Sodium Nitroprusside Stimulates Growth and Shoot Regeneration in Chrysanthemum

Muthukrishnan Arun,AUNG HTAY NAING,전수민,TRINH NGOC AI,Thanda Aye,김창길 한국원예학회 2017 Horticulture, Environment, and Biotechnology Vol.58 No.1

In this study, we demonstrate that the nitric oxide (NO) donor sodium nitroprusside (SNP) improvesplant regeneration in chrysanthemums. Internode explants of three different chrysanthemum cultivars, ‘White ND’,‘White wing × Peach ND’, and ‘Hunt × Lemon ND’, were cultured on shoot induction medium (SIM) containingvarious concentrations of N6–benzyladenine (2.22 or 4.44 μM BA) and SNP (0.83–6.71 μM) individually andin combination. Most combinations of BA and SNP significantly improved the morphogenetic potential of internodeexplants and enhanced shoot regeneration in all three chrysanthemum cultivars compared to treatment with BAalone. The cultivar ‘White wing × Peach ND’ displayed the highest regeneration response (98.3%) and shoot regenerationrate (27.3 shoots/explant) in SIM containing optimal BA (4.44 μM) and SNP (0.83 μM) concentrations within30 days of culture. Individual shoots of 'White wing × Peach ND' were transferred to root induction medium(RIM) containing various concentrations of SNP (0.83–6.71 μM) alone. Shoots rooted in the presence of SNPresulted in healthy plantlets within 30 days of culture with improved root (number of roots/shoot, root length,and fresh weight) and shoot (number of leaves, shoot length, and fresh weight) growth characteristics comparedto the control. In addition, the regeneration procedure described in this study only requires a short duration (60 days)to obtain rooted plantlets from internode explants of chrysanthemums. Our results suggest that supplementationof chrysanthemum regeneration medium with SNP enhances shoot regeneration and improves plant growth,overcoming problems associated with propagation and genetic transformation.

Application of sonication in combination with vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars.

Arun, Muthukrishnan,Subramanyam, Kondeti,Mariashibu, Thankaraj Salammal,Theboral, Jeevaraj,Shivanandhan, Ganeshan,Manickavasagam, Markandan,Ganapathi, Andy Humana Press 2015 Applied biochemistry and biotechnology Vol.175 No.4

<P>Soybean is a recalcitrant crop to Agrobacterium-mediated genetic transformation. Development of highly efficient, reproducible, and genotype-independent transformation protocol is highly desirable for soybean genetic improvement. Hence, an improved Agrobacterium-mediated genetic transformation protocol has been developed for cultivar PK 416 by evaluating various parameters including Agrobacterium tumefaciens strains (LBA4404, EHA101, and EHA105 harboring pCAMBIA1304 plasmid), sonication duration, vacuum infiltration pressure, and vacuum duration using cotyledonary node explants of soybean prepared from 7-day-old seedlings. The transformed plants were successfully developed through direct organogenesis system. Transgene expression was assessed by GUS histochemical and gfp visual assays, and integration was analyzed by PCR and Southern blot hybridization. Among the different combinations and durations evaluated, a maximum transformation efficiency of 18.6 % was achieved when the cotyledonary node explants of cv. PK 416 were sonicated for 20 s and vacuum infiltered for 2 min at 250 mmHg in A. tumefaciens EHA105 suspension. The amenability of the standardized protocol was tested on four more soybean cultivars JS 90-41, Hara Soy, Co 1, and Co 2 in which all the cultivars responded favorably with transformation efficiency ranging from 13.3 to 16.6 %. The transformation protocol developed in the present study would be useful to transform diverse soybean cultivars with desirable traits.</P>

Combinatorial expression of transcription factor genes <i>B</i>-<i>Peru</i> and <i>mPAP1</i> enhances anthocyanin accumulation in transgenic <i>Petunia hybrid</i>

Ai, Trinh Ngoc,Arun, Muthukrishnan,Naing, Aung Htay,Lim, Sun-Hyung,Kim, Chang Kil Elsevier 2016 Scientia horticulturae Vol.200 No.-

<P><B>Abstract</B></P> <P>The present study aimed to determine the role of transcription factors (bHLH and MYB) in enhancing anthocyanin production in <I>Petunia</I> ‘Mirage rose’. Initially, we optimized an <I>Agrobacterium</I>-mediated transformation protocol to over-express transcription factors (<I>B-Peru</I>, <I>mPAP1</I>, and <I>B-Peru</I> + <I>mPAP1</I>) in <I>Petunia.</I> Phosphinothricin (PPT) concentrations of 0.5, 1.0, and 1.5mgl<SUP>−1</SUP> were found to be ideal for the selection and regeneration of transformed shoots at different developmental stages. Clavamox (250mgl<SUP>−1</SUP>) efficiently eliminated <I>Agrobacterium</I> after co-cultivation (2 days) and favored maximum shoot regeneration. In addition, incubation for 30min in <I>Agrobacterium</I> suspension increased the number of transformed cells and resulted in improved regeneration in the selection medium. The transformed plants were successfully developed through a direct organogenesis system. The transformed plants were selected using BASTA<SUP>®</SUP> and the presence of transgenes was assessed using PCR. Visible anthocyanin accumulation was evident only in plantlets (shoots, stem, leaves, and roots) carrying the gene combination <I>B-Peru</I> <I>+</I> <I>mPAP1</I>. The expression of biosynthetic genes involved in the flavonoid pathway was analyzed using quantitative real-time PCR. Expression levels of <I>PAL</I>, <I>CHS</I>, <I>CHI</I>, <I>F3H</I>, <I>DFR</I>, and <I>ANS</I> were higher in the young and mature leaves of plants transformed with <I>B-Peru</I> <I>+</I> <I>mPAP1</I> compared to those transformed with <I>B-Peru</I>, <I>mPAP1,</I> and/or non-transformed plants. Furthermore, the highest level of anthocyanin was recorded in the leaves, stem, and roots of plants transformed with <I>B-Peru</I> <I>+</I> <I>mPAP1</I>. These results validate the combinatorial requirement of <I>B-Peru</I> and <I>mPAP1</I> to enhance anthocyanin content in <I>Petunia hybrida</I> ‘Mirage rose’.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We report an improved <I>Agrobacterium</I>-mediated transformation protocol for <I>Petunia</I>. </LI> <LI> PPT used throughout the selection procedure reduced the total number of escapes. </LI> <LI> Clavamox controlled <I>Agrobacterium</I> after co-cultivation and supported regeneration. </LI> <LI> The protocol was utilized to transfer anthocyanin regulatory genes into <I>Petunia</I>. </LI> <LI> Co-expression of <I>B-peru</I> and <I>mPAP1D</I> significantly improved anthocyanin in <I>Petunia</I>. </LI> </UL> </P>

Comparision of Anthocyanin Accumulation Levels in Petunia ‘Mirage Rose’ Hybrid using DifferentAnthocyanin Regulatory Genes by Agrobacterium Transformation

Ai Trinh Ngoc,Arun Muthukrishnan,Chang Kil Kim 한국원예학회 2015 한국원예학회 학술발표요지 Vol.2015 No.5

Effect of Sucrose on Anthocyanin Production of Transgenic Petunia ‘Mirage Rose’ from Different Anthocyanin Regulatory Genes

Trinh Ngoc Ai,Muthukrishnan Arun,Chang Kil Kim 한국원예학회 2015 한국원예학회 학술발표요지 Vol.2015 No.10

Expression of <i>RsMYB1</i> in <i>Petunia</i> enhances anthocyanin production in vegetative and floral tissues

Ai, Trinh Ngoc,Naing, Aung Htay,Arun, Muthukrishnan,Jeon, Su Min,Kim, Chang Kil International Society for Horticultural Science 2017 Scientia horticulturae Vol. No.

<P><B>Abstract</B></P> <P>Anthocyanin production enhanced by heterologous expression of R2R3 MYB transcription factors has been studied. However, little is known about validated information on the ability of <I>RsMYB</I> (an MYB gene from radish) to enhance anthocyanin production in a heterologous system. In the present study, heterologous expression of <I>RsMYB1</I> in <I>Petunia hybrida</I> ‘Mirage Rose’ enhanced anthocyanin production in vegetative and floral tissues such as leaves, stems, roots, and petals by transcriptional activation of anthocyanin biosynthetic genes and endogenous antocyanin regulatory genes. Line PM6 expressed higher transcript levels of <I>RsMYB1</I> than line PM2 and regulated transcript levels of the investigated genes largely than line PM2, whereas those regulated in wild type (WT) plants were the lowest. In addition, transcript levels of the genes detected using qualitative real-time polymerase chain reaction were found to be higher in petals, followed by leaves, stems and roots. Taken together, our results suggest that <I>RsMYB1</I> enhances anthocyanin production in vegetative and floral tissues of this cultivar, thus, we expect that heterologous expression of <I>RsMYB1</I> would help to modify flower color of other ornamental plants as well.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Expression of <I>RsMYB1</I> in <I>Petunia</I> enhanced anthocyanin production in vegetative and floral tissues. </LI> <LI> RsMYB1 enhances transcript levels of several anthocyanin biosynthetic genes and endogenous genes. </LI> <LI> Transcript levels of <I>RsMYB1</I> and investigated genes were found to be higher in leaves, flowers, followed by stems and roots. </LI> </UL> </P>