Efficient regeneration system from wheat leaf base segments

Efficient plant regeneration system from leaf base segments of wheat (Triticum aestivum L.) was developed. The factors affecting the callus formation and regeneration capacity of leaf segments of two genotypes; Bobwhite and Pavon 76, were investigated. The highest number of somatic embryos (SE) was obtained on Murashige and Skoog medium supplemented with 2 mg dm⁻³ 2,4-dichlorophenoxyacetic acid + 1 mg dm⁻³ naphthalenacetic acid (14.7 SE per segment). Highest frequency of embryogenic callus (96 %) and somatic embryo formation (24.3 SE per segment) were achieved in the first segments. The highest plantlet regeneration was obtained after transfer of embryogenic calli to regeneration medium supplemented with 1 mg dm⁻³ kinetin (6.3 plantlets per segment).     

Establishment of a highly efficient regeneration system from leaf base segments of oat (Avena sativa L.)

A reliable and efficient protocol for the regeneration of fertile plants derived from leaf base segments of young in-vitro-grown oat seedlings has been developed successfully. Callus induction and shoot regeneration were achieved when the basal region of young seedlings was cultured on auxin-containing medium. Callus induction efficiencies as well as regeneration frequencies were correlated with the developmental stage and the genotype of the explants. In five different genotypes of oat, an average of 25 plants per explant could be produced and for the most responsive genotype more than 50 regenerants per explant could be regenerated reproducibly. This high regeneration potential makes oat leaf bases a very attractive target for transformation. Received: 6 May 1997 / Revision received: 10 August 1997 / Accepted: 15 September 1997     

马铃薯叶片高效再生体系的建立

以4个马铃薯栽培品种为试材,进行了叶片离体再生研究,结果表明:东农303、鄂1号叶片愈伤组织诱导的最佳培养基为MS+6-BA2.5mg·L-1+NAA0.2mg·L-1;费乌瑞它为MS+6-BA2.0mg·L-1+NAA0.1mg·L-1;夏波帝为MS+6-BA2.0mg·L-1+NAA0.2mg·L-1,愈伤组织的诱导率均可达100%.诱导不定芽分化的最佳培养基分别是:费乌瑞它、鄂1号为MS+6-BA2.5mg·L-1+GA35.0mg·L-1;东农303为MS+6-BA1.0mg·L-1+IAA0.1mg·L-1+GA32.5mg·L-1;夏波帝为MS+6-BA2.5mg·L-1+IAA0.5mg·L-1+GA32.5mg·L-1,其不定芽分化率分别达94.3%、100%、100%和90%.     

A highly efficient plant regeneration system through multiple shoot differentiation from commercial cultivars of barley (Hordeum vulgare L.) using meristematic shoot segments excised from germinated mature embryos

A fast and highly efficient short-term in vitro regeneration system was developed for barley (Hordeum vulgare L.) based on readily available explants. Clumps of multiple shoots and buds suitable for transformation were obtained 9–10 weeks after culture initiation from model and current commercial cultivars. Meristematic shoot segments (MSSs) excised from mature embryo-derived seedlings and subsequently cultured on MS-based medium containing 2 mg/l Picloram and 3 mg/l thidiazuron (TDZ) differentiated up to ten multiple shoots after 3–4 weeks with no or very little callus formation. Sectors of the already multiplied shoot clumps were further multiplied on proliferation-maintenance medium containing 2 mg/l Picloram and 2.5 mg/l TDZ. Biweekly subcultures resulted in a continuous process of multiplication of these highly differentiating green sectors without any loss of morphogenic potential. The differentiated small shoots and shoot buds gave rise to normal shoots on medium with 0.1 mg/l Picloram and 1 mg/l TDZ. After rooting on basal medium with 0.5 mg/l or 1 mg/l IBA the plants were transferred to soil and showed normal growth and fertility compared to the seed-grown plants. All of the genotypes tested formed multiple shoots. The percentage of relative MSS multiplication was 63–83%, and the average number of multiplied shoots per MSS ranged from 16 to 34 among the genotypes after 9–11 weeks.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - Dicamba 3,6-Dichloro-2-methoxybenzoic acid - IBA Indole-3-butyric acid - MSS Meristematic shoot segment - NAA -Naphthaleneacetic acid - Picloram 4-Amino-3,5,6-trichloropicolinic acid - TDZ Thidiazuron     

Oat leaf base: tissue with an efficient regeneration capacity

Summary An efficient short term regeneration system using seedling derived oat (Avena sativa) leaf tissue has been developed. Callus derived from the leaf base showed a higher response of plant regeneration than callus initiated from mesocotyls and more mature parts of the leaves. A correlation between the nuclear DNA content of the donor material, as analysed with flow cytometry, and its ability to form callus was observed. Somatic embryogenesis was histologically recognised from callus derived from tissue close to the apical meristem. Plant regeneration media with various concentrations of auxin were tested. Callus from three different cultivars had a similar regeneration potential with an optimal regeneration frequency of 60%. About 2 months after inoculation regenerated plantlets could be moved to a greenhouse for cultivation.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 6-diamidino-2-phenylindole - IAA indole-3-acetic acid - KT kinetin - MS Murashige and Skoog's medium - NAA naphthalene acetic acid     

An efficient genotype-independent method for regeneration of potato plants from leaf tissue

The regeneration of plants from leaf explants of a number of potato cultivars using a number of published one-, two- and three-step methods was assessed. A method using a pretreatment with high levels of auxin and cytokinin coupled with silver thiosulphate in the regeneration medium proved the most rapid and efficient for the eight cultivars examined.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IAA indoleacetic acid - MS Murashige and Skoog - NAA naphthaleneacetic acid - STS silver thiosulphate     

Direct plant regeneration from cultured young leaf segments of sugarcane

Young leaf segments (1.0–1.5 cm) excised from spindle explants of three commercial sugarcane varieties viz. Co J 64, Co J 83 and Co J 86 were cultured on different media compositions based on Murashige and Skoog (1962) salts. Cultured explants exhibited swelling followed by direct shoot regeneration on media containing naphthaleneacetic acid, in all the three varieties. Highest frequency 83.12% shoot regeneration occurred on Murashige and Skoog medium supplemented with naphthaleneacetic acid (5.0 mg l⁻¹) and kinetin (0.5 mg l⁻¹) in variety Co J 83. Medium devoid of naphthaleneacetic acid and supplemented with only kinetin did not induce direct shoot regeneration in any of the varieties thus tried. Subsequently profuse rooting of shoots was observed on the same medium and complete plantlets were recovered within 6 weeks. The plantlets were hardened and transferred to soil. Tissue culture derived field-grown plants were normal and exhibited faster growth and better tillering. This developed single step method of direct plant regeneration can be used for rapid mass cloning and genetic transformation of sugarcane.     

In vitro regeneration of Rubus from leaf and stem segments

Various media, sourees of explant and Rubus genotypes of diverse origin were assessed for their ability to regenerate whole plants in vitro. Regenerants were produced from leaf discs and from both peeled and unpeeled internodal stem segments but not from epidermal peelings. Hormone type and concentration, amount of sucrose, absence of activated charcoal, presence of light and for leaf discs their orientation with adaxial surface uppermost were factors crucial for plantlet regeneration, and genotypes differed considerably in their capacity to regenerate.     

Enhancement of plant regeneration from embryogenic callus of commercial barley cultivars

Genotypic restrictions on plant regeneration from cultured cells have hindered the genetic transformation of most barley cultivars. Optimizing culturing protocols for specific cultivars of commercial interest may facilitate their genetic transformation. Plant regeneration from embryogenic callus of `Harrington', `Morex', and `Hector' as affected by certain protocol modifications was examined in replicated experiments. Regeneration was improved for all cultivars by separately autoclaving certain components of the culture media and by reducing the amount of embryogenic callus cultured per petri dish. Regeneration improvements in response to various concentrations of copper and 2,4-dichlorophenoxyacetic acid were more genotype specific. This study suggests that the development and use of genotype-specific protocols can enhance plant regeneration. Enhancements in plant regeneration are expected to facilitate the transformation of commercial barley germplasm. Received: 11 August 1997 / Revision reveived: 2 March 1998 / Accepted: 20 March 1998     

Somatic embryogenesis and plantlet regeneration from leaf segments of Piper colubrinum

Direct somatic embryogenesis from in vitro-cultured leaf segments of multiple disease-resistant pepper, Piper colubrinum Link is reported. Somatic embryos were initiated on Murashige and Skoog's (1962) basal medium containing 2.2 μM benzyladenine+0.46 μM kinetin and multiplied profusely through secondary embryogenesis on the same medium. Some 91% somatic embryos converted into plantlets on MS medium supplemented with 4.4 μM benzyladenine+0.23 μM kinetin and plantlets developed on half-strength MS+2.4 μM indole-3-butyric acid. Plantlets were hardened, transferred to soil, and 100% of plants survived. Various developmental stages of somatic embryogenesis were studied using histological methods. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plant regeneration from barley cultivars grown in Spain

Thirty-two barley cultivars grown in Spain, 18 of the two-row type and 14 of the six-row type, were screened for plant regeneration from cultured immature embryos. Although there was much variation in regeneration capacity among the cultivars, plants were obtained from all cultivars except Almunia. No statistical differences were found in the percentage of regeneration between two- and six-row types. The influence of the auxins 2,4-dichlorophenoxyacetic acid, dicamba, and picloram on the induction and maintenance of embryogenesis and regeneration capacity after 3–4 months in culture, were evaluated for cultivars Cobra, Hop and Reinette. Hop had the highest rates of maintenance of embryogenic capacity and plant regeneration. The medium containing dicamba gave the best embryogenic callus induction, maintenance and regeneration. Five regeneration media, differing in growth regulators and micronutrient composition, as well as partial desiccation of the calli before regeneration, were tested. The regeneration medium containing 10 μm copper sulfate gave the best results. Regeneration frequencies after 3–4 months in culture of cultivar Hop were raised from 59.5 to 93.7% in this medium. Silver nitrate and partial desiccation of the calli also enhanced plant regeneration, but the medium containing 10 μm of silver nitrate reduced root formation. Received: 30 October 1997 / Revision received: 3 April 1998 / Accepted: 17 April 1998     

An efficient method for in vitro regeneration from immature inflorescence explants of Canadian wheat cultivars

Fertile, green plants were regenerated from immature inflorescence explants from each of four Canadian wheat cultivars. The cultivars were representative of four classes of Canadian wheat. Explants from immature inflorescences of three size ranges were cultured on two types of media: MSI/MSR, which contains 1650 mg l^-1 NH₄NO₃and sucrose as a carbon source, and BII/BIR, which contains 250 mg l^-1 NH₄NO₃and maltose as a carbon source. Regeneration from all cultivars was significantly better on BII/BIR media than on MSI/MSR media. On BII/BIR media, `AC Karma', `Plenty', and `Fielder' gave the highest number of shoots per 10 explants, where the explants were derived from immature inflorescences 5.1 to 10.0 mm in length. 'Columbus' did not regenerate on MSI/MSR medium, and regenerated poorly on BII/BIR medium. Differences were found between cultivars with regard to the number of regenerant plants produced with the best treatments: `Plenty' produced 16.1 shoots per 10 explants, `AC Karma' 12.4, `Fielder' 6.4, and `Columbus' 2.2.     

Adventitious shoot regeneration from leaf explants of southern highbush blueberry cultivars

Protocols were developed to optimize adventitious shoot regeneration from four southern highbush blueberry cultivars. Leaf explants from 6 week-old shoots of the four cultivars were excised and cultured on woody plant medium each containing thidiazuron (4.54 or 9.08 μM), zeatin (18.2 μM), or zeatin riboside (5.7 or 11.4 μM) either separately or in combination with α-naphthaleneacetic acid at 2.69 μM. Optimum medium for shoot regeneration was genotype-dependent. Efficient regeneration was obtained at frequencies of 88.9% for ‘Jewel’, 87.8% for ‘Emerald’, 53.3% for ‘Jubilee’ and 87.8% for ‘Biloxi’. Leaf explants of newly developed shoots from the cultures having undergone five subcultures had higher regeneration frequencies than those having undergone two subcultures. Regenerated shoots, 80–100% for each cultivar, rooted in 8 weeks after transplantation to soil. The regeneration systems described have potential use in genetic transformation of southern highbush blueberry cultivars.     

Efficient regeneration of fertile barley plants from callus cultures of several Nordic cultivars

The regeneration potential of three major Estonian barley cultivars was tested and compared to that of the Finnish cultivar Kymppi. Two different regeneration systems were used. The first was characterized by the high maltose concentration (60 g l(-1)) and by the use of 2,4D together with two different combinations of amino acids in the callus induction medium followed by the regeneration medium containing BAP (2 mg l(-1)) and 2,4D (0.2 mg l(-1)). The second exploited callus induction medium that contained Dicamba, lower concentrations of maltose (30 g l(-1)) and higher concentrations of myo-inositol and thiamine and different set of amino acids and regeneration medium that contained higher concentrations of Cu2+ and inorganic nitrogen accompanied by lower concentrations of NH4+ and BAP, when compared to the first regeneration system. The second regeneration system used produced significantly higher rates of callus induction, callus growth and regeneration of plantlets. However, it yielded also many albino plants (up to 51%), whereas the first regeneration system used did not produce practically any albino plants. No major genotype-dependent differences were observed in comparison between two regeneration systems - in both systems higher regeneration potential of Anni, Elo and Kymppi contradicted to the low regeneration potential of Teele. It is concluded that the continuous somatic embryogenesis on the regeneration medium allows the regeneration of many plants from the same callus over long periods of time and makes available highly efficient regeneration protocols for Estonian and Finnish barley cultivars.     

草木樨状黄芪高频离体再生体系的建立

以草木樨状黄芪无菌苗茎切段为材料,在含１－２mg/L2,4-D和０－０．５mg/L６ＢＡ的MS培养基上培养获得大量愈伤组织,愈伤组织诱导率在９５％以上,愈伤组织在附加０．２mg/LＫＴ,１mg/L６ＢＡ,０或０．５mg/LＮＡＡ,５００mg/LＣＨ 和２００mg/L ＹＥ的ＭＳ培养基上诱导丛生芽,并进而发育成苗。苗的分化频率为１００％。分化苗或其茎的切段在不国源植物激素的１／２ＭＳ培养基上可出现根的分化,分化频率达９０％以上,再生植株经炼苗后移栽成活率达８０％以上。     

简单快捷建立高频黄瓜子叶离体再生体系

以１０种我国市场上常见的黄瓜品种为材料,建立了一个简单高效的黄瓜子叶离体再生系统。从种子萌发到获得再生植株仅需６周。子叶外植体在ＭＳ＝０．５ｍｇ／Ｌ ＢＡ培养基上不经愈伤组织诱导阶段而直接走器官发生途径。４～５天的子叶外 植体的不定芽发生能力最强。供试１０种基因型中,“碧春”和“甜翠绿”的再生率高达１００％。待不定芽长度超过１．０ｍｃ时,直接将不定芽转移到ＭＳ培养基生根成苗,幼苗移栽温室后正常结实     

In vitro plant regeneration from cotyledon and hypocotyl segments in two bell pepper cultivars

In vitro plant regeneration has been obtained from Capsicum annuum cvs. Pico and Piquillo. Shootbuds were induced from hypocotyl and cotyledon segments after 15–20 days of culture on MS basal medium supplemented with IAA and BAP or Zeatin. Shoot-buds grew into rosettes that rooted in MS plus NAA (0.1 mg/l) and IBA (0.05 mg/l) after 15 days. The small plantlets were successfully transferred to pots with a mixture of peat and perlite and maintained under greenhouse conditions. Elongation took place when the plantlets were growing in the greenhouse.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog (1962) - NAA alpha-naphthalenacetic acid - Z Zeatin     

An efficient regeneration system via somatic embryogenesis in olive

Olive is one of the most important oil crops in the Mediterranean area. Biotechnological improvement of this species is hampered by the recalcitrant nature of olive tissue regeneration in vitro. In this investigation, we have developed an efficient regeneration system for juvenile olive explants via somatic embryogenesis. Embryogenic cultures were obtained at a rate of 25% by culturing isolated radicles from mature seeds in a modified olive medium (OMc) containing 2.5 μM 6-(dimethylallylamino) purine (2iP) and 25 μM indole-3-butyric acid (IBA) over 3 weeks and later transferring to the same medium without 2iP and with a lower IBA concentration. Two different basal formulations, OMc and olive cyclic embryogenesis medium (ECO) [1/4 OM macroelements, 1/4 Murashige and Skoog (MS) microelements and 1/2 OM vitamins supplemented with 550 mg l⁻¹ glutamine], were tested for embryogenic callus proliferation and maturation. The growth rate of embryogenic calli was similar in both media. However, the regeneration of mature embryos, achieved by culturing embryogenic masses in the same medium without hormones and supplemented with 1 g l⁻¹ activated charcoal, was significantly higher when embryos were cultured in the ECO basal formulation. Pre-culturing embryogenic masses in liquid medium for up to 4 weeks did not affect subsequent callus proliferation in solid medium. The maturation rate of small globular somatic embryos, 1–3 mm size, obtained after filtering liquid cultures through a 3 × 3 mm mesh, was also similar to control embryos cultured in solid medium. To improve the maturation and germination rates, the effect of culturing globular somatic embryos on semi-permeable cellulose acetate membranes was also tested. Membrane treatments reduced the regeneration of mature embryos from 56.5% in the control treatment to 40.6% when the membrane was applied during the first half of the 8-week maturation phase and to 18% when the membrane was applied during last 4 weeks of the maturation period. However, membrane treatments significantly enhanced the conversion of mature embryos to plants, increasing the embryo conversion rate from 1.5% in the control to an average value of 37.8% in the membrane treatment. Cotyledonary embryos that were matured on the membranes showed lower values of water and solute potential than controls, indicating that this treatment exerted a controlled desiccation rate that enhanced the recovery of plants.