Assessment of factors affecting in vitro shoot regeneration from axillary bud explant of Camptotheca acuminata

Axillary buds from 3-yr.-old seedlings of Camptotheca acuminata in the greenhouse were cultured on the different basal media with different concentrations of growth regulators for shoot regeneration for studying the effects of different basal media, different concen- trations of growth regulators (BA or TDZ), sucrose, agar and pH value on shoot regeneration from axillary bud. The results showed that B5 and WPM media were the optimal basal media and the optimal phyotohormone was BA of 1.0 mg/L or TDZ of 0.1mg/L; The concentrations of sucrose of 30g/L and agar of 6g/L were most suitable for the shoot regeneration; pH value from 5.8 to 6.6 were broadly effective, but the best at pH 5.8.

Effects of Thidiazuron,basal medium and light quality on adventitious shoot regeneration from in vitro cultured stem of Populus alba×P. berolinensis

The effect of Thidiazuron （TDZ）, basal media and light quality on adventitious shoot regeneration from in vitro cultured stem of Populus albaxP berolinensis were determined to establish a high efficiency shoot regeneration system from stem explants of P. alba^P berolinensis. Stems ofPopulus alba^P berolinensis were collected from cultured shoots in vitro derived from dormancy buds of 3-year-old seedlings. The stem explants were cultured on MS medium containing 0.02-mg·L-1NAA （naphthaleneacetic acid）, and 0.1, 0.3, 0.5 and 1.0 mg·L-1 concentrations of TDZ to determine the effect of TDZ on shoot regeneration. Three basal media, i.e. MS, woody plant medium （WPM） and B5, were used to test their influences of different media on adventitious shoot regeneration. Green, red, blue and yellow plastic films in comparison with florescent light as control were used to observe their effects on shoot regeneration. The results showed that differ- ent concentrations of TDZ had an evident influence on shoot regeneration. Lower concentration of TDZ （0.1 mg·L-1） resulted in more ad- ventitious shoot regeneration and higher concentration of TDZ （〉0.1 mg·L-1） inhibited shoot regeneration. Among different media, MS medium exhibited a high efficiency for shoot regeneration, followed by WPM medium, while B5 medium inhibited shoot regeneration. Normal light and yellow light exhibited better effects on shoot regeneration, compared with other light.

Adventitious Shoot Regeneration from Leaf of Uenishiwase Persimmon

Effects of basal mediums, hormones and their concentrations on the shoot regeneration fromleaf of sweet persimmon (Diospyros kaki Thunb. cv. Uenishiwase) were studied by orthogonaldesign trial. The result showed that modified Murashige and Skoog [MS (1/2 N)] was the mostoptimum for the regeneration and 1/2 MS was better than MS. Shoot percentage in the mediumcontaining 4.0 mg L-1 ZT(zeatin) was much higher than that of other two concentrations, amongwhich 2.0 mg L-1 ZT was much better than 1.0 mg L-1 ZT and shoot percentage in the concentrationof 1.0 mg L-1 ZT was only 4%. There were no any beneficial effects when supplementing IAA in themedium. Shoot percentage and average shoots per explants were dramatically decreased in the 2.0 mgL-1 IAA. Data in the orthogonal trial indicated that ZT was the most effective factor in theshoot regenerating of Uenishwase persimmon and basal medium was important too, but IAA had noany beneficial effects at all. In the orthogonal trial, the best result was achieved in MS (1/2 N) medium containing 4.0 mg L-1 ZT and 1.0 mg L-1 IAA, in which shoot percentage and averageshoots per explants were 86% and 2.2, respectively.

Influences of Genotype and Explant on Callus Induction and Shoot Regeneration in Sunflower （Helianthus annuus L.）

In vitro regeneration was performed with the aim of developing efficient callus and shoot regeneration from different explants of sunflower （Helianthus annuus L.）. The 6 genotypes （RHAI0, RHA 14, RHA15, PR6404, N Record 109/Sanay 3-5, N Record 109/Sera） were used as plant materials. The roots, hypocotyls and cotyledons were excised from 4 day-old seedlings and cultured on embryo induction medium （EIM） supplemented with Benzylaminopurine （BAP）, Naphthaleneacetic acid （NAA） and Gibberellic acid （GA3）. The experiments were kept in 18/6 hour light/dark photoperiod at 26± 2 ℃for four weeks. The rates of callus and root organ formation on explants were 67%-100% and 7%-31% respectively, depending on the genotype. Root explants produced statistically high callus formation （2.6 score） compared to cotyledon （2.0 score） and hypocotyl explants （2.5 score） at all sunflower genotypes used. The highest shoot regeneration was obtained from RHAI 5 （7%） while PR6404 （100%） produced the highest callus formation.

EXPRESSION OF TOMATO ANTISENSE ACC SYNTHASE GENE IN TRANSGENIC TOBACCO AND ITS ROLE IN SHOOT FORMATION

An ACC synthase cDNA isolated from tomato (Lycopersicum esculentum Mill.) fruit was constructed in antisense orientation under the transcriptional control of CaMV 35S promoter and then introduced into tobacco (Nicotiana tabacum L.) . PCR amplification demonstrated the integration of this antisense gene in tobacco genomes. Northern hybridization and reverse transcription-PCR analyses indicated the expression of this heterologous antisense gene in the transgenic tobacco tissues, which caused a decrease in the ethylene production, particularly when shoot regeneration exhibited. The ability of shoot regeneration of the transgenic plant during the culture process was enhanced remarkably as compared with that of the control. These results indicate at the molecular level that ethylene may play a regulatory role in shoot formation.

One Rapid and Efficient Method for Isolation of Total RNA from Shoots Regenerated in vitro of Populus suaveolens

Total RNA was isolated from shoots regenerated in vitro of Populus suaveolens by the modified method of CTAB, and two clear bands of rRNA (28S and 18S) were observed in agarose electrophoresis. In addition, the values of OD260/OD280 and OD260/OD230 of extracted RNA were 2.12 and 2.23 respectively. The results show that RNA is little decomposed and the purity of RNA is high. Moreover, RNA isolated by the modified method of CTAB reagent had been successfully used for reverse transcription of P. suaveolens cDNAs and ideal special band was observed.

Influences of antibiotics on plantlet regeneration via organogenesis in loblolly pine (Pinus taeda L.)

Three antibiotics ampicillin, carbenicillin, and cefotaxime were evaluated for their effects on induction, growth, and differentiation of organogenic calli, as well as rooting of regenerated shoots of three loblolly pine (Pinus taeda L.) genotypes. Of the antibiotics administered, cefotaxime maximally increased the frequency of callus formation and growth rate of organogenic calli, carbenicillin maximally increased the frequency of shoot regeneration and the average number of adventitious shoots per piece of organogenic callus, ampicillin maximally decreased the rooting frequency of regenerated shoots and mean number of roots per regenerated shoot, in comparison with antibiotic-free media. Compared with the control, ampicillin minimally increased the frequency of callus formation, cefotaxime minimally increased the frequency of shoot regeneration, and carbenicillin mini-mally decreased the rooting frequency of regenerated shoots in three loblolly pine genotypes tested. All three antibiotics in-creased the frequencies of callus formation and shoot regeneration, and reduced the rooting frequency of regenerated shoots suggested that the establishment of an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integra-tion of foreign genes into loblolly pine need to select a suitable antibiotic. This investigation could be useful for optimizing genetic transformation of conifers.

Regeneration of Lo Han Kuo (Siraitia grosvenori) in vitro by Direct Organogenesis

The traditional technique for asexual propagation of Siraitia grosvenori in vitro is adopted widely by pressing the vine, which has a high risk of carrying viral diseases and limits the production of promoted cultivars. So this paper reported in vitro regeneration of S. grosvenori by testing for shoot induction from various explant sources such as leaf, petiole and stem. Several phytohormone combinations of TDZ, BA and IAA were examined for shoot regeneration and NAA or IBA for rooting. The highest shoot induction rate （100% of regeneration frequency and 15.3 shoots per explant） in leaf was obtained by incubation on MS medium supplemented with 0.5 mg·L^-1 TDZ, 2.0 mg·L^-1 BA and 0.5 mg·L^-1 IAA; unlike shoot regeneration in leaves, the most efficient bud inductions in petiole and stem explants were initiated on MS medium containing 0.2 mg·L^-1 TDZ, 2.0mg·L^-1 BA and 0.5 mg·L^-1 IAA, in addition, adventitious buds in petiole and stem explants needed to be transformed to MS medium for development; optimal root was obtained when shoots were cultured on 1/2MS medum supplemented with 0.5 mg·L^-1 NAA or 0.5 mg·L^-1 IBA.

The nitrate-responsive transcription factor Md NLP7 regulates callus formation by modulating auxin response

Under appropriate culture conditions,plant cells can regenerate new organs or even whole plants.De novo organ regeneration is an excellent biological system,which usually requires additional growth regulators,including auxin and cytokinin.Nitrate is an essential nutrient element for plant vegetative and reproductive development.It has been reported that nitrate is involved in auxin biosynthesis and transport throughout the growth and development of plants.In this study,we demonstrated that the ectopic expression of the MdNLP7 transcription factor in Arabidopsis could regulate the regeneration of root explants.MdNLP7 mainly participated in the regulation of callus formation,starting with pericycle cell division,and mainly affected auxin distribution and accumulation in the regulation process.Moreover,MdNLP7 upregulated the expression of genes related to auxin biosynthesis and transport in the callus formation stage.The results demonstrated that MdNLP7 may play a role in the nitrate-modulated regeneration of root explants.Moreover,the results revealed that nitrate–auxin crosstalk is required for de novo callus initiation and clarified the mechanisms of organogenesis.

Establishment of Embryogenic Cultures and Efficient Plant Regeneration System from Explants of Forced Softwood Shoots of Teak (Tectona grandis L.)

The present study highlights an efficient plant regeneration system in teak(Tectona grandis L.) using forced softwood shoots as an initial plant material. Forced softwood shoots of teak were cut to prepare shoot tip, nodal and internodal explants and cultured on Murashige and Skoog(MS) medium + NAA(1, 3, 6, 10, and 15 μmol · L-1) or TDZ(0.001, 0.01, 0.1, 1, 4, 8, 10, 12 μmol · L-1) for callus induction. Such calluses were further grown on the same levels of TDZ or 0.4, 1, 4, 8, 10 μmol · L-1 BA + 1 μmol · L-1 IBA or GA3. Callus induction was the highest with4.55 cm3 callus volume and 5.75 g dry weight at 0.1 μmol · L-1 TDZ from shoot tips after 35 days. Embryogenic calluses were then shifted to 6, 8 or12 μmol · L-1 TDZ + 2 μmol · L-1 BA or IBA along with 5 mmol · L-1 ascorbic acid(AA) for shoot regeneration from embryogenic cultures. The highest embryogenesis(100%) with 36.4 globular and 5.5 heart-shaped embryo-like structures was obtained at 8 μmol · L-1 TDZ + 2 μmol · L-1 BA after 63 days. Such cultures when further maintained on the same medium up to 150 days resulted in 100% shoot regeneration with 16.4 mean shoots.Shoots were elongated up to 50 mm on agar medium + 8 μmol · L-1 BA + 1 μmol · L-1 GA3. An efficient rooting response(70%) was achieved having4.50 mean number and 49.10 mm root length at 8 μmol · L-1 IBA + 8 μmol · L-1 NAA + 0.1% activated charcoal after 36 days. Rooted shoots were acclimatized in glasshouse, achieving 56.6% plantlet survival.

Cytosine methylation of the FWA promoter promotes direct in vitro shoot regeneration in Arabidopsis thaliana

Epigenetic modifications within promoter sequences can act as regulators of gene expression.Shoot regeneration is influenced by both DNA methylation and histone methylation,but the mechanistic basis of this regulation is obscure.Here,we identified 218 genes related to the regeneration capacity of callus that were differentially transcribed between regenerable calli(RC)and non-regenerable calli(NRC)in Arabidopsis thaliana.An analysis of the promoters of five of the differentially expressed genes(FWA,ACC1,TFL1,MAX3,and GRP3)pointed to an inverse relationship between cytosine methylation and transcription.The FWA promoter was demethylated and highly expressed in NRC,whereas it was methylated and expressed at low levels in RC.Explants of the hypomethylation mutants fwa-1 and fwa-2 showed strong levels of FWA expression and regenerated less readily than the wild type,suggesting that FWA inhibits direct in vitro shoot regeneration.WUSCHEL-RELATED HOMEOBOX 9(WOX9),which is required for shoot apical meristem formation,was directly repressed by FWA.Overexpressing WOX9 partly rescued the shoot regeneration defect of fwa-2 plants.These findings suggest that cytosine methylation of the FWA promoter forms part of the regulatory system governing callus regenerability and direct in vitro shoot regeneration.

ARR12 promotes de novo shoot regeneration in Arabidopsis thaliana via activation of WUSCHE Lexpression

Auxin and cytokinin direct cell proliferation and differentiation during the in vitro culture of plant cells, but the molecular basis of these processes, especially de novo shoot regeneration, has not been fully elucidated. Here, we describe the regulatory control of shoot regeneration in Arabidopsis thaliana(L.) Heynh, based on the interaction of ARABIDOPSIS RESPONSE REGULATOR12(ARR12) and WUSCHEL(WUS). The major site of ARR12 expression coincided with the location where the shoot apical meristem(SAM) initiated. The oar12 mutants showed severely impaired shoot regeneration and reduced responsiveness to cytokinin; consistent with this, the overexpression of ARR12 enhanced shoot regeneration.Certain shoot meristem specification genes, notably WUSCHEL(WUS) and CLAVATA3, were significantly downregulated in the arr1z explants. Chromatin immunoprecipitation(ChIP) and transient activation assays demonstrated that ARR12 binds to the promoter of WUS. These observations indicate that during shoot regeneration, in vitro, ARR12 functions as a molecular link between cytokinin signaling and the expression of shoot meristem specification genes.

AtPRMT5 Regulates Shoot Regeneration through Mediating Histone H4R3 Dimethylation on KRPs and Pre-mRNA Splicing of RKP in Arabidopsis

Protein arginine methylation plays important roles in diverse biological processes, but its role in regulating shoot regeneration remains elusive. In this study, we characterized the function of the protein arginine methyltransferase AtPRMT5 during de novo shoot regeneration in Arabidopsis. AtPRMT5 encodes a type II protein arginine methyltransferase that methylates proteins, including histories and RNA splicing factors. The frequency of shoot regeneration and the number of shoots per callus were decreased in the atprmt5 mutant compared with those in the wild type. Chromatin immunoprecipitation analysis revealed that AtPRMT5 targets KIP-RELATED PROTEINs （KRPs）, which encode the cyclin-dependent kinase inhibitors that repress the cell cycle. During shoot regeneration, the KRP transcript level increased in the atprmt5 mutant, which resulted from reduced histone H4R3 methylation in the KRP promoter. Overexpression of KRP significantly reduced the frequency of shoot regeneration and shoot number per callus. Furthermore, abnormal pre-mRNA splicing in the gene RELATED TO KPC1 （RKP）, which encodes an ubiquitin E3 ligase, was detected in the atprmt5 mutant. RKP functions in regulating KRP protein degradation, and mutation in RKP inhibited shoot regeneration. Thus, AtPRMT5 regulated shoot regeneration through histone modification-mediated KRP transcription and RKP pre-mRNA splicing. Our findings provide new insights into the function of protein arginine methylation in de novo shoot regeneration.

Tissue Culture Technology for Populus × euramericana Hybrids: Effect of Genotype on in Vitro Regeneration

In order to select a suitable genotype for the studies on genetic transformation, the difference among three genotypes (DN04-18, DN04-25 and DN04-26) of Populus × euramericana (Populus deltoides × P. nigra) in terms of shoot regeneration was investigated in this paper. An efficient in vitro micropropagation method was successfully established for genotype DN04-18. The high frequency shoot regeneration (90.0%) from leaf discs was obtained in genotype DN04-18 on the modified Murashige and Skoog (mMS) medium with 30 g/L sucrose and 5.5 g/L agar (pH: 5.8) which was supplemented with 0.8 mg/L 6-benzyladenine (6-BA), while the shoot regeneration rates of DN04-25 and DN04-26 were below 40.0% even on the same medium. The optimum rooting medium was 1/2 mMS medium supplemented with 0.3-0.4 mg/L indole-3-butyric acid (IBA) for three genotypes. The results showed that genotype is a key factor for restricting shoot regeneration in establishing regeneration system of P. × euramerican hybrids. This micropropagation system of DN04-18 is expected to be suitable for the studies on gene transformation to improve drought tolerance of P. × euramerican.

Nitric Oxide Mediates Cytokinin Functions in Cell Proliferation and Meristem Maintenance in Arabidopsis

Cytokinin and nitric oxide （NO） have been characterized as signaling molecules to trigger cell division in tissue culture. Here, we show that the hypocotyl and root explants of Arabidopsis NO-deficient mutant nosl/noal exhibit severe defects in callus induction and shoot regeneration in response to cytokinin. Accordingly, depletion of NO caused by a NO scavenger leads to a severe inhibitory effect on callus induction. Moreover, cytokinin-induced NO production is impaired in nosl/noal in which cytokinin-triggered activation of cell cycle gene CYCD3;1 is inhibited, indicating that NO may act downstream of cytokinin in the control of cell proliferation through CYCD3;1. This hypothesis is further con- firmed by the genetic evidence that constitutive expression of CYCD3;1 complements the defects of nosl/noal mutant in meristematic activity in shoot, root, and floral tissues as well as in cytokinin-induced callus initiation and shoot regen- eration. Furthermore, we show that NO deficiency caused by loss of NOSl/NOA1 impairs cellular development such as the duration of the mitotic phase and timing of the transition to endocycles in nosl/noal mutant leaves, which can be reverted by constitutive expression of CYCD3;1. Taken together, these results demonstrate that NO mediates transcrip- tional activation of CYCD3,1 in regulating the mitotic cycles in response to cytokinins.