Improved shoot development and rooting from mature cotyledons of sunflower

Regeneration and development of shoots from sunflower cotyledons were improved by optimizing explant age, plant growth regulator concentrations, and duration of exposure to plant growth regulators during shoot initiation, development and rooting. Shoot initiation required only a brief exposure to auxin and cytokinin, and minimizing the duration of exposure to high levels of plant growth regulators improved shoot development. Rooting was improved in terms of both the number of shoots that rooted and the time required for rooting by incorporating activated charcoal in the lower layer of a 2-layer rooting medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

A liquid cytokinin pulse induces adventitious shoot formation from Douglas-fir cotyledons

Summary The effects of high-concentration, 2-h liquid pulses of N⁶-benzylaminopurine (BA) and thidiazuron (TD) on adventitious bud and shoot formation were tested in cotyledons of Douglas-fir (Pseudotsuga menziesii). Seedling age proved important; on average, cotyledons from the youngest seedlings formed 10-fold more buds than cotyledons from the oldest seedlings. Optimal cytokinin concentrations for the youngest cotyledons were 400 and 800 M BA, and 100 and 200 M TD. Shoots developed best from buds induced with 300, 400, and 800 M BA. Four gelling agents were tested; BRL agarose yielded more than three times the number of buds, and Gelrite nearly twice the number of buds, as either Sigma agar or Difco Bacto-Agar. One of the best treatments (400 M BA, agarose) yielded more cotyledons with buds, and more buds per cotyledon, than when cytokinins were incorporated into the growth medium.Abbreviations BA N⁶-benzylaminopurine - TD thidiazuron - SH Schenk and Hildebrandt (1972) - NAA 1-naphthaleneacetic acid Paper 2691 of the Forest Research Laboratory, Oregon State University, Corvallis. Mention of trade names or commercial products does not constitute endorsement or recommendation for use by the authors or Oregon State University.     

An efficient method for adventitious shoot regeneration from stem-segment explants of gypsophila

An efficient adventitious shoot regeneration procedure was developed for Gypsophila paniculata L. Using cultivar Arbel, shoot regeneration from the three upper internodes of the stem was monitored on MS media supplemented with different cytokinins (thidiazuron, benzyladenine, kinetin or zeatin) and an auxin (naphthaleneacetic acid). Thidiazuron was found to be the most efficient cytokinin, with up to 100% of the explants forming shoots, at an average of up to 19 shoots per explant being regenerated. The highest percentage of shoot formation was observed in the stem explants originating from the first internode, with all cytokinins tested. The adventitious origin of shoots regenerated from stem explants was confirmed by scanning electron microscopy. The regeneration procedure was found to be applicable to five other gypsophila cultivars (Perfecta, Golan, Gilboa, Flamingo and Tavor). Regenerating plants were successfully transferred to soil, and did not differ in flower color, size or shape from standard vegetatively propagated plants derived from cuttings. This revised version was published online in June 2006 with corrections to the Cover Date.     

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

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

Dark pretreatment improves adventitious shoot organogenesis from cotyledons of diploid watermelon

The influence of light incubation during embryo germination on shoot organogenesis from cotyledons of four diploid watermelon [Citrullus lanatus (Thumb.) Matsum. & Nakai cultivars was examined. Germinating embryos in darkness significantly improved the number of explants that produced harvestable shoots during the 6 week incubation period on shoot regeneration medium under a 16-h photoperiod. The percentage of explants with shoots more than doubled for `Crimson Sweet' and was about 1.5-fold greater for `Sweet Gem' and `Yellow Doll' when embryos were germinated in darkness. The percentage of explants with shoots was not significantly improved for `Minilee' by pretreating seedlings in darkness. This study demonstrates that optimal shoot regeneration can be obtained by germinating embryos in darkness before preparing cotyledon explants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Gelling agents, silver nitrate and sequestrene iron influence adventitious shoot and callus formation from Rubus leaves

Summary Blackberry (Rubus spp.) cultivars Chester Thornless, Kotata, Marion, and Navaho were evaluated for morphogenic potential using the top one to four leaves from in vitro-grown shoots. In each experiment adventitious shoots were regenerated via organogenesis without an intervening callus phase. Rooting was spontaneous when shoots were transferred to a medium without growth regulators or with indole-3-butyric acid (IBA). Three gelling treatments (agar, Gelrite, and a combination) calibrated for equal gel firmness [110 g/(1.1 cm)² π] did not affect explant regeneration or the number of shoots per explant, but did affect callus production. Significantly more callus (P≤0.05) was induced on regeneration medium (RM) with 10 μM N⁶-benzyladenine (BA), 0.5 μM IBA, and 0.19% Gelrite, than on medium containing either 0.71% agar, or the 0.35% agar/0.11% Gelrite combination. Explants on RM with 5 μM IBA produced significantly more callus, but fewer shoots, compared to zero or 0.5 μM IBA treatments for all gel treatments. Adding 200 mg l⁻¹ sequestrene iron [sodium ferric ethylene diamine di-(O-hydroxyphenylacetate)] at the first transfer onto RM induced more shoots per explant than the control, but did not influence the amount of callus produced. Sequestrene iron in the second transfer on RM significantly increased the regeneration (caulogenesis) frequency from 30 to 40% for ‘Marion’ and from 23 to 43% for ‘Kotata’. Silver nitrate significantly reduced callus production for all treatments, but did not affect the frequency of caulogenesis or the number of shoots per explant. Part of a thesis submitted by C. W. V. Tsao in partial fulfillment of the requirements for the MS degree. The use of trade, names in this publication does not imply endorsement by the US Department of Agriculture or Oregon State University.     

Regeneration of almond from immature seed cotyledons

Adventitious shoots were regenerated from immature cotyledons of four almond cultivars (`Ne Plus Ultra', `Nonpareil', `Carmel' and `Parkinson'). Open-pollinated fruit were collected from orchard-grown trees 100–115 days after full bloom. The proximal ends of the cotyledons were excised and the embryonic axes discarded. The effects of different concentrations of thidiazuron (TDZ) and indole-3-butyric acid (IBA) and the presence or absence of light for the first 7 days of culture were determined. Shoot regeneration rates were highest for cotyledons cultured for 8 weeks on Murashige and Skoog (MS) basal medium containing TDZ (10.0 M), followed by 4 weeks on medium without plant growth regulators. Regeneration levels were further improved if cotyledons were maintained in darkness for the first 7 days. IBA (0.5 M) significantly reduced the development of adventitious shoots. The frequency of cotyledons that developed adventitious shoots under the optimum tested conditions for `Ne Plus Ultra', `Nonpareil', `Carmel', and `Parkinson' were 80.0%, 73.3%, 100.0% and 86.7%, respectively.     

In vitro adventitious shoot regeneration from leaves of Prunus spp.

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

High frequency adventitious shoot induction and plant regeneration from leaves of statice

An efficient regeneration system for large-scale propagation of statice (Limonium altaica cv. Emille) was developed using leaves from mature plants. Leaf segments (5×5 mm sections) were cultured on Murashige and Skoog's medium supplemented with N⁶-benzyladenine (BA) and thidiazuron (TDZ) individually and in combination with indole-3-acetic acid (IAA) and α-naphthaleneacetic acid (NAA). Prolific direct adventitious shoot regeneration occurred on most of the media. The best response in terms of frequency of shoot regeneration (99.5%) and number of shoots per explant (112 shoots per explant) was observed on medium supplemented with 2.85 μM IAA and 1.14 μM TDZ. The shoots rooted easily on half strength MS medium and MS medium with indole-3-butyric acid. In vitro propagated plants could be transferred to soil with survival rates of more than 95%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Factors effecting adventitious shoot regeneration from leaf explants of quince (Cydonia oblonga)

A procedure for adventitious shoot regeneration from leaf explants of quince (Cydonia oblonga Mill.) using thidiazuron (TDZ) was developed. Excised leaves of cultures grown on Murashige and Skoog (MS) medium containing 5 M benzyladenine (BA) and 0.9% Gibco Phytagar were used. Several experiments were conducted to determine optimum concentrations of thidiazuron, -naphthaleneacetic acid (NAA) and sucrose. When the medium contained 1.5 M TDZ and 2.5 M NAA, 85% of the discs regenerated shoots with an average of eight shoots per leaf disc. An incubation period of three weeks in the dark was necessary for optimum shoot regeneration. Leaves excised from four to six-week-old cultures gave a higher percent shoot regeneration than leaves from cultures older than six weeks. Regeneration percentages were significantly reduced when sucrose concentration in the medium was less than 3%. A significantly higher percentage of shoots regenerated when leaf discs were placed on the regeneration medium abaxial side down as compared to the adaxial side.Regenerated shoots were cultured on MS medium containing 5 M BA and rooted on half-strength MS medium containing 10 M NAA. Rooted plantlets were acclimatized to greenhouse conditions for evaluation of any somaclonal variation. The importance of these findings are discussed in relation to in vitro improvement of plants.Abbreviations BA benzyladenine - MS Murashige & Skoog (1962) salt mixture - NAA -naphthaleneacetic acid - TDZ thidiazuron (N-phenyl-N'-1,2,3,-thiadiazol-5-ylurea) Approved for publication by the Director, West Virginia Agric. and For. Expt. Sta. as Scientific Article No.2346     

Improved shoot organogenesis from hypocotyl segments of lingonberry (<Emphasis Type="Italic">Vaccinium vitis-idaea</Emphasis> L.)

Summary An improved protocol for shoot regeneration from hypocotyl segments of seedlings from open-pollinated seeds of lingonberry (Vaccinium vitis-idaea L.) cultivars, ‘Ida’, ‘Splendor’, and ‘Erntesegen’, and a native clone from Newfoundland was developed. The effect of thidiazuron (TDZ) on adventitious bud and shoot formation from apical, central, and basal segments of the hypocotyl was tested. Highly regenerative callus was obtained from hypocotyl segments on modified Murashige and Skoog (MMS) medium containing 5–10 μM TDZ. A maximum of 10 buds and 12 shoots per apical segment for seedlings of cultivar ‘Ida’ regenerated on MMS containing 10 μM TDZ. Callus and bud regeneration frequency, callus growth, and number of buds and shoots per regenerating explant depended not only on the specific segment of the hypocotyl, but also on parental genotype. Inhibition of shoot elongation by TDZ was overcome by transferring shoot cultures to a shoot proliferation medium containing 1–2 μM zeatin. The optimal concentration of sucrose for shoot elongation was 20 gl⁻¹. Shoots were rooted ex vitro on a 2 peat: 1 perlite (v/v) medium after dipping in 0.8% indole-3-butyric acid, and rooted plants acclimatized readily under greenhouse conditions.     

Screening of soybean, Glycine max L.) Merrill, lines for somatic embryo induction and maturation capability from immature cotyledons

Summary Seventeen breeding lines of soybean, Glycine max (L.) Merrill, and cv. Jack, from relative maturity groups 0.3–7.5 were assessed for their ability to undergo somatic embryogenesis. The goal of this study was to determine which lines had high embryogenic capacity. We also sought to understand the relationship between relative maturity and embryogenesis. Embryos from immature cotyledons were initiated on solid MS medium with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D). Qualitative and quantitative measures of initiation, proliferation, differentiation, and maturation were recorded. The breeding lines differed significantly with respect to percent induction, number of embryos induced, and quality of induced embryos. After 1 mo, of proliferation, two early maturing lines, the control, Jack, and NK-5, had the best overall performance. High percent response of proliferating embryos was positively associated with lower maturity groups. Relatively high concentrations of 2,4-D (compared with that used in prolifcrating medium, e.g., 226 μM; 50 mg l⁻¹) in the initiating medium reduced numbers of embryo clusters per cotyledon initiated and percent initiation, and the concentration of 2,4-D affected the proliferation of somatic embryos in a breeding line-dependent manner. The breeding lines differed significantly in the time to produce mature somatic embryos. There was a positive correlation between immature embryo quality and number of differentiated somatic embryos produced. Retired.     

Immunolocalization of glyoxysomal malate synthase from soybean cotyledons (Glycine max L)

Summary— Malate synthase (MS; EC 4.1.3.2), an enzyme specific to the glyoxylate cycle, was studied in cotyledons of dark-grown soybean (Glycine max L) seedlings with light and electron microscopy techniques. Immunogold localization confirmed biochemical evidence that MS from soybean is a glyoxysomal matrix enzyme.     

Combination of thidiazuron and 2-isopentenyladenine promotes highly efficient adventitious shoot regeneration from cotyledons of mature sunflower (Helianthus annuus L.) seeds

Genetic improvement of sunflower (Helianthus annuus L.) through the use of biotechnological tools requires a reliable in vitro shoot regeneration system. Tissue culture protocols reported to date for sunflower suffer from low efficiency, poor reproducibility, genotype dependence and a tendency for flowering in vitro. The present study describes an efficient protocol system for shoot regeneration via direct adventitious shoot organogenesis from cotyledons of mature seeds of sunflower. About 169 media combinations comprising 12 different growth regulator combinations in various concentrations were assessed for induction of shoots from cotyledons derived from mature seeds and also from seedling tissues of 2?C20-day-old seedlings. Appearance of shoots from seedling tissues was sporadic and the frequency of shoot regeneration was low. Cotyledon explants from mature seeds were consistent with regard to frequency of adventitious shoot regeneration and number of shoots per explant. A high frequency (93.86?%) of adventitious shoot regeneration was obtained within 2?weeks of culture initiation on Murashige and Skoog (MS) medium supplemented with 9.84???M 2-isopentenyladenine (2-iP), 2.85???M indole-3-acetic acid (IAA) and 0.45???M thidiazuron (TDZ). Use of 2-iP in the shoot induction and elongation media prevented precocious flowering. Statistical analysis revealed significant effects of explant orientation, age of seedlings, and genotype on adventitious organogenesis. Maximum shoot regeneration was obtained when cotyledons from 0 and 1-day-old seedlings were placed with their adaxial surface in contact with the medium surface. The protocol developed was tested on 42 genotypes and found to be applicable to a wide range of genotypes. Histological studies indicated that the shoots originated predominantly through adventive organogenesis from the sub-epidermal and cortical regions.     

Adventitious shoot production from immature embryos of white clover

Cotyledonary-stage embryos of Haifa white clover, collected 13 days after cross-pollination, were induced to form adventitious shoots primarily from the hypocotyl region. The culture medium used for the production of adventitious shoots contained 5 M thidiazuron and 0.5 M -naphthaleneacetic acid. Numerous shoot meristems were produced within the first week, discrete shoots developed by week three, small plantlets by week eight, and whole plants in soil by week ten. 95–100% of all embryos, regardless of genotype, produced adventitious shoots within four weeks with an average production of 17.5 shoots per embryo. The majority of shoots (on average 77%) were easily converted to whole plants in soil. The white clover regeneration system described is prolific, rapid and effective on a large number of genotypes.Abbreviations BA N⁶-benzylaminopurine - MS medium Murashige & Skoog medium (1962) - NAA -naphthaleneacetic acid - thidiazuron N-phenyl-N-1,2,3-thiadiazol-5-ylurea     

High frequency of adventitious shoot regeneration from commercial cultivars of evening primrose (Oenothera spp.) using thidiazuron

Evening primrose (Oenothera spp.) is grown commercially for its seed oil that contains gamma linolenic acid (GLA), a valuable food supplement and pharmaceutical. There is considerable interest in the potential of genetic engineering to improve yields of GLA in evening primrose, and attention has focused on the current state of tissue‐culture knowledge in this species which is a prerequisite for genetic transformation. Published protocols for the regeneration of plants from leaf or cotyledon material of Oenothera spp. are available, but these prove unsatisfactory when applied to commercial cultivars used in this study. An efficient method for regenerating three commercial cultivars of evening primrose Rigel, Merlin and Vulcan was developed using thidiazuron (TDZ) as a growth regulator. Explants from one month old seedlings were cultivated in vitro; a large number of buds were induced directly from strips of leaves, cotyledons and stems when cultured on Murashige and Skoog (MS) basal medium containing TDZ and indole‐butyric acid (IBA). Shoots that were excised and placed onto MS basal medium, supplemented with IBA, rooted with 85–90% efficiency. Plantlets were transferred to soil after 6–8 wk. TDZ stimulated the regeneration process, and its effects were enhanced when combined with IBA or indole‐3‐acetic acid (IAA). The methods developed may be a useful advance toward improvement of this oil seed crop through genetic modification.     

Effect of thidiazuron on adventitious shoot regeneration from seedling explants of Nothapodytes foetida

Summary Regeneration of adventitious shoots from the medicinal plant Nothapodytes foetida (Weight) Sleumer Syn. Mappia foetida (family Ieacinaceceae) has been achieved using different seedling explants. Direct, regeneration of shoot buds was observed in Murashige and Skoog's (MS) basal medium supplemented with various concentrations of thidiazuron. The optimum levels of thidiazuron concentrations were 0.91–4.45 μM. Leaf explants formed more shoots followed by hypocotyls or cotyledons. The shoot buds elongated and rooted on MS basal medium with N⁶-benzyladenine (0.88–2.22 μM) and indole-3-butyric acid (0.49 μM).     

Culture of cotyledons of Douglas-fir on a medium for the induction of adventitious shoots induces rapid changes in polypeptide profiles and mRNA populations

Cotyledons of 3- to 4-week-old seedlings of Douglas-fir [ Pseudotsuga menziesii (Mirb). Franco] were treated with shoot induction medium (SIM) containing 5 μ M 6N-benzylaminopurine (BAP) and 5 n M naphthaleneacetic acid (NAA). Fresh weight, dry weight and soluble protein levels were not altered within the first 48 h of SIM treatment. SIM-treated cotyledons were labelled in vivo with ³⁵SO₄^2-, and TCA-insoluble proteins were analyzed electrophoretically by a 2-dimensional system consisting of non-equilibrium electrophoresis (NEPHGE) followed by sodium dode-cylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). A basic polypeptide with a relative molecular weight of 14.5 kDa was detected 32 h after induction and after 48 h a number of polypeptides in the 14 to 35 kDa range were induced. Translation products of poly-A⁺ RNA isolated from cotyledons treated with SIM for 4, 16, 32 and 48 h were analyzed by using 2-dimensional NEPHGE-SDS-PAGE. Both qualitative and quantitative differences in the translation products were observed at all time points investigated. Expression of a specific RNA coding for a 30 kDa polypeptide was demonstrated as early as 4 h after culture on SIM. This RNA was also present at 16 h, but decreased with longer SIM treatments. Thus, culture of excised cotyledons on a medium inducing the formation of adventitious shoots results in rapid quantitative and qualitative changes in the polypeptide composition and translatable RNA population prior to morphological evidence of shoot induction.     

Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi

Camellia nitidissima Chi (Theaceae) is a world-famous economic and ornamental plant with golden-yellow flowers. It has been classified as one of the rarest and most endangered plants in China. Our objective was to induce somatic embryogenesis, shoot organogenesis and plant regeneration for C. nitidissima. Three types of callus (whitish, reddish and yellowish) were induced from immature cotyledons on improved woody plant medium (WPM) with different plant growth regulators (PGRs). Among the callus, whitish callus was induced by 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and reddish and yellowish callus were induced by strongly active cytokinins, thidiazuron (TDZ) or 6-benzylaminopurine (BAP), singly or combined with weakly active auxin, α-naphthaleneacetic acid (NAA). The embryogenic callus could differentiate into somatic embryos, nodular embryogenic structures (large embryo-like structures) or adventitious shoots depending on the PGR used in WPM. BAP was best for adventitious buds and zeatin was best for somatic embryogenesis while kinetin (Kt) was best for the formation of nodular embryogenic structures. The three regeneration pathways often occurred in the same embryogenic callus clumps. Most shoots (80.0%) developed roots in WPM supplemented with 24.6 μM IBA and 0.3 μM NAA while 47.5% of somatic embryos could germinate directly and develop into plantlets on induction medium supplemented with 0.9 μM BAP and 0.1 μM NAA. The nodular embryogenic structures could be sub-cultured and cyclically developed in one of two differentiation pathways: shoot organogenesis or somatic embryogenesis. Plantlets derived from shoot buds rooted and somatic embryos germinated when transplanted into soil in a greenhouse; 66.7% of plantlets from shoot culture and 78.6% of plantlets from somatic embryos survived after 8 weeks’ acclimatization.     

In vitro adventitious bud regeneration from internode segments of beech

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