Callus Induction and Plant Regeneration from Mature Seeds of Perennial Ryegrass

Objective The aim was to explore callus induction and plant regeneration of perennial ryegrass, as well as provide the foundation for transgenic research on perennial ryegrass.[ Methed] Mature seeds of perennial ryegrass were used as explants to study the effects of different hormone compositions on callus induction, proliferation and plant differentiation. Result The result showed that the induction rate achieved its highest on 2,4-D of 8 mg/L combining with 6-BA of 0.025 mg/L, which was up to 56.42%. Callus were differentiated after two to three generations, the highest differentiation rate 34.14% was achieved in the medium contained MS medium with 6-BA of 2 mg/L, and the differentiation rate was obviously affected by the callus condition after proliferation. The root inducing medium, containing 0.5 mg/L NAA and MS medium with half of macroelement, gained 98% root inducing rate. Conclusien A high frequency genetic regeneration system was established.

Transcript Profiling Reveals Abscisic Acid,Salicylic Acid and Jasmonic-Isoleucine Pathways Involved in High Regenerative Capacities of Immature Embryos Compared with Mature Seeds in japonica Rice

Induced pluripotent cell mass plays a role in genetic transformation mediated by Agrobacterium. Mature seeds are more recalcitrant to the induction of suitable calli than immature embryos in rice, but the exact molecular mechanisms involved remain elusive. In this study, the morphological structure of calli induced from mature seeds and immature embryos were observed under a scanning electron microscope using a paraffin embedded technique. Meanwhile, a total of 2 173 up- and down-regulated genes were identified in calli induced from mature seeds and immature embryos by RNA-seq technique and furtherly confirmed by quantitative real-time PCR. The results revealed the remarkable morphological differences in calli induced from mature seeds and immature embryos, and plant hormone signal transduction and hormone biosynthesis pathways, such as abscisic acid, salicylic acid and jasmonic-isoleucine, were found to play roles in somatic embryogenesis. This study provided comprehensive gene expression sets for mature seeds and immature embryos that were served as an important platform resource for further functional studies in plant embryogenesis.

Callus induction and plant regeneration from mature bluegrass (Poa pratensis L.) seeds

A protocol was discussed for high efficient plant regeneration from seven bluegrass （Poa pratensis L.） cultivars via an in- direct callus induction and somatic embryogenesis method. Mature seeds were used as explants for callus initiation. Callus induction and proliferation efficiencies were investigated on NB, modified MS （MMS） and MS media, supplemented with 2.0 mg·L^-1 2,4-dichlorophenoxyacetic acid （2,4-D）. The MMS medium performed best. Based on the MMS medium, direct and indirect callus induction effects of bluegrass from mature seeds were compared at the range of 1-5 mg·L^-1 2,4-D contained in the medium. Under the direct callus induction method, the most suitable 2,4-D concentrations varied among cultivars. Under the indirect callus induction method, a significantly high callus induction frequency （93.33%-98.33%） was obtained and there were barely any statistically sig- nificant differences among the tested genetically diverse cultivars. Somatic embryos were promoted on the MMS medium supple- mented with 3 mg·L^-1 2,4-D, 0.1 mg·L^-1 kinetin and 0.8 mg·L^-1 CuSO4. Embryogenetic calli developed into plantlets on the MMS medium containing different concentrations of thidiazuron （TDZ）, and the differentiation frequencies varied in the range from 20.15% to 77.65%. The 0.25 mg·L^-1 TDZ was generally the most suitable concentration for the tested cultivars.

Genetic Analysis and QTL Mapping of Mature Seed Culturability in Indica Rice

Genetic segregation analysis for mature seed culturability was conducted using recombinant inbred lines derived from a cross of indica rice, Yangdao 6 and Pei＇ai 64s. Three indices of seed culturability, the frequency of callus induction, the frequency of brown callus and the increase of callus weight were investigated. A combined genetic map constructed with simple sequence repeat （SSR）, sequence tag site （STS）, cleaved amplified polymorphic sequences （CAPS） and single nucleotide polymorphism （SNP） markers covered a total distance of 1 732.5 cM, averaging approximately 12 cM between two neighboring loci. Three QTLs on chromosomes 7, 7 and 10 were detected for the frequency of callus induction; three QTLs on chromosomes 6, 7 and 9 were detected for the frequency of brown callus; and two QTLs on chromosomes 5 and 7 were detected for the increase of callus weight. Common QTLs mapped at the interval flanking RM5481 and RM6835 on chromosome 7 were identified to be involved in the frequency of callus induction and the frequency of brown callus, explaining 7.29% and 12.52% of phenotypic variation, respectively. A total of 14 epistatic effects were detected for the three indices of mature seed culturability. ~

Dominance of Brassica and No Effects of Raphanus in Mature Seed Production in Intergeneric Hybrid between Brassica rapa ssp. Pekinensis and Raphanus

We succeeded in producing mature seed from a line of Brassica rapa ssp. pekinensis that had been hybridized with Raphanus sativus var. major. Our focus was on dominance of B. rapa ssp. pekinensis;radish (R. sativus var. major) had no influence. Marker tests for similarity showed that the original CR291M-64 x HwiM-2 hybrid was an inbred CR291M-64, rather than a genuine cross;this appears to have resulted from weak self-incompatibility in this strain. The plants from the mature seed bloomed with reddish flowers differently shown up to present. The intergeneric hybrid between Brassica inbred and Raphanus hybrid was very weak in strength compared to the Brassica inbred which was self-pollinated even though the cause of the weak was not identified. The hybrids between Brassica hybrid, dominant and elite recessive, and Raphanus can be developed in large quantities using mature hybrid seed without resorting to ovule culture techniques.

GAMT2 Encodes a Methyltransferase of Gibberellic Acid That is Involved in Seed Maturation and Germination in Arabidopsis

Salicylic acid methyltransferase （SAMT）, benzoic acid methyltransferase （BAMT） and theobromine methyltransferase （TH） （henceforth, SABATH） family proteins belong to a unique class of mehtyltransferase that can methylate small molecular compounds Including indole-3-acidic acid （IAA）, salicylic acid （SA） and jasmonic acid （JA）, in plants. Here we report that the GAMT2 protein, which has 34.2% similarity with IAMT1 in the amino acid sequence, can methylate gibberellic acid （GA）. Biolnformatics analysis suggests that GAMT2 may be able to methylate one molecule larger than SA. GAMT2 is predominantly expressed in the developing seed embryo and endosperm in Arabidopsis. During seed germination, the expression of GAMT2 decreases until the cotyledons expand out of the seed coat. Overexpression of GAMT2 in Arabidopsis resulted in multiple phenotypes, including dwarfism, retarded growth, late flowering, and reduced fertility, which are similar to the phenotypes of GA-deficient mutants. Seed germination assay showed that GAMT2 overexpression in plants was hypersensitive to GA biosynthesis inhibitor （ancymidol） and abscisic acid （ABA） treatments, whereas the GAMT2 null mutant （SALK_075450） was slightly Insensitive to such treatments, suggesting that GAMT2 may methylate GA or ABA. Enzyme activity analysis indicated that GAMT2 was able to methylate GA3 into Methyi-GA3 in vitro, but could not methylate ABA. Microarray analysis on GAMT2 overexpression plants suggested that Methyl-GA may be an Inactive form of GA in Arabidopsis. These data suggest that GAMT2 Is Involved in seed maturation and germination by modulating GA activity.

Retarded Embryo Development 1(RED1) regulates embryo development,seed maturation and plant growth in Arabidopsis

Plant seeds accumulate large amounts of protein and carbohydrate as storage reserves during matura- tion. Thus, understanding the genetic control of embryo and seed development may provide bioengi- neering tools for yield improvement. In this study, we report the identification of Retarded Embryo Development1 （RED1） gene in Arabidopsis, whose two independent T-DNA insertion mutant lines, SALK_085642 （red1-1 ） and SALK_022583 （red1-2）, show a retarded embryo development phenotype. The embryogenesis process ceases at the late heart stage in red1-1 and at the bent-cotyledon stage in red1-2, respectively, resulting in seed abortion in both lines. The retarded embryo development and seed abortion phenotypes reverted to normal when REDI complementation constructs were introduced into mutant plants. Small redl-2 homozygous plants can be successfully rescued by culturing immature seeds, indicating that seed abortion likely results from compromised tolerance to the desiccation process associated with seed maturation. Consistent with this observation, redl-2 seeds accumulate less protein, and the expression of two late embryo development reporter transgenes, LEA：：GUS and β-con- glycinin：：GUS, was significantly weak and started relatively late in the redl-2 mutant lines compared to the wild type. The REDI gene encodes a plant specific novel protein that is localized in the nucleus. These results indicate that RED1 plays important roles in embryo development, seed maturation and plant growth.

The Pumilio RNA-binding protein APUM24 regulates seed maturation by fine-tuning the BPM-WRI1 module in Arabidopsis

Pumilio RNAbindingproteinsparticipateinmes-senger RNA(mRNA)degradation and t ranslational repression,but their roles in plant development are largely unclear.Here,we show that Arabidopsis PUMILIO PROTEIN24(APUM24),an atypical Pumiliohomology domaincontaining protein,plays an im-portant part in regulating seed maturation,a major stage of plant development.APUM24 is strongly expressed in maturing seeds.Reducing APUM24 expression resulted in abnormal seed maturation,wrinkled seeds,and lower seed oil contents,and APUM24 knockdown resulted in lower levels of WRINKLED 1(WRI1),a key transcription factor con-trolling seed oil accumulation,and lower expression of WRI1 target genes.APUM24 reduces the mRNA stability of BTB/POZMATH(BPM)family genes,thus decreasing BPM protein levels.BPM is responsible for the 26S proteasomemediated degradation of WRI1 and has important functions in plant growth and development.The 3′untranslated regions of BPM family genes contain putative Pumilio response elements(PREs),which are bound by APUM24.Re-duced BPM or increased WRI1 expression rescued the decient seed maturation of apum242 knock-down mutants,and APUM24 overexpression re-sulted in increased seed size and weight.Therefore,APUM24 is crucial to seed maturation through its action as a positive regulatornetuning the BPMWRI1 module,making APUM24 apromising target for breeding strategies to increase crop yields.

The COP9 SIGNALOSOME Is Required for Postembryonic Meristem Maintenance in Arabidopsis thaliana

Cullin-RING E3 ligases （CRLs） regulate different aspects of plant development and are activated by modification of their cullin subunit with the ubiquitin-like protein NEDD8 （NEural precursor cell expressed Developmentally Down-regulated 8） （neddylation） and deactivated by NEDD8 removal （deneddylation）. The CONSTITUTIVELY PHOTOMORPHOGENIC9 （COP9） signalosome （CSN） acts as a molecular switch of CRLs activity by reverting their neddylation status, but its contribution to embryonic and early seedling development remains poorly characterized. Here, we analyzed the phenotypic defects of csn mutants and monitored the cullin deneddylation/neddylation ratio during embryonic and early seedling development. We show that while csn mutants can complete embryogenesis （albeit at a slower pace than wildtype） and are able to germinate （albeit at a reduced rate）, they progressively lose meristem activity upon germination until they become unable to sustain growth. We also show that the majority of cullin proteins are progressively neddylated during the late stages of seed maturation and become deneddylated upon seed germination. This developmentally regulated shift in the cullin neddylation status is absent in csn mutants. We conclude that the CSN and its cullin deneddylation activity are required to sustain postembryonic meristem function in Arabidopsis.