates successfully differentiated into plantlets which later grew to maturity under greenhouse conditions.     

Role of exogenous reduced nitrogen and sucrose in rapid high frequency somatic embryogenesis in Medicago sativa

The effect of exogenously supplied reduced nitrogen and sucrose on high-frequency somatic embryogenesis in petiole-derived tissue cultures of a diploid and a tetraploid regenerable clone of Medicago sativa ssp. falcata was investigated. There was an absolute requirement for ammonium during embryo induction and differentiation, with 5mM being the optimum for induction and 10–20 mM the optimum for differentiation of somatic embryos. Exogenous amino acids were not essential for differentiation and often even inhibitory, except 1 or 2 g/l casein hydrolysate or 4.4 mM glutamine with 3.1 mM proline which, under certain conditions, resulted in increases of 20–30% in the number of embryos obtained. High and low sucrose concentrations inhibited somatic embryogenesis and there was no reason to deviate from the 3% (0.088 M) sucrose level commonly used in plant tissue culture media. Selected clones from three M. sativa cultivars showed a response similar to the highly regenerable ssp. falcata clone F1.1.     

Plant regeneration from protoplasts of mango (Mangifera indica L.) through somatic embryogenesis

 This report describes a protocol for the regeneration of plants from protoplasts isolated from proembryogenic masses (PEMs) in a suspension culture derived from the nucellar callus of mango (Mangifera indica L. cv 'Amrapali'). The maximum yield (24.6±1.1×10⁶), with 81.04±4.1% viable protoplasts per gram PEMs, was obtained with an enzyme mixture containing 1.2% cellulase, 1.0% hemicellulase and 0.6% pectinase. An optimum density of 5×10⁴ cultured protoplasts per milliliter culture medium was required for the highest frequency (88.89±5.40%) of division. Dividing protoplasts developed into microcalli that proliferated on medium supplemented with growth regulators (auxins or kinetin alone, or auxins with kinetin) and produced somatic embryos after transfer to a growth regulator-free medium. The protocallus on 2,4-D-containing medium produced the maximum number (102.50±6.93) of somatic embryos. Maturation of somatic embryos depended upon the presence, and the nature and combination of growth regulators in the medium during proliferation of the callus. The mature somatic embryos germinated and developed into plants that were transferred to soil. Received: 1 April 1999 / Revision received: 27 July 1999 / Accepted: 23 August 1999     

Maturation and conversion ofLiriodendron tulipifera somatic embryos

Summary Embryogenic suspension cultures of the hardwood forest tree yellow-poplar (Liriodendron tulipifera) have the potential to produce millions of plantlets. However, low conversion frequencies limit the realization of this potential. Using 4 embryogenic yellow-poplar lines, we first tested the ability of somatic embryos, selected for their similarity to mature zygotic embryos, to convert to plantlets, then tested physical and chemical treatments for their effects on promoting maturation of somatic embryos and subsequent plantlet production. Embryos selected based on resemblance to mature zygotic embryos and transferred to a hormone-free basal medium without casein hydrolysate (CH) produced plantlets at a frequency of 63%. Populations of synchronized somatic embryos were obtained by repeated fractionation of liquid medium-cultured proembryogenic masses (PEMs) on stainless steel sieves. These fractionated embryos failed to mature properly when cultured in liquid basal medium, however. Development of embryos cultured in basal medium supplemented with 5×10⁻⁷ M abscisic acid (ABA) was slowed and embryos appeared to mature properly, with separated cotyledons and little precocious germination. However, ABA-treated embryos only rarely converted to plantlets, possibly due to residual effects of the ABA. PEMs fractionated on sieves, transferred to filter paper and placed on solidified basal medium gave a 60–70% synchronous population of mature embryos 10–12 days following plating. Mature embryos transferred to basal medium without CH converted at a frequency of 72%. The percentage of all embryos differentiating from PEMs on filter paper that formed plantlets was 32%. This material is based upon work supported by the U. S. Department of Agriculture Cooperative State Research Service under Agreement No. 85-FSTY-9-0117.     

In vitro culture of immature tassels of an inbred field variety of Zea mays,cv. Oh43

The medium and conditions which permit in vitro culture of immature tassels of Zea mays cv. Oh43 are reported. Final fresh weight, total number of spikelets and the number of normal spikelets were enhanced by optimal concentrations of sucrose (0.3 M), kinetin (10^–7 M) and casein hydrolysate (30 mg l^–1) when added to the Murashige and Skoog salts, White's vitamins and glycine and inositol. A single cultured tassel produces up to 200 normal spikelets which contain anthers bearing germinable pollen.     

Somatic embryogenesis and regeneration of triploid plants in endosperm cultures of Acacia nilotica

Immature endosperm of Acacia nilotica formed a nodular callus on MS medium supplemented with 2,4-D, BAP and CH. In the third passage on this medium, in the dark, the callus differentiated somatic embryos. The embryos germinated on MS only after 15 d pre-treatment on modified MS medium in which major salts were replaced by those of major salts of B₅ medium and supplemented with glutamine, CH and CW. Triploid nature of the somatic embryos was confirmed by Feulgen cytophotometry.Abbreviations ABA abscisic acid - AC activated charcoal - BAP 6-benzylaminopurine - B₅ Gamborg et al. (1968) medium - CH casein hydrolysate - CW coconut water - d days - MS Murashige and Skoog (1962) medium - PEG 4000 polyethylene glycol - MW 3500–4000 - 2,4-D 2,4-dichlorophenoxyacetic acid     

Proliferation and differentiation from endosperms of Carthamus tinctorius

The endosperms of Carthamus tinctorius cv. HUS-305, excised at globular to heart-shaped stages of zygotic embryo development, were cultured on Murashige and Skoog’s medium (MS) supplemented with different concentrations of 6-benzylaminopurine (BAP), kinetin, thidiazuron (TDZ), 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthalene-acetic acid (NAA). The highest incidence of callusing was on 2,4-D supplemented media. However, embryos differentiated only from the calli developed on media supplemented with BAP, kinetin or TDZ with the last eliciting maximum embryogenic response. The addition of a reduced nitrogen source, casein hydrolysate to MS medium supplemented with BAP and/or NAA, did not stimulate the response. However, adenine sulphate (100 mg dm⁻³) promoted the induction of somatic embryos. Upon transfer to MS basal medium or the same supplemented with 0.61 μM gibberellic acid (GA₃), plumular poles of few embryos elongated resulting in the development of shoots.     

Improvement of somatic embryogenesis in highland-papaya cell suspensions

Axillary buds (2 mm) from 3-year-old Carica pubescens Lenné et Koch (highland papaya) fruit-bearing plants grown in the greenhouse were cultivated in NN-medium supplemented with different growth regulators naphthaleneacetic acid and indoleacetic acid in combination with Zeatin, benzyladenine, Kinetin and thidiazuron. Several responses were observed within 2–3 months; namely, sprouting of the preformed axillary buds, bud branching into multiple shoots, callus formation at the basal end of the explant and somatic embryogenesis in the preformed callus. Somatic embryogenesis was frequent in most of the tested growth regulator combinations, with the exception of thidiazuron which showed no effect. A much higher yield of somatic embryos could be obtained in suspensions. Somatic embryogenesis was enhanced by the occurence of adventive embryogenesis on single embryos as globular embryo clusters. This was observed in cell suspensions initially grown in a WPM-medium with 2,4-dichlorophenoxyacetic acid, or in combination with benzyladenine or zeatin, for 6 days, then maintained in a growth regulator-free medium under continuous agitation (50 RPM) on an orbital shaker for 3 months. Single cells grown in the absence of 2,4-dichlorophenoxyacetic acid did not initiate embryogenesis and de-differentiated into callus. Plantlets were recovered after transfer of mature embryos from cell suspensions into Magenta flasks. In a second subculture, adventitious embryogenesis occurred spontaneously in clusters at the globular embryo stage under the same growth conditions, yielding a high number of embryos. The culture conditions described above allowed initiation of a large number of somatic embryos directly from cell suspensions through adventive somatic embryogenesis and indirectly from callus on axillary buds.Abbreviations 2,4-d dichlorophenoxyacetic acid - CH casein enzymatic hydrolysate - BA benzyladenine - FAA formalin:acetic acid:alcohol - Glu l-glutamine - IAA indoleacetic acid - NAA naphthaleneacetic acid - NN Nitsch and Nitsch-medium (1969) - TDZ thidiazuron - SD standard deviation     

Somatic embryogenesis and plant regeneration inAzadirachta indica A. Juss

Summary Media for induction of somatic embryogenesis from immature cotyledonary tissues ofAzadirachta indica (Neem) were determined. Callus was initiated on Murashige and Skoog medium supplemented with 0.5 mg·liter⁻¹ of indol-3 acetic acid, 1.0 mg·liter⁻¹ of 6-benzyl amino purine, and 1000 mg·liter⁻¹ of casein hydrolysate. Effect of kinetin was also studied for embryo induction. Carbohydrate source in the form of sucrose and glucose alone and in combination was tested for embryogenic efficiency. Seventy percent embryos showed germination. Healthy plants were potted in sand and soil. Histologic studies confirmed indirect somatic embryogenesis.     

In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls

A high-efficiency two-step culture procedure for direct somaticorganogenesis in loblolly pine (Pinus taeda L.) resulting inthe formation of multiple shoot structures induced on cotyledons andhypocotyls of mature zygotic embryos is described. Mature zygoticembryos of eight genotypes of loblolly pine were used as explants toinduce direct somatic organogenesis with this two-step culture method,involving the induction and the differentiation of direct adventitiousshoots. After mature zygotic embryos of eight genotypes of loblolly pinewere cultured on induction medium containing 2,4-dichlorophenoxyaceticacid (2,4-D) or -naphthaleneacetic acid (NAA), 6-benzyladenine(BA), and kinetin for 2–3 weeks, embryos were transferred todifferentiation medium. Adventitious shoot regeneration via directsomatic organogenesis with the frequency of 8.7–27.8% wasobtained from mature zygotic embryo cultures of the genotypes tested.The highest mean number of 32.6 adventitious shoots per mature zygoticembryo was produced from genotype La. The tissue culture protocol of invitro shoot regeneration via direct somatic embryogenesis was optimizedafter examining the periods of the induction culture, chillingtreatment, glutamine concentration, and basic medium levels. Rooting wasachieved on TE medium supplemented with 0.5 mg/l indole-3-butyric acid(IBA), 0.5 mg/l gibberellic acid (GA₃), and 1 mg/l6-benzyladenine (BA), and regenerated plantlets were established insoil. These results suggested that adventitious shoot regeneration viadirect somatic organogenesis could be useful for clonal micropropagationof some genotypes of loblolly pine and for establishing a transformationsystem of this coniferous species.     

Somatic Embryogenesis from 20 Open-Pollinated Families of Portuguese Plus Trees of Maritime Pine

Immature zygotic embryos from 20 open-pollinated (OP) families of maritime pine (Pinus pinaster) plus trees were screened for their somatic embryogenic capacity. The best time for zygotic embryo collection was between 30th June and 16th July 1999 when most embryos were at a pre-cotyledonary stage of development. The somatic embryogenesis (SE) initiation frequency was highest on DCR basal medium with 13.6 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 µM 6-benzylaminopurine (BAP) supplemented with L-glutamine and casein hydrolysate. On this medium, initiation frequencies among OP families ranged from 4.6 to 49.1%. Initiation of embryogenic cell lines from all 20 OP families was possible only on DCR based medium, but the addition of L-glutamine and casein hydrolysate significantly increased the number of zygotic embryos producing SE. Most families showed a similar behaviour on different initiation media; however, a few exceptions were observed. Further development of somatic embryos on maturation medium, consisting of DCR with 120 µM abscisic acid (ABA), 100 g l^–1 polyethylene glycol (PEG) and 10 g l^–1 gellan gum, occurred in 29% of 896 embryogenic lines representing all 20 OP families. However, development into cotyledonary somatic embryos was observed in only 11% of the cell lines, but this still represented 18 OP families.     

GROWTH IN VITRO OF TISSUES ISOLATED FROM NORMAL STEMS AND INSECT GALLS

Pelet , F., A. C. Hildebrandt , A. J. Riker, and F. Skoog . (U. Wisconsin, Madison.) Growth in vitro of tissues isolated from normal stems and insect galls . Amer. Jour. Bot. 47(3) : 186—195. Illus. 1960.–In a preliminary analysis of the nature of gall formation induced by insects, a comparative study has been made of the in vitro growth and nutrition of plant tissues derived from insect galls and from normal plants. Grape, elm, poplar, and willow tissues were grown on a standard medium, modified White's nutrient medium, with coconut milk and/or various growth factors added. Satisfactory growth was obtained over a temperature range from 16° to 36°C. but was generally optimal at 28°—32°C. The optimum pH was generally 4.0—4.5, but a pH of 6.0 or 7.0 gave better growth when the medium contained 2,4-dichlorophenoxyacetic acid. Detailed nutritional studies were limited to grape tissue. Excised stems and excised galls induced by Phylloxera vastatrix Planch, were grown on the basal medium with vitamins and supplemented with naphthaleneacetic acid, indoleacetic acid, kinetin, casein hydrolysate, yeast extract, adenine and a few amino acids added in various combinations. Growth (fresh weight) was measured after a 6-week growth period. When these substances were added singly the optimal concentrations and the quality of growth of stem explants were as follows: with adenine (40 mg./l.) or kinetin (1 mg./l.), growth poor; with NAA (1 mg./l.) or IAA (2 mg./l.), growth fair; and with the only concentration of a powdered casein hydrolysate (3 g./l.), growth good. Gall explants responded more readily to kinetin or adenine but did not form callus in the presence of casein hydrolysate alone. Stem tissues formed both roots and callus, whereas gall tissues formed only callus. The same substances were tested in various combinations. NAA and kinetin provided for moderate, continuous growth, and excellent growth if casein hydrolysate and adenine also were added to the medium. The NAA requirements were markedly reduced in the grape tissues which had been subcultured for 1 or 4 years on coconut milk medium. Friable tissue types were inhibited by the adenine and casein hydrolysate combinations. They grew through 1 passage only on basal medium and then died if not supplied with NAA and kinetin. Firm tissues responded favorably, although irregularly, to casein hydrolysate and adenine. It was concluded that although nutrient requirements varied with tissues derived from insect galls and from normal plants, they also varied with the time of cultivation in vitro. The induction of galls by Phylloxera was not a permanent change in growth factor requirements comparable to that conferred by the crown gall bacteria. In attempts to grow the insect in sterile culture in vitro 5 successive generations of phylloxera were reared on callus tissue.