Propagation of the tropical tree,Leucaena leucocephala K67, by in vitro bud culture

A tissue culture method is described for clonal multiplication of Leucaena leucocephala K67 using single lateral bud explants from 2–3 m tall greenhouse grown trees. N-6 benzyladenine (BA: 3.0 mg.1^-1) and napthaleneacetic acid (NAA: 0.05 mg.1^-1) in Murashige & Skoog's (MS) medium were found to be best suited for multiple shoot differentiation in 4–5 week old cultures. Analysis of variance of the main treatment effects of BA and NAA on shoot parameters showed that BA significantly (P=0.001) affected shoot development while NAA did not. A shoot multiplication rate of 22±3.63 shoots per bud explant was obtained in 150 days on 1/2 strength MS medium with 3.0 mg.1^-1 BA and 0.05 mg.1^-1 NAA. Shoots developed adventitious roots within 15 days in 1/2 strength MS medium containing indole-3-butyric acid (IBA: 3.0 mg.1^-1) and Kinetin (0.05 mg.1^-1). Eighty percent of the transplanted plantlets are being grown in greenhouse conditions.     

The Influence of NAA, BA and Temperature on Shoot and Root Development from Begonia×cheimantha Petiole Segments Grown in vitro

The effect of exogenously supplied NAA and BA on the shoot and root formation in isolated petiole segments of Begonia×cheimantha was determined in vitro on a modified White medium at a constant temperature of 24°C. The best development of normally appearing plants was obtained on media containing 0.01 mg × 1^?1 of NAA and 0.5 to 1.0 mg × 1^?1 of BA. Lower concentrations of BA yielded no shoots, higher concentrations promoted shoot formation, but the shoots were abnormal with malformed leaves. Lower concentrations of NAA resulted in poorer survival rate and no roots, with higher concentrations of NAA many roots developed, but these were thickened and their longitudinal growth inhibited. Temperature proved to be of utmost importance for the induction of shoot formation. Thus significantly fewer shoots were formed at the higher temperature (25°C) than at lower temperatures (15 to 20°C). Temperature immediately after initial transfer was of greatest importance: 25°C, during the first week followed by low temperature, produced very few shoots.     

Rapid and stable propagation ofOrnithogalum umbellatum L. in long term culture

Callus cultures were raised from bulb scale segments ofOrinthogalum umbellatum L. (Liliaceae), on a Murashige and Skoog (1962) medium (MS) with 8 mg/l naphthaleneacetic acid (NAA). Bulbous shoots developed from calli after 2 months using MS medium with 2 mg/l NAA and 0.5 mg/l N⁶ - benzyladenine (BA). Shoots were also induced directly from scales of regenerated bulb used as secondary explants on MS medium supplemented with 0.5 mg/l BA. Shoots developed roots in 1/2 - strength MS medium. Regenerants multiplied rapidly in 1/2-MS liquid medium. Chromosome instability was reduced in callus grown on 2 mg/l NAA compared to callus grown on 8 mg/l NAA. Callus retained regeneration potential for 5 years in this modified MS medium. The chromosome analysis of regenerants dervied from callus, even from long term culture of 5 years, revealed only diploid cells with normal karyotype comprising 2n=46 chromosomes. Stable nature of callus and regenerants were further confirmed by cytophotometry. This procedure can be applied for securing stable regenerants on a mass scale inO. umbellatum.     

Micropropagation of Spilanthes acmella Murr.

Multiple shoots of Spilanthes acmella Murr. were induced from hypocotyl segments obtained from 1-week-old seedlings on Murashige and Skoog's (MS) medium containing benzyladenine (BA), isopentenyl adenine, and naphthaleneacetic acid (NAA). High frequency shoot proliferation (95 %) and maximum number of shoots per explant (10 ± 0.6) were recorded with 0.5 mg dm^–3 BA in combination with 0.1 mg dm^–3 NAA. A proliferation was achieved by repeatedly subculturing the nodal segments on shoot multiplication medium. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing indole-3-butyric acid (1.0 mg dm^–3). 95 % of the plantlets were successfully acclimatized and established in soil. Transplanted plantlets showed normal flowering without any morphological variation.     

Optimisation of plantlet regeneration from leaf and nodal derived callus of <Emphasis Type="Italic">Vanilla planifolia</Emphasis> Andrews

An indirect in vitro plant regeneration protocol for Vanilla planifolia has been established. Juvenile leaf and nodal segments from V. planifolia were used as explants to initiate callus. Nodal explants showed better callus initiation than juvenile leaf explants, with 35.0% of explants forming callus when cultured on Murashige and Skoog (MS) basal medium supplemented with 2.0 mg/l 1-naphthylacetic acid (NAA) and 1.0 mg/l 6-benzyladenine (BA). Almost 10.0% of juvenile leaf explants were induced to form callus on the MS basal medium containing 2.0 mg/l NAA and 2.0 mg/l BA, whereas no callus formed in the presence of any concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and BA. After 8 weeks, callus generated was transferred to MS basal medium containing 1.0 mg/l BA and 0.5 mg/l NAA. A mean number of 4.2 shoots per callus was produced on this medium, with a mean length of 3.8 cm after 8 weeks of culture. Roots formed on 88.3% of plantlets when they were cultured on MS medium supplemented with 1.0 mg/l NAA, with a mean length of 4.4 cm after 4 weeks of culture. Of the rooted plantlets, 90.0% survived acclimatisation and were making new growth after 4 weeks.     

Transformation of sweetgum via microprojectile bombardment of nodule cultures

Summary A sweetgum (Liquidambar styraciflua) nodule culture system was developed and integrated with genetic transformation by microprojectile bombardment. Nodule cultures were established from seedling hypocotyls and proliferated in liquid medium containing 0.1 mg (0.45 μM) thidiazuron (TDZ) per 1 and 0.01 mg (0.045 μM) 2,4-dichlorophenoxyacetic acid (2,4-D) per 1. Shoots differentiated from the nodules in liquid media containing (per 1) 1 mg (4.4 μM) benzyladenine (BA), 0.5 mg (2.2 μM) BA, and 0.01 mg (0.054 μM) naphthaleneacetic acid (NAA), or 0.5 mg BA, 0.01 mg NAA, and 0.05 mg (0.23 μM) TDZ under the light. Differentiating shoots required 4 wk of dark treatment for further development on semisolid medium containing 1 mg BA per 1. Elongated shoots were harvested and the basal ends were soaked in a solution containing 10 mg (49.2 μM) indole-3-butyric acid (IBA) per 1 before being planted in potting mix for ex vitro rooting. Roots formed and leaves expanded in 2 wk. Sweetgum nodules were stably transformed by microprojectile bombardment with a 7.4-kb plasmid, pTRA 140, harboring CaMV 35S-HPH and CaMV 35S-GUS. Evidence that nodules growing in the presence of hygromycin B were stably transformed was provided by polymerase chain reaction analysis and β-glucuronidase activity. Sweetgum shoots differentiated in liquid medium in the presence of hygromycin B. Shoots transferred to solid medium lacking hygromycin B elongated and displayed β-glucuronidase activity in their expanding leaves and stems. Southern analysis confirmed the presence of the GUS gene in nodules and shoots. Transgenic shoots initiated roots and showed leaf expansion 2 wk after being planted in potting mix.     

Rhizome induction and plantlet regeneration of Cymbidium goeringii from flower bud cultures in vitro

Apical flower buds of Cymbidium goeringli Reichenbach fil. (ca 2 mm long) exeised from infloreseences (ca 5 cm long) were explanted on modified Murashige & Skoog medium (=MS medium) supplemented with N⁶-benzyladenine (BA) and -naphthaleneacetic acid (NAA). Within 107 days of culture, swelling growth, chlorophyll synthesis, and subsequent rhizome differentiation were observed. MS medium containing 0.1 mg l^-1 BA and 10 mg l^-1 NAA was found to be optimal for initiating rhizome development and subsequent plantlet regeneration.Explants cultured on MS medium supplemented with 1 mg l^-1 NAA alone formed a mass of rhizome branches. Multiple shoots of rhizome branches were induced from apical segments when rhizomes were transferred to MS medium containing 0.1 mg l^-1 BA and 10 mg l^-1 NAA.Abbreviations NAA -naphthaleneacetic acid - BA N⁶-benzyladenine     

红豆草愈伤组织原生质体再生植株

红豆草又名驴豆,是一种耐旱性较强的多年生豆科牧草。其蛋白质含量高,作为饲料适口性好,并且不发生臌胀病,因此被看作是进行体细胞杂交,改良其它豆科牧草的理想亲本。然而要实现这一目标,必须掌握原生质体培养成再生植株的最适条件和规律。曾有过叶片原生质体培养的报道。本研究用我国西北地区种植的红豆草愈伤组织分离原生质体,并培养形成再生植株。     

金鱼草下胚轴不同部位切段形态发生能力的研究

金鱼草（ＡｎｔｉｒｈｉｎｕｍｍａｊｕｓＬ．）下胚轴不同部位切段的形态发生能力有很大差异。在外源添加ＢＡ,ＮＡＡ的情况下,金鱼草下胚轴近基部切段培养物的芽、根发生明显高于其上各部位切段,以近基部切段为外植体培养时,其形态发生能力以ＢＡ和ＮＡＡ配合为好,最适剂量为ＢＡ１０ｍｇ／Ｌ＋ＮＡＡ０１５ｍｇ／Ｌ。     

深山含笑的组织培养和快速繁殖

组织培养深山含笑的实验结果表明：利用休眠芽和种子萌发时实生苗的上胚轴及下胚轴为外植体均能诱导出愈伤组织和不定芽,其中无菌实生苗的上胚轴最易诱导出不定芽,无菌实生苗的下胚轴最易诱导出愈伤组织。外体植体在ＭＳ＋１．０－２．０ｍｇ Ｌ＾－１ ２,４－Ｄ培养基上只产生愈伤组织;在ＭＳ＋３．０ｍｇ Ｌ＾－１ ＢＡ＋０．２ｍｇ Ｌ＾－１ ＮＡＡ培养基上产生较多的不定芽和较多的愈伤组织;在ＭＳ＋２．０ｍｇ Ｌ＾     

Root and shoot initiation by leaf,stem, and storage root explants of sweet potato

Explants from stem, leaf, and storage root tissue of sweet potato (Ipomoea batatas L.) cv. Jewel, were placed on media conaining 0.1, 1.0, and 10 mg/1NAA with 0.1, 1.0, or 10 mg/1BA in a factorial experiment. Some callus formed in every treatment, but the best callus growth was on media containing 1.0 mg/1NAA and 10 mg/1BA. Roots formed over a range of treatments but were most prolific on the medium containing 1.0 mg/1NAA and 0.1 mg/1BA. Some de novo formed roots subsequently produced shoot buds in culture. Shoot formation increased the longer the original explants remained in culture without subculture. Roots could be subcultured indefinitely on agar solidified medium, but shoot regeneration did not occur after two subcultures. Shoot formation was greatest when the roots were subcultured on medium containing 1.0 mg/NAA and 0.1 mg/1BA. The cultivar Caromex followed the same regeneration pathway, but the number of shoots formed was considerably less. Regeneration in both Jewel and Caromex explants was enhanced by light.Paper No. 8292 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned.This work was done as a partial requirement for the M.S. degree at North Carolina State University.     

Plant Regeneration from Protoplasts of Coriandrum sativum

Calli produced from stem segments of seedling of Coriandrum satwum which were cultured on MS agar medium containing NAA 1.0mg/L. The embryogenic cell colony suspension was estabilished on MS liquid medium containing NAA 1.0mg/L%2,4-D 0.2mg/L+BA 0.5 mg/L. The cell suspension culture was used for protoplast preparation. Protoplasts were obtained in the enzyme mixture containing 2.0% Onozuka R-10, 1.0% pectinase, 0.5% snailase, 0.5% dextran sulfate potassium Salt, 0.6mol/L mannital CPW solution at pH 5.8 and 25℃. Cultured in a KM8P liquid medium containing NAA 1.0mg/L+2,4-D 0.2mg/L+6-BA 0.5 mg/L, glucose 0.4mol/L and CM 20mi/L; the protoplasts entered the stage of derision after three days, cell clusters formed in 10 days and calli formed after about 50 days. When the calli were transferred to MS agar medium containing many growth substances, they differentiated into embryoids, and then developed into plantlet with many green leaves and roots on the 1/2 MS agar medium.     

唐菖蒲花色突变株开花后子房组织培养与植株再生的研究

以唐菖蒲花色突变株开花后的子房为外植体,接种于MS+1.0mg/L BA+0.01-0.1mg/L NAA或MS+1.0mg/LBA+1.0mg/L KT+0.01-0.1mg/LNAA的培养基中,从膨大的组织表面直接诱导不定芽.膨大的组织或带不定芽的组织每隔周转入1/2MS_1.0mg/L BA+0.01-0.1mg/L ANN培养基中,不断地诱导产生不定芽,分化的不定芽转入不含激素的1/2MS培养基中,形成幼苗、然后生根,最后可形成小球茎.幼苗转入1/2MS+0.1mg/L NAA培养基中,幼苗生根,再可形成小球茎.1个唐葛蒲突变株子房,经6—7个月培养,获得了上千株试管苗.     

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.     

In vitro plant regeneration from leaf explants of Ophiorrhiza japonica

An efficient in vitro plant regeneration system from leaves of Ophiorrhiza japonica Blume was established for the first time. Callus formation rate was more than 90.4 % from leaf segments on Murashige and Skoog (MS) supplemented with either α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzyladenine (BA). The highest shoot regeneration (78.9 %) was achieved on MS medium containing 2.0 mg dm⁻³ BA and 0.2 mg dm⁻³ NAA, with an average of 9.4 shoots developed per leaf segment. Shoot regeneration was also improved when the leaf explants were cultured in MS basal medium supplemented with 0.5 % (m/v) polyvinylpyrrolidone (PVP). The leaf explants from seedlings with age of about 18–27 d showed the highest shoot regeneration. The regenerated shoots were rooted on half-strength basal MS medium supplemented with 0.5 mg dm⁻³ indole-3-butyric acid (IBA), which averagely produced 24.8 roots per shoot. The plantlets were transferred to soil, where 100 % survived after 1 month of acclimatization.     

Plant regeneration through callus cultures derived from immature-cotyledon explants of oleaster (Elaeagnus angustifolia L.)

Efficient plant regeneration was achieved from callus derived from immature-cotyledon explants of oleaster (Elaeagnus angustifolia L.). Calli were obtained on MS media containing 3% sucrose and different concentrations of TDZ. The highest rate of green, compact and nodular callus was formed on MS medium supplemented with 1 mg/l of TDZ. Shoot organogenesis was achieved when the callus was transferred onto MS media containing 3% sucrose and BA alone (05–4 mg/l) or BA (0.5 and 1 mg/l) combined with NAA or IAA (0.5 and 1 mg/l). Maximum organogenesis was obtained with 1 mg/l BA in combination with 0.5 mg/l NAA. Rooting of the shoots was achieved on MS medium supplemented with 0.2 mg/l IBA. Regenerated plantlets were acclimatized and successfully transplanted to soil.     

Direct somatic embryogenesis and plantlet regeneration from seedling leaves of winged bean,Psophocarpus tetragonolobus (L.) DC

Excised seedling leaf segments of winged bean [Psophocarpus tetragonolobus (L.) DC.] underwent direct somatic embryogenesis under appropriate incubation conditions. Initiation and development of the somatic embryos occurred using a two-step culture method. The culture procedure involved incubation for 28 days on MS basal medium supplemented with 0.1–0.5 mg/l NAA and 1.0–2.0 mg/l BA (induction medium) before transfer to MS medium supplemented with 0.1 mg/l IAA and 2.0 mg/l BA (embryo development medium). The initial exposure to low levels of NAA coincident with high levels of BA in the induction medium was essential for embryogenic induction. Maximum embryogenesis (43.3%) was obtained with 0.2 mg/l NAA and 2.0 mg/l BA, and at least 14 days on induction medium were required prior to transfer to the embryo development medium. The conversion frequency of cotyledonary embryos was 53.3% upon culture on MS medium containing 0.1 mg/l ABA for 7 days followed by transfer to MS medium supplemented with 0.1 mg/l IBA and 0.2 mg/l BA. Following conversion, the regenerated plantlets were transferred to soil and showed normal morphological characteristics.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - ABA abscisic acid     

马可波罗百合的组织培养和离体快繁

以马可波罗百合的鳞片、茎段和茎尖为外植体 ,成功建立了快速无性繁殖系。诱导鳞片产生丛芽的最佳培养基为 :MS +0 .3～ 0 .8mg/LBA +0 .0 5mg/LNAA ;茎尖的最佳诱导培养基为 :MS +2mg/LBA +0 .0 5mg/LNAA ;茎段的最佳诱导培养基为 :MS +0 .8mg/LBA +0 .1mg/LNAA。丛芽增殖培养基 :MS +0 .2mg/LBA +0 .1mg/LNAA和MS +0 .2mg/LBA +0 .1mg/LIAA。生根诱导最佳培养基为 1 /2MS +0 .2mg/LKT +0 .0 5～ 0 .5mg/LNAA。     

Production of plantlets from Aegle marmelos nucellar callus

Techniques have been developed for the regeneration of Aegle marmelos from nucellar explants. Slow-growing calli were induced from nucellar explants excised from 90–120 d-old developing fruits. The medium consisted of Murashige and Skoog formulation containing 40 g/l sucrose, 400 mg/l casein hydrolysate, 5 mg/l 1-naphthaleneacetic acid and 1 mg/l kinetin. The basal medium with high concentration (1–5 mg/l) of N6-benzyladenine (BA) and low concentration (0.1 mg/l) of NAA was suitable for regeneration of shoots from 3-month-old calli. Addition of 1 mg/l gibberellic acid (GA3) favoured shoot growth. Callus-derived shoots produced roots and developed into plantlets when transferred to half-strength MS medium supplemented with 0.5 mg/l indole-3-butyric acid (IBA) and 0.5 mg/l NAA. Approximately 5 months were required for the full regenerative process.     

绞股蓝茎尖组织培养和植株再生研究

以绞股蓝茎尖作为外植体,接种于ＭＳ＋１ｍｇ／Ｌ ＢＡ＋０．１ｍｇ／Ｌ ＮＡＡ的培养基中,茎尖逐渐形成芽苗,芽苗分离后接种于ＭＳ＋１ｍｇ／Ｌ ＢＡ＋０．１ｍｇ／Ｌ ＮＡＡ的培养基中进行增殖培养,同时从增殖的芽苗上不断地诱导出幼苗,将幼苗分离转入不含激素的ＭＳ培养基中,幼苗生根,形成完整的植株。