Induction of Transgenic Hairy Roots in Trigonella foenumgraceum Co-cultivated with Agrobacterium rhizogenes Harboring a GFP Gene

Zeynab Shahabzadeh,Bahram. Heidari,Reza Faramarzi Hafez 한국작물학회 2013 Journal of crop science and biotechnology Vol.16 No.4

An important feature of A. rhizogenes-induced hairy roots is their unique ability for investigation of gene function and productionof secondary metabolites such as diosgenin in fenugreek. In order to evaluate the transformation frequency and the efficiency oftransgenic hairy root induction, leaf and stem explants from two fenugreek ecotypes, Karaj and Bushehr, were infected with threeconcentrations of OD600= 0.8, 1.2, and 1.6 of A. rhizogenes strain K599 harboring a GFP gene. Regardless of ecotype, the ability ofstem explants for the induction of hairy roots (8.09) and the transformation frequency (81.3%) was higher compared with leafexplants with the values of 5.97 and 71.88%, respectively. The number of transgenic GFP-positive hairy roots ranged from 4.2 to13.5 in the Karaj ecotype and 3.8 to 9.9 in Bushehr. The highest transgenic hairy root (8.76), the transformation frequency (79.76%),and the growth rate of transgenic roots (0.77 d-1) were obtained from infection with K599 at OD600= 1.2, while the lowest belonged tothe bacterial concentration of OD600=1.6. Although the ecotype Bushehr had lower total roots (7.53) and transgenic hairy roots (6.08),it showed higher transformation frequency (79.56%) than Karaj (73.63%). Therefore, the results indicate the importance of genotype,type of explant and bacterial concentration in breeding for induction of transgenic hairy roots and consequently, production of secondarymetabolites in fenugreek.

Regenerating Salt Tolerant Saffron (Crocus sativus) Using Tissue Culture with Increased Pharmaceutical Ingredients

Shahabzadeh, Zeynab,Heidari, Bahram,Dadkhodaie, Ali 한국작물학회 2013 Journal of crop science and biotechnology Vol.16 No.3

Due to its vegetative reproduction, saffron has a narrow genetic base and induced in vitro variations provide opportunities for expanding new cultivars. The objectives of this study were to evaluate sodium azide-induced variations in saffron's corm culture in order to increase salt tolerance and pharmaceutical ingredients. Corm explants from the well-known ecotypes, Estahban and Kashmar, were subjected to various concentrations of sodium azide ($NaN_3$) (0.09, 0.12, and 0.22 mg $L^{-1}$) and NaCl (1.5, 2.5, and 4.0 dS equivalent to 0.07, 0.12, and 0.20 g NaCl in 100 mL water) in Murashige and Skoog medium supplemented with 1 mg $L^{-1}$ 2-4-D, 1 mg $L^{-1}$ BAP, and 30 g $L^{-1}$ sucrose and in a second pot culture experiment. The active pharmaceutical ingredients (crocin, picrocrocin, and safranal) were measured by high-performance liquid chromatography (HPLC). Variations in sodium azide-treated plants were more broadened for callus fresh weight (0.57 - 7.57 g), embryo weight (1.24 - 10.29 g), and regenerated seedlings (3.0 - 21.25) compared with those (0.12 - 3.77 g, 0.56 - 4.56 g, and 0.25 - 11.50, respectively) that were not treated with sodium azide. Under 0.20% salt, flowering failed in some of plants developed from sodium azide-untreated corms. HPLC analysis indicated wider ranges for crocin (11.92 - 18.03 mg $g^{-1}$), picrocrocin (8.99 - 14.76 mg $g^{-1}$), and safranal (2.13 - 7.36 mg $g^{-1}$) in sodium azide-treated plants compared to the ranges (0.0 - 16.1, 0.0 - 12.5, and 0.0 - 6.66 mg $g^{-1}$, respectively) in untreated plants. From a breeding perspective, induced variations found in this study would be useful to improve saffron's quality and salt tolerance.

Regenerating Salt Tolerant Saffron (Crocus sativus) Using Tissue Culture with Increased Pharmaceutical Ingredients

Zeynab Shahabzadeh,Bahram. Heidari,Ali Dadkhodaie 한국작물학회 2013 Journal of crop science and biotechnology Vol.16 No.3

Due to its vegetative reproduction, saffron has a narrow genetic base and induced in vitro variations provide opportunities for expanding new cultivars. The objectives of this study were to evaluate sodium azide-induced variations in saffron’s corm culture in order to increase salt tolerance and pharmaceutical ingredients. Corm explants from the well-known ecotypes, Estahban and Kashmar, were subjected to various concentrations of sodium azide (NaN3) (0.09, 0.12, and 0.22 mg L-1) and NaCl (1.5, 2.5, and 4.0dS equivalent to 0.07, 0.12, and 0.20 g NaCl in 100 mL water) in Murashige and Skoog medium supplemented with 1 mg L-1 2-4-D,1 mg L-1 BAP, and 30 g L-1 sucrose and in a second pot culture experiment. The active pharmaceutical ingredients (crocin, picrocrocin,and safranal) were measured by high-performance liquid chromatography (HPLC). Variations in sodium azide-treated plants were more broadened for callus fresh weight (0.57 - 7.57 g), embryo weight (1.24 - 10.29 g), and regenerated seedlings (3.0 - 21.25)compared with those (0.12 - 3.77 g, 0.56 - 4.56 g, and 0.25 - 11.50, respectively) that were not treated with sodium azide. Under 0.20% salt, flowering failed in some of plants developed from sodium azide-untreated corms. HPLC analysis indicated wider ranges for crocin (11.92 - 18.03 mg g-1), picrocrocin (8.99 - 14.76 mg g-1), and safranal (2.13 - 7.36 mg g-1) in sodium azide-treated plants compared to the ranges (0.0 - 16.1, 0.0 - 12.5, and 0.0 - 6.66 mg g-1, respectively) in untreated plants. From a breeding perspective,induced variations found in this study would be useful to improve saffron's quality and salt tolerance

Induction of Transgenic Hairy Roots in Trigonella foenum-graceum Co-cultivated with Agrobacterium rhizogenes Harboring a GFP Gene

Shahabzadeh, Zeynab,Heidari, Bahram,Hafez, Reza Faramarzi 한국작물학회 2013 Journal of crop science and biotechnology Vol.16 No.4

An important feature of A. rhizogenes-induced hairy roots is their unique ability for investigation of gene function and production of secondary metabolites such as diosgenin in fenugreek. In order to evaluate the transformation frequency and the efficiency of transgenic hairy root induction, leaf and stem explants from two fenugreek ecotypes, Karaj and Bushehr, were infected with three concentrations of $OD_{600}=0.8$, 1.2, and 1.6 of A. rhizogenes strain K599 harboring a GFP gene. Regardless of ecotype, the ability of stem explants for the induction of hairy roots (8.09) and the transformation frequency (81.3%) was higher compared with leaf explants with the values of 5.97 and 71.88%, respectively. The number of transgenic GFP-positive hairy roots ranged from 4.2 to 13.5 in the Karaj ecotype and 3.8 to 9.9 in Bushehr. The highest transgenic hairy root (8.76), the transformation frequency (79.76%), and the growth rate of transgenic roots ($0.77d^{-1}$) were obtained from infection with K599 at $OD_{600}=1.2$, while the lowest belonged to the bacterial concentration of $OD_{600}=1.6$. Although the ecotype Bushehr had lower total roots (7.53) and transgenic hairy roots (6.08), it showed higher transformation frequency (79.56%) than Karaj (73.63%). Therefore, the results indicate the importance of genotype, type of explant and bacterial concentration in breeding for induction of transgenic hairy roots and consequently, production of secondary metabolites in fenugreek.

Molecular characterization of tall fescue germplasm using SNP markers: population structure, linkage disequilibrium, and association mapping of yield-related traits

Shahabzadeh Zeynab,Darvishzadeh Reza,Mohammadi Reza,Jafari Morad,Alipour Hadi 한국식물생명공학회 2023 Plant biotechnology reports Vol.17 No.2

Tall fescue (Festuca arundinacea) from Poaceae family is an allohexaploid (2n=6x=42) and cross pollinated plant that is used throughout the world as a forage plant. This research was conducted to identify the genomic regions associated with 10 agro-morphological traits using 15,881 SNP markers in a diverse tall fescue germplasm. UPGMA, Structure, and PCA analyses illustrated high genetic differentiation between forage, turf, and hybrid tall fescue samples. Correcting r2 for population structure and kinship effects revealed the fact that the r2v (corrected for kinship) is much smaller than r2 (uncorrected) illustrating the necessity of removing the effect of coancestry to reduce the inflation of r2 estimates. Analysis of linkage disequilibrium revealed that the amount of SNP pairs in LD differs between sub-genomes and chromosomes and any increase in the distance among SNPs reduces the amount of SNP pairs in LD. A total of 111 and 17 marker-trait associations were identified via GLM and MLM methods for the investigated traits, respectively. Most of the significant MTAs belonged to plant height and days to anthesis. Gene ontology results revealed that 34% of MTAs were located in protein-coding regions. Most of the MTAs were located on chromosomes 1B and 1D. The MTAs for plant height and days to anthesis located within the coding regions can be utilized in genome-based breeding in tall fescue breeding programs.

Investigation of the Efficiency of Direct and Indirect Regeneration in Evening Primrose (Oenothera biennis)

Hafez, Reza Faramarzi,Shahabzadeh, Zeynab,Heidari, Bahram,Ghadimzadeh, Morteza 한국작물학회 2013 Journal of crop science and biotechnology Vol.16 No.4

As a medicinal plant, the importance of evening primrose (Oenothera biennis L.) is due to its unsaturated fatty acids in the seeds and roots, and also oenotherine and comfarol in the leaves. Low germination and difficulties in seed production are the main problems encountered with growing this plant in the field. As an alternative approach, an in vitro experiment was set up for the evaluation of evening primrose production via direct and indirect regeneration of the cultivars NC-1 and VNK. For callogenesis and direct regeneration, the explants from the apical bud and petiole were cultured on MS medium supplemented with 0.25, 0.75, and $1.25mg\;L^{-1}$ of both BAP and Kinetin (KIN). Indirect regeneration was performed by placing apical buds, petioles, and leaf explants on MS medium supplemented with 0.5 and $1mg\;L^{-1}$ 2,4-D and 0.5, 1, and $1.25mg\;L^{-1}$ of both BAP and KIN. The highest shoot induction from direct regeneration was obtained with apical bud explants of VNK treated with $0.75mg\;L^{-1}$ BAP. The highest callus weight (3.17 g) obtained from indirect regeneration was with petiole explants treated with $1mg\;L^{-1}$ 2, 4-D and $1mg\;L^{-1}$ BAP in VNK cultivars. The highest number of torpedo embryogenic clusters (23.8) was obtained from the VNK petiole explants treated with $0.5mg\;L^{-1}$ 2, 4-D and $1.25mg\;L^{-1}$ BAP. BAP had higher positive effects on in vitro production of evening primrose than KIN in both direct and indirect regeneration. In general, results indicated that VNK was more potent for regeneration than NC-1 and concentrations of $0.75mg\;L^{-1}$ BAP for direct and $0.5mg\;L^{-1}$ 2, 4-D and $1.25mg\;L^{-1}$of BAP for indirect regeneration had a higher efficiency for increasing in vitro production of evening primrose.

Investigation of the Efficiency of Direct and Indirect Regeneration in Evening Primrose (Oenothera biennis)

Reza Faramarzi Hafez,Bahram Heidari,Zeynab Shahabzadeh,Bahram. Heidari 한국작물학회 2013 Journal of crop science and biotechnology Vol.16 No.4

As a medicinal plant, the importance of evening primrose (Oenothera biennis L.) is due to its unsaturated fatty acids in the seedsand roots, and also oenotherine and comfarol in the leaves. Low germination and difficulties in seed production are the main problemsencountered with growing this plant in the field. As an alternative approach, an in vitro experiment was set up for the evaluationof evening primrose production via direct and indirect regeneration of the cultivars NC-1 and VNK. For callogenesis and directregeneration, the explants from the apical bud and petiole were cultured on MS medium supplemented with 0.25, 0.75, and 1.25 mgL-1 of both BAP and Kinetin (KIN). Indirect regeneration was performed by placing apical buds, petioles, and leaf explants on MSmedium supplemented with 0.5 and 1 mg L-1 2,4-D and 0.5, 1, and 1.25 mg L-1 of both BAP and KIN. The highest shoot inductionfrom direct regeneration was obtained with apical bud explants of VNK treated with 0.75 mg L-1 BAP. The highest callus weight(3.17 g) obtained from indirect regeneration was with petiole explants treated with 1 mg L-1 2, 4-D and 1 mg L-1 BAP in VNK cultivars. The highest number of torpedo embryogenic clusters (23.8) was obtained from the VNK petiole explants treated with 0.5 mg L-12, 4-D and 1.25 mg L-1 BAP. BAP had higher positive effects on in vitro production of evening primrose than KIN in both direct andindirect regeneration. In general, results indicated that VNK was more potent for regeneration than NC-1 and concentrations of 0.75mg L-1 BAP for direct and 0.5 mg L-1 2, 4-D and 1.25 mg L-1of BAP for indirect regeneration had a higher efficiency for increasing invitro production of evening primrose