Direct regeneration of Drosera from leaf explants and shoot tips

An efficient protocol for the micropropagation of Drosera anglica, D. binata and D. cuneifolia is described. Proliferation was obtained from leaf segments and shoot tips, which served as initial explants. The regeneration capacity of explants was influenced by factors such as nutrient media, concentrations of growth regulators and the type of medium (liquid or solid). The highest number of plants regenerating from D. binata explants was obtained on the growth regulator-free Vacin and Went medium. In the case of D. anglica the highest proliferation rate was obtained on the Fast medium supplemented with 0.05 M 6-benzyladenine (BA) and 0.005 M -naphthaleneacetic acid (NAA), whereas for D. cuneifolia the optimal regeneration medium proved to be 1/2 MS with the growth regulator supplementation estimated at 0.2 M BA and 0.2 M NAA. Liquid media significantly increased the regeneration potential of D. anglica and D. binata explants.     

Effect of genotype,explant, subculture interval and environmental conditions on regeneration of shoots from in vitro monoploids of a diploid potato species,Solanum phureja Juz. & Buk.

In vitro anther-derived monoploids (2n=x=12) of Solanum phureja were compared for shoot regeneration from leaf and stem explants under various environmental conditions. Monoploids from the same or different diploid clones varied for frequency and earliness of shoot regeneration and number of shoots formed per explant. Leaf explants regenerated at higher frequencies than stem explants. Explants from stock plantlets subcultured at a 2- or 4-week interval regenerated earlier and at a higher frequency than those from plantlets subcultured at longer intervals. Regeneration frequency and number of shoots per explant were greater when explants were incubated at 20°C compared to 25°C. Explants from stock plantlets maintained under a 16 h as opposed to an 11 h photoperiod exhibited increased shoot regeneration; however, neither photoperiod nor the maintenance temperature of the stock plantlets influenced regeneration frequency. Genotypic differences were observed for the frequency of chromosome doubling among regenerated shoots whereas temperature treatments had no influence on chromosome doubling.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA -naphthale-neacetic acid     

Micropropagation of selected somaclones ofBegonia andSaintpaulia

Begonia x elatior plantlets which regenerated from leaf disk callus showed variations in plant morphology, number of flowers per plant, and flower size. Variations in flowering period, number of flowers per plant, and flower morphology were observed in Saintpaulia ionantha L. plants directly regenerated from leaf disk explants. The cytokinins, benzylaminopurine and zeatin, tested in the culture medium did not affect the basic plant characteristics including flower colour which remained stable in both species. Micropropagation of selected somaclones having the desirable trait of high number of flowers per plant was stable in the MV2 and MV3 generations.     

Somatic embryogenesis,organogenesis, and regeneration from leaf callus culture of <Emphasis Type="Italic">Decalepis hamiltonii</Emphasis> Wight &; Arn., an endangered shrub

Summary A novel protocol has been developed for inducing somatic embryogenesis from leaf cultures of Decalepis hamiltonii. Callus was obtained from leaf sections in Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA)+N⁶-benzyladenine (BA) or 2,4-dichlorophenoxyacetic acid (2,4-D)+BA. Nodular embryogenic callus developed from the cut end of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. Upon subsequent transfer of explants with primary callus onto MS media containing zeatin and/or gibberellic acid (GA₃) and BA, treatment with zeatin (13.68μM) and BA (10.65 μM) resulted in the induction of the highest number of somatic embryos directly from nodular tissue. The maturation of embryos took place along with the induction on the same medium. Embryogenic calluses with somatic embryos were subcultured onto MS basal medium supplemented with 4.56μM zeatin+10.65 μM BA. After 4wk, more extensive differentiation of somatic embryos was observed. The mature embryos developed into complete plantlets on growth regulator-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis from leaf explants of Decalepis hamiltonii, which has not been reported previously. By using this protocol, complete plantlets could be regenerated through indirect somatic embryogenesis or organogenesis from leaf explants in 12–16 wk.     

东北地区被子植物新资料

东北地区位于中国东北部,地处欧亚大陆东缘,地域辽阔,环境复杂,植被类型多样,是我国北方植物种类较为丰富、植物区系起源演变发展的重要地区。该研究根据野外实地调查及资料分析,报道了东北地区2种新分布植物,分别是野生堇菜(Viola arvensis)、线叶毛膏菜(Drosera anglica)。野生堇菜为东北地区新归化种,分布于哈尔滨市帽儿山,该种在我国台湾地区有分布,原产于非洲北部、亚洲西南部和欧洲;线叶毛膏菜为中国新记录种,分布于东北长白山区。该属在东北地区还有1种,即圆叶茅膏菜(D.rotundifolia)。两者的主要区别:前者叶为线状匙形,花柱3,每个花柱2分裂至中部;后者叶片近圆形,花柱3,每个花柱深裂至基部。凭证标本保存于东北林业大学植物标本室(NEFI)。这些新资料种的发现,对于丰富东北地区植物种类及对本地区植物多样性的研究具有重要意义。     

Somatic embryogenesis and plant regeneration of Gladiolus (Gladiolus hort.)

Summary Friable embryogenic callus and somatic embryos of 4 Gladiolus cultivars were obtained on Murashige and Skoog (MS) medium with various concentration of auxins from the following explants: corm slices, young leaf bases and whole, intact plantlets. Somatic embryos transferred on MS hormone-free medium regenerated into plantlets. All plantlets obtained through embryogenesis did not differ phenotypically from the parental clones. The embryogenic friable callus has been maintained for over 2 years in culture and has retained a very high regeneration capacity.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KIN kinetin - NAA naphthaleneacetic acid - MS Murashige and Skoog Medium (1962) - E embryogenic callus - NE non-embryogenic callus     

Shoot regeneration of dwarf dogwood (Cornus canadensis L.) and morphological characterization of the regenerated plants

Dwarf dogwoods (or the bunchberries) are the only suffrutex in Cornaceae. They are attractive ground cover ornamentals with clusters of small flowers surrounded by petaloid bracts. Little has been reported on plant regeneration of dogwoods. As a step toward unraveling the molecular basis of inflorescence evolution in Cornus, we report an efficient regeneration system for a dwarf dogwood species C. canadensis through organogenesis from rejuvenated leaves, and characterize the development of the plantlets. We used the nodal stem segments of vegetative branches as explants. Micropropogated shoots were quickly induced from axillary buds of nodes on an induction medium consisting of basal MS medium supplemented with 4.44 μM BAP and 0.54 μM NAA. The new leaves of adventitious shoots were used as explants to induce calli on the same induction medium. Nearly 65% of leaf explants produced calli, 80% of which formed adventitious buds. Gibberellic acid (1.45 μM) added to the same induction medium efficiently promoted quick elongation of most adventitious buds, and 0.49 μM IBA added to the basal MS medium promoted root formation from nearly 50% of the elongated shoots. The growth of plantlets in pot soil was characterized by the development of functional woody rhizomes, which continuously developed new aboveground vegetative branches, but not flowering branches, within the past 12 months. Potential reasons causing the delay of flowering of the regenerated plants are discussed. The establishment of this regeneration system facilitates developing a genetic transformation system to test candidate genes involved in the developmental divergence of inflorescences in Cornus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation of an endangered medicinal plant Saussurea involucrata Kar. et Kir.

An efficient micropropagation system for Saussurea involucrata Kar. et Kir., an endangered Chinese medicinal plant, has been developed. Shoot organogenesis occurred from S. involucrata leaf explants inoculated on medium with appropriate supplements of plant growth regulators. 66.0% of shoot regeneration frequency and 5.2 shoots per leaf explant were achieved when cultured on a medium containing 10 μM 6-benzylaminopurine (BAP) and 2.5 μM 1-naphthaleneacetic acid (NAA). Shoot organogenesis was improved further when the leaf explants were pre-incubated at low temperature, and 80.6% of shoot regeneration frequency was recorded with 9.3 shoots per leaf explant at 4°C by 5-day pretreatment period. Up to 87.0% of the regenerated shoots formed complete plantlets on a medium containing 2.5 μM indole-3-acetic acid (IAA) within 28 days, and 85.2% of the regenerated plantlets survived and grew vigorously in greenhouse condition. The phytochemical profile of the micropropagated plants was similar to that of wild plants. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of the elite medicinal plant.     

Plant regeneration from seedling explants and cotyledon protoplasts ofGlycine argyrea tind

Summary Plants have been regenerated from nodular, green callus derived from cotyledon, petiole and leaf lamina explants ofG. argyrea, a perennial relative of the soybean (G. max). The degree of response obtained was governed primarily by the genotype used, accession G1626 proving the most responsive. Shoots were also recovered from about 6.0% of cotyledon protoplasts of this genotype. The implications of these results are discussed in relation to genetic manipulations using this species.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis> Wright,an important pharmaceutical crop

Dioscorea zingiberensis Wright has been cultivated as a pharmaceutical crop for production of diosgenin, a precursor for synthesis of various important steroid drugs. Because breeding of D. zingiberensis through sexual hybridization is difficult due to its unstable sexuality and differences in timing of flowering in male and female plants, gene transfer approaches may play a vital role in its genetic improvement. In this study, the Agrobacterium tumefaciens-mediated transformation of D. zingiberensis was investigated with leaves and calli as explants. The results showed that both leaf segments and callus pieces were sensitive to 30 mg/l hygromycin and 50–60 mg/l kanamycin, and using calli as explants and addition of acetosyringone (AS) in cocultivation medium were crucial for successful transformation. We first immersed callus explants in A. tumefaciens cells for 30 min and then transferred the explants onto a co-cultivation medium supplemented with 200 μM AS for 3 days. Three days after, we cultured the infected explants on a selective medium containing 50 mg/l kanamycin and 100 mg/l timentin for formation of kanamycin-resistant calli. After the kanamycin-resistant calli were produced, we transferred them onto fresh selective medium for shoot induction. Finally, the kanamycin resistant shoots were rooted and the stable incorporation of the transgene into the genome of D. zingiberensis plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The method reported here can be used to produce transgenic D. zingiberensis plants in 5 months and the transformation frequency is 24.8% based on the numbers of independent transgenic plants regenerated from initial infected callus explants.     

Plantlet regeneration through different morphogenic pathways in pommelo tissue culture

Pommelo (Citrus grandis Osbeck) plantlets were regenerated through different morphogenic pathways in culture. Multiple shoot regeneration through de novo organogenesis was obtained with epicotyl segments and root cultures. Shoot regeneration was observed in 84% of the midtal epicotyl segments cultured in Murashige and Skoog's medium (MS) with 2.2 M benzyladenine (BA) and 83% of the middle and proximal epicotyl segments cultured on basal medium. Isolated root segments cultured on medium containing 0.089 M BA showed best shoot regeneration at 71% with an average of 3.3 shoots per segment. Callus tissues derived from cotyledon and leaf explants regenerated shoots on BA-enriched medium. Shoots were also obtained at high frequencies from shoot-tip and nodal explants. Roots developed when regenerated shoots were excised and cultured on half strength MS medium with 2.5 M indolebutyric acid.Abbreviations BA 6-Benzyladenine - IBA Indole-3-butyric acid - MS Murashige and Skoog medium - NAA I-Naphthaleneacetic acid - 2,4-d 2,4-Dichlorophenoxyacetic acid     

Direct plant regeneration from leaf explants of Plumbago species

Direct plant regeneration was achieved from leaf explants of Plumbago rosea and Plumbago zeylanica on Murashige and Skoog (MS) medium supplemented with 6.7 M 6-benzylaminopurine (BA), 1.4 M indole-3-acetic acid (IAA), 370 M adenine sulfate (Ads) and 3% (w/v) sucrose. The shoot initials developed within 2–3 weeks on the leaf margin as well as from the cut surface of the leaf. High frequency shoot-bud regeneration was achieved on similar medium in subsequent subcultures. The semi-mature leaves produced more shoot-buds as compared to the younger leaves. Mature leaves did not show any response for shoot bud initiation. More than 85% of the semi-mature explants produced shoot-buds per leaf explant within 4 weeks of culture. Shoots rooted on half-strength basal MS medium supplemented with 1.2 M indole-3-butyric acid (IBA) and 2% (w/v) sucrose; approximately 90% of the in vitro raised plantlets survived in the greenhouse. The regenerated plantlets looked morphologically similar to the mother plants. This protocol might be useful for genetic improvement programs.     

Plant regeneration of <Emphasis Type="Italic">Erigeron breviscapus</Emphasis> (vant.) Hand. Mazz. and its chromatographic fingerprint analysis for quality control

An efficient micropropagation system for Erigeron breviscapus (vant.) Hand. Mazz., an important medicinal plant for heart disease, has been developed. Shoot organogenesis occurred from E. breviscapus leaf explants inoculated on a medium supplemented with a combination of plant growth regulators. On average, 17 shoots per leaf explant were produced after 30 days when they were cultured on MS basal salts and vitamin medium containing 5 μM 6-benzylaminopurine (BAP) and 5 μM 1-naphthaleneacetic acid (NAA). All the regenerated shoots formed complete plantlets on a medium containing 2.5–10 μM indole-3-butyric acid (IBA) within 30 days, and 80.2% of the regenerated plantlets survived and grew vigorously in field conditions. Based on the variation in common peaks and the produced amount of the most important bioactive component, scutellarin, a high performance liquid chromatography (HPLC) fingerprinting system was developed for quality control of these micropropagated plants. Chemical constituents in E. breviscapus micropropagated plants varied during plant development from regeneration to maturation, the latter of which showed the most similar phytochemical profile in comparison with mother plants. The regeneration protocol and HPLC fingerprint analysis developed here provided a new approach to quality control of micropropagated plants producing secondary metabolites with significant implications for germplasm conservation.     

Callus induction and plant regeneration from excised zygotic embryos of the seed-propagated yams Dioscorea composita Hemsl. and D. cayenensis Lam.

A tissue culture method for regeneration of plantlets from calluses of Dioscorea composita Hemsl. and Dioscorea cayenensis L. is described. Zygotic embryos were used as initial explants. Calluses were obtained on Murashige & Skoog basal medium supplemented with 18 M 2,4-D and plantlets were regenerated on media containing 0.1 M zeatin and 3.3 mM glutamine according to previously described protocols [3]. Inclusion of 0.3% (w/v) activated charcoal in media did not increase callusing. Regeneration of plantlets from D. cayenensis calluses occurred only at low levels of 2,4-D (2.25 M) contained in the media tested. The results indicated that there were genotype-dependent differences between the yam species in their ability to regenerate plantlets in vitro.     

High frequency plant regeneration from protoplasts in cotton <Emphasis Type="Italic">via</Emphasis> somatic embryogenesis

A highly reproducible system for efficient plant regeneration from protoplast via somatic embryogenesis was developed in cotton (Gossypium hirsutum L.) cultivar ZDM-3. Embryogenic callus, somatic embryos and suspension culture cells were used as explants. Callus-forming frequency (82.86 %) was obtained in protoplast cultures from suspension culture cells in KM₈P medium with 0.45 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.93 μM kinetin (KIN), 1.5 % glucose and 1.5 % maltose. Protocolonies formed in two months with plating efficiency of 14 %. However, the callus-forming efficiencies from other two explants were low. The calli from protoplast culture were transferred to somatic embryo induction medium and 12.7 % of normal plantlets were obtained on medium contained 3 % maltose or 1 % of each sucrose + maltose + glucose, 2.46 μM indole-3-butyric acid (IBA) and 0.93 μM KIN. Over 100 plantlets were obtained from protoplasts derived from three explants. The regenerated plants were transferred to the soil and the highest survival rate (95 %) was observed in transplanting via a new method.     

Agrobacterium tumefaciens-mediated transformation of embryogenic tissue and transgenic plant regeneration in Chamaecyparis obtusa Sieb. et Zucc.

A genetic transformation procedure for Chamaecyparis obtusa was developed after co-cultivation of embryogenic tissues with disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the sgfp (synthetic green fluorescent protein) visual reporter and nptII (neomycin phoshotransferase II) selectable marker genes. The highest transformation frequency was 22.5 independent transformed lines per dish (250 mg embryogenic tissue) following selection on kanamycin medium. Transgenic plantlets were regenerated through the maturation and germination of somatic embryos. The intensity of GFP fluorescence, observed under a fluorescence microscope, varied from very faint to relatively strong, depending on the transgenic line or part of the transgenic plant. The integration of the genes into the genome of regenerated plantlets was confirmed by Southern blot analysis.     

Characterization of progeny of Coleus blumei following an in vitro selection for salt tolerance

An in vitro selection method was developed for Coleus blumei to enhance salt tolerance of this amenity species. Leaf disc explants were incubated on a Murashige & Skoog medium containing benzylaminopurine, 2 mg l^-1, and napthalene acetic acid, 1 mg l^-1, which initiated both callus and plantlets from the explants. A large number of explants were incubated on this differentiating medium containing 90 mM NaCl, which inhibited over 90% of plantlet formation. Surviving plantlets. were grown to maturity, when apical cuttings were taken and propagated. Plants were also allowed to flower and set seed. Cuttings from the selected regenerated plants showed consistently better growth in the presence of NaCl than unselected cuttings. Seed progeny of selected plants also showed more vigorous growth in the presence and absence of NaCl than progeny from unselected plants. The in vitro selection was compared with the results of an earlier in vivo selection to assess the contribution from tissue culture derived somaclonal variation. Progeny from the in vitro selection showed a higher level of tolerance than progeny from the in vivo selection.     

Plant regeneration from aged-callus of the woody ornamental species Lonicera japonica cv. “Hall's Prolific”

Summary Callus cultures were initiated from in vitro grown leaf, stem and root segments of Lonicera japonica “Hall's Prolific”, on a medium containing 10.7 μM α-naphthtylacetic acid and 2.7 μM benzyladenine, while media with 2,4-dichlorophenoxyacetic acid led to a rapid necrosis of explants. Shoot regeneration from true-callus (i.e. without any part of the original explant) was achieved for the three different source tissues within 12 weeks. The highest rate of regeneration was obtained by using benzyladenine (4.4 to 44.4 μM) as the sole hormone in the medium. The regenerated shoots were readily elongated and rooted on the same medium as used for multiplication, and plantlets were subsequently transferred to greenhouse conditions, where nearly 100% of them were successfully acclimatized. This is the first example of plant regeneration from aged (≥6 month-old) true-callus of a woody ornamental species.     

Molecular analysis of micropropagated neem plants using aflp markers for ascertaining clonal fidelity

Summary The importance of neem (Azadirachta indica A. Juss.) as a medicinal tree species has been acknowledged worldwide. Superior trees with desired traits such as high azadirachtin content have been identified and micropropagated. Somaclonal variants that may arise in vitro, however, pose limitations to large-scale micropropagation. It is, therefore, imperative to establish genetic uniformity of such plantlets by ensuring strict quality checks at various stages of in vitro culture. This is the first study that evaluates the applicability of amplified fragment length polymorphism (AFLP) markers in establishing clonal fidelity of tissue culture(TC)-raised neem plants. Seven AFLP primer combinations generated a total of 334 amplified fragments across the mother plant, TC progenies, and other neem accessions that were included as controls. Two hundred and thirty-nine amplified fragments were monomorphic across the mother tree and its TC progenies. No extra band was detected in the TC plantlets that was absent in the mother tree, indicating that the TC plantlets regenerated through nodal explants are indeed true-to-type. Ninety-five AFLP fragments were detected in the controls, which allowed their discrimination from the elite mother tree and its TC progenies. Similarity matrix based on Jaccard's coefficient revealed that the pair-wise value between the mother tree and its TC plantlets was ‘1’, indicating perfect similarity. Phenetic dendrogram based on UPGMA (unweighted pair group method of arithmetic averages) analysis further confirmed the true-to-type nature of TC progenies, since a tie was observed between the mother tree and its TC plantlets. On the contrary, the control neem accessions were distinct from the mother and its TC progenies. AFLP markers proved to be an ideal tool for routine analysis and certification of genetic fidelity of micropropagated plants prior to commercialization, especially in tree species because of their long generation time.