Plant Productions

Optimization of antibiotics during co-culture of Petunia hybrida leaf explants and Agrobacterium tumefaciens to increase gene transgenic efficiency

Azadeh Khadem; zahra sargazi moghaddam; Mahboobeh yazdi; Mahdiyeh Kharrazi; Ahmad Sharifi

Volume 46, Issue 3 , December 2023, , Pages 353-366

https://doi.org/10.22055/ppd.2024.43645.2099

Abstract

IntroductionGene transfer by the Agrobacterium tumefaciens is one of the well-known methods for the production of the transgenic plants. The success rate of this method depends on the ... Read More IntroductionGene transfer by the Agrobacterium tumefaciens is one of the well-known methods for the production of the transgenic plants. The success rate of this method depends on the growth of the bacteria and the regeneration of the plant explants. Among these variables, the antibiotics functioning as bacterial growth regulators have mainly inhibitory effects on the plant growth. Therefore, the introduction of a successful gene transfer method which is mediated by Agrobacterium requires the identification of an antibiotic with an inhibitory effect on the growth of the bacteria and also has a minimal effect on the regeneration of the explants and the growth of the transgenic plants. In this regard, the present research was conducted with the aim of optimizing the antibiotic used in co-culture in order to increase the transgenic yield of Petunia hybrida. For this purpose, a variety of antibiotics in different concentrations were tested.Materials and MethodsThe growth rate of A. tumefaciens in the different medium culture with two suitable pH for the bacterial growth (7) and plant growth (5.7) in the presence of 500 mg/L of the cefotaxime antibiotic as a common antibiotic for controlling bacterial growth was investigated. Sensitivity tests were run. The second experiment was conducted to investigate the effect of five different antibiotics in three concentration on the regeneration of the petunia leaf explants. Results and DiscussionThe results of the bacterial sensitivity test showed that the type and concentration of salt and the pH of the medium culture significantly affect the growth rate of the bacteria. So that the highest rate of bacterial growth was observed in LB and MH medium culture at pH=7, but on the contrary, no growth was observed in MS medium culture. In addition, two antibiotics of cefotaxime and ceftriaxone at 700 mg/L concentration had the highest inhibitory effect on the bacterial growth. The results of this experiment showed that different antibiotics have different effects on the regeneration of explants and the growth of the seedlings. According to the results, the highest volume of callus and also the highest percentage of regeneration were observed in the presence of ceftizoxime and coamoxiclav antibiotics in all three concentrations and cefepime at 300 mg/L. The highest number of the seedlings was also observed in the treatment of ceftizoxime at concentrations of 300 and 500 mg/L with an average of 7.70 seedlings. In relation to the seedling growth traits, the highest number and length of root per seedling were observed in ceftizoxime at 300 mg/l and cefotaxime at the same concentration, respectively. The highest dry weight belonged to the grown seedlings in the presence of co-amoxiclav antibiotic followed by the ceftizoxime (300 mg/L).Conclusion The overall results of these experiments show that the seedlings which are grown in the presence of ceftizoxime antibiotic had a good growth rate, while the use of this antibiotic was effective in inhibiting the growth of A. tumefaciens. Therefore, we recommend that the use of ceftizoxime antibiotic in the co-culture process will be effective in increasing the efficiency of the gene transfer. Further studies will solidify these results..

Optimization of in vitro Cultural Conditions of Haworthia (Haworthia Cooperi Baker)

Mahdiyeh Kharrazi; Maryam Moradian; Nasim Safari; Azadeh Khadem; Ahmad Sharifi

Volume 45, Issue 1 , May 2022, , Pages 53-66

https://doi.org/10.22055/ppd.2021.36744.1971

Abstract

IntroductionHaworthia cooperi is a type of ornamental succulent that is propagated by separating the offsets and leaf cuttings. Due to the small number of produced offsets by the mother ... Read More IntroductionHaworthia cooperi is a type of ornamental succulent that is propagated by separating the offsets and leaf cuttings. Due to the small number of produced offsets by the mother plant, the rate of propagation in this way is low, so micropropagation can be considered as an effective and efficient way to propagate this ornamental plant. Materials and MethodsFor this purpose, in the present study, the growth response of Haworthia cooperi plants to in vitro culture conditions in 1398 in the research laboratory of the Department of Ornamental Plants Biotechnology was investigated. This research was conducted in the form of three separate experiments based on a completely randomized design with three replications. In the first experiment, the effects of cytokinin type (BA and KIN) and concentration of cytokinin (0, 1, 2 and 3 mg/l) on leaf explant regeneration were evaluated. In the second experiment, the effects of culture medium type (MS and B5) and concentration of culture medium (0.5 and 1) on plantlet propagation were investigated. In the third experiment, plantlet rooting rate was evaluated under the influence of auxin type (NAA, IBA and IAA) and concentration (0, 0.5, 1 and 1.5 mg/l) of auxin. Results and DiscussionThe results showed that application of BA and KIN hormones increased the number of regenerated plantlets. The highest number of plantlets (5.50) was recorded in the MS medium containing 3 mg/l BA with 0.5 mg/l NAA. Plantlets cultured in the hormone-free culture medium had no regenerated symptoms. The number of plantlets increased with increasing the concentration of both hormones, but BA hormone in the culture medium had a greater effect on this factor. Lack of cytokinin hormone application in culture medium led to root production; however, the use of BA and KIN hormones in the culture medium reduced the number of produced roots by plantlets. The highest number of roots was recorded in hormone-free culture medium and the lowest number was recorded in the high concentration of BA hormone. The reduction in the number of roots in the culture medium containing BA was greater than the culture medium containing KIN. The of the second experiment revealed that use of MS culture medium showed better performance compared to B5 culture medium. Thus, the highest dry weight of plantlet and roots was obtained at full concentration of MS culture medium. The results of rooting stage showed that the presence of NAA hormone in the culture medium increased rooting in the plant. However, the produced roots in this culture medium were so thick that these roots are not suitable for plant growth in the acclimation stage. Therefore, considering the number and length of produced roots, the optimal culture medium for plantlet rooting is ½ MS culture medium containing 1 mg/l IAA hormone. Determining the best type of culture medium and optimizing it for regeneration, proliferation and rooting is one of the important factors in success in vitro culture conditions. Based on the results of the current study, application of MS medium containing 3 mg/l BA with 0.5 mg/l NAA was so effective for regeneration phase of this ornamental plant. Also, the efficiency of MS culture medium in the process of propagation of this plant is most likely related to the suitability of the components of this culture medium. In general, due to the presence of plant growth regulators such as NAA, IBA and IAA in the culture medium, rooting is induced in plantlets. In the present study, the use of IAA hormone at a concentration of 1 mg/l was a suitable hormonal combination for rooting in this ornamental plant. ConclusionBased on the results of the present study, the application of MS culture medium containing 3 mg/l BA with 0.5 mg/l NAA is recommended for regeneration and proliferation of tissue culture plantlets of Haworthia cooperi. It is also recommended to use ½ MS culture medium containing 1 mg/l of IAA hormone for rooting of tissue culture plantlets.

Effect of Kind and Plant Growth Regulator Composition on Micropropagation of Three Begonia Species

Fateme Hosseini; Nasrin Moshtaghi; Ahmad Sharifi; Abdolreza Bagheri; Hasan Marashi; Fateme Keykha Akhar

Volume 44, Issue 1 , June 2021, , Pages 25-36

https://doi.org/10.22055/ppd.2019.27321.1660

Abstract

AbstractBackground and ObjectivesIntroduction: Begonia is one of the most important ornamental plants around the world. Begonia species are grown for their attractive leaves and flowers ... Read More AbstractBackground and ObjectivesIntroduction: Begonia is one of the most important ornamental plants around the world. Begonia species are grown for their attractive leaves and flowers in indoor and outdoor conditions. The tissue culture technique is an alternative method for the propagation of begonia species. It can overcome the difficulties of vegetative propagation and lead to high-quality and uniform planting material under disease-free conditions irrespective of the season and weather. In begonia tissue culture, different species showed diverse responses to the medium composition, especially plant growth regulators. Also, lack of information on large scale micropropagation of begonia in Iran highlights research on optimization of begonia species propagation on in vitro culture condition. Thus, in this study the effect of plant growth regulators on shoot regeneration and proliferation of three begonia species (B. elatior‌‌, B. soli-mutata, and B. tiger), the impact of Auxin and sucrose on rooting of B. elatior‌‌and diferent pot substrate on acclimation were survived. Materials and MethodsIn this experiment, petioles of three begonia species (B. soli-mutata, B. elatior, and B. tiger) were used as explant sources. So explants were washed under tab water for 30 mins and sterilized with 1.5% sodium hypochlorite for 15 mins. After sterilization, thin Cell Layers (TCL) with 2 mm thickness were prepared from petiols and cultured in MS medium with different concentrations (0.2, 1, 2 mg/L) of kinetin (kin) or thidiazuron (TDZ) in combination with NAA (0, 0.2 mg/L). Next, the effect of auxin type (1 mg/L of IAA, IBA, and NAA) and sucrose concentration (30 and 40 g/l) on rooting of B. elatior plantlets were determined on in vitro condition. For plantlet acclimation, sand, CocoPeat perlite mixture (1:1 ratio), and Peat moss as pot substrate were evaluated. Results The results showed the shoot regeneration response of begonia species to the plant growth regulators of the medium, and there was a significant difference between them in all of the species adding the Auxin to the medium increased shoot regeneration. In B. elatior and B. tiger lacking of NAA to the medium caused no shoot regeneration. Maximum shoot regeneration (100%) was achieved in B. soli-mutata species in medium containing 1 or 2 mg/L of kin and 0.2 mg/l NAA, in B. elatior at 1 or 2 mg/L of TDZ in combination with 0.2 mg/l NAA and in B. tiger at 2 mg/L of both plant growth regulator (TDZ and Kin) and 0.2 mg/l NAA. DiscussionFor rooting B. elatior, MS medium containing 1 mg/L IAA plus 30 g/L sucrose was the best and favorite acclimation was acheived in cocopeat perlite mixture and peat moss pot substrate.

Effect of Medium, Iron-Chelating Agent and Plant Growth Regulator on Micropropagation of Pomegranate (Punica granatum L.)

Tayyabeh Eshaghi Sanayi; Mohammad Zare Mehrjerdi; Ahmad Sharifi

Volume 43, Issue 2 , September 2020, , Pages 309-322

https://doi.org/10.22055/ppd.2019.27232.1655

Abstract

Abstract Background and ObjectivesPomegranate (Punica granatum L.) is a fruit tree that originated from Iran and has been cultivated since ancient times. Classical propagation ... Read More Abstract Background and ObjectivesPomegranate (Punica granatum L.) is a fruit tree that originated from Iran and has been cultivated since ancient times. Classical propagation methods of pomegranate like stem cutting and air-layering have seasonal timing and a higher risk of pathogens contamination. Micropropagation by tissue culture could be considered a method for production of steady and pathogen free plants in this species. However, factors such as lack of rooting and browning due to the oxidation of phenolic components under invitro culture can limit its micropropagation efficiency. To overcome these problems, the effects of various factors, including media, iron-chelating agent, and plant growth regulators, were examined on the pomegranate propagation through in vitro conditions. Materials and MethodsTo obtain sterile explants, stem cuttings of the pomegranate cultivar Shishe Cap Ferdos were treated with different disinfection treatments. Then, the effect of DKW, MS and WPM basal media and two types of iron chelating agents (EDDHA and EDTA) were evaluated to decrease the secretion of phenolic compound from explant and subsequent browning. In the next step, the effect of plant growth regulators treatments, including 1-2 mg/l of BA in combination with 0.2 mg/l of various auxins (IAA, NAA or IBA) on shoot development and proliferation was studied. After that, rooting of regenerated plantlets was explored at WPM medium supplemented with 0.5 or 1 mg/l IAA, IBA or NAA. Finally, pomegranate plantlets were transferred to different soil mixtures, including eat moss and perlite (2:1), cocopeat and perlite (2:1), peat moss and vermiculite (2:1) and cocopeat and vermiculite (2: 1) for acclimation. ResultsThe results indicated that sterilization with 2% sodium hypochlorite for 15 minutes followed by washing with 1g/l carbendazim for 10 minutes, and finally culturing the explants in the medium containing 1 g/l carbendazim had the best effect on reducing explant contamination. Among different basal media and iron chelating agents, WPM resulted in the lowest explant browning. Also, EDDHA was more efficient compared to the EDTA so that explant browning in WPM media containing Fe-EDDHA reduced by 55.8% compared to DKW containing Fe-EDTA. In addition, WPM basal medium supplemented with 2 mg/l BA and 0.2 mg/l IAA induced higher shoot proliferation in node explant of pomegranate. For rooting treatments, after 45 days, WPM supplemented with 1 mg/l IAA induced higher rooting percentage, root length and number in pomegranate shoots. Among different acclimatizing treatments, plantlets that grew on peat moss and perlite (2: 1) had the highest leaf number and stem height. DiscussionOur results suggested that the medium composition and chelating agent affected pomegranate explants browning. EDDHA appears to have a positive effect on reducing browning by chelating copper, which is a phenol oxidase cofactor. Also, EDDHA was more efficient compared to the EDTA.

Effect of hormonal compositions on micropropagation of fifteen cultivars of Gerbera (Gerbera jamesonii Bolus ex Hooker f.)

M. Kharrazi; A. Sharifi; F. Keykha Akhar; A. Bagheri; Y. M. Moradian

Volume 40, Issue 4 , March 2018, , Pages 91-102

https://doi.org/10.22055/ppd.2018.13443

Abstract

Background and objectives Gerbera is one of the most important ornamental plants that used as cut flower and potted plant. The common methods of propagation does not have performance ... Read More Background and objectives Gerbera is one of the most important ornamental plants that used as cut flower and potted plant. The common methods of propagation does not have performance for supplying the global demand of this ornamental plant. So the most common method for commercial propagation of gerbera is micropropagation. Material and methods In this study, effect of different hormonal treatments and cultivars on micropropagation and rooting of gerbera shoot tip explant was evaluated. Shoot tip explants were first washed with running tap water for 30 min then they were surface sterilized by dipping in 1.5% sodium hypochlorite solution for 5 min and rinsed with sterile distilled water, followed by immersing in 0.1 % mercuric chloride solution for 5 min. After Sterilization with mercuric chloride solution, capitulum was washed with sterile distilled water. Subsequent washing was done with sterile distilled water three times. Sterilization with sodium hypochlorite solution and mercuric chloride and final rinse with sterile distilled water were done under laminar air flow hood. In the proliferation phase, MS medium containing 1 mg/l BA or KIN in combination with 0.1 mg/l IAA or hormone-free medium, 3% sucrose, 8 g/l agar was used. For rooting of propagated plantlets, ½ MS medium containing 1 mg/l IBA, IAA, 2IP or ½ MS hormone-free medium, 3% sucrose, 8 g/l agar was used. After 4 weeks different parameters such as number of plantlet, height of plantlet, number of main root, root length, number of secondary root were measured. Cultivars that was used in this experiment are as follows: 1: ‘Aventura’, 2: ‘Barones’, 3: ‘Mayfair’, 4: ‘Cacharelle’, 5: ‘Real’, 6: ‘Sorbet’, 7: ‘Dalma’, 8: ‘Goldy’, 9: ‘Panama’, 10: ‘Lancaster’, 11: ‘Dune’, 12: ‘Bellavue’, 13: ‘Blinddate’, 14: ‘Applause’, 15: ‘Sunway’. Results and discussion The results showed that in all studied cultivars, the medium containing BA lead to producing the highest number of plantlet. So using the medium containing BA in propagation phase is recommended for obtaining the highest number of plantlet. Also the results showed that the rooting of gerbera plantlets was affected by cultivar and suitable hormonal composition for rooting of each cultivar was different. However, the appropriate acclimation of produced plantlets is so important. Although the length and number of roots in ½ MS medium was at the average of other treatments, but propagated plantlets from this medium, had a suitable acclimation capacity. Moreover, additional cost for utilization of rooting hormones was reduced and therefore commercial production cost decreased. So application of this medium for rooting of different gerbera cultivars is recommended. Finally acclimation of rooted plantlet was done in cocopeat and perlite medium, with 95% success.

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Author = Ahmad Sharifi

Optimization of antibiotics during co-culture of Petunia hybrida leaf explants and Agrobacterium tumefaciens to increase gene transgenic efficiency

Abstract

Optimization of in vitro Cultural Conditions of Haworthia (Haworthia Cooperi Baker)

Abstract

Effect of Kind and Plant Growth Regulator Composition on Micropropagation of Three Begonia Species

Abstract

Effect of Medium, Iron-Chelating Agent and Plant Growth Regulator on Micropropagation of Pomegranate (Punica granatum L.)

Abstract

Effect of hormonal compositions on micropropagation of fifteen cultivars of Gerbera (Gerbera jamesonii Bolus ex Hooker f.)

Abstract