Factors affecting Agrobacterium tumefaciens-mediated genetic transformation of Lycium barbarum L.

Summary Using the system for genetic transformation and transgenic plant regeneration via somatic embryogenesis (SE) of Lycium barbarum established in this laboratory, this study reports the optimization of the factors affecting the efficiency of transformation, including pre-culture period, leaf explant source, use of acetosyringone, strains and density of Agrobacterium, and temperature of co-cultivation. The optimized transformation protocol for L. barbarum included preculture of leaf explants from 3-wk-old seedlings for 3 d on the medium for callus induction followed by inoculation with Agrobacterium strain EHA101 (pIG121 Hm), co-cultivation for 3d at 24°C, and transfer to the selection regeneration medium with 50 mg l^−1 kanamycin (Kan). Using this protocol, 65% L. barbarum explants gave rise to Kan-resistant and GUS-positive calli. In addition, the expression of introduced transgene (npt II) in clonal progeny was verified by formation of calli and somatic embryos from leaf segments of nine transgenic plants grown on the Kan-containing medium. All explants formed calli at 50 mg l^−1 Kan and seven out of nine transgenic plants were found to possess callus-forming capacity even at 100 mg l^−1 Kan. These calli also possessed higher SE potential on SE medium supplemented with 25 mg l^−1 Kan.     

Growth regulators affect primary and secondary somatic embryogenesis in Madagaskar periwinkle (<Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don) at morphological and biochemical levels

An efficient somatic embryogenesis system has been established in Catharanthus roseus (L.) G. Don in which primary and secondary embryogenic calluses were developed from hypocotyls and primary cotyledonary somatic embryos (PCSEs), respectively. Two types of calluses were different in morphology and growth behaviour. Hypocotyl-derived embryogenic callus (HEC) was friable and fast-growing, while secondary callus derived from PCSE was compact and slow-growing. HEC differentiated into somatic embryos which proliferated quickly on medium supplemented with NAA (1.0 mg l⁻¹) and BA (1.5 mg l⁻¹). Although differentiation and proliferation of somatic embryos were faster in primary HEC, maturation and germination efficiency were better in somatic embryos developed from primary cotyledonary somatic embryo-derived secondary embryogenic callus (PCSEC). At the biochemical level, two somatic embryogenesis systems were different. Both primary and secondary/adventive somatic embryogenesis and the role of plant growth regulators in two modes of somatic embryo formation have been discussed.     

利用mRNA差别显示技术分析枸杞体细胞胚发生早期基因的差别表达

本研究选用枸杞体细胞胚发生体系中的继代愈伤组织(对照)、胚性愈伤组织和早期胚体为实验材料,提取细胞总RNA,在12种锚定真核生物mRNA3'末端的OligodT12VN中,随机选用OligodT12GA为引物合成了以上三种材料的cDNA第一链,以此cDNA为模板,用随机引物进行PCR扩增,选择差别表达的片段。我们选用了OPA、OPH、OPK和OPB四组的60个随机引物对所得的c DNA进行了PCR扩增,得到了三个在体细胞胚发生早期组织中基因特异表达的片段。结果表明,在体细胞胚发生早期有胚胎发生特异性基因的表达,而且这种特异表达的基因在继代愈伤组织中没有表达,说明植物的体细胞胚发生过程就是细胞内基因差别表达的结果。 Abstract:Embryogenic calli and early embryo can be obtained from both auxin and auxin-free medium.The analysis of differential gene expression in early somatic embryogenesis has been hindered by above-mentioned material.The modifications of the recently described mRNA differential display method were reported and differential gene expression in early slmatic embryogenesis was analyzed.We have obtained three differential bands of cDNA in early somatic embryogenesis.The results indicate that gene expression has temperal and spalil order in early somatic embryogenesis of Lycium barbarum L.Plant somatic embryogenesis is the results of differential gene expression in cell.     

Callus production, somatic embryogenesis and plant regeneration of Lycium barbarum root explants

A new micropropagation system for Lycium barbarum (L.) was developed using root explants as starting material. Callus can be produced from root explants on Murashige and Skoog (MS) medium containing 0.2 mg dm⁻³ 2,4-dichlorophenoxyacetic acid. After three subcultures on the same medium, callus was then transferred onto the MS medium supplemented with 500 mg dm⁻³ lactalbumin hydrolysate to induce somatic embryogenesis (SE). After 20 d, about 60 somatic embryos per 0.25 g(f.m.) of embryogenic callus were obtained but only about 10 % of embryos converted into plantlets. After acclimated in the greenhouse, all of the randomly selected plantlets had survived and were similar phenotypically to zygotic seedlings. In addition, the effects of irradiance, photoperiod, growth regulators, explant age and cold treatment on SE of root-derived callus were evaluated.     

Expression of abundant mRNAs during somatic embryogenesis of white spruce [Picea glauca (Moench) Voss]

Embryogenic tissues of white spruce [Picea glauca (Moench) Voss] remain in an early developmental stage while cultured on 2,4-dichlorophenoxyacetic acid and N⁶-benzyladenine, but develop to cotyledonary embryos when these phytohormones are replaced by abscisic acid. Twenty-eight cDNAs were isolated from cotyledonary embryos by differential screening against immature embryo and non-embryonic tissues. Temporal expression patterns of these cDNAs during ABA-stimulated somatic embryo development were observed. This showed that clones could be allocated to various groups, including embryo-abundant, embryo-maturation-induced, and those whose expression was modulated during embryo development, germination or in non-embryogenic tissues. Expression corresponding to these cDNA clones showed that there were various responses to exogenous ABA or polyethylene glycol during a period of 48 h. Analyses of DNA and predicted amino acid sequence revealed that 12 of 28 cDNA clones had no known homologues, while others were predicted to encode different late-embryogenesis-abundant proteins, early methionine-labelled proteins, storage proteins, heat-shock proteins, glycine-rich cell wall protein, metallothionein-like protein and some other metabolic enzymes.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - ABA abscisic acid - BA N⁶-benzyladenine - cDNA complementary deoxyribonucleic acid - Em early methionine-labelled - HSP heat-shock protein - LEA late embryogenesis abundant - PEG polyethylene glycol The authors thank Mr. Terry Bethune for his assistance, and Dr. Larry Pelcher, Mr. Barry Panchuk and Mr. Don Schwab for DNA sequencing and primer synthesis. This is National Research Council of Canada publication number 38929.     

PCR-generated cDNA library of transition-stage maize embryos: cloning and expression of calmodulin genes during early embryogenesis

One hundred maize zygotic embryos microdissected at the transition stage were used to construct a cDNA library after non-selective PCR (NS-PCR) amplification of whole cDNA populations. The library contains 2.3 × 10⁵ recombinants and two different calmodulin cDNAs were cloned using a heterologous probe from petunia. Calmodulin expression was confirmed throughout maize embryogenesis at the mRNA, amplified cDNA and protein levels. Sequence analysis suggests a maize origin for both clones and negligible nucleotide changes linked to PCR. This library is the first described for early plant embryos and represents a breakthrough to isolate genes involved in embryo differentiation.     

Somatic embryogenesis and plant regeneration in elite genotypes of Picea koraiensis

Picea koraiensis, called Korean spruce, is an evergreen tree and found mostly in northeast Asia. In this study, plant regeneration via somatic embryogenesis from open-pollinated immature zygotic embryos of nine genotypes of elite trees was established. Immature zygotic embryos were cultured onto RJW medium modified from 505 medium with 21.48 μM NAA, 2.22 μM BA, and 2.32 μM KT. The average frequency for all nine genotypes was 74.2%. Embryogenic calluses of the nine genotypes of elite trees were subcultured on RJW basal medium containing 8.06 μM NAA, 1.11 μM BA, and 1.16 μM kinetin. The calluses of three lines, 3^#, 9^#, and 2^#, were actively proliferated but others were not. Somatic embryogenesis was induced from the embryogenic callus in genotypes of 3^#, 9^#, and 2^# on RJW medium with ABA and 60 g l⁻¹ sucrose. Cotyledonary somatic embryos were subjected to a drying process. The drying of embryos by uncapping the culture bottle for 5 days on a clean bench resulted in a high frequency of germination of somatic embryos (87% in RJW medium). However, plantlet conversion from germinated embryos was greatly reduced and the optimal medium for plant conversion was 1/2 WPM or 1/2 BMI medium. In conclusion, we have, for the first time, established a plant regeneration system via somatic embryogenesis in the Korean spruce, which can be applied for rapid micropropagation of elite trees.     

High frequency somatic embryogenesis and plant regeneration in root explant cultures of carnation

Root explants excised from carnation plants maintained in vitro formed off-white, friable calluses after three weeks of culture on Murashige and Skoog (MS) medium supplemented with 1 mg l⁻¹ thidiazuron (TDZ) and 1 mg l⁻¹ α-naphthalaneacetic acid (NAA). These calluses were subsequently transferred to MS basal medium where, after an additional four weeks of culture, approximately 50% of the calluses formed somatic embryos. However, calluses formed on root explants that had been cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid did not produce somatic embryos upon transfer to MS basal medium. Somatic embryos developed into plantlets and subsequently were grown to maturity. These results indicate that root explants have a high competence for somatic embryogenesis in carnation. J. Seo and S.W. Kim contributed equally to this work.     

Proliferation and maintenance of embryogenic capacity in elm embryogenic cultures

Summary This paper investigates maintenance and proliferation of somatic embryogenesis systems for Ulmus minor and U. glabra. Proliferation occurred with subculture of embryogenic calluses. The calluses were mainly formed by friable nodules composed of meristematic cells organized into proembryogenic cell masses (PEMs) and thin-walled vacuolated parenchymatic cells. Cotyledonary embryos, with procambial strands and differentiation of their vascular tissues as well as visible root meristems, were identifiable after 18d of culture on a proliferation medium with 0.44 μM benzyladenine (BA). The shoot meristem was only occasionally well developed. Somatic embryo multiplication from elm embryogenic calluses is a clearly asynchronic system, and PEMs as well as embryos at all stages of development are observed simultaneously at the end of subculture period. Factors affecting the proliferation of elm embryogenic callus, such as culture medium, carbon source and genotype, were studied. Basal medium (MS) or medium supplemented with 0.44 μM BA produced the highest number of somatic embryos. Somatic embryo production was higher with sucrose or glucose than with maltose, and significant differences were also found among the four embryogenic lines tested. The use of liquid medium with filter paper support is an essential step for the survival of isolated somatic embryos during the germination stage. The addition of 0.22 μM BA′ to liquid MS medium was the best treatment for germination and plantlet conversion of elm somatic embryos.     

Somatic embryogenesis in leaf callus from a mature Quercus suber L. Tree

Summary Somatic embryos were obtained from a 60-yr-old Quercus suber L. tree. Leaf explants were cultivated on Murashige and Skoog medium with 30 gl⁻¹ sucrose, 3 gl⁻¹ gelrite, pH adjusted to 5.8, and different growth regulator combinations. Callus induction took place at 24±1°C in the dark during the first 3 wk. After 3 mo, calluses that showed embryogenic structures were transferred to the same medium without growth regulators. Somatic embryogenesis was only observed in calluses induced on E3 medium (supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid and 9.0 μM zeatin). On average, 7.5% of the initial explants formed embryogenic calluses in this medium. Somatic embryo proliferation was high due to secondary embryogenesis. On average, 10% of the somatic embryos germinated and 40% of these germinated embryos converted into plants. Plants were elongated on the same medium without growth regulators and acclimated to greenhouse conditions.     

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.     

Inherited chilling tolerance in somatic hybrids of transgenic Hibiscus rosa-sinensis x transgenic Lavatera thuringiaca selected by double-antibiotic resistance

Summary Improvement of Hibiscus rosa-sinensis for increased frost tolerance has been attempted through somatic hybridization with the frost tolerant Lavatera thuringiaca. Cell suspensions from Hibiscus and Lavatera were transformed with A. tumefaciens harboring plasmids containing selectable genes coding for kanamycin and hygromycin resistance, respectively. We provided evidence that H. rosa-sinensis and L. thuringiaca were transformed by strong selection of transformed calluses in medium containing antibiotics, by GUS activity determination in protein extracts and by molecular confirmation of chromosomal integration and expression of the selectable genes. Protoplasts isolated from a kanamycinresistant Hibiscus callus and from a hygromycin-resistant Lavatera callus were fused and selected in medium containing both antibiotics. We determined unambiguously that the regenerated double-antibiotic resistant clones obtained are indeed somatic hybrids through analysis of acid phosphatase zymograms and nuclear DNA content. Plant regeneration through somatic embryogenesis was accomplished from both isolated protoplasts and transgenic calluses of L. thuringiaca. However, regeneration from the double-antibiotic resistant fusant calluses was unsuccessful. Analysis of the somatic hybrids at the callus level showed that chilling and freezing tolerance are governed by independent genetic components. The somatic hybrids displayed significant improvement for chilling tolerance at conditions lethal to H. rosa-sinensis, although frost tolerance was not expressed.     

An alternative method for the genetic transformation of sweet organce

Summary An alternative method for transforming sweet organe [Citrus sinensis (L.) Osbeck] has been developed. Plasmid DNA encoding the non-destructive selectable marker enhanced green fluorescent protein gene was introduced using polyethylene glycol into protoplasts of ‘Itaborai’ sweet organe isolated from an embryogenic nucellar-derived suspension culture. Following protoplast culture in liquid medium and transfer to solid medium, transformed calluses were identified via expression of the green fluorescent protein, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. Transgenic plantlets were recovered from germinating somatic embryos and by in vitro rooting of shoots. To expedite transgenic plant recovery, regenerated shoots were also micrografted onto sour orange seedling rootstocks. Presence of the transgene in calluses and regenerated sweet organe plants was verified by gene amplification and Southern analyses. Potential advantages of this transformation system over the commonly used Agrobacterium methods for citrus are discussed.     

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.     

Plant regeneration via somatic embryogenesis from in vitro tissue culture in two Tunisian Cucumis melo cultivars Maazoun and Beji

Hypocotyl, cotyledon and zygotic embryo explants from two Tunisian Cucumis melo L. cultivars Beji and Maazoun, cultured on the MS medium added with 2,4-D (0.25–1 mg l⁻¹) and BA (0.10–0.50 mg l⁻¹), produce calluses with somatic embryos after 3 weeks of culture. For Beji c.v. the highest percentage (62.50%) of embryogenesis was observed for cotyledons. The average embryo number per callus was 10.40. Embryogenesis induction for zygotic embryos reached 33.50% with 29 embryos per callus. The embryogenesis ability of hypocotyls did not exceed 12.50% (2.50 embryos per callus). Somatic embryogenesis for Maazoun c.v. explants was less efficient. Embryos formation was observed only for cotyledons (29%) and zygotic embryos (25%). Cotyledonary staged embryos, when transferred to hormone free MS medium, germinated. The maximum germination rates were 51.50 and 44.50%, respectively for Maazoun and Beji c.v. The highest percentage (36.50%) of survival plants was noted for Beji c.v. Regenerants were diploids (2n = 2x = 24) and morphologically similar to their parents issued from seeds.     

Plant regeneration from zygotic embryo-derived embryogenic cell suspension cultures of Ranunculus kazusensis

Culture conditions for high frequency plant regeneration via somatic embryogenesis from cell suspension cultures of Ranunculus kazusensis are described. Zygotic embryos formed white nodular structures and pale-yellow calluses at a frequency of 84.9% when cultured on half-strength Schenk and Hildebrandt (SH) medium supplemented with 0.1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). However, the frequency of white nodular structure and off-white callus formation decreased with an increasing concentration of 2,4-D up to 10 mg l⁻¹, when the frequency reached 25%. Cell suspension cultures were established from zygotic embryo-derived pale-yellow calluses using half-strength SH medium supplemented with 0.1 mg l⁻¹ of 2,4-D. Upon plating onto half-strength SH basal medium, over 90% of cell aggregates gave rise to numerous somatic embryos and developed into plantlets. Regenerated plantlets were successfully transplanted to potting soil and grown to maturity at a survival rate of over 90% in a growth chamber. The plant regeneration system established in this study can be applied to mass propagation and conservation of this species.     

In vitro regeneration in Trifolium. 1. Direct somatic embryogenesis in T. rubens (L.)

The origin and development of zygotic and somatic embryos of Trifolium rubens L. was studied with the aid of paraffin sections and light microscopy. Zygotic embryos were collected, fixed and prepared daily from one to ten days after cross-pollination. Somatic embryos were obtained by plating petiole sections on modified L2 medium with 0.015 mgl^-1 picloram and 0.1 mgl^-1 6-BAP. Cultured petioles were collected and fixed daily from one to 25 days after plating. Two regions in the vascular bundle sheath of cultured petioles gave rise to callus. The first region was adjacent to the phloem fibers and produced friable callus. The second region gave rise to compact callus that was connected to the fascicular cambium. Somatic embryos originated from single cells in the cortex directly without intervening callus formation and from single cells in the friable callus. In addition, embryos arose from meristematic regions in compact callus. Many early stages of embryogenesis (one, two and four-celled stages) were observed in the cortex and friable callus. Zygotic embryogenesis in Trifolium differs from other legumes in that the suspensor is short and has a broad attachment. This arrangement was observed in zygotic embryos of T. rubens and in many somatic embryos. However, a continuum of somatic embryogenesis was observed where some young embryos had a Trifolium suspensor-like arrangement while others were attached to a long narrow suspensor-like structure more characteristic of Medicago.     

Direct somatic embryogenesis from mature embryos of sandalwood

Plants were regenerated from mature zygotic embryos of sandalwood (Santalum album L.) through direct somatic embryogenesis. Somatic embryos were formed directly without any intervening callus phase on zygotic embryos plated on Murashige and Skoog (MS) medium containing thidiazuron or benzylaminopurine. Individual somatic embryos were then isolated and transferred to MS medium without cytokinin on which they formed secondary embryos in repetitive cycles with or without the addition of indole acetic acid to the medium. Conversion of somatic embryos into plantlets was achieved by isolating somatic embryos with distinct cotyledons and reculturing them onto half-strength MS medium with GA₃ (1.4 M). Recovered plantlets were acclimatised and grown in the greenhouse. This is the first report on in vitro regeneration via direct somatic embryogenesis of sandalwood.     

Direct and indirect somatic embryogenesis on cotyledon explants of <Emphasis Type="Italic">Quassia amara</Emphasis> L., an antileukaemic drug plant

Summary In vitro propagation of Quassia amara L. (Simaroubaceae) was attempted using mature and juvenile explants. Attempts to establish in vitro culture using leaf and internode explants from a plant more than 15yr old were unsuccessful due to severe phenolic exudation. Plant regeneration through direct and indirect somatic embryogenesis was established from cotyledon explants. Murashige and Skoog (MS) medium with 8.9 μM N⁶-benzyladenine (BA) and 11.7 μM silver nitrate induced the highest number (mean of 32.4 embryos per cotyledon) of somatic embryos. Direct somatic embryogenesis as well as callus formation was observed on medium with BA (8.9–13.3 μM). Semi-mature pale green cotyledons were superior for the induction of somatic embryos. Embryos developed from the adaxial side as well as from the point of excision of the embryonic axis. More embryos were developed on the proximal end compared to mid and distal regions of the cotyledons. Subculture of callus (developed along with the somatic embryos on medium with BA alone) onto medium containing 8.9 μM BA and 11.7 μM silver nitrate produced a mean of 17.1 somatic embryos. Primary somatic embryos cultured on MS medium with 8.9 μM BA and 11.7μM silver nitrate produced a mean of 9.4 secondary somatic embryos. Most of the embryos developed up to early cotyledonary stage. Reduced concentration of BA (2.2 or 4.4 μM) improved maturation and conversion of embryos to plantlets. Ninety percent of the embryos converted to plantlets. The optimized protocol facilitated recovery of 30 plantlets per cotyledon explant within 80d. Plantlets transferred to small cups were subsequently transferred to field conditions with a survival rate of 90%.