Soybean hairy roots produced in vitro by Agrobacterium rhizogenes-mediated transformation

Soybean is one of the world's most important oil and protein crops. Efficient transformation is a key factor for the improvement of soybean by genetic modification. We describe an optimized protocol for the Agrobacterium rhizogenes-mediated transformation of soybean and the induction of hairy root development in vitro. Cotyledons with 0.5-cm hypocotyls were cut from 5-day-old seedlings and used as explants. After infection and co-cultivation,hairy roots were produced in induction culture medium after 10–12 days. Using this method, 90%–99% of the infected explants of five different cultivars produced hairy roots within one month. Observations using reporter constructs showed that 30%–60% of the hairy roots induced were transformed. Based on high transformation efficiency and short transformation period, this method represents an efficient and rapid platform for study of soybean gene function.

Elevated methionine content in soybean seed by overexpressing maizeβ-zein protein

Soybean provides superior and readily available protein for human and livestock.However,nutritional value of soybean is limited due to the deficiency of an essential amino acid,methionine.To improve total methionine content of soybean,a methionine-rich seed storage protein,β-zein,was introduced into soybean cultivar’Jack’under the control of legumin B4 promoter or Ca MV 35S promoter.Totally 4 T3transgenic lines exhibited higher expression levels of foreign genes,and legumin B4 promoter directed a stronger accumulation ofβ-zein protein than Ca MV 35S promoter.Compared to wild type plant,total methionine content in transgenic soybean seeds significantly increased by up to approximately 15%.Although the introduction ofβ-zein gene improved total methionine content,the level was negligible compared to native soybean storage proteins,implying that the inadequate soluble methionine is the limiting factor.Based on these observations,a new strategy for simultaneously increasing the"source"and"sink"of methionine metabolism is proposed to further improvement of total methionine content in soybean seed.

Identification of transgene insertions in two genetically modified soybeans using high throughput next generation sequencing

Genetically modified(GM) organisms are widely adopted. However, their safety assessments and control are still of special concern to the public. Identifying and localizing transgene insertion is an essentially prerequisite step. In this study, 2 independent transgene soybean lines were selected(LB4-AtDCGS-1-20-5-2 and CGS-ZG11) as typical cases. Both lines contained expression cassette of At-DCGS that encoding a feedback-insensitive cystathionine gamma-synthase to produce higher level methionine(Met). LB4-AtDCGS-1-20-5-2 was whole genome sequenced with one paired-end 500 bp library and two mate-paired 1 kb and 2 kb libraries using Illumina HiSeq sequencing platform. CGS-ZG11 was sequenced with only one paired-end 500 bp library. Both genomes were assembled,and 2 scaffold sequences(1 for each line) were screened out by aligning with transgene.Then the transgene insertion and its flanking regions in soybean genome were further identified and confirmed by PCR cloning and Sanger sequencing. Results showed that these 2 transgene lines had single copy of inserted transgene. Their transgene insertion contents were identified, which facilitates further safety assessment. These results indicated that genome assembly using high throughput sequencing is a powerful tool for identifying transgene insertions, even with limited knowledge.

Content Determination about the Pharmacological Constituents of Salvia miltiorrhiza Cultivated in Jizhou District

[Objectives] To evaluate the quality of Salvia miltiorrhiza cultivated in Jizhou District. [Methods]With Salvia miltiorrhiza cultivated in Jizhou District as test material,HPLC was used to study the content of its pharmacological constituents( tanshinone II A,tanshinone I and danshinolic acid B). [Results] For Salvia miltiorrhiza cultivated in Jizhou District,tanshinone II A content was 0. 014%,tanshinone I content was 0. 4212%,and danshinolic acid B content was 3. 018%,higher than the standard specified in Pharmacopoeia( 2015).[Conclusions]The quality of Salvia miltiorrhiza met the requirements of medication.

Nitrate confers rice adaptation to high ammonium by suppressing its uptake but promoting its assimilation

Dear Editor,earEaltor,Nitrogen(N)is the most important macronutrient driving plant growth and development.For higher plants,inorganic N including nitrate(NO_(3)^(-))and ammonium(NH_(4)^(+))are predominant N sources(Hu et al.,2023).Nitrate needs to be firstly reduced into ammonium to implement its assimilation,thus requiring a higher energy consumption than ammonium,making ammonium more cost effective for plants.However,ammonium usually causes severe growth retardation of plants under high concentration,which is known as ammonium toxicity.The concentrations of nitrate and ammonium greatly vary in different soil environments.Nitrate is the major inorganic N form in dry land,while ammonium accounts for thehighest proportion of inorganic N in the paddy field,where nitrification is suppressed(Haynes and Goh,1978).Although nitrogen is generally one of the most important contributing factors for yield increase,irrational fertilization strategies can cause negative effects.

Nitrogen assimilation in plants:current status and future prospects

Nitrogen(N)is the driving force for crop yields;however,excessive N application in agriculture not only increases production cost,but also causes severe environmental problems.Therefore,comprehensively understanding the molecular mechanisms of N use efficiency(NUE)and breeding crops with higher NUE is essential to tackle these problems.NUE of crops is determined by N uptake,transport,assimilation,and remobilization.In the process of N assimilation,nitrate reductase(NR),nitrite reductase(Ni R),glutamine synthetase(GS),and glutamine-2-oxoglutarate aminotransferase(GOGAT,also known as glutamate synthase)are the major enzymes.NR and Ni R mediate the initiation of inorganic N utilization,and GS/GOGAT cycle converts inorganic N to organic N,playing a vital role in N assimilation and the final NUE of crops.Besides,asparagine synthetase(ASN),glutamate dehydrogenase(GDH),and carbamoyl phosphate synthetase(CPSase)are also involved.In this review,we summarize the function and regulation of these enzymes reported in three major crops—rice,maize,and wheat,also in the model plant Arabidopsis,and we highlight their application in improving NUE of crops via manipulating N assimilation.Anticipated challenges and prospects toward fully understanding the function of N assimilation and further exploring the potential for NUE improvement are discussed.

Modulation of nitrate-induced phosphate response by the MYB transcription factor RLI1/HINGE1 in the nucleus

The coordinated utilization of nitrogen(N)and phosphorus(P)is vital for plants to maintain nutrient balance and achieve optimal growth.Previously,we revealed a mechanism by which nitrate induces genes for phosphate utilization;this mechanism depends on NRT1.1B-facilitated degradation of cytoplasmic SPX4,which in turn promotes cytoplasmic-nuclear shuttling of PHR2,the central transcription factor of phosphate signaling,and triggers the nitrate-induced phosphate response(NIPR)and N-P coordinated utilization in rice.In this study,we unveiled a fine-tuning mechanism of NIPR in the nucleus regulated by Highly Induced by Nitrate Gene 1(HINGE1,also known as RLI1),a MYB-transcription factor closely related to PHR2.RLI1/HINGE1,which is transcriptionally activated by PHR2 under nitrate induction,can directly activate the expression of phosphate starvation-induced genes.More importantly,RLI1/HINGE1 competes with PHR2 for binding to its repressor proteins in the nucleus(SPX proteins),and consequently releases PHR2 to further enhance phosphate response.Therefore,RLI1/HINGE1 amplifies the phosphate response in the nucleus downstream of the cytoplasmic SPX4-PHR2 cascade,thereby enabling fine-tuning of N-P balance when nitrate supply is sufficient.