Epigenetic variation in the callus of <Emphasis Type="Italic">Brassica napus</Emphasis> under different inducement conditions

Tissue culture, a traditional technique broadly used for the genetic transformation and functional verification of target genes, induces epigenetic variations in transgenic acceptors of plants. This study compared the DNA methylation patterns during the callus formation of Brassica napus induced by different concentrations of 6-BA and 2,4-D through methylation-sensitive amplification polymorphism. The highest induction rate (85%) was observed in the hypocotyls cultured with 0.1 mg/L 2,4-D and the lowest methylation rate (25.09%) was detected in the hypocotyls cultured with 1.0 mg/L 6-BA. The methylation rates of the callus cultured with 0.2 and 0.05 mg/L 2,4-D were 29.99 and 28.31%, respectively. The callus induction rates were reduced to 79 and 80%. The methylation rates of the callus induced by 2.0 and 0.5 mg/L 6-BA were 28.17 and 33.98%, respectively. The callus induction rates were reduced to 76 and 74%. The expression analysis of methyltransferase under different induction conditions agreed with methylation modifications; therefore, the effects of hormones on callus induction may be partially indicated by methylation changes in B. napus genome.     

Response of light- and dark-grown callus of atrazine-resistant and susceptible rapeseed (Brassica napus) to varying concentrations of atrizine

Callus cultures initiated with tissue of triazine-resistant and susceptible lines of Brassica napus L. were cultured in the presence of atrazine (10^-5 to 10^-4 M) under a 16-h photoperiod or in continuous darkness. On atrazine-free media callus growth was characteristically greater for the light-grown cultures. On media containing atrazine, growth of callus from triazine-resistant and susceptible rapeseed cultured in the dark did not differ significantly. Addition of atrazine to the culture medium resulted in a significant reduction in the growth of light-grown callus of the susceptible genotype to a level equivalent to that of the dark-grown cultures. Atrazine had no effect on the growth of light-grown callus initiated with triazine-resistant rapeseed.     

大花金挖耳愈伤组织诱导与增殖

以大花金挖耳无菌苗的子叶、下胚轴和根为外植体,进行愈伤组织诱导与增殖研究。结果表明:大花金挖耳无菌苗的根是诱导愈伤组织的理想外植体;其愈伤组织诱导的最适培养基为:B5 3.0mg/L NAA 0.2mg/L6-BA,诱导率可达100%;愈伤组织的增殖在45g/L的蔗糖、pH5.7、光照12h/d培养条件下可延迟愈伤组织褐化出现的时间,并维持其良好的组织结构,愈伤组织的最适继代周期为30~40d。     

Solasodine production in rapidly proliferating tissue cultures of Solanum laciniatum ait

Callus and suspension cultures, established from seedling and leaf explants of Solanum laciniatum Ait were analysed for solasodine using a spectrophotometric assay. Solasodine concentration in both types of culture ranged from 0.5 – 1.0 mg/g dry wt., with a small number of callus explants containing higher concentrations. There was no overall fall in concentration as a result of serial subculture, and in suspension cultures the level remained constant throughout a single passage. Solasodine concentration was enhanced by the induction of organogenesis in both primary leaf explants and callus. ABA, added at 0.04 mg 1^?1, increased solasodine yield in callus cultures whilst CEPA, at concentrations of 10 mg 1^?1 and higher, inhibited production. Dark grown callus contained significantly more solasodine than light grown.     

Detection of DNA methylation pattern in thidiazuron-induced blueberry callus using methylation-sensitive amplification polymorphism

During the normal developmental process, programmed gene expression is an essential phenomenon in all organisms. In eukaryotes, DNA methylation plays an important role in the regulation of gene expression. The extent of cytosine methylation polymorphism was evaluated in leaf tissues collected from the greenhouse grown plants and in in vitro-derived callus of three lowbush and one hybrid blueberry genotypes, using methylation-sensitive amplification polymorphism (MSAP) technique. Callus formation started from the leaf segments after 4 weeks of culture on a thidiazuron (TDZ) containing medium. Maximum callus formation (98 %) was observed in the hybrid blueberry at 1.0 mg dm^-3 TDZ. Although noticeable changes in cytosine methylation pattern were detected within the MSAP profiles of both leaf and callus tissues, methylation events were more polymorphic in calli than in leaf tissues. The number of methylated CCGG sites varied significantly within the genotypes ranging from 75 to 100 in leaf tissues and from 215 to 258 in callus tissues. Differences in the methylation pattern were observed not only in a tissue-specific manner but also within the genotype in a treatment specific manner. These results demonstrated the unique effect of TDZ and the tissue culture process on DNA methylation during callus development.     

Rapid changes in amplification and methylation pattern of genomic DNA in cultured carrot root explants (Daucus carota L.)

Summary Rapid genomic DNA variation due to methylation and copy number alteration was observed in carrot root explants 6 h after inoculation and during a 36-h period of exponential callus growth. De novo methylation and amplification of restricted BspNI fragments of low molecular weight occurred before cell cycle activation and should, therefore, be independent of progression through the S-phase of the cell cycle. Growth regulators seemed to influence the amplification pattern indirectly by regulating cell division activity. In exponentially growing callus tissue the copy number of most of the repetitive fragments was dramatically reduced. It is presumed that this reduction in the copy number of repetitive fragments is characteristic of rejuvenilization. 3-Indole-acetic-acid (IAA) and inositol in the medium increased the degree of unspecific genomic DNA methylation in growing rhizogenic carrot callus tissue in the absence of kinetin, which inhibits root induction at that stage. A possible relation to the induction of rhizogenesis is considered. The observed reduction in number of repetitive restriction fragments and the increase in DNA methylation are gross changes covering the total genome. The results are discussed in relation to the controversy concerning the general biological significance of the methylation and amplification of DNA sequences.     

Influence of 5′-azacitidine on promoting recovery of cell competence for shoot organogenesis in <Emphasis Type="Italic">Arabidopsis</Emphasis>

In Arabidopsis, adventitious shoots are formed at a high frequency when the calli are induced from roots or hypocotyls cultured on callus induction medium (CIM) and then transferred to shoot induction medium (SIM). The prolonged duration of culture on CIM decreased the frequency of shoot regeneration. However, when 5′-azacitidine (AzaC), an inhibitor of DNA methylation, was added to CIM, the excess culturing on CIM did not decrease the frequency of shoot regeneration. The level of methyl cytosine was up-regulated when hypocotyl explants were cultured on CIM for 2 weeks. We examined the expression patterns of genes that are involved in the formation or regeneration of shoots. Prolonged duration of culture on CIM up-regulated the CUC1, CLV1, CLV3, ESR1, and WUS mRNA levels, and the addition of AzaC to CIM reduced their expression levels. Our results suggest that an increase in DNA methylation decreased the shoot-forming ability and that AzaC can partially recover this ability.     

Callus induction and plant regeneration from different explant types of Miscanthus x ogiformis Honda ‘Giganteus’

Different explants of Miscanthus x ogiformis Honda Giganteus were tested in order to develop an efficient tissue culture system. Shoot apices, leaf and root sections from in vitro-propagated plants, and leaf and immature inflorescence sections from 6-month-old greenhouse-grown plants were used. The explants were cultured on Murashige and Skoog medium supplemented with 4.5, 13.6, 22.6 or 31.7 M 2,4-dichlorophenoxyacetic acid. Three types of callus were formed but only one was embryogenic and regenerated plants. Callus induction and formation of embryogenic callus depended on the type and developmental stage of the explants. Shoot apices formed the highest percentage of embryogenic callus. There was a difference in the formation of embryogenic callus between leaf explants from in vitro-propagated shoots and greenhouse-grown plants. The best results were obtained from newly formed leaves of in vitro-propagated shoots and older leaves of greenhouse-grown plants. Immature inflorescences smaller than 2.5 cm produced a higher percentage of embryogenic callus than larger more mature inflorescences. Embryogenic callus derived from immature inflorescences had the highest regeneration capacity. Differences in 2,4-dichlorophenoxyacetic acid concentrations had no significant effect on callus induction, embryogenic callus formation and plant regeneration.Abbreviations MS Murashige & Skoog - 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzyladenine - NAA 1-naphthaleneacetic acid - PPFD photosynthetic photon flux density     

朱砂根愈伤组织培养及悬浮细胞系建立

以朱砂根(Ardisia crenata Sims.)无菌苗的茎段、叶片、胚轴和胚根为外植体进行愈伤组织诱导研究。结果表明:胚根在含有2,4-D的培养基中的诱导率最高,在添加5 mg L-1 AgNO3的MS+2,4-D 0.5 mg L-1+KT 0.01 mg L-1培养基中继代培养的增殖系数高达8倍以上。培养中获得了5种类型的愈伤组织(I-白色湿软状、Ⅱ-白色冰砂状、Ⅲ-淡黄色颗粒状、Ⅳ-黄绿色块状和V-绿色块状),其中Ⅱ和Ⅲ型愈伤组织可以成功建立悬浮细胞系,用M S+2,4-D 0.5 mg L-1+KT 0.01 mg L-1培养基进行固-液轮回培养,可以较好地保持悬浮细胞系。     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying -glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies.     

Different methylation pattern of melon satellite DNA sequences in hypocotyl and callus tissues

Satellite DNA sequences from Cucumis melo have been examined with respect to modification at CCGG sequences in hypocotyls and in callus tissues. For this purpose, restriction fragments given by HpaII and MspI were compared (both enzymes recognize CCGG sequences but have different sensitivity to methylation at this site). Whereas the methylation level of satellite DNA sequences is on average higher in hypocotyls than in callus tissues, the comparison of partially methylated repeat units of satellite DNA reveals that in callus tissues, all methylated restriction sites are doubly methylated.     

扁桃愈伤组织诱导及分化的研究

以扁桃优良品种'Naporeil'的茎段、叶片和花药作为外殖体,分别对其进行愈伤组织诱导和分化研究,以筛选愈伤组织的最佳诱导增殖培养基、分化培养基和生根培养基.结果表明,该品种以茎段、花药作为外殖体最易诱导获得愈伤组织,叶片不适宜作为外殖体诱导愈伤组织;愈伤组织的最佳诱导增殖培养基均为B5+0.5 mg/L 2,4-D+0.2 mg/L 6-BA+1.0 mg/L NAA,愈伤组织诱导率为100%,增殖倍数最高可达7倍;茎段愈伤组织的分化培养基为MS+0.2 mg/L NAA+0.8 mg/L 6-BA+0.5 mg/L ZT,分化率为71%;花药愈伤组织未见分化.由茎段愈伤组织再分化获得的不定芽在1/2MS+0.5 mg/L IBA培养基上诱导生根,并给以黑暗预处理可使生根率达80%以上.     

Plant regeneration from callus and explants of Sesbania spp.

Root, hypocotyl and cotyledon explants of Sesbania bispinosa, Sesbania cannabina, Sesbania formosa, and Sesbania sesban were cultured on Murashige and Skoog medium with benzyladenine (BA; 2.22, 4.44, 8.88 M) in combination with 2,4-dichlorophenoxyacetic acid (2,4-d; 2.26, 4.52, 9.05 M), indolebutyric acid (IBA; 0.25, 0.49, 4.92 M) or naphthaleneacetic acid (NAA; 2.69, 5.37, 10.74 M). Although all explant types developed some callus, callus occurred earliest and continued to grow fastest with hypocotyls. Media including 2.4-d or NAA gave the fastest growing callus. Callus was subcultured up to 10 times at 20-day intervals and retained a rapid growth rate. Shoots regenerated readily from both hypocotyls or cotyledons but not from roots. Shoot organogenesis was most frequent with IBA (0.25–4.92 M) in combination with BA (4.44–8.88 M) and did not occur with 2,4-d. With each species at least one medium induced shoot differentiation from more than 50 percent of the callus pieces. With one exception, media containing IBA that induced shoot organogenesis on explants also did so in callus, but media containing NAA, even when effective with explants, did not cause differentiation of callus. Shoots that differentiated were excised and cultured on MS medium without growth regulators or with IBA (2.46, 4.92, 9.84 M). Roots developed after 3–8 days on an appropriate rooting medium, often without IBA. Rooted plantlets were transplanted to pots in a greenhouse and developed into normal plants. Suitable media and protocols for initiating and subculturing callus and regenerating whole plants in vitro from callus and explants have thus been established for four species of Sesbania.     

牛蒡组织培养和高频植株再生的研究

通过对牛蒡(A rctium lapp a L.)不同外植体、不同激素配比的比较研究,建立了牛蒡离体培养高效植株再生体系.牛蒡子叶与下胚轴切段在含2.0 m g/L 2,4-D和0.5~2.0 m g/L BA的M S培养基中愈伤组织诱导率可以达到87%~100%;在1.0~3.0 m g/L NAA和0.5~2.0 m g/L BA的M S培养基上通过愈伤组织间接分化或外植体直接分化形成不定芽,其中愈伤组织分化率可达100%;下胚轴的分化率明显高于子叶,在1.0 m g/L NAA和1.0 m g/L BA的M S培养基上下胚轴直接分化率达77.3%.组织学观察发现牛蒡再生有器官发生和体细胞胚发生两种途径.将生长状态良好的不定芽转至含1.0 m g/L IBA和1.0 m g/L NAA的1/2 M S培养基上生根,移栽,成活率达到93.3%.从诱导愈伤组织到组培苗在珍珠岩中过渡成活,大约需要13周.组培苗次年开花并结实,生长形态特征正常.     

唐古特大黄组织培养技术的研究

试验选用唐古特大黄(Rheum tunguticum Maxim．ex Regel．)种了萌发的无菌苗及无菌苗子叶、下胚轴、胚根和幼根作为材料,研究唐古特大黄不同外植体的离体培养技术。结果表明,唐古特大黄的无菌苗和无菌苗子叶、下胚轴、胚根和幼根都可以作为离体培养的良好外植体。唐古特大黄的最适分化培养基足：B5 NAA0．1mg／L 6-BA3mg／L;最适乍根培养素是：1／2MS NAA1mg／L 3%蔗糖或1／2MS NAA0．5mg／L 3％蔗糖;愈伤组织诱导培养基是：MS 2,1-D 1mg／L NAA1mg/L 6BA1mg／L。     

Induction of mitosis in polytene nuclei and hormonal effect on nuclear changes during callus initiation in diploid Urginea indica Kunth. (liliaceae)

Bulb scale and inflorescence explants of Urginea indica Kunth. (2n=20) were cultured in vitro on modified Murashige & Skoog's medium with different hormonal composition. Media containing 2,4-dichlorophenoxy-acetic acid (2,4-D) (2 and 4 mgl^–1) and -naphthalene-acetic acid (NAA) (2 mgl^–1) could induce callus in inflorescence explants. Combination of 2,4-D (4 mgl^–1) + NAA (2 mgl^–1) + Kinetin (2 mgl^–1) only could induce callus formation in scale explants. The bulb scale explants contained mostly diploid cells while the inflorescence explants contained cells with nuclear DNA content ranging from 2C to 64C. The lowest karyological heterogeneity was recorded in callus derived from bulb scale and in callus derived from inflorescence induced with NAA. The highest variability was recorded on media with 2,4-D alone. Induction of division, probably of the pre-existing polytenic nuclei in the inflorescence explant, has been suggested to be the cause of origin of polyploid cells in such cases.     

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.     

Shoot regeneration from mesophyll protoplasts and leaf explants of Rehmannia glutinosa

Mesophyll protoplasts obtained from leaves of shoot cultures of Rehmannia glutinosa were cultured in Murashige and Skoog (1962) liquid or liquid-over-agar medium containing 2.0 mg L^?1 naphthaleneacetic acid and 0.5 mg L^?1 benzylamino purine. An amino acid mixture of glutamine, arginine, glycine, and aspartic acid promoted sustained protoplast division, with an average plating efficiency of 27%. Protoplast-derived colonies formed callus which readily regenerated shoots on fransfer to Murashige and Skoog based agar medium with 2.0 mg L^?1 indoleacetic acid and 1.0 mg L^?1 benzylamino purine. Leaf explants also showed a marked capacity for shoot regeneration in culture.     

Plant regeneration from apple seedling explants and callus cultures

Leaf, cotyledon, and hypocotyl explants were obtained from 3-week-old seedlings of open-pollinated ‘Golden Delicious’ (Malus domestica bork H.) grown in vitro. They were placed on modified Murashige and Skoog (MS) medium containing B5 vitamins, sucrose and agar, supplemented with 6-benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA), and maintained at 25°C±2 in the light or in the dark to assess morphogenetic responses. Leaf and cotyledon explants cultured in the dark for an initial 3 weeks, then transferred to light for 4 weeks, produced 5- to 20-fold more adventitious shoots than those cultured for 7 weeks in the light. Conversely, light did not significantly influence the number of adventitious shoots formed on hypocotyl explants. Five-minute daily exposures of leaf explants to red light (651 nm) suppressed adventitious shoot formation by 80%; five-minute exposure to far-red light (729 nm) immediately following the red light counteracted the red suppression. Seedling explants, immature fruit halves and immature embryos were also cultured on Schenk and Hildebrandt (SH) medium containing 2, 4-dichlorophenoxyacetic acid (2, 4-D), p-chlorophenoxyacetic acid (CPA) and kinetin. Light inhibited callus formation on leaf and cotyledon explants, but not on hypocotyl explants. The derived callus was placed on MS + BAP or MS + BAP + NAA for shoot regeneration. Both shoots and roots regenerated from callus placed in the dark but not in the light; the frequency of shoot regeneration was 5% or less. Regenerated shoots were rooted on MS macronutrient salts (1/3 concentration), micronutrients, i-inositol, thiamine HCl, sucrose and agar with or without indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or NAA under a light intensity of 5.0 W.m^-2 (16 h per day). Auxin concentration strongly influenced root morphology.     

Plant regeneration via somatic embryogenesis from protoplasts of six explants in Coker 201 (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>)

Fertile regenerated plants were obtained from protoplasts via somatic embryogenesis in Coker 201 (Gossypium hirsutum L.). Protoplasts were isolated from six different explantsleaves, hypocotyls, young roots, embryogenic callus, immature somatic embryos and suspension cultures and cultured in liquid thin layer KM8P medium. Callus-forming percentage of 20–50% was obtained in protoplast cultures from embryogenic callus, immature embryos and suspension cultures, and visible callus formed within 2 months. Callus-forming percentage of 5–20% in protoplast cultures from young roots, hypocotyls and leaves, and visible callus formed in 3 months. NAA 5.371 μM/kinetin 0.929 μM was effective to stimulate protoplast division and callus formation from six explants. Percentage of callus formation in the medium with 2,4-D 0.452 μM/kinetin 0.465 μM was over 40% from suspension cultures and immature embryos, 25% from embryogenic callus and 10% from hypocotyls. Callus from protoplasts developed into plantlets via somatic embryogenesis. Over 100 plantlets were obtained from protoplasts derived from 6 explants. Ten plants have been transferred to the soil, where they all have set seeds.