BeMADS1 is a key to delivery MADSs into nucleus in reproductive tissues-De novo characterization of Bambusa edulis transcriptome and study of MADS genes in bamboo floral development.

BACKGROUND: The bamboo Bambusa edulis has a long juvenile phase in situ, but can be induced to flower during in vitro tissue culture, providing a readily available source of material for studies on reproductive biology and flowering. In this report, in vitro-derived reproductive and vegetative materials of B. edulis were harvested and used to generate transcriptome databases by use of two sequencing platforms: Illumina and 454. Combination of the two datasets resulted in high transcriptome quality and increased length of the sequence reads. In plants, many MADS genes control flower development, and the ABCDE model has been developed to explain how the genes function together to create the different whorls within a flower. RESULTS: As a case study, published floral development-related OsMADS proteins from rice were used to search the B. edulis transcriptome datasets, identifying 16 B. edulis MADS (BeMADS). The BeMADS gene expression levels were determined qRT-PCR and in situ hybridization. Most BeMADS genes were highly expressed in flowers, with the exception of BeMADS34. The expression patterns of these genes were most similar to the rice homologs, except BeMADS18 and BeMADS34, and were highly similar to the floral development ABCDE model in rice. Transient expression of MADS-GFP proteins showed that only BeMADS1 entered leaf nucleus. BeMADS18, BeMADS4, and BeMADS1 were located in the lemma nucleus. When co-transformed with BeMADS1, BeMADS15, 16, 13, 21, 6, and 7 translocated to nucleus in lemmas, indicating that BeMADS1 is a key factor for subcellular localization of other BeMADS. CONCLUSION: Our study provides abundant B. edulis transcriptome data and offers comprehensive sequence resources. The results, molecular materials and overall strategy reported here can be used for future gene identification and for further reproductive studies in the economically important crop of bamboo.

[Probiotics improve obesity-associated dyslipidemia and insulin resistance in high-fat diet-fed rats].

OBJECTIVE: To evaluate the effect of probiotics (bifidobacterium breve and lactobacillus acidophilus) on serum lipid, serum insulin and insulin resistance in high-fat diet (HFD)-induced obese rats. METHODS: Fifty male Sprague-Dawley rats were randomly assigned to a control (n=10) and a high fat diet groups (n=40) and were fed with standard diet and HFD respectively. Four weeks later, thirty-six HFD-induced obese rats were randomly administered with normal saline (NS), bifidobacterium breve and lactobacillus acidophilus daily (n=12 each). Four weeks later, body lengths, body weights and abdomen circumference of rats were measured, blood lipid, glucose and insulin levels were measured, and Lee's index and insulin resistance index were calculated. RESULTS: Body weight, abdomen circumference, Lee's index, fasting glucose, triglyceride (TG), total cholesterol (TC), low density lipoprotein (LDL) in the NS-treated HFD group were significantly higher than the control group (P<0.05). The bifidobacterium breve and lactobacillus acidophilus-treated groups had significantly lower levels of body weight, abdomen circumference, Lee's index, fasting glucose, TC, TG and LDL than the NS-treated HFD group (P<0.05), but the levels of the parameters in the bifidobacterium breve and lactobacillus acidophilus-treated groups were significantly higher than the control group (P<0.05). High density lipoprotein (HDL) and insulin sensitivity index in the NS-treated HFD group were significantly lower than the control group (P<0.05). Bifidobacterium breve and lactobacillus acidophilus treatment dramatically increased HDL levels and insulin sensitivity index compared with the NS-treated HFD group (P<0.05), although the levels of the two parameters did not reach to the levels of the control group. There were significant differences in the levels of fasting insulin, insulin resistance index and insulin secretion index between the bifidobacterium breve and lactobacillus acidophilus groups (P<0.05). CONCLUSIONS: Lactobacillus acidophilus and bifidobacterium breve can decrease serum levels of lipid and glucose and improve insulin resistance in obese rats. Bifidobacterium breve seems to be more effective on attenuating insulin resistance than lactobacillus acidophilus.

[Research on the features of DNA methylation in leaves of different chronological ages of Phyllostachys heterocycla var. pubescens based on the method of MSAP].

In order to probe the features of DNA methylation for bamboo stand with different chronological ages, the technique of methylation-sensitive amplified polymorphism (MSAP) was employed to detect DNA methylation in the paper. Experiment material is Moso bamboo (Phyllostachys heterocycla var. pubescens) leaves with 3 various chronological ages (5, 31, and > 60 years after seed germination). During the procedure of genome DNA extration and MSAP analysis, total 35 pairs of MSAP primers were amplyfied. The results showed that MSAP value for bamboo with those three chronological ages were respectively 24.44%, 28.21% and 32.12%, and full-methylation ratios were 16.57%, 19.41% and 21.23%. Meanwhile, the value of variable sites for methylation reached 52.3% and for demethylation was 10.3%. Therefore, it could be concluded that with ages increasing MSAP value rising for Moso bamboo. Moreover the result of variance analysis for methylation ratio indicated that no significant (P = 0.307 > 0.05) difference among individuals with the same ages, while significant (P < 0.001) difference exsited among different chronological ages. Throuygh ANOVA it showed that 6 pairs (E3/HM2, E3/HM6, E3/HM7, E4/HM5, E4/HM6 and E5/HM5) of primers had obvious influence on DNA methylation for ones with different chronological ages and could be used for further research.

Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application.

In the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system, protoplasts are not only useful for rapidly validating the mutagenesis efficiency of various RNA-guided endonucleases, promoters, sgRNA designs, or Cas proteins, but can also be a platform for DNA-free gene editing. To date, the latter approach has been applied to numerous crops, particularly those with complex genomes, a long juvenile period, a tendency for heterosis, and/or self-incompatibility. Protoplast regeneration is thus a key step in DNA-free gene editing. In this report, we review the history and some future prospects for protoplast technology, including protoplast transfection, transformation, fusion, regeneration, and current protoplast applications in CRISPR/Cas-based breeding.

Flowering of Woody Bamboo in Tissue Culture Systems.

Flowering and subsequent seed set are not only normal activities in the life of most plants, but constitute the very reason for their existence. Woody bamboos can take a long time to flower, even over 100 years. This makes it difficult to breed bamboo, since flowering time cannot be predicted and passing through each generation takes too long. Another unique characteristic of woody bamboo is that a bamboo stand will often flower synchronously, both disrupting the supply chain within the bamboo industry and affecting local ecology. Therefore, an understanding of the mechanism that initiates bamboo flowering is important not only for biology research, but also for the bamboo industry. Induction of flowering in vitro is an effective way to both shorten the flowering period and control the flowering time, and has been shown for several species of bamboo. The use of controlled tissue culture systems allows investigation into the mechanism of bamboo flowering and facilitates selective breeding. Here, after a brief introduction of flowering in bamboo, we review the research on in vitro flowering of bamboo, including our current understanding of the effects of plant growth regulators and medium components on flower induction and how in vitro bamboo flowers can be used in research.

Correlation between DNA methylation and chronological age of Moso bamboo (Phyllostachys heterocycla var. pubescens).

BACKGROUND: Chronological age is the primary consideration when studying the physiological development, aging, and flowering of bamboo. However, it's difficult to determine bamboo's chronological age if the time of germination is unknown. To investigate the chronological age of bamboo from the genomic DNA methylation profile, methylation-sensitive amplification polymorphism (MSAP) was employed to analyze the genomic DNA methylation of Moso bamboo (Phyllostachys heterocycla var. pubescens) from stands of nine germination-ages, using six primer pairs which have previously been shown to yield methylation rates that reflect the age of Moso bamboo. RESULTS: The results showed that the total genomic DNA methylation rates in Moso bamboo at different chronological ages were significantly different, and the increase in genomic DNA methylation rate was consistent with the increase of chronological age. Six primer pairs displayed different genomic DNA methylation rates in Moso bamboo of nine age's group; however, a significantly positive correlation existed among these primer pairs. An integrated index was obtained by performing principal component analysis on the six primer pairs to represent the genomic DNA methylation levels in Moso bamboo of various chronological ages, and a quadratic curve between the chronological age and genomic DNA methylation levels was obtained. CONCLUSIONS: Such a relationship between DNA methylation and its chronological age may serve a reference for its aging study in Moso bamboo.

Protocol for callus induction and somatic embryogenesis in Moso Bamboo.

Moso bamboo [Phyllostachys heterocycla var. pubescens (Mazel ex J. Houz.) Ohwi] is one of the most important forest crops in China and the rest of Asia. Although many sympodial bamboo tissue culture protocols have been established, there is no protocol available for plantlet regeneration as indicated by callus induction for monopodial bamboos, such as Moso bamboo. In the present report, embryogenic callus induction, embryoid development, and germination were established for Moso bamboo from zygotic seed embryos. Callus was initiated from zygotic embryos after 10-20 d culture on MS media supplemented with 4.0 mg/L 2, 4-D and 0.1 mg/L zeatin (ZT). About 50% of the explants produced calli, and nearly 15% of the calli were found to be embryogenic in nature. These embryogenic calli can be subcultured for proliferation in the Murashige and Skoog media (MS) supplemented with 0.5-2.0 mg/L 2, 4-D. These calli were found to have maintained their capacity for regeneration even after one year of subculture. The viable somatic embryoids regenerated in medium containing 5.0-7.0 mg/L ZT. Nearly 5% of the calli were found capable of regenerating into plantlets directly in MS medium containing 5.0-7.0 mg/L ZT. Root growth was more pronounced when the plantlets were transferred to medium containing 2.0 mg/L NAA. After 30 days of subculture, the plantlets were transferred to a greenhouse.